Note: Descriptions are shown in the official language in which they were submitted.
CA 02668580 2009-06-10
COMPOUNDS, METHODS, AND TREATMENTS
FOR ABNORMAL SIGNALING PATHWAYS
FOR PRENATAL AND POSTNATAL DEVELOPMENT
CROSS-REFERENCE TO RELATED APPLICATIONS
100011 This application is a continuation-in-part of co-pending US Patent
Application Serial
No. 11/591,398, filed November 1, 2006 and US Ser. No. 12/001,869, filed
December 13, 2007,
each of which is incorporated by reference in their entirety herein.
STATEMENT REGARDING FEDERALLY SPONSORED
RESEARCH OR DEVELOPMENT
[00021 Not Applicable
FIELD OF THE INVENTION
[00031 The present invention relates to the field use of inositol
stereoisomers and derivatives
thereof, especially phosphorylated and carboxylated derivatives thereof (the
invention
compounds, some of which are novel compounds) in a wide range of disease
states and medical
conditions. The invention further relates to use of the invention compounds to
modulate
signaling pathways in the development and growth of various cells. Still
further, the invention
relates to reducing the incidence of fetal defects due to aberrant pattern
formation during
gestation. Still further, the invention relates to prevention of fetal alcohol
syndrome with
invention compounds and combinations. This invention also relates to novel
inhibitors of alpha
Tumor Necrosis Factor and protein kinases, pharmaceutical compounds containing
inhibitors,
1
CA 02668580 2009-06-10
and methods for preparing these inhibitors. They can be useful for the
treatment of hepatitis A,
B, C,D, the treatments of chronic inflammation, chronic inflammatory arthritis
such as
rheumatoid arthritis, other chronic active diseases, chrohn's disease,
inflammatory bowel
disease, cancer, autoimmune diseases like lupus, sjrogren's, mixed connective
tissue disease, and
other pro-inflammatory cytokine-mediated diseases.
[0004] The invention still further relates to combination therapy of one or
more of the
invention compounds with a substantial range of additional compounds, such as
anti-cancer
therapeutic agents; etc. as further detailed herein (generally as means of
reducing tumor load or
distant metastasis, or as a synergistic inhibitor) as well as agents which
prevent or diminish the
aberrant cell from obtaining drug resistance; estrogenic or antiandrogenic
therapeutic substances
(generally as a means of inhibiting the response of breast tissue to estrogen
excess insult
(absolute or relative estrogen excess as compared to normal
estrogenic/androgenic substance
balance); folic acid or other folate sources (primarily with respect to
reducing the incidence of
fetal malformations), etc. as further detailed herein.
[00051 Still further, the invention relates to reducing the emergence of
resistance to interferon
alpha therapy, to normalizing abnormal activity of TNF as well as kinases
active in chronic-
active hepatitis (Janus Kinase [JAK] and TYK2) activated upon INF-alpha
binding, as well as
conditions resulting from such abnormal kinase activity. The methods and
compositions can also
be used to treat abnormal kinase activity in cancers. These include:
phosphatidylinositol 3-
kinase (PI3K-AKT Mtor signaling pathway)Ras/MAPK-erk 1/2 pathway,Rapamycin
pathway,
Insulin-like growth factor(S) signaling, protein kinases including
serine/threonine kinases such
2
CA 02668580 2009-06-10
as Raf kinases, such as aurora kinases, protein kinase such as MEK, and
tyrosine
kinases, epidermal growth factor receptor family (EGFR), platelet-derived
growth factor receptor
family (PDGFR), vascular endothelial growth factor receptor (VEGFR) family,
nerve growth
factor receptor family (NGFR), fibroblast growth factor receptor family (FGFR)
insulin receptor
family, ephrin receptor family, Met family, Ror family, c-kit family, Src
family, Fes family, Fak
family, Syk/ZAP-70 family, Tec family and Ab 1 family. Furthermore, these
compounds and
methods can be used to upregulate the p27kip 1 and P21 cip 1 and to down
regulate or inhibit Ap- l
and ppRb.
[00061 More specifically, in some embodiments, the present invention further
relates to the
phosphatidylinositol/PI3K signaling pathways for prevention or correction of
improper signaling
in these pathways. In still other embodiments, the invention relates to
compositions for the
prevention and/or minimization of fetal malformations, some of which are due
to sonic hedgehog
(Shh) and/or other hedgehog variants such as Indian (Ihh) and Desert (Dhh),
etc. signaling
defects; for the prevention and/or inhibition of breast cancer and metastases
thereof some of
which are due to one or more of sequela of estrogen exposure or estrogen
surplus exposure
(whether during hormonal therapy (males or females) or birth control use) or
super-active
estrogen receptors, or due to excess number of estrogen receptors (receptor
expansion), or
excessively sensitive estrogen receptors in mammary epithelial breast tissue
(whether due to
derangements in signaling pathways or other bases such as estrogen receptor
overexpression in
certain predisposed phenotypes, whether due to developmental, or to
environmental, or
endogenous exposures). The invention further relates to increasing the
therapeutic efficacy of
anti-cancer agents, especially those related to the prevention or treatment of
breast and prostate
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cancers, and the prevention or reduction of aberrant cells becoming resistant
to one or more anti-
cancer agents.
[0007] In yet other aspects of the invention, it relates to manipulating cell
growth and
differentiation in culture for implantation of such cells. In this context,
the invention relates to
treatments for regenerating neural tissue, hepatic tissue, pancreatic tissue,
intestinal tissue,
spleenic tissue, cardiac tissue, among others. In still other embodiments, the
invention relates to
regulating or inhibiting growth of cells and therefore finds use in the
treatment of excessive or
inappropriate hair growth conditions, psoriasis, actinic keratosis, acne,
miscellaneous dermatitis
conditions, etc. In yet other embodiments, the invention relates to inducing
an anti-angiogenic
state in local and distant metastatic tumors.
[0008] The invention further relates to correcting the inherent mechanism of
tumor stem cell
autoregulation. Yet another embodiment of the invention relates to decreasing
the risk of deep
vein thrombosis (DVT's) and Pulmonary emboli (PE's) while using
chemotherapeutic agents,
antiestrogens such as tamoxifen etc., hormonal therapies such as androgen
ablative therapies, or
estrogenic hormone therapy. The invention still further relates to reducing
the numbers and size
of tumors locally or distant especially in breast cancers, but also cancers
originating from blood,
colon, lung, liver, pancreas,cervix, prostate, skin, and soft tissue.
[0009] The invention further relates to preventing breast cancer or precursors
thereof in utero.
4
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BACKGROUND OF THE INVENTION
[00101 The present invention relates to compounds that are of the inositol
family and for many
of the uses set forth herein specifically the D-chiroinositol family of
compounds. Most are
derivatives of the basic parent of the family, but in some indications, even
the basic parent was
not previously known or thought to be useful in the contexts indicated. As
indicated in the above
fields of the invention, the invention is applicable to a wide array of
utilities a number of which
are addressed separately in this background section below.
[00111 I. Fetal Malformation
[00121 Fetal malformations are a continuing medical problem in serious need of
prevention and
treatment. These malformations can result in innocuous defects that pose no
health or
psychological issues, to those that pose primarily social or psychological
issues (such as webbed
digits, etc.), to those that pose medical issues of varying degrees of
severity. Some of the more
medically severe malformations include neural tube defects (such as, among
others, anencephaly
where the brain is underdeveloped or there is an incomplete skull,
encephalocele, where there is
a hole in the skull through which tissue protrudes, and spina bifida, where a
portion of the spine
is exposed) to cranio-facial defects (such as, among others, cleft lip and
cleft palate) to
imperforate anus (where the anal opening doesn't form properly leaving no exit
for intestinal
contents, or intestinal/rectal emptying into inappropriate structures such as
the bladder, ureter,
uterus or vagina). Other birth defects like neural derived brain tumors are
not so evident
immediately after birth and rarely if ever seen at birth. For example, the
onset of pediatric
tumors like primitive neuroectodermal tumors of the central nervous system
(PNET'S) is
CA 02668580 2009-06-10
insidious. There is considerable controversy regarding the histiogenesis of
these tumors, but a
genetic loci of interest in the pathnogenesis of these central nervous system
derived tumors
includes Shh gene pathways.
[00131 Cleft palate children were found to have low red blood cell zinc levels
and low myo-
inositol levels (Krapels, et al: Myo-inositol, glucose and zinc status as risk
factors for non-
syndromic cleft lip with or without cleft palate in offspring: a case-control
study, BJOG. 2004
Jul; 111(7):661-8). Impairment of the Folbpl gene function adversely impacts
the expression of
several critical signaling molecules. Mis-expression of these molecules,
perhaps mediated by
Shh may potentially contribute to the observed failure of the neural tube and
the development of
craniofacial defects in the mutant mice (Birth Defects Research, Wiley
Interscience, 2003).
[00141 IV. Signaling Pathways
[0015] In molecular biology, 'signal transduction' refers to any process by
which a cell converts
one kind of signal or stimulus into another. Most processes of signal
transduction involve
ordered sequences of biochemical reactions inside the cell, which are carried
out by enzymes,
activated by second messengers, resulting in the signal transduction pathway.
These processes
are usually quick, lasting milliseconds in the case of sodium, potassium and
calcium ion fluxes,
or minutes for the activation of protein and lipid-mediated kinase cascades.
However, some
signaling cascade events can take days and many hours. (As is the case with
gene expression), to
complete. The number of proteins and other molecules participating in the
events involving
6
CA 02668580 2009-06-10
signal transduction increases as the process starts from the initial stimulus,
resulting in a "signal
cascade," that usual begins with a relatively small stimulus that elicits a
larger response. This is
referred to as" amplification of the signal" where the signal spreads across a
spatial concentration
gradient. The continued cell growth and/or death of a cell are some of the
cellular responses to
extracellular stimulation that require signal transduction as well. Gene
activation leads to further
cellular effects, like activation of transcription factors which are also the
result of another signal
transduction cascade. This cascade leads to activate yet more genes.
[00161 Most mammalian cells require constant growth factor stimulation to
control not only
cell division but also survival. In the absence of signal transduction,
programmed cell death
ensues in most cells. Such requirements for extra-cellular stimulation are
necessary for
controlling cell behavior in the multi-cellular organisms. Signal transduction
pathways are
known to be so central to biological processes that it is not surprising that
a large number of
diseases have been attributed to their disregulation and cross talk. Discussed
below is how signal
transduction can disrupt genes that can lead to various diseases.
[00171 Disruptions of genes in one pathway can also have deleterious effects
in other pathways
and may result in serious dysmorphogensis or cancer years later. For a better
understanding of
this aspect of the present invention, a basic overview of some of the genes
and signaling
pathways that may be affected by consequences of alterations in genes, their
products, and
specific exposures is in order. This also requires some understanding of the
pathways involved
in formation, embryonic development, and cancer.
7
CA 02668580 2009-06-10
100181 During embryogenesis a simple, patterned body plan is established. To
establish this
organization, the cells of the embryo need to become specified and must
differentiate into cell
types in an integrated manner. The genetic regulation of this process is
addressed here. In
animals, cell-cell communication involving extracellular signals and cell
surface-bound
receptors plays an important role in cell fate decisions during embryogenesis
(Johnston and
Nilsslein-Volhard, 1992). Probably the best characterized signaling events in
embryo patterning
are those that involve sonic hedgehog (Shh). Mutations in the Shh genes and
the genes that
encode its downstream intracellular signaling pathway and subsequent responses
can set the
pattern for various birth defects and cancers in both prenatal and post natal
development (Mol
Med Today, 1998). Pattern formation is the activity by which embryonic cells
form ordered
spatial arrangements of differentiated tissues. The physical complexity of
higher organisms
arises during embryogenesis through the interplay of cell-intrinsic lineage
and cell-extrinsic
signaling. Inductive interactions are involved in embryonic patterning in
vertebrate development
from the earliest establishment of the body plan, to the patterning of the
organ systems, to the
generation of diverse cell types during tissue differentiation (Davidson, E.,
(1990) Development
108: 365-389; Gurdon, J. B., (1992) Cell 68: 185-199; Jessell, T. M. et al.,
(1992) Cell 68: 257-
270). The effects of developmental cell interactions are varied. Typically,
responding cells are
diverted from one route of cell differentiation to another by inducing cells
that differ from both
the uninduced and induced states of the responding cells (inductions).
Sometimes cells induce
their neighbors to differentiate like themselves (homeogenetic induction); in
other cases a cell
inhibits its neighbors from differentiating like itself. Cell interactions in
early development may
be sequential or convergent, such that an initial induction between two cell
types leads to a
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CA 02668580 2009-06-10
progressive amplification of diversity. Moreover, inductive interactions occur
not only in
embryos, but in adult cells as well, and can act to establish and maintain
morphogenetic patterns
as well as induce differentiation in cell types (J. B. Gurdon (1992) Cell
68:185-199). These cells
can also act to establish and maintain morphogenetic patterns as well as
induce differentiation (J.
B. Gurdon (1992) Cell 68:185-199). The involvement of signal transduction
pathways in the
inhibition of gene expression and the occurrence of frequent allelic deletions
in humans and
animals support a tumor suppressor function for these pathways. Their role in
the regulation of
embryonic pattern formation known to be involved in cell signaling and
intercellular
communication provides a possible mechanism of fetal malformations, birth
defects,
autoimmune inflammatory diseases, inflammation, drug resistance, anti-
angiogenesis, and
cancers.
[00191 Notwithstanding the above, there is still a tremendous amount that is
still not fully
understood in the art about the nature of the all the mechanisms involved in
the etiology of these
malformations and diseases and how to appropriately intervene to reduce or
prevent the
occurrence of such defects. Thus, we are also investigating the possibility
that many signaling
pathways comprise multiple steps and cross convergences along the feedback
loop.
Nevertheless, we are closer to prevention of these diseases, and better
treatment options that will
eventually improve the quality of many patients' lives. The role of D-chiro-
inositol its
steroisomers and/or its phosphates and other derivatives among these pathways
mentioned
above, and their likely cross-talk in regulation of embryonic patterning,
cancer, inflammatory
autoimmune diseases, inflammation is now addressed in this application.
9
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[00201 III. Fetal Alcohol Syndrome
[00211 Fetal alcohol syndrome (FAS) is one of the most common, preventable
causes of
devastating congenital structural birth defects to major organs like the
brain, heart and kidneys
and to varying defects of the face and limbs. It may occur as often as neural
tube defects.
According to recent literature, at least 33% to 50% of children born to
chronic alcoholic mothers
show some signs of fetal alcohol syndrome. Fetal alcohol syndrome remains a
significant
psychosocial and clinical challenge. While there have been great strides in
preventing neural
tube defects with folic acid, there are no known truly preventative strategies
for preventing fetal
alcohol syndrome related birth defects. One recent article (Li, et al; Fetal
alcohol exposure
impairs hedgehog cholesterol modification and signaling, Laboratory
Investigation (2007) 87,
231-240 (1/22/2007)) discusses results in the zebrafish that exposure of
zebrafish embryos to low
levels of alcohol during gastrulation blocks modification of Sonic hedgehog by
cholesterol and
lead to permanent developmental defects that resemble fetal alcohol syndrome
defects, and
further that administration of cholesterol rescues the Sonic hedgehog function
and prevents the
defects in zebrafish. Unfortunately, cholesterol has a significant number of
problems of its own
that make it generally an unacceptable therapy for use in the human population
and other
treatment avenues are desired.
[00221 Ofori, et al Risk of congenital anomalies in pregnant users ofstatin
drugs, British
Journal of Clinical Pharmacology, vol. 64, No. 4, October 2007, pp 496-509,
states that because
cholesterol is known to be essential for fetal development, statins , which
inhibit cholesterol
production, have been considered potential teratogens, and therefore have been
contraindicated
CA 02668580 2009-06-10
in pregnancy. Ofori states that from their database of 288 they did not find
evidence of increased
risk in women who filled prescriptions for statins during pregnancy as
compared to those who
stopped statin use before pregnancy (between 1 year and 1 month before
preganancy) and
compared to women who used fibrates during pregnancy based on live births.
They do state
however, that conclusions remain uncertain due to the lack of data about non-
live births. There
were no matched controls in this study either.
[0023] A posting on www.medpa eg today.com/OBGYN/Pregnancy/11317 published
October
14, 2008, by Gever, et al Statins May Prevent Some Miscarriages indicates that
antiphospholipid
antibody seen with patients with autoimmune inflammatory diseases induced
miscarriages may
be prevented by administration of the statins simvastatin and pravastatin.
Thus, statin use in
pregnancy in those showing predisposition to or symptoms of antiphospholipid
antibodies is
desirable. (See also Weiler, Tracing the molecular pathogenesis
ofantiphospholipid syndrome,
J. Clin. Invest. 118(10): 3276-3278 (2008).)
[00241 Autoimmune and inflammatory Diseases
100251 The present invention also relates to inflammatory autoimmune diseases
that are
triggered and promoted by abnomal kinase activity due to defective cell
signaling. Interferon
alpha (IFN-alpha) has been used for a decade to treat viral hepatitis, is a
disease that effects
approximately 350 million per year worldwide, more than half of those viral
hepatitis patients
respond poorly to drugs. It appears that high levels of tumor necrosis factor
(TNF) correlate
highly with resistance to interferon alpha therapy. There is an urgent need to
find appropriate
11
CA 02668580 2009-06-10
treatments for interferon resistance without adding more side effects to the
already compromised
liver. The methods and compounds are provided for treating diseases associated
with abnormal
activity of protein kinases. The method comprises administration of a specific
or selective AKT
inhibitor (for example D-3-Deoxy-2-O-methyl-D-Chiro- inositol-[1-(R)-2-methoxy-
3-
(octadecyloxx propyl HPO4-1 (hydrogen phosphate)) with interferon alpha(IFN-
alpha) to a
patient in therapeutically effective amounts; and administering a kinase
inhibitor to the patient in
therapeutically effective amount, such that the in vivo activity of the high
levels of tumor
necrosis factor (TNF) is reduced relative to that prior to the treatment so in
order to overcome
resistance to INF-alpha therapy. Interferon-alpha is currently the only well-
established treatment
for chronic-active viral hepatitis that affects millions world wide. It is
thought that the activity of
interferon is mediated by inducing expression of antiviral proteins and
modulation of the
immune system. Even though interferon has been used widely for many years for
the treatment
of viral hepatitis, most of these patients respond poorly. The mechanism
underlying this problem
remains unclear at this point, although both genetic and molecular
implications must be
considered. There have been many attempts to improve patient response to
interferon by
combining interferon with other anti-viral agents (i.e.,Ribivarin). It appears
that only a small
number of patients responded to this attempt to increase the efficacy of
combined interferon and
Ribivarin. Thus, it is an urgent medical need to develop a new combination of
compounds to
improve interferon alpha response in patients with chronic-active hepatitis.
[00261 A number of acute and chronic inflammatory diseases have also been
associated with
very high amounts of circulating inflammatory cytokines and interleukin 1,6,8.
These cytokines
include but not limited to Tumor necrosis factor (TNF)-a cytokine involved in
systemic
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CA 02668580 2009-06-10
inflammation. TNF also causes apoptotic cell death, cellular proliferation,
inflammation,
differentiation, tumorigenesis, and viral replication. But TNF's primary role
is in the regulation
of immune type of cells and their dysregulation and overproduction have been
implicated in
those diseases desrcribed above. Interleukin 1 beta, interleukin 6,
interleukin8 are also other
types of cytokines responsible for inflammatory response in a variety of human
diseases.
[0027] The phosphoinositide 3-kinase (PI3K)/Akt signaling pathway plays a
pivotal role in
cellular proliferation and growth signaling as described above. It involves
the downstream
activation of the protein kinase Akt. Ptdlns (3,4) P2(biphosphate) and
ptdlns(3,4,5)P3(triphosphate) lipids produced by P13K are able to bind Akt
recruiting it to the
plasma membrane where PDK1 and PDK2 phosphorylate it to its active form. This
allows Akt
to target proteins involved in cell death. Due to the constitutive activation
of P13K pathway in
many cancers and immune mediated signaling pathways this pathway may produce
systemic
autoimmunity and resistance to interferon alpha for people with chronic,
active viral hepatitis
and chronic autoimmune inflammatory disease. Therefore, the inventor's
research is helping to
elucidate the role of this pathway and how perturbations that leads to a
disregulated activation in
this pathway. Inhibition of this pathway with combination therapy is a novel
treatment for
cancer and inflammatory disease states.
[0028] The phosphatidylinositol 3-[OH] kinase (PI3K)/Akt-( Protein kinase B)
pathway is also
important for survival such as platelet derived growth factor (PDGF), nerve
growth factor (NGF)
and insulin-like growth factor-1 (IGF-1), promote cell survival under various
conditions by
inducing the activity of P13K. Activated P13K leads to the production of
phosphatidylinositol
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CA 02668580 2009-06-10
(3,4,5)-triphosphate (Ptdlns(3,4,5)-P3), a second messenger which in turn
binds to, and promotes
the activation of, the serine/threonine kinase Akt, which contains a
pleckstrin homology (PH)-
domain(Franke et al, Cell,88:435-437, 1997).
[00291 Recent Literature analysis of AKT levels in autoimmune inflammatory
diseases protein
Kinase B (Akt) appears to be overexpressed or hyperactive in a significant
number of
inflammatory autoimmune diseases such as Rheumatoid arthritis, Systemic lupus,
Inflammatory
Bowel Diseases, Chron's disease and others. It is also postulated here that
high levels of tumor
necosis factor is mediated by dyregulated PI3k signaling which can lead to
Inteferon alpha
resistance seen in many patients with viral hepatitis. Furthermore, PTEN is a
tumor suppressor
gene mutated in many human sporadic cancers and hyperactive immune systems.
Those with
hereditary cancer syndromes such as Bannayan-Zonana syndrome and Cowden
syndromes are
purportedly pten deficient (Lieaw et al. nature genetics 16:64-67.
Functionally, PTEN is a dual
protein and lipid phosphatase enzyme (Li et al. science 275:1943-1947, 1997)
and the major
substrate of PTEN is phosphatidyl inositol-3,4,5-triphosphate (PIP3), a second
messenger
molecule then produced through P13K signaling activation induced by growth
factor stimulation.
PIP3 activates the serine-threonine kinase PKB/Akt which is involved in anti-
apoptosis,
proliferation, and oncogenesis. PTEN negatively regulates cell survival by
dephosphorylating
PIP3 by enzymatic activation. Recent literature cited studies that showed that
a null mutation of
Pten in mice is lethal during embryogenesis. In a study, it was demonstrated
that PKB/Akt
pathway is hyperactivated in the absence of Pten). Furthermore, Pten+i mice
frequently develop
T cell lymphomas, and endometrial, prostatic, and breast cancers. Autoimmune
disorders are
also prevalent in Pten deficient mice. In T cell-specific Pten-deficient mice,
it showed that
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CA 02668580 2009-06-10
CD4+ lymphomas and autoimmune disorders were prevalent. These observations
support the
role of PI3k/Akt signaling pathways play an importamt role for regulating cell
survival,
apoptosis in transforming cells into cancerous, metastatic tumors.
[00301 Three members of the Akt/PKB subfamily of second-messenger regulated
serine/threonine protein kinases have been identified and termed
Aktl/PKB.alpha.,
Akt2/PKB.beta., and Akt3/PKB.gamma., respectively. The isoforms are
homologous,
particularly in regions encoding the catalytic domains. Akt/PKBs are activated
by
phosphorylation events occurring in response to PI3K signaling. PI3K
phosphorylates membrane
inositol phospholipids, generating the second messengers phosphatidyl-inositol
3,4,5-
trisphosphate and phosphatidylinositol 3,4-bisphosphate, which have been shown
to bind to the
PH domain of Akt/PKB. The current model of Akt/PKB activation proposes
recruitment of the
enzyme to the membrane by 3'-phosphorylated phosphoinositides, where
phosphorylation of the
regulatory sites of Akt/PKB by the upstream kinases occurs (B. A. Hemmings,
Science 275:628-
630 (1997); B. A. Hemmings, Science 276:534 (1997); J. Downward, Science
279:673-674
(1998)).
[00311 Phosphorylation of Aktl/PKB.alpha. occurs on two regulatory sites,
Thr308 in the
catalytic domain activation loop and on Ser473 near the carboxy terminus
(D. R. Alessi et
al. EMBO J. 15:6541-6551 (1996) and R. Meier et al. J. Biol. Chem. 272:30491-
30497 (1997).
Equivalent regulatory phosphorylation sites occur in Akt2/PKB.beta. and
Akt3/PKB.gamma..
The upstream kinase, which phosphorylates Akt/PKB at the activation loop site
has been cloned
and termed 3'-phosphoinositide dependent protein kinase 1 (PDK1). PDKI
phosphorylates not
CA 02668580 2009-06-10
only Akt/PKB, but also p70 ribosomal S6 kinase, p90RSK, serum and
glucocorticoid-regulated
kinase (SGK), and protein kinase C (B. A. Hemmings, Science 276:534 (1997); J.
Downward,
Science 279:673-674 (1998).
[00321 Inhibition of Akt activation and activity can be achieved by inhibiting
P13K with
inhibitors already known in the prior art such as LY294002 and wortmannin.
However, some
research has suggested PI3K inhibition has the potential to indiscriminately
affect not just all
three Akt isozymes but also other PH domain-containing signaling molecules
that are dependent
on Ptdlns(3,4,5)-P3, such as the Tec family of tyrosine kinases. Furthermore,
it has been
disclosed that Akt can be activated by growth signals that are independent of
PI3K.
[00331 At the same time, Akt activity can be inhibited by blocking the
activity of the upstream
kinase PDKI. No specific PDKI inhibitors have been disclosed. However, a
recent study
interestingly suggested that Ins(1,3,4,5,6)P5 and InsP6, as well as
Ins(1,3,4,5)P4, were found to
bind PDKI with high affinity, with K, values of 20-60 nM. In contrast, Ins
(1,4,5)P3 binding to
PDKI could not be detected. The hydrogen bonds to the Dl-phosphate appear to
play a crucial
role in mediating binding of inositol phosphates to PDKI . Furthermore, they
next investigated
whether Ins(1,3,4,5,6)P5 and InsP6 could compete with Ptdlns(3,4,5)P3 in
regulating the
activation of PKB by PDKI in an in vitro assay. The results showed with a dose
given of 300
M InsP6 or Ins(1,3,4,5,6)P5 inhibited the activation and phosphorylation of
PKB in the presence
of Ptdlns(3,4,5)P3, by over 80%. InsP6 or Ins(1,3,4,5,6)P5 did not directly
inhibit PDKI catalytic
activity, as the phosphorylation of the PDKtide peptide substrate by PDKI was
not affected by
D6-phosphorylated inositol phosphates. It has also been reported that
increasing extracellular
16
CA 02668580 2009-06-10
Ins(1,3,4,5,6)P5 in cells lowered PKB activation, through an undefined
mechanism (Piccolo et al,
2004). It is the inventor's belief that different inositol polyphosphates
interact differently. Some
agozine the activity in the presence of other substrates while others
antagonize the activation of
these signaling pathways. EMBO J. 2004 October 13; 23(20): 3918-3928.
100341 The inventor does not believe that the inhibition of PDK1 would result
in inhibition of
multiple protein kinases whose activities depend on PDK1, such as atypical
Protein Kinase C
PKC isoforms, SGK, and S6 kinases if the different invention compounds were
utilized in a
selective, specific manner in the presence of different substrates. For
example, phosphorylation
of PKC by PDK-1 may not require the PH domain of PDK-1 and may not be affected
by 3'-
phosphoinositides. Thus, despite what earlier literature suggests, the
inventor believes that
perhaps not all of these enzymes are dependent upon phosphoinositde
phosphorylation and that
there may be some other lipid signaling pathway present that is involved in
cross- talk.
[00351 Therefore, further embodimets of the invention compounds are envisioned
to be
selective for inhibiting different isoenzymes. The compounds of the instant
invention are
inhibitors of the activity of Akt and PDK1 are thus useful in the treatment of
cancer, in particular
cancers associated with irregularities in the activity of Akt and/or GSK3.
Such cancers include,
but are not limited to ovarian, pancreatic and breast cancer.
[00361 In an embodiment of the invention, the instant compound is a selective
inhibitor whose
inhibitory efficacy is dependent on the PH domain. In this embodiment, the
compound exhibits a
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CA 02668580 2009-06-10
decrease in in vitro inhibitory activity or no in vitro inhibitory activity
against truncated Akt
proteins lacking the PH domain.
[0037] In a further embodiment, the instant compound is selected from the
group of a selective
inhibitor of Aktl, a selective inhibitor of Akt2 and a selective inhibitor of
both Aktl and Akt2.
[0038] In another embodiment, the instant compound is selected from the group
of a selective
inhibitor of Aktl, a selective inhibitor of Akt2, a selective inhibitor of
Akt3 and a selective
inhibitor of two of the three Akt isoforms.
[0039] In another embodiment, the instant compound is a selective inhibitor of
all three Akt
isoforms, but is not an inhibitor of one, two or all of such Akt isoforms that
have been modified
to delete the PH domain, the hinge region or both the PH domain and the hinge
region.
[0040] In another embodiment with a different inositol pyrophosphate or
inositol
polyphosphate (or further derivatives of either), the invention is a selective
inhibitor of PDK1.
[0041] The present invention is further directed to a method of inhibiting Akt
activity which
comprises administering to a mammal in need thereof a pharmaceutically
effective amount of the
instant compound to be be protective against TNF mediated cell apoptosis as
well as sensitize
cells to interferon alpha through modulations of the P13K signaling activity.
18
CA 02668580 2009-06-10
[00421 The compounds of the instant invention are inhibitors of the activity
of PI3-
kinase/PDK1/AKT- dependent signaling pathway are thus useful in the treatment
of, cancers
associated with irregularities in the activity of Akt and/or GSK3. Such
cancers include, but are
not limited to ovarian, pancreatic and breast cancer. They are also useful in
the treatment of
inflammatory conditions, and in the treatment of elevated Tumor Necrosis
Factor in chronic
inflammatory states such as like Rheumatoid Arthritis, Systemic Lupus
Erythematosus, Hepatitis
(A, B, C, and/or D), Ankylosing Spondylitis, Inflammatory Bowel Disease and
Psoriasis, but not
limited to only these inflammatory conditions.
[00431 Chronic inflammation has been implicated in the pathogenesis of many
severe
autoimmune disorders, as well as in diabetes, pulmonary diseases, and cancer.
Inflammation also
accompanies most solid cancers. Some reseachers investigated the role of the
major
proinflammatory cytokine tumor necrosis factor a(TNFa) in the malignancy of
Pancreatic ductal
adenocarcinoma (PDAC) cells in vitro and in vivo. In vitro, TNFa strongly
increased
invasiveness of Colo357, BxPc3, and PancTuI cells and showed only moderate
antiproliferative
effect. TNFa treatment of mice bearing orthotopically growing PDAC tumors led
to dramatically
enhanced tumor growth and metastasis. Notably, they found that PDAC cells
themselves secrete
TNFa. Although inhibition of TNF with infliximab or etanercept only
marginally affected
proliferation and invasiveness of PDAC cells in vitro. In severe combined
immunodeficient mice
with orthotopically growing Colo357, BxPc3, or PancTul tumors, human-specific
anti-TNF
antibody infliximab reduced tumor growth and metastasis by about 30% and 50%,
respectively.
Infliximab and etanercept reduced the number of liver metastases by 69% and
42%, respectively,
as well as volumes of recurrent tumors by 73 % and 51 %. Thus, tumor cell-
derived TNFa
19
CA 02668580 2009-06-10
appears to play a profound role in malignancy of PDAC, and inhibition of TNFo
represents a
promising therapeutic option for cancers that are caused by chronic
inflammation that is one
cause of cancer or solid tumors that transform to metastatic tumors. [Cancer
Res
2008;68(5):1443-50]
100441 V. Invention Compound and Folate
[0045] In one aspect of the invention, the invention further relates to D-
chiroinositol and
derivatives thereof, more specifically D-chiro-inositol, phosphates thereof,
and other derivatives
of each as more fully detailed below. In addition, this aspect of the
invention also relates to
folates. The number of births presenting with spina bifida has been reduced in
recent years in
patients at risk of having such defects by having adequate folate levels in
the mother just before
and during the first trimester of pregnancy. More specifically, if a woman
takes folic acid before
conception and during early pregnancy, the risk of the fetus developing a
neural tube defect is
reduced by about 70%. Unfortunately, folate supplementation still does not
prevent all such
cases, and presumbably, the impairment of the Folbpl due to aberrant sonic
hedgehog signaling
likely mediates the acquisition of folate resistance of the mother. The
remaining 30% risk is still
substantial. In a Research Review from Neurosciences and Mental Health 2005
from Great
Ormond Street Hospital, the use of inositol in combination with folate therapy
is mentioned as
being explored. However, no particular type of inositol is mentioned nor is
any dosage amount
or regimen.
CA 02668580 2009-06-10
100461 Inositol prevents expression of a genetic model of neural tube defects
in mice; Nutrition
Reviews, May 1997 reports that myo-inositol reduced the incidence of neural
tube defects in
mouse models that are folate resistant. The curly tail model is particularly
resistant to folate
therapy. (Human Reproduction, Vol. 17, No. 9, 2451-2458) and frequently used
to test for
activity in these conditions. Cogram et al, Human Reproduction, Vol. 17, No.
9, 2451-2458,
states that D-chiro-inositol alone reduced frequency of spina bifida in this
model to a greater
extent than myo-inositol alone. Without being bound thereto, it is the
inventor's belief that in the
present invention D-chiroinostitol (or a phosphorylated or other derivative
thereof, preferably
combinations of two or more selected from D-chiroinositol, its phosphates, or
other derivatives
thereof) stimulates these signaling mechanisms, activating certain isoforms of
protein kinases
that appear to be required for neural tube defects. Other relevant literature
includes: Frederick, et
al; An essential role for an inositol polyphosphate multikinase, Ipk2, in
mouse embryogenesis
and second messenger production, PNAS June 14, 2005, Vol 102, No. 24, 8454-
8459; Riobo, et
al. Phosphoinositide 3-kinase and Akt are essential for sonic Hedgehog
signaling, PNAS March
21, 2006, Vol. 103, No. 12, 4505-45 10.
[00471 Meyers, et al; Folic Acid Supplementation and Risk for Imperforate Anus
in China;
American Journal of Epidemiology, Vol. 154, No. 11: 1051-1056, 2001 reports on
a public
health campaign in China in 1993 to 1995, where women were requested to take
400 mg folic
acid, with or without other vitamins daily from their pre-marital examination
through the end of
their first trimester of pregnancy. The rate of imperforate anus was
calculated to be 3.1 per
10,000 births for those not taking folic acid compare to 1.6 per 10,000 births
for those taking
folic acid. The authors conclude that folic acid may reduce imperforate anus
risk.
21
CA 02668580 2009-06-10
[00481 In addition, Mo et al, Anorectal malformations Caused by Defects in
Sonic Hedgehog
signaling, American Journal of Pathology 2001, 159, 765-774 report on a mutant
mouse with
various defects in the Sonic Hedgehog signaling pathway that presents with a
number of distal
hindgut defects that appear to the authors to mimic human anorectal
deformations. An excellent
review of inositol and some of its phosphates is given in Fisher, et al;
Inositol and higher inositol
phosphates in neural tissues: homeostasis, metabolism and functional
significance; Journal of
Neurochemistry, Vol 82, 736 August 2002.
[00491 In some embodiments of the present invention, folic acid of the formula
0 OH
OH
HN
0
0
HN
N
I
I' N
H2NN I OH
and (D)-chiro-inositol or a phosphatidyl-chiro-inositol or a complex of the
present invention are
linked via a covalent bond, preferably with an ester bond formed between a
carboxyl of the folic
acid and hydroxyl of the inositol. Alternatively, with the phosphorylates or
other derivatives of
the invention compounds in which the invention compounds have a free acidic
group (the -OH
of a phosphate, sulfate, sulfonate, or carbonate, the linkage can be an ester
between a free
22
CA 02668580 2009-06-10
hydroxy group of the folic acid and the acidic group of theinvention compound,
or further an
acid anhydride between the folic acid acidic group and the invention compound
acidic group,
although the acid anhydrides are more prone to rapid hydrolysis. All of these
compounds having
an invention compound component and a folic acid component are further
compounds within the
invention. The inventor is using the folic acid as guiding media to target
tumor cells with the
invention compounds as described. Folic acid polysaccharides (where the
polysaccharide does
not contain an inositol) are analogously suitable and part of the invention
forming Folic acid
polysaccharide-D chiro inositol complexes.
[0050] V. Inositols
[0051] Inositols are a group of compounds that have the following structure
R1 R3
R11 R5
R2 R4
R9 R7 R6
R12
R10 R8
where each of the R groups is either H or OH, but each carbon of the ring has
one H and one OH.
The most common form is myo-inositol, which is available to some degree from
dietary sources.
Myo-inositol requires that all of Ri, R3, R5, R8, R9, and R12 are OH and R2,
R4, R6, R7, RIO,
and R i I are all hydrogen. Epi-inositol and scyllo-inositol are the other two
most abundant forms
23
CA 02668580 2009-06-10
(each being substantially less than the myo-inositol in terms of abundance). D-
chiro-inositol is
not available from dietary sources and is the isomer where R1, R3, R6, R8, R9,
and R12 are OH
and R2, R4, R5, R7, R10, and RI 1 are hydrogen. In other words, D-chiro-
inositol differs from
myo-inositol in the inversion of R5/R6.
[00521 There are a total of ten isomers of inositol, and for those that have
found potential
medicinal or nutritional use, many of the uses are truly limited to particular
isomers and/or
phosphates thereof (where one or more of the hydroxyl groups are
phosphorylated), while for
other uses (such a in connection with blood glucose regulation) more than one
inositol isomer
has been found useful or is projected to be useful. For example, recently
scyllo inositol has been
found to prevent the accumulation of amyloid [3 deposits and improved
cognitive ability in
Alzheimer's patients. (McLaurin, et al, Inositol Stereoisomers Stabilize an
Oligomeric
Aggregate ofAlzheimer Amyloid beta Peptide and Inhibit A beta-induced
Toxicity, J. Biol.
Chem., Vol. 275, Issue 24, 18495-18502, June 16, 2000; and Research News from
Howard
Hughes medical Institute June 11, 2006 A Sweet Solution to Alzheimer's
Disease?) Myo-inositol
was found not to be effective in this condition. Scyllo-inositol worked when
given before
symptoms appeared as well as after symptoms appeared in this indication, while
epi-inositol only
worked at all when given before disease onset. Interestingly, scyllo-inositol
has been reported to
be an "inositol" uptake inhibitor causing similar fetal development defects in
non-hyperglycemic
pregnancies as seen in hyperglycemic pregnancies (Cederberg; Oxidative Stress,
antioxidative
defense, and Outcome in Experimental Diabetic pregnancy; Comprehensive
Summaries of
Uppsala Dissertations from the Faculty of medicine 1008, AUU Uppsala 2001, pp.
1-66). Myo-
inositol has been found useful in treating panic attacks (Levine, et al,
Double-blind, placebo-
24
CA 02668580 2009-06-10
controlled, crossover trial for inositol treatment for panic disorder, Am J
Psychiatry 1995; 152;
1084-1086).
[0053] One of the more prominent uses for myo-inositol has been for blood
sugar regulation.
Recently, D-chiro-inositol has been proposed for insulin resistance patients
(Lamer, D-Chiro-
Inositol - Its functional role in Insulin Action and its Deficit in Insulin
Resistance, International
Journal of Experimental Diabetes Research 3 (2002), 47-60) on the theory that
such patients
have a defect in epimerization of the myo-inositol to the D-chiro-inositol and
that the D-chiro-
inositol is the active moiety in this regard. As stated above, scyllo-inositol
actually resulted in an
increase in fetal defects in non-diabetic pregnancies. Phosphoinositide
derangement and poor
maternal metabolic turnover carries a relative risk in diabetic pregnancies
for giving birth to a
baby with lethal congenital anomalies like sirenomelia (Tahna, Davari et al,
2002). This defect
is similar to those seen in anorectal malformation spectrum of defects.
OBJECTS OF THE INVENTION
[0054] It is therefore an object of the invention to provide a method of
preventing or reducing
the rate of birth defects.
[0055] It is therefore an object of the invention to use folic acid as guiding
media for the
invention compounds to target tumor cells with the invention compounds as
described within the
claims where the Folic acid receptor alpha is always expressed on a tumor. The
complex will be
taken into the cell membrane by endocytosis and thereby anchor itself to a
glycerophosphatide.
CA 02668580 2009-06-10
The invention will also be made with a folic acid/inositol-PEG-liposome that
will target tumor
cell better than than a liposome by itself.
[00561 It is therefore an object of the invention to provide a method of
treatment of women
pre-pregnancy to prevent or reduce the chance of fetal malformations by
administering D-chiro-
inositol or a phosphate derivative thereof or other derivative thereof.
[00571 It is another object of the invention to provide a method of treatment
of women during
the first trimester of pregnancy to prevent or reduce the chance of fetal
malformations by
administering D-chiro-inositol or a phosphate derivative thereof or other
derivative thereof.
[00581 It is another object of the invention to provide co-therapy for women
pre-pregnancy
with both a folate source and D-chiro-inositol or a phosphate derivative
thereof or other
derivative thereof.
[00591 It is another object of the invention to provide a method of treatment
of women during
the first trimester of pregnancy to prevent or reduce the chance of fetal
malformations by co-
administering D-chiro-inositol or a phosphate derivative thereof or other
derivative thereof and a
folate source.
26
CA 02668580 2009-06-10
[00601 It is yet another object of the invention to treat women who are taking
birth control pills
but who might nonetheless become pregnant by including D-chiro-inositol (or a
phosphate
thereof or other derivative thereof) and optionally a folate source into the
pills that do not contain
an estrogenic substance.
[00611 It is yet another object of the invention to treat women who are taking
birth control pills
but who might nonetheless become pregnant by including D-chiro-inositol (or a
phosphate
thereof or other derivative thereof) and optionally a folate source into each
of the pills in the
birth control pill packet.
[00621 It is yet another object of the invention to treat women who are taking
birth control pills
and who may have excess estrogen insult with hyperactive/sensitive estrogen
receptor(ER)
positive breast tissue by including D-chiro-inositol (or a phosphate thereof
or other derivative
thereof) and optionally a folate source into each of the pills in the birth
control pill packet.
[00631 It is another object of the invention to prevent or reduce the rate of
birth defects
associated with in utero fetal mis-mapping.
[00641 It is yet another object of theinvention to prevent or reduce the rate
of birth defects
associated with improper signaling in at least one of the sonic hedgehog,
smoothened, and gli
pathways.
27
CA 02668580 2009-06-10
[0065] It is still another object of the invention to prevent or reduce the
rate of birth defects due
to a kinase disfunction in at least one of the inositol pathways.
[0066] It is another object of the invention to provide a method of preventing
or reducing birth
defects associated with cholesterol reduction medications.
[0067] Yet another object of the invention is the prevention or reduction of
the rate of birth
defects associated with fetal alcohol syndrome.
[0068] It is still another object of the invention to provide a fixed
combination formulation
comprising (A) at least one compound selected from (a) a folate or folic acid,
(b) a hormone or
steroid used in birth control, hormone replacement, and androgenablative
therapy, (c) a
cholesterol lowering medication, or (d) an anticancer medication, together
with (B) at least one
compound selected from an inositol, a phosphorylated inositol, and derivatives
thereof as defined
herein.
[0069] It is still another object of the invention to treat women who are on
estrogenic hormone
therapy and who may have estrogen-receptor (ER) and/or, ErbB receptor
overexpression
phenotype mediated by the (PI3K-Akt) pathway by administering as co-therapy
with said
estrogenic hormone therapy D-chiro-inositol (or a phosphate thereof or other
derivative thereof)
thereby blocking the downstream signaling elements resulting in cell cycle
arrest in the G1
phase, thereby downregulating these important receptors (Breast cancer
research 2004, 6: 219-
224).
28
CA 02668580 2009-06-10
[00701 It is still another object of the invention to treat women who are on
estrogenic hormone
therapy and who may have estrogen-receptor and/or, ErbB receptor
overexpression phenotype
mediated by the (PI3K-Akt) pathway by administering as a single composition
said estrogenic
hormone therapy drug and D-chiro-inositol (or a phosphate or other derivative
thereof or other
derivative thereof).
[00711 It is still another object of the invention to treat women who are on
anti-androgenic
hormone therapy and who may have estrogen-receptor and/or, ErbB receptor
overexpression
phenotype mediated by the (PI3K-Akt) pathway-receptor overexpression phenotype
by
administering as co-therapy with said anti-androgenic hormone therapy D-chiro-
inositol (or a
phosphate or other derivative thereof).
[00721 It is still another object of the invention to treat women who are on
anti-androgenic
hormone therapy and who may have estrogen-receptor and/or, ErbB receptor
overexpression
phenotype mediated by the (PI3K-Akt) pathway by administering as a single
composition said
anti-androgenic hormone therapy dug and D-chiro-inositol (or a phosphate or
other derivative
thereof).
[0073) It is still another object of the invention to treat men who are on
estrogenic hormone
therapy and who may have estrogen-receptor and/or, ErbB receptor
overexpression phenotype
29
CA 02668580 2009-06-10
mediated by the (PI3K-Akt) pathway by administering as co-therapy with said
estrogenic
hormone therapy D-chiro-inositol (or a phosphate or other derivative thereof).
[00741 It is still another object of the invention to treat men who are on
estrogenic hormone
therapy and who may may have estrogen-receptor and/or, ErbB receptor
overexpression
phenotype mediated by the (PI3K-Akt) pathway by administering as a single
composition said
estrogenic hormone therapy dug and D-chiro-inositol (or a phosphate or other
derivative
thereof).
[00751 It is still another object of the invention to treat men who are on
anti-androgenic
hormone therapy and who may have estrogen-receptor and/or, ErbB receptor
overexpression
phenotype mediated by the (PI3K-Akt) pathway by administering as co-therapy
with said anti-
androgenic hormone therapy D-chiro-inositol (or a phosphate or other
derivative thereof).
[00761 It is still another object of the invention to treat men who are on
anti-androgenic
hormone therapy and who may have estrogen-receptor and/or, ErbB receptor
overexpression
phenotype mediated by the (PI3K-Akt) pathway by administering as a single
composition said
anti-androgenic hormone therapy drug and D-chiro-inositol (or a phosphate or
other derivative
thereof).
[00771 It is still a further object of the invention to reduce or prevent
fetal malformation
occurrence where the fetal malformation is a neural tube defect, a cranio-
facial defect, an
CA 02668580 2009-06-10
anorectal malformation spectrum, caudal regression syndrome, neuralectoderm
derived pediatric
tumors, etc.
[00781 It is still another object of the invention to provide a method of
modulating the
phosphatidylinositol/PI3K signaling pathway with compounds and/or therapy of
the present
invention.
100791 A still further object of the present invention is to provide a method
of modualting the
sonic hedgehog, the receptors patched and smoothened, and GL1,2,3
transcription family
pathway with compounds and/or therapy of the present invention.
[00801 Another object of the invention is to provide a method of prevention or
amelioration or
treatment of a phosphatidylinositol/PI3K signaling pathway signaling defect
with the compounds
and/or therapies of the present invention.
[00811 Thus, it is an object of the invention to correct the inherent
mechanism of stem cell
autoregulation.
[0082) Another object of the invention is to increase the efficacy of standard
chemotherapeutic
agents.
31
CA 02668580 2009-06-10
100831 Yet another object of the invention is to provide a method of
prevention or amelioration
or treatment of a defect in the signaling pathway associated with sonic
hedgehog, the receptors
patched and/or smoothened, and/or GL 1,2,3 transcription family signaling
pathway with the
compounds and therapies of the present invention.
[0084] Yet another object of the invention is to provide a method or treatment
for anti-
angiogenic activity to reduce tumor incidence and/or tumor load.
[0085] Still another object of the invention is to induce antiangiogenesis in
localized or distant
metastasized tumors.
[0086] Yet another object of the invention is to decrease the risk of deep
vein thrombosis
(DVT's) while using chemotherapeutic agents, however administered, including
oral, parenteral,
or transdermal, birth control pills or hormonal products.
[0087] Yet another object of the invention is to provide a method or treatment
for
antiangoigenic activity to reduce Deep Vein Thrombosis (DVT's), pulmonary
emboli (PE's) etc.
utilizing the therapies and/or compounds of the present invention whether
administered
parenterally, orally, transdermally or other suitable administration mode.
[0088] Yet another object of the invention is to provide a method or treatment
for increasing
the chemotherapeutic efficacy by synergistic action of the current isomer (or
phosphate or other
32
CA 02668580 2009-06-10
derivative or of two or more of the isomer, a phosphate thereof or other
derivative thereof) with
standard chemotherapeutic agents in cancer treatments, especially breast,
prostate, blood, colon,
lung, liver, pancreatic, cervix, skin, and soft tissue cancers.
[00891 Still another object of the invention is to reduce the potential
hazardous risk of
tamoxifen-associated cardiovascular disease.
[00901 Yet another object of the invention is to reduce the numbers and size
of tumors locally
or distant especially in breast cancers, but also cancers originating from
blood, colon, lung, liver,
cervix, prostate, skin, and soft tissue.
[00911 Still another object of the invention is manipulating cell growth for
the regeneration of
neural, hepatic, pancreatic, intestinal, spleenic, and/or cardiac tissue.
[0092] Still another object of the invention is the treatment of tissue
necrosis factor related
conditions.
[00931 Yet another object of the invention is the treatment of conditions
associated with
abnormal kinase activity.
[0094] Yet another object of theinvention is to protect estrogen sensitive
tissue from excess
estrogen insult, whether such excess insult is due to absolute estrogen excess
or relative estrogen
33
CA 02668580 2009-06-10
excess with respect to normal estrogen/androgen balance, whether endogenous or
exogenously
derived.
[0095] A further object of the invention is the prevention of or reduction in
the amount or rate
of emergence of resistence of tumorigenic cells to anticancer agents.
[00961 A still further object of the invention is the administration of the
compound of the
invention, with or without additional therapeutic agents in a polymer matrix
or bound to a
polymer as a depot or implant.
[0097] An even further object of the invention is to inhibit cell growth in
the treatment of
psoriasis, actinic keratosis, acne, dermatitis, conditions of inappropriate or
excess hair growth,
and/or cosmetic purposes.
[0098] Another object of the inention is to provide methods and compositions
for obtaining at
least one of Shh loss-of-function or patched or smoothened gain-of-function by
administration of
at least one inositol isomer (other than D-chiroinositol or myo-inositol),
phosphorylate, or
derivative thereof. Still further objects of the invention will be apparent to
those of ordinary
skill.
(0099] It is therefore an object of the invention to provide novel compounds
that are inhibitors
of P13k-Akt signaling pathway.
34
CA 02668580 2009-06-10
[01001 It is also an object of the present invention to provide pharmaceutical
compositions that
comprise the novel compounds that are specific inhibitors of Akt/PKB.
[01011 It is also an object of the present invention to provide a method for
treating cancer that
comprises administering such inhibitors of Akt/PKB activity.
[01021 It is therefore an object of the invention to provide novel compounds
that are inhibitors
of overactive proinflammatory cytokines.
101031 It is also an object of the present invention to provide a method for
treating hepatitis
serous A,B,C,D that comprises administering such inhibitors of Tumor Necrosis
Factor.
[01041 It is also an object of the invention to improve the response to alpha
interferon therapy
by combining the invention compound with interferon.
[01051 It is another object of the invention to alleviate anemia due to
enlarged spleen problems
associated with chronic active hepatitis.
[01061 Yet it is another object of the invention to inhibit overexpression of
SOC3 and SHP2.
[01071 It is another object of the invention to upregilate p27kipl to overcome
Herceptin
resistance.
CA 02668580 2009-06-10
[01081 It is another object of the invention to upregulate P21cip for cell
cycle arrest in cancer
and inflammation.
[01091 Yet it is another object of the invention to down regulate or inhibit
Ap-1 , thus
inhibiting AP- I transcription factor causes blockade of multiple signal
transduction pathways
and inhibits cancer growth.
[01101 It is another object of the invention to inhibit or downregulate ppRb
thereby inhibiting
tumor survival factor.
[01111 Still other objects of theinvention will be apparent to those of
ordinary skill upon
reading the following specification.
SUMMARY OF THE INVENTION
[01121 These and other objects of the invention can be achieved via the
administration, to an
appropriate patient, of a compound which is a member of the family of D-
chiroinositol,
phosphorylates, and other derivatives thereof. In some uses, the objects can
also be achieved
with a broader array of inositol based compounds, their phosphorylates, and
other derivatives
thereof.
[01131 The foregoing fetal malformation prevention objects and others are
achieved by treating
women of child bearing years with D-chiro-inositol (and/or a phosphate or
other derivative
36
CA 02668580 2009-06-10
thereof) and optionally a folate source, optimally from pre-conception through
at least the first
trimester of pregnancy. Inclusion of the D-chiro-inositol along with birth
control pills has the
added benefit that stores of D-chiro-inositol (and/or phosphates and/or other
derivatives thereof)
and folate are high in women taking birth control pills even before they
discontinue such
treatment or become pregnant notwithstanding being on such therapy. A further
benefit of such
inclusion is that D-chiro-inositol (or a phosphate or other derivative
thereof) also downregulates,
modulates, or antagonizes estrogen-receptor and/or, ErbB receptor
overexpression phenotypes in
breast tissue.
[01141 The breast cancer avoidance objects of the invention are achieved by
administering D-
chiro-inositol (with or without folate) to patients who are known to have or
are suspect of having
estrogen-receptor and/or, ErbB receptor overexpression phenotypes that is
sensitive to estrogenic
substance exposure or to anti-androgenic therapy(which may ultimately result
in estrogenic
excess). The breast cancer avoidance (prevention) objects of the invention can
be achieved in
both men and women. While the benefits may be greater with the folate in many
of the
foregoing, the benefits can also be achieved even in the absence of the folate
component in many
of the present invention objects, and unless specifically excluded, or
required by the context to
be excluded, the folate free methods and treatments are included within the
scope of the
invention. Other objects of the invention with respect to Shh loss-of-function
and/or smoothened
or patched gain-of-function and the sequelae thereof are also achieved by
administration of an
inositol isomer other than D-chiroinositol and myo-inositol, phosphorylates,
pyrophosphorylates,
and other derivatives thereof and such non-D-chiroinositol non-myoinositol
based compounds
are also part of the invention.
37
CA 02668580 2009-06-10
[0115] One aspect of the present invention makes available methods and
compositions for
inhibiting certain receptors in cell pathway activation. In certain
embodiments, the subject
methods can be used to counteract the phenotypic effects of unwanted
activation of the pathway.
For example, the subject method can involve contacting a cell (in vitro or in
vivo) with the
compositions (defined infra), such as a D-chiroinositol or a phosphate or
other derivative thereof
in an amount sufficient to antagonize a dependent defective pathway
activation.
[0116] The invention objects involving modulating receptor-dependent pathway
activation can
be achieved by, for example, contacting a cell (in vitro or in vivo) with an
agonist (defined infra)
in an amount sufficient to activate a dependent pathway activation pathway.
[0117] In general the above-mentioned inositols are selected from D-
chiroinositol, their
phosphorylates, and other derivatives of either as described further herein.
For some of the
embodiments, the inositol compound may also be a different isomer of inositol,
its
phosphorylates and derivatives of either. These compounds (based on the D-
chiroinositol
structure) are described more fully in co-pending US Patent Applications Ser.
No. 11/591,398,
filed Nov. 1, 2006 and US 12/001,869, filed Dec 13, 2007. Those compounds, as
well as their
corresponding analogs that differ by being based on myo-inositol or other
inositol isomer rather
than being based on D-chiroinositol, (whether or not further detailed herein
are incorporated
herein by reference) may be chosen as the inositol component in addition to
any of the inositol
component compounds specifically set forth herein. The optional folate
component is also
38
CA 02668580 2009-06-10
detailed in these two co-pending applications and that disclosure is also
incorporated herein by
reference as the optional folate component in addition to any specific
disclosure set forth herein.
[0118] The subject compounds may be formulated as a pharmaceutical preparation
comprising
a pharmaceutically acceptable excipient. Antagonists of the invention and/or
preparations
comprising them may be administered to a patient to treat conditions involving
unwanted cell
proliferation, e.g., cancer and/or tumors (such as, without limitation,
medulloblastoma,
rhabdomyosarcomas, adenocarcinomas, basal cell carcinoma, etc, non-malignant
hyperproliferative disorders, etc). Receptor agonists such as those for
smoothened or G-protein
coupled receptors can also be used to regulate the growth and differentiation
of normal tissues.
In certain embodiments, such compounds or preparations are administered
systemically and/or
locally, e.g., topically, transdermally, or as an injected depot or an implant
in and/or around
tumor site after excision or incisional biopsies.
BRIEF DESCRIPTION OF THE DRAWING
[0119] Not Applicable
DETAILED DESCRIPTION OF THE INVENTION
[0120] I. General
[0121] A. Definitions
39
CA 02668580 2009-06-10
[0122] I. Biological and Medicinal Terms
[0123] For convenience, certain terms employed in the specification, examples,
and appended
claims are collected here.
[0124] The phrase "aberrant modification or mutation" of a gene refers to such
genetic lesions
as, for example, deletions, substitution or addition of nucleotides to a gene,
as well as gross
chromosomal rearrangements of the gene and/or abnormal methylation of the
gene. Likewise,
mis-expression of a gene refers to aberrant levels of transcription of the
gene relative to those
levels in a normal cell under similar conditions, as well as non-wild-type
splicing of mRNA
transcribed from the gene.
[0125] "Basal cell carcinomas" exist in a variety of clinical and histological
forms such as
nodular-ulcerative, superficial, pigmented, morphealike, fibroepithelioma and
nevoid syndrome.
Basal cell carcinomas are the most common cutaneous neoplasms found in humans.
The majority
of new cases of nonmelanoma skin cancers fall into this category.
[0126] "Burn wounds" refer to cases where large surface areas of skin have
been removed or
lost from an individual due to heat and/or chemical agents.
[0127] The term "carcinoma" refers to a malignant new growth made up of
epithelial cells
tending to infiltrate surrounding tissues and to give rise to metastases.
Exemplary carcinomas
CA 02668580 2009-06-10
include: "basal cell carcinoma", which, is an epithelial tumor of the skin
that, while seldom
metastasizing, has potentialities for local invasion and destruction;
"squamous cell carcinoma",
which refers to carcinomas arising from squamous epithelium and having cuboid
cells;
"carcinosarcoma", which include malignant tumors composed of carcinomatous and
sarcomatous
tissues; "adenocystic carcinoma", carcinoma marked by cylinders or bands of
hyaline or
mucinous stroma separated or surrounded by nests or cords of small epithelial
cells, occurring in
the mammary and salivary glands, and mucous glands of the respiratory tract;
"epidermoid
carcinoma", which refers to cancerous cells which tend to differentiate in the
same way as those
of the epidermis; i.e., they tend to form prickle cells and undergo
comification; "nasopharyngeal
carcinoma", which refers to a malignant tumor arising in the epithelial lining
of the space behind
the nose; and "renal cell carcinoma", which pertains to carcinoma of the renal
parenchyma
composed of tubular cells in varying arrangements. Other carcinomatous
epithelial growths are
"papillomas", which refers to benign tumors derived from epithelium and having
a
papillomavirus as a causative agent; and "epidermoidomas", which refers to a
cerebral or
meningeal tumor formed by inclusion of ectodermal elements at the time of
closure of the neural
groove.
101281 The "corium" or "dermis" refers to the layer of the skin deep to the
epidermis,
consisting of a dense bed of vascular connective tissue, and containing the
nerves and terminal
organs of sensation. The hair roots, and sebaceous and sweat glands are
structures of the
epidermis which are deeply embedded in the dermis.
41
CA 02668580 2009-06-10
[0129] "Dental tissue" refers to tissue in the mouth which is similar to
epithelial tissue, for
example gum tissue. The method of the present invention is useful for treating
periodontal
disease.
[0130] "Dermal skin ulcers" refer to lesions on the skin caused by superficial
loss of tissue,
usually with inflammation. Dermal skin ulcers which can be treated by the
method of the
present invention include decubitus ulcers, diabetic ulcers, venous stasis
ulcers and arterial
ulcers. Decubitus wounds refer to chronic ulcers that result from pressure
applied to areas of the
skin for extended periods of time. Wounds of this type are often called
bedsores or pressure
sores. Venous stasis ulcers result from the stagnation of blood or other
fluids from defective
veins. Arterial ulcers refer to necrotic skin in the area around arteries
having poor blood flow.
[0131] The term "ED50" means the dose of a drug which produces 50% of its
maximum
response or effect.
[0132] An "effective amount" of, e.g., a receptor antagonist, with respect to
the subject method
of treatment, refers to an amount of the antagonist in a preparation which,
when applied as part
of a desired dosage regimen brings about, e.g., a change in the rate of cell
proliferation and/or the
state of differentiation of a cell and/or rate of survival of a cell according
to clinically acceptable
standards for the disorder to be treated or the cosmetic purpose.
42
CA 02668580 2009-06-10
[01331 The terms "epithelia", "epithelial" and "epithelium" refer to the
cellular covering of
internal and external body surfaces (cutaneous, mucous and serous), including
the glands and
other structures derived therefrom, e.g., corneal, esophegeal, epidermal, and
hair follicle
epithelial cells. Other exemplary epithlelial tissue includes: olfactory
epithelium, which is the
pseudostratified epithelium lining the olfactory region of the nasal cavity,
and containing the
receptors for the sense of smell; glandular epithelium, which refers to
epithelium composed of
secreting cells; squamous epithelium, which refers to epithelium composed of
flattened plate-like
cells. The term epithelium can also refer to transitional epithelium, like
that which is
characteristically found lining hollow organs that are subject to great
mechanical change due to
contraction and distention, e.g., tissue which represents a transition between
stratified squamous
and columnar epithelium.
[01341 The term "epithelialization" refers to healing by the growth of
epithelial tissue over a
denuded surface.
[01351 The term "epidermal gland" refers to an aggregation of cells associated
with the
epidermis and specialized to secrete or excrete materials not related to their
ordinary metabolic
needs. For example, "sebaceous glands" are holocrine glands in the corium that
secrete an oily
substance and sebum. The term "sweat glands" refers to glands that secrete
sweat, situated in the
corium or subcutaneous tissue, opening by a duct on the body surface.
43
CA 02668580 2009-06-10
101361 The term "epidermis" refers to the outermost and nonvascular layer of
the skin, derived
from the embryonic ectoderm, varying in thickness from 0.07-1.4 mm. On the
palmar and
plantar surfaces it comprises, from within outward, five layers: basal layer
composed of
columnar cells arranged perpendicularly, prickle-cell or spinous layer
composed of flattened
polyhedral cells with short processes or spines; granular layer composed of
flattened granular
cells; clear layer composed of several layers of clear, transparent cells in
which the nuclei are
indistinct or absent; and horny layer composed of flattened, cornified non-
nucleated cells. In the
epidermis of the general body surface, the clear layer is usually absent.
[01371 "Excisional wounds" include tears, abrasions, cuts, punctures or
lacerations in the
epithelial layer of the skin and may extend into the dermal layer and even
into subcutaneous fat
and beyond. Excisional wounds can result from surgical procedures or from
accidental
penetration of the skin.
101381 The "growth state" of a cell refers to the rate of proliferation of the
cell and/or the state
of differentiation of the cell. An "altered growth state" is a growth state
characterized by an
abnormal rate of proliferation, e.g., a cell exhibiting an increased or
decreased rate of
proliferation relative to a normal cell.
[01391 The term "hair" refers to a threadlike structure, especially the
specialized epidermal
structure composed of keratin and developing from a papilla sunk in the
corium, produced only
by mammals and characteristic of that group of animals. Also, "hair" may refer
to the aggregate
44
CA 02668580 2009-06-10
of such hairs. A "hair follicle" refers to one of the tubular-invaginations of
the epidermis
enclosing the hairs, and from which the hairs grow. "Hair follicle epithelial
cells" refers to
epithelial cells which surround the dermal papilla in the hair follicle, e.g.,
stem cells, outer root
sheath cells, matrix cells, and inner root sheath cells. Such cells may be
normal non-malignant
cells, or transformed/immortalized cells.
[01401 The term "hedgehog antagonist" refers to an agent which potentiates or
recapitulates the
bioactivity of patched, such as to repress transcription of target genes.
Preferred hedgehog
antagonists can be used to overcome a ptc loss-of-function and/or a smoothened
gain-of-
function, the latter also being referred to as smoothened antagonists. The
term 'hedgehog
antagonist' as used herein refers not only to any agent that may act by
directly inhibiting the
normal function of the hedgehog protein, but also to any agent that inhibits
the hedgehog
signalling pathway, and thus recapitulates the function of ptc.
[01411 The term "hedgehog gain-of-function" refers to an aberrant modification
or mutation of
a ptc gene, hedgehog gene, or smoothened gene, or a decrease (or loss) in the
level of expression
of such a gene, which results in a phenotype which resembles contacting a cell
with a hedgehog
protein, e.g., aberrant activation of a hedgehog pathway. The gain-of-function
may include a
loss of the ability of the ptc gene product to regulate the level of
expression of Ci genes, e.g.,
Gli 1, Gli2, and Gli3. The term 'hedgehog gain-of-function' is also used
herein to refer to any
similar cellular phenotype (e.g., exhibiting excess proliferation) which
occurs due to an alteration
anywhere in the hedgehog signal transduction pathway, including, but not
limited to, a
modification or mutation of hedgehog itself. For example, a tumor cell with an
abnormally high
CA 02668580 2009-06-10
proliferation rate due to activation of the hedgehog signalling pathway would
have a 'hedgehog
gain-of-function' phenotype, even if hedgehog is not mutated in that cell.
[01421 As used herein, "immortalized cells" refers to cells which have been
altered via
chemical and/or recombinant means such that the cells have the ability to grow
through an
indefinite number of divisions in culture.
[01431 "Internal epithelial tissue" refers to tissue inside the body which has
characteristics
similar to the epidermal layer in the skin. Examples include the lining of the
intestine. The
method of the present invention is useful for promoting the healing of certain
internal wounds,
for example wounds resulting from surgery.
[01441 The term "keratosis" refers to proliferative skin disorder
characterized by hyperplasia of
the horny layer of the epidermis. Exemplary keratotic disorders include
keratosis follicularis,
keratosis palmaris et plantaris, keratosis pharyngea, keratosis pilaris, and
actinic keratosis.
[01451 The term "LD50" means the dose of a drug which is lethal in 50% of test
subjects.
101461 The term "nail" refers to the horny cutaneous plate on the dorsal
surface of the distal
end of a finger or toe.
46
CA 02668580 2009-06-10
[01471 The term "patched loss-of-function" refers to an aberrant modification
or mutation of a
ptc gene, or a decreased level of expression of the gene, which results in a
phenotype which
resembles contacting a cell with a hedgehog protein, e.g., aberrant activation
of a hedgehog
pathway. The loss-of-function may include a loss of the ability of the ptc
gene product to
regulate the level of expression of Ci genes, e.g., Glil, Gli2 and Gli3. The
term 'ptc loss-of-
function' is also used herein to refer to any similar cellular phenotype
(e.g., exhibiting excess
proliferation) which occurs due to an alteration anywhere in the hedgehog
signal transduction
pathway, including, but not limited to, a modification or mutation of ptc
itself. For example, a
tumor cell with an abnormally high proliferation rate due to activation of the
hedgehog signalling
pathway would have a 'ptc loss-of-function' phenotype, even if ptc is not
mutated in that cell.
[01481 A "patient" or "subject" to be treated by the subject method can mean
either a human or
non-human animal.
[01491 The term "prodrug" is intended to encompass compounds which, under
physiological
conditions, are converted into the therapeutically active agents of the
present invention. A
common method for making a prodrug is to include selected moieties which are
hydrolyzed
under physiological conditions to reveal the desired molecule. In other
embodiments, the prodrug
is converted by an enzymatic activity of the host animal.
[01501 As used herein, "proliferating" and "proliferation" refer to cells
undergoing mitosis.
47
CA 02668580 2009-06-10
[0151] Throughout this application, the term "proliferative skin disorder"
refers to any
disease/disorder of the skin marked by unwanted or aberrant proliferation of
cutaneous tissue.
These conditions are typically characterized by epidermal cell proliferation
or incomplete cell
differentiation, and include, for example, X-linked ichthyosis, psoriasis,
atopic dermatitis,
allergic contact dermatitis, epidermolytic hyperkeratosis, and seborrheic
dermatitis. For
example, epidermodysplasia is a form of faulty development of the epidermis.
Another example
is "epidermolytis", which refers to a loosened state of the epidermis with
formation of blebs and
bullae either spontaneously or at the site of trauma.
[0152] As used herein, the term "psoriasis" refers to a hyperproliferative
skin disorder which
alters the skin's regulatory mechanisms. In particular, lesions are formed
which involve primary
and secondary alterations in epidermal proliferation, inflammatory responses
of the skin, and an
expression of regulatory molecules such as lymphokines and inflammatory
factors. Psoriatic
skin is morphologically characterized by an increased turnover of epidermal
cells, thickened
epidermis, abnormal keratinization, inflammatory cell infiltrates into the
dermis layer and
polymorphonuclear leukocyte infiltration into the epidermis layer resulting in
an increase in the
basal cell cycle. Additionally, hyperkeratotic and parakeratotic cells are
present.
[0153] The term "skin" refers to the outer protective covering of the body,
consisting of the
corium and the epidermis, and is understood to include sweat and sebaceous
glands, as well as
hair follicle structures. Throughout the present application, the adjective
"cutaneous" may be
used, and should be understood to refer generally to attributes of the skin,
as appropriate to the
context in which they are used.
48
CA 02668580 2009-06-10
[0154] The term "smoothened gain-of-function" refers to an aberrant
modification or mutation
of a smo gene, or an increased level of expression of the gene, which results
in a phenotype
which resembles contacting a cell with a hedgehog protein, e.g., aberrant
activation of a
hedgehog pathway. While not wishing to be bound by any particular theory, it
is noted that ptc
may not signal directly into the cell, but rather interact with smoothened,
another membrane-
bound protein located downstream of ptc in hedgehog signaling (Mango et al.,
(1996) Nature
384: 177-179). The gene smo is a segment-polarity gene required for the
correct patterning of
every segment in Drosophila (Alcedo et al., (1996) Cell 86: 221-232). Human
homologs of smo
have been identified. See, for example, Stone et al. (1996) Nature 384:129-
134, and GenBank
accession U8440 1. The smoothened gene encodes an integral membrane protein
with
characteristics of heterotrimeric G-protein-coupled receptors; i.e., 7-
transmembrane regions.
This protein shows homology to the Drosophila Frizzled (Fz) protein, a member
of the wingless
pathway. It was originally thought that smo encodes a receptor of the Hh
signal. However, this
suggestion was subsequently disproved, as evidence for ptc being the Hh
receptor was obtained.
Cells that express Smo fail to bind Hh, indicating that smo does not interact
directly with Hh
(Nusse, (1996) Nature 384: 119-120). Rather, the binding of Sonic hedgehog
(SHH) to its
receptor, PTCH, is thought to prevent normal inhibition by PTCH of smoothened
(SMO), a
seven-span transmembrane protein. Recently, it has been reported that
activating smoothened
mutations occur in sporadic basal cell carcinoma, Xie et al. (1998) Nature
391: 90-2, and
primitive neuroectodermal tumors of the central nervous system, Reifenberger
et al. (1998)
Cancer Res 58: 1798-803.
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CA 02668580 2009-06-10
[01551 The term "therapeutic index" refers to the therapeutic index of a drug
defined as
LD50/ED50.
[01561 As used herein, "transformed cells" refers to cells which have
spontaneously converted
to a state of unrestrained growth, i.e., they have acquired the ability to
grow through an indefinite
number of divisions in culture. Transformed cells may be characterized by such
terms as
neoplastic, anaplastic and/or hyperplastic, with respect to their loss of
growth control.
[01571 2. Chemical Terms
[01581 An "aliphatic group" includes straight chain or branched chain, or
cyclic hydrocarbons
(other than aromatic groups), the aliphatic group having up to 30 carbon atoms
(preferably up to
20 carbon atoms, more preferably up to 10 carbon atoms, even more preferably
up to 7 carbon
atoms, most preferably up to 5 carbon atoms, especially methyl, ethyl, propyl,
and butyl for
straight chain saturated variants) and the corresponding branched analogs and
the unsaturated
analogs of each and 3-10 carbon atoms in the corresponding cyclic aliphatic
rings
(cycloaliphatic), more preferably 5, 6, or 7 ring members, Each aliphatic
group may be
unsubstituted or substituted with one or more substituents as detailed below.
Furthermore, each
of the above groups can be interrupted by one or more heteroatoms selected
from nitrogen,
sulfur, oxygen, and phosphorous, excepting peroxy (-0-0-). The cycloaliphatic
rings may have
two, three, or four rings fused together, each ring independently having
definitions in accordance
with this paragraph. Each aliphatic group and cycloaliphatic ring may be
independently
CA 02668580 2009-06-10
unsubstituted or substituted in accordance witht the definitions below,
however, if a definition
results in a continuous loop, only three loop circuits at most are permitted.
[0159] An "aromatic group" or "aryl group" as used herein includes (unless
specifically
excluded or the context requires exclusion) heteroaryls, and each ring of
which has 6 to 8 ring
members per aromatic ring and may be fused to aromatic or aliphatic rings,
each of which is
unsubstituted or substituted with one of more substituents as set forth more
fully below.
Heteroaryls correspond to carbocyclic aryls except that they have one or more
ring members
selected from nitrogen, oxygen, or sulfur. Each aromatic group may be
independently
unsubstituted or substituted in accordance witht the definitions below,
however, if a definition
results in a continuous loop, only three loop circuits at most are permitted.
[0160] Substituents for the above aliphatic and aromatic groups may include,
without
limitation, those from the following group (A): halogen (prefereably fluorine,
chlorine, bromine,
or iodine, more preferably fluorine or chlorine), hydroxyl, trihalomethyl
(especially
trifluoromethyl), cyano, carbonyl, derivatized carbonyl (such as carboxylic
acid, alkoxycarbonyl,
(optionally N- substituted with alkyl or acyl)aminocarbonyl, formyl),
C2_7acyl, C2_7 acyloxy,
thiocarbonyl, analogous derivitized forms thereof to the derivatized carbonyl
in which the doubly
bound oxygen is replaced by sulfur, the corresponding -C(S)SH group and and
their derivatized
counterparts, phosphoryl, phosphate, phosphonate, phosphinate, amido, amidine,
imine, cyano,
nitro, azido, sulfhydryl, alkyl thio, sulfate, sulfonate, sulfamoyl,
sulfonamide, sulfonyl,
heterocyclyl (aka heterocycloaliphatic), aralkyl, aromatic group, or
heteroaromatic group, each
51
CA 02668580 2009-06-10
of which can be substituted further not from the above subtituent list, but
from one or more
substituents selected from the following group (B) consisting of amino, azido,
imino, amido,
phosphoryl (including phosphonate and phosphinate), sulfonyl (including
sulfate, sulfonamide,
sulfamoyl, and sulfonate), ether, alkylthio, carbonyl (including ketone,
aldehyde, carboxylate,
and ester), trihalomethyl (especially trifluoromethyl), cyano, alkoxy,
hydroxy, and the like, and
each of these further group (B) substituents may be still further substituted
with groups selected
from group (B). In any case where the above substituent requires a group to be
specified that is
not so specified above or below (for example an amino, an ether, an ester,
etc. where the
remainder of the group cannot be determined from the above or below), the
preferred group is,
without being limited thereto, an alkyl of up to 7 carbon atoms or if results
in a cyclical unending
definitional loop, such loop terminates after no more than three cycles
thereof in either a
hydrogen or alkyl of up to 7 carbon atoms). These substituents may also be a
replacement for
one or more of the hydrogen atoms on the inositol ring hydroxy groups or a
hydroxyl group
indicated wihin the substituents set forth in this definition (provided that
no peroxy groups result)
and/or one or more of the hydrogen atoms that are on the inositol ring
directly and/or or a
hydroxyl group indicated wihin the substituents set forth in this substituent
definition provided
that if an unending loop results, such loop terminates after no more than
three cycles thereof in
either a hydrogen or alkyl of up to 7 carbon atoms.
[01611 In addition to the general definition above, the term "alkylthio"
refers to an alkyl group,
as defined above, having a sulfur radical attached thereto. In preferred
embodiments, the
"alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl,
and -S-(CH2)m Ra,
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CA 02668580 2009-06-10
wherein in is 0-8, preferably 0-4 and Ra is aryl, cycloalkyl, cycloalkenyl,
heterocyclyl, or
polycyclyl. Representative alkylthio groups include methylthio, ethylthio, and
the like.
[0162] In addition to the general definition above, the terms "amine" and
"amino" are art-
recognized and refer to both unsubstituted and substituted amines, e.g., a
moiety that can be
represented by the general formula:
NRc Rd Rc
R or + I
b R b
wherein Rb, R, and Rd each independently represent a hydrogen, an alkyl, an
alkenyl, -(CH2)m
Ra, or carbonyl or Rb and R, taken together with the N atom to which they are
attached complete
a heterocycle having from 4 to 8 atoms in the ring structure; Ra represents an
aryl, a cycloalkyl, a
cycloalkenyl, a heterocycle or a polycycle; and in is zero or an integer in
the range of 1 to 8. In
preferred embodiments, only one of Rb, R, and Rd can be a carbonyl, e.g., they
and the nitrogen
together do not form an imide. In even more preferred embodiments, each
independently
represent a hydrogen, an alkyl, an alkenyl, or -(CH2)m Ra. Thus, the term
"alkylamine" as used
herein means an amine group, as defined above, having a substituted or
unsubstituted alkyl
attached thereto, i.e., at least one of Rb and Rc is an alkyl group.
53
CA 02668580 2009-06-10
[01631 The term "amido" is art-recognized as an amino-substituted carbonyl and
includes a
moiety that can be represented by the general formula:
O
AN 11 Rb
I
RC
Rb and R, are as defined above. Preferred embodiments of the amide will not
include imides
which may be unstable.
[01641 The term "aralkyl", as used herein, refers to an alkyl group
substituted with an aryl
group (e.g., an aromatic or heteroaromatic group).
[01651 The term "aryl" as used herein includes 5-, 6-, and 7-membered single-
ring aromatic
groups that may include from zero to four heteroatoms, for example, benzene,
pyrrole, furan,
thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine,
pyrazine, pyridazine and
pyrimidine, and the like. Those aryl groups having heteroatoms in the ring
structure may also be
referred to as "aryl heterocycles" or "hetroaryls" or "heteroaromatics." The
aromatic ring can be
substituted at one or more ring positions with such substituents as described
above, for example,
halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,
alkoxyl, amino, nitro,
sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl,
carboxyl, ether,
alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl,
aromatic or
54
CA 02668580 2009-06-10
heteroaromatic moieties, -CF3, -CN, or the like. The term "aryl" also includes
polycyclic ring
systems having two or more cyclic rings in which two or more carbons are
common to two
adjoining rings (the rings are "fused rings") wherein at least one of the
rings is aromatic, e.g., the
other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls
and/or heterocyclyls.
[0166] The term "carbocycle", as used herein, refers to an aromatic or non-
aromatic ring in
which each atom of the ring is carbon.
[0167] The term "carbonyl" is art-recognized and includes such moieties as can
be represented
by the general formula:
O O
AX 11 Re or X'J~ Re
wherein X is a bond or represents an oxygen or a sulfur, and Re represents a
hydrogen, an alkyl,
an alkenyl, -(Xa)n-(CH2)m Ra or a pharmaceutically acceptable salt, where X,
in and Ra are as
defined above Xa is a bond or represents an oxygen or a sulfur, and n is 0 or
1, but required to be
0 when Re is bound to an oxygen. When X is oxygen and Re is -(Xa)n -(CH2)n,-Ra
and Xa is also
oxygen, the substituent is a carbonate. Where X is oxygen and Re is not
hydrogen, the formula
represents an "ester". Where X is oxygen, and Re. is as defined above, the
moiety is referred to
herein as a carboxyl group, and particularly when Re is a hydrogen, the
formula represents a
"carboxylic acid" or a formate respectively. In general, where the oxygen atom
of the above
CA 02668580 2009-06-10
formula is replaced by sulfur, the formula represents a "thiocarbonyl" group.
On the other hand,
where X and Xa are each a bond, and Re. is not hydrogen, the above formula
represents a
"ketone" group. Where X is a bond, and Re is hydrogen, the above formula
represents an
"aldehyde" group.
101681 The term "heteroatom" as used herein means an atom of any element other
than carbon
or hydrogen. Preferred heteroatoms are nitrogen, oxygen, phosphorus, sulfur,
and selenium,
more preferably nitrogen, oxygen, phosphorus, and sulfur; most preferably
nitrogen, oxygen, and
sulfur.
[1691 The terms "heterocyclyl" or "heterocyclic group", notwithstanding and
without limitation
to prior definitions of these terms herein, refer to 3- to 1 0-membered ring
structures, more
preferably 3- to 7-membered rings, whose ring structures include one to four
heteroatoms.
Heterocycles can also be polycycles. Heterocyclyl groups include, for example,
thiophene,
thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin,
pyrrole, imidazole,
pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine,
indolizine, isoindole,
indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine,
naphthyridine,
quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,
phenanthridine, acridine,
pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,
phenoxazine,
pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine,
lactones, lactams
such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The
heterocyclic ring
can be substituted at one or more positions with such substituents as
described above, as for
example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,
amino, nitro, sulfhydryl,
56
CA 02668580 2009-06-10
imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl,
ether, alkylthio,
sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or
heteroaromatic moiety, -CF3, -
CN, or the like.
[01701 Also for purposes of this invention, the term "hydrocarbon" is
contemplated to include
all permissible compounds having at least one hydrogen and one carbon atom. In
a broad aspect,
the permissible hydrocarbons include acyclic and cyclic, branched and
unbranched, carbocyclic
and heterocyclic, aromatic and nonaromatic organic compounds which can be
substituted or
unsubstituted. Preferably hydrocarbons contain only hydrogen and carbon unless
modified to
indicate some other type of atom is present.
[01711 The term "lower" in connection with an aliphatic group means up to 7
carbons,
preferably up to 5 carbons, more preferably up to 4 carbons.
[01721 A "phosphonamidite" can be represented in the general formula:
Rf Rf
I I
Q \ or QIF,01K9
/- Rc Rc
Rb Rb
wherein Rb and R. are as defined above, Q2 represents 0, S or N, Rf.
represents a lower alkyl or
an aryl, and Rg represents H, lower alkyl, or aryl.
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CA 02668580 2009-06-10
[01731 A "phosphoramidite" corresponds to the above phosphonoamidite except
that Rf is
replaced by =O:
101741 A "phosphoryl" can in general be represented by the formula:
Q,
-P-
O-Rg
wherein Q1 is 0 or S and Rg is hydrogen, alower alkyl, or an acyl.
[01751 The phrase "protecting group" as used herein means temporary
substituents which
protect a potentially reactive functional group from undesired chemical
transformations.
Examples of such protecting groups include esters of carboxylic acids, silyl
ethers of alcohols,
and acetals and ketals of aldehydes and ketones, respectively. The field of
protecting group
chemistry has been reviewed (Greene, T. W.; Wuts, P. G. M Protective Groups in
Organic
Synthesis, 2nd ed.; Wiley: New York, 1991).
[01761 As used herein, the term "substituted" is contemplated to include all
permissible
substituents of organic compounds. In a broad aspect, the permissible
substituents include
acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic,
aromatic and
nonaromatic substituents of organic compounds. Illustrative substituents
include, for example,
those described herein above. The permissible substituents can be one or more
and the same or
different for appropriate organic compounds. For purposes of this invention,
the heteroatoms
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CA 02668580 2009-06-10
such as nitrogen may have hydrogen substituents and/or any permissible
substituents of organic
compounds described herein which satisfy the valences of the heteroatoms. This
invention is not
intended to be limited in any manner by the permissible substituents of
organic compounds.
[0177] It will be understood that "substitution" or "substituted with"
includes the implicit
provison that such substitution is in accordance with permitted valence of the
substituted atom
and the substituent, and that the substitution results in a stable compound,
e.g., which does not
spontaneously undergo transformation such as by rearrangement, cyclization,
elimination, etc.
[01781 As used herein, the definition of each expression, e.g., alkyl, m, any
particular R group,
etc., when it occurs more than once in any structure, is intended to be
independent of its
definition elsewhere in the same structure.
[0179] The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognized and
refer to
trifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, and
nonafluorobutanesulfonyl
groups, respectively. The terms triflate, tosylate, mesylate, and nonaflate
are art-recognized and
refer to trifluoromethanesulfonate ester, p-toluenesulfonate ester,
methanesulfonate ester, and
nonafluorobutanesulfonate ester functional groups and molecules that contain
said groups,
respectively.
[0180] The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,
phenyl,
trifluoromethanesulfonyl, nonafluorobutanesulfonyl, p-toluenesulfonyl and
methanesulfonyl,
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CA 02668580 2009-06-10
respectively. A more comprehensive list of the abbreviations utilized by
organic chemists of
ordinary skill in the art appears in the first issue of each volume of the
Journal of Organic
Chemistry; this list is typically presented in a table entitled Standard List
of Abbreviations. The
abbreviations contained in said list, and all abbreviations utilized by
organic chemists of ordinary
skill in the art are hereby incorporated by reference.
101811 3. Compounds
[01821 In the following discussion, for the invention compounds, reference
will be made to D-
chiroinositol, its phosphorylates, and derivatives of either, but it should be
understood that the
present invention, unless specifically indicated otherwise, or the context
requires or the
avoidance of prior art requires, includes the corresponding compounds based on
any other
inositol isomer. Where exclusion is not explicit in this specification, but is
required due to prior
art, the invention compounds are to be deemed to exclude such prior art
compounds in particular,
and preferably exclude compounds having the same substituent pattern exactly
as the prior art
compounds but based on an inositol isomer other than D-chiroinositol or
myoinositol, or
preferably having the same substituent pattern as the prior art compound but
based on an inositol
isomer other than D-chiroinositol, or preferably having the same substituents
but in the same
pattern or a different pattern from that in the prior art molecule based on
the same inositol isomer
as the prior art compound, or preferably having the same substituents but in
the same pattern or a
different pattern from that in the prior art molecule based on an inositol
isomer other than D-
chiroinositol or myo-inositol, or preferably having the same substituents but
in the same pattern
or a different pattern from that in the prior art molecule based on an
inositol isomer other than D-
CA 02668580 2009-06-10
chiroinositol. These exclusions are on a utility-by-utility and composition-by-
composition basis
so that exclusion of a compound for one utility does not exclude the compound
from being
within the invention for another utility or exclusion of a compound in one
formulation does not
exclude the compound for being within the invention for another compsition.
Every recitation of
compounds in this specification as part of the invention is deemed to be the
specific recitation as
well as the more general recitiations to include the corresponding compounds
based on inositol
isomers other than the one specifically mentioned subject to the exclusions
set forth in this
paragraph, and on the location of prior art that requires exclusion of one or
more compounds oin
a particular use or composition, application of the various exclusions above
to the particular
recitation in the particular context is deemed to have been specifically
recited herein.
[01831 4. Uses
[01841 In one aspect, the present invention relates to the discovery that
signal transduction
pathways regulated by either phosphatidylinositol and/or by Shh and its
constituents (patched
(ptc), gli and/or smoothened) can be inhibited, at least in part, by D-
chiroinositol and/or
derivatives thereof (as set forth more fully below). While not wishing to
bound by any particular
theory, the activation of the receptor proteins is believed to be the
mechanism by which these
agents act. For example, the ability of these agents to inhibit proliferation
or patched loss-of
function (ptclof) cells may, be due to the ability of such molecules to
interact with hedgehog,
patched, or smoothened, or at least to interfere with the ability of those
receptor proteins to
activate a hedgehog, ptc, and/or smoothened-mediated signal transduction
pathway. Again,
without being bound thereto, it is the inventor's belief that D-chiroinostitol
(or a phosphorylated
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CA 02668580 2009-06-10
or other derivative thereof, preferably combinations of two or more selected
from D-
chiroinositol, its phosphates, or other derivatives thereof) stimulates these
signaling mechanisms,
activating certain isoforms of protein kinases which kinases appear to be
involved in neural tube
defects prevention.
[0185] It is, therefore, specifically contemplated that these small molecules
(D-chiroinositol
and its derivatives) which interfere with aspects of signal transduction
activity will likewise be
capable of inhibiting proliferation (or other biological consequences) in
cells. In preferred
embodiments, the subject inhibitors are organic molecules having a molecular
weight less than
2500 amu, more preferably less than 1500 amu, and even more preferably less
than 750 amu, and
are capable of modulating (inhibiting or activating) at least some of the
biological activities of
hedgehog proteins, protein kinases and preferably specifically in target
cells.
[0186] Thus, the methods of the present invention include the use of D-chiro
insoitol and/or
derivatives thereof (optionally with folate sources) which agonize (mimic) the
inhibition of
certain receptor complexes of hedgehog signaling, such as by inhibiting
activation of
downstream components of the signal pathway, in the regulation of repair
and/or functional
performance of a wide range of cells, tissues and organs. For instance, the
subject method has
therapeutic and cosmetic applications ranging from regulation of neural
tissues, bone and
cartilage formation and repair, regulation of spermatogenesis, ovulation,
regulation of smooth
muscle, regulation of lung, liver and other organs arising from the primitive
gut, regulation of
hematopoietic function, regulation of skin and hair growth, etc. Moreover, the
subject methods
can be performed on cells which are provided in culture (in vitro), or on
cells in a whole animal
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CA 02668580 2009-06-10
(in vivo). See, for example, PCT publications WO 95/18856 and WO 96/17924 (the
specifications of which are expressly incorporated by reference herein).
[0187] In a preferred embodiment, the subject method can be to treat
epithelial cells. For
instance, the subject method can be used in treating or preventing basal cell
carcinoma or other
hedgehog pathway-related disorders.
[0188] In another preferred embodiment, the subject method can be used as part
of a treatment
regimen for malignant medulloblastoma and other primary CNS malignant
neuroectodermal
tumors. Additional exemplary cancers in which the present invention is of use
includes those in
the following table
ORGAN OR TISSUE SPECIES CELL LINE
1. BLOOD Human Erythrolekemia
K562 cell line
K562 + human bone marrow
2.Colon Human Adenocarcinoma
HT-29cell line
3. Lung Rat Tracheal epithelium
+B{a}P
4. Liver Human HepG2 cells
5. Mammary Human Adenocarcinoma
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MCF-7, MDA-MB 231 cells
6. Cervix(uterine) Human HeLa cells
7.Skin Mouse JB6 cells
Mouse HEL-30 cells
8. Soft tissue Mouse 3T3 fibroblast
Human Rabdomyosarcoma, RD cells
(Shamsuddin Abul& Vucenik, Ivana, Current Cancer Therapy Reviews, 2005, 1,259-
269).
[01891 In another aspect, the present invention provides pharmaceutical
preparations
comprising, as an active ingredient which is D-chiroinositol or a derivative
thereof with or
without a folate source, a receptor antagonist/agonist, or protein modulator,(
antagonist/agonist),
a kinase antagonist or agonist formulated in an amount sufficient to inhibit,
in vivo, proliferation
or other biological consequences of aberrant PI3K/Shh cellular signaling,
especially ptc loss-of-
function, hedgehog gain-of-function, or smoothened gain-of-function.
[0190) The subject treatments can be effective for both human and animal
subjects. Animal
subjects to which the invention is applicable extend to both domestic animals
and livestock,
raised either as pets or for commercial purposes. Non-limiting examples are
dogs, cats, cattle,
horses, sheep, hogs, and goats.
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[0191] The present invention is further a method of treatment so as to avoid
or reduce the
incidents of fetal malformations and the avoidance or reduction of activation
of breast cancer (or
breast cancer precursor condition) in either men or women. The treatment and
compositions can
be administered to men or women are on estrogenic hormonal therapy or anti-
androgenic
hormonal therapy (which results in an estrogenic/androgenic balance of surplus
of estrogenic-
receptor effects) or to those with known or suspect highly estrogen sensitive
epithelial and/or
mammary breast tissue.
[0192] One of the significant aspects of the invention is the prevention of or
reductionof the
risk of birth defects associated with fetal alcohol syndrome, low cholesterol
levels during
pregnancy, and/or protein kinase overactivity. The method involves
administering to a woman
during her pregnancy a compound selected from the group consisting of an
inositol, a
phosphorylate thereof, a derivative of either (as described more fully below),
or mixtures thereof.
The administration during the first trimester is of particular value,
especially because the fetus is
most sensitive to aberrations at this time. As such, it is preferred that the
women be treated
prophylactically in order to assure treatment when she may not realize she is
pregnant and
therefore may be likely to be consuming alcohol or be treated with cholesterol
lowering
medications without thinking of the issues of impact on a fetus.
[0193] Without being bound to theory, it is the inventor's belief that
exposure to alcohol during
the critical period during first trimester embryonic development may inhibit
efficient
transcription of genes, especially those related to expression of proteins
involved in proper
mapping during fetal development. This in turn leads to impaired signal
transduction during
CA 02668580 2009-06-10
fetal mapping. Also, mutations in genes that encode specific kinases that
produce phosphate-
responsive promoters for proper gene expression may also be impaired during
alcohol exposure.
It is also the inventor's belief that alcohol induces hyperactive active
states of gene encoding
phosphate kinases which may contribute to defective or loss of shh signaling.
Therefore,
supplementing alcohol exposed fetuses by administering at least one of an
inositol (especially
myo-inositol and/or D-chiroinositol, particularly D-chiroinositol) and/or a
phosphorylate thereof
(especially a polyphosphorylate thereof as further defined herein) and/or a
derivative of the
foregoing (as further defined herein) optionally in the presence of a folate
source (preferably
folic acid) (to inhibit hyperactive states of protein kinases) should restore
proper signaling and
prevent major birth defects related to fetal alcohol exposure. In addition,
supplementing the
embryos with these compounds compounds will also rescue the loss of proper shh
gene
expression and/or inhibit hyperactive states of certain protein kinases that
impair transcription
factors. This leads to the use of these compounds for the prevention of these
common congenital
defects related to fetal alcohol syndrome.
[01941 Again, without being bound to theory, the pressent inventor believes
that the low
cholesterol levels also result in a negative impact on signaling and is the
basis for the thought
that statins might be teratogenic and therefore contraindicated during
pregnancy. The inventor
believes that supplementation with the above inositols (the free inositols,
phosphorylates thereof
and/or derivatives of either or mixtures thereof) optionally in the presence
of a folate source
(preferably folic acid) can restore (at least in part) proper mapping function
in those individuals
who have low cholesterol levels, either naturally or have low cholesterol
levels due to use of
cholesterol lowering medications such as statins and/or fibrates.
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101951 In the foregoing situations, the use of the above compounds (inositols,
phosphorylates
thereof, and/or other derivatives of either as defined herein) are useful for
the prevention or
reduction in incidence of fetal alcohol syndrome defects (FASD) as well as
safeners for the use
of alcohol or chloesterol lowering medications in women who are pregnant as
well as those who
may be pregnant and not yet be aware of the pregnancy.
101961 Administration of the above compounds is clearly indicated when the
patient is a
women of child bearing age and is known to be pregnant. It is especially of
use when the patient
is known to be a frequent user of alcohol and is not a candidate to adhere to
an alcohol free
pregnancy. However, since it is unclear as to whether even infrequent alcohol
use can give rise
to FASD, it is preferable to generally use the above compounds in a
prophylactic general manner
with all women who are pregnant so as to minimize the rate of FASD overall. In
addition, since
a particularly sensitive time period for the fetus is the early stages of
pregnancy (first trimester,
especially the first month) and this is the precise period when a woman is
least likely to know
she is pregnant, it is the most likely time when fetal injury can occur due to
alcohol and/or
depressed cholesterol. Thus, the inventor believes that the above compounds
should be used
prophylacticly in all women of child bearing age who utilize alcohol on a
regular basis and in all
women of child bearing age who are either on cholesterol lowering medications
or who naturally
have abnormally low cholesterol levels.
[01971 The subject methods and compounds may be used to regulate proliferation
and/or
differentiation of cells in vitro and/or in vivo, e.g., in the formation of
tissue from stem cells, or
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CA 02668580 2009-06-10
to prevent the growth of hyperproliferative cells to illustrate but a few
uses. For example,
according to the present invention, large numbers of non-tumorigenic neural
progenitor cells can
be perpetuated in vivo and their differentiation and proliferative rates can
be amplified preferably
in the presence of growth factors by contacting the cells with the subject
compound.
[01981 II, Particulars
[01991 A. Compounds
[02001 Certain compounds of the present invention may exist in particular
geometric or
stereoisomeric forms. The present invention contemplates all such compounds,
including, cis-
and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-
isomers, especially the
racemic mixtures (racemates) thereof, and other mixtures thereof, as falling
within the scope of
the invention, except that chiro inositol and the phosphates and derivatives
thereof used in the
present invention have the inositol ring (i.e. a six carbon 6 membered ring,
with each carbon
having one H and one OH in the underivitized form) and especially in the form
of D-
chiroinositol. In some embodiments this limitation to the D-chiro form of
inositol (its
phosphorylates and derivatives of either) is to the exclusion of the other
isomeric forms of
inositol regardless of the actual naming convention of the complete molecule.
In other
embodiments of the invention, the invention requires an inositol, a
phosphorylate thereof or a
derivative of either without being limited to the D-chiroinositol structural
form. In some
contexts, this will be specifically pointed out and in others, as indicated in
the definitions section
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CA 02668580 2009-06-10
of this specification, the exclusion of compounds will be on the basis that
such compounds are
part of the prior art in a limited number of contexts. Additional asymmetric
carbon atoms may
be present in a substituent such as an alkyl group. All such isomers, as well
as mixtures thereof,
are intended to be included in this invention.
102011 For convenience, the structure of D-chiroinositol is as follows and the
naming
convention used herein follows that shown, regardless of whether the IUPAC
naming convention
differs for this particular compound or its derivatives:
OH OH
H 1 2 H
H H
6 3-
OH H OH
OH 5 4
H OH
In the substituted compounds of the invention, to the extent that the
compounds retain either
hydroxyl groups or have hydroxyl groups in which the hydrogen of the hydroxyl
has been
replaced, the retention of the special arrangement of the shown hydroxyl
groups by the retained
hydroxyls or the inositol ring carbon-Oxygen bond of the derivatized compound
or the inositol
ring carbon-carbon or other heteroatom bond replacing one or more of the D-
chiroinositol
hydroxyl groups defines a D-chiroinositol compound. Replacement of the
inositol ring
hydrogens will not affect the consideration of the compound as a D-
chiroinositol derivative for
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CA 02668580 2009-06-10
this application. Thus, in each of the exemplary compounds below where the
hydroxy group or
derivatized hydroxy group arrangement mimics D-chiro inositol, it is a D-
chiroinositol
OH OH
OH F H
1 2 H
H H F
1 2 H 6 3
H H : OH H
6 3 OH 5 OH
4
OH IH/ OH
OH 5 41~ H OH
H OH
nositol
a D-chiroinositol derivative of a D-chiroinositol
nv
of the invention the e invention
OH OH
/alkyl
H 1 2 H O
a D-chiroinositol
H H of the invention
6 3: H \ /O
OH H p-,P OH
OH 5 4 \O
H OH
OH OH
H 1 2 H / alkyl
H H
a D-chiroinositol
6 3 : of the invention
OH H O O
OH 5 4Y
OH
H OH 0
OH H
H 1 2 H
H F NOT a D-chiroinositol
6 3
OH H OH
OH 5 4V
H OH
CA 02668580 2009-06-10
compound of the invention. Where a hydroxyl is replaced by a hydrogen and the
other hydrogen
is replaced by another group (see the final structure above of this
paragraph), then it is not a D-
chiroinositol derivative for the present invention, although it is a
derivitized inositol and may be
within the scope of compounds for other aspects of the invention. However,
where both the
hydrogen and the hydroxyl of a particular D-chiroinositol ring carbon atom are
replaced by
substituents (see the structures below), both are to be considered D-
chiroinositol derivatives for
purposes of the invention regardless of the actual naming convention name,
except in the case
where one or both are found in the prior art, in which case the prior art
compound is not to be
considered a D-chiroinositol derivative for the present invention and is
subject to the
exclusionary aspects indicated above.
OH F OH
NHZ
H 1 2. H H
1 2- H
H H2N H F
6 3: 6 3-
= 05 4H OH _ 05 H OH
OH OH 4
H OH H
OH
[0202] If, for instance, a particular enantiomer of a compound of the present
invention is
desired, it may be prepared by asymmetric synthesis, or by derivation with a
chiral auxiliary,
where the resulting diastereomeric mixture is separated and the auxiliary
group cleaved to
provide the pure desired enantiomers. Alternatively, where the molecule
contains a basic
functional group, such as amino, or an acidic functional group, such as
carboxyl, diastereomeric
salts may be formed with an appropriate optically active acid or base,
followed by resolution of
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CA 02668580 2009-06-10
the diastereomers thus formed by fractional crystallization or chromatographic
means well
known in the art, and subsequent recovery of the pure enantiomers.
[02031 Contemplated equivalents of the compounds described above include
compounds which
otherwise correspond thereto, and which have the same general properties
thereof (e.g., the
ability to inhibit hedgehog signaling), wherein one or more simple variations
of substituents are
made which do not adversely affect the efficacy of the compound. In general,
compounds which
themselves are not inositols, inositol phosphorylates or other inositol
derivatives are not
considered equivalents for the presnt invention. The compounds of the present
invention may be
prepared by the methods illustrated in the general reaction schemes as, for
example, described
below, or by modifications thereof, using readily available starting
materials, reagents and
conventional synthesis procedures. In these reactions, it is also possible to
make use of varients
which are in themselves known, but are not mentioned here.
[02041 For purposes of this invention, the chemical elements are identified in
accordance with
the Periodic Table of the Elements, CRC Handbook of Chemistry and Physics,
82th Ed., 2001-
2002, inside cover.
[02051 B. Exemplary Compounds of the Invention.
[02061 As described in further detail below, it is contemplated that some of
the subject
methods can be carried out using any of D-chiroinositol, D-chiroinositol
phosphates, or a variety
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CA 02668580 2009-06-10
of different D-chiroinositol derivatives or mixtures thereof which can be
readily identified, e.g.,
by such drug screening assays as described herein. Other aspects of the
invention can be carried
out by these same D-chiroinositols as well as the corresponding inositols
which are based on
inositol isomers other than the D-chiro form. These other insoitol forms when
underivitized,
differ from D-chiroinositol only in the particular orientation of the hydroxy
groups.
[0207] D-chiro-inositol is a compound of the structure I
OH OH
H 1 2 H
H H
6 3
OH H OH
4
OH
H OH
D-chiro inositol is not present in dietary sources and is derived from soil.
Any such compound
available to the body must be made by conversion of other sources, either
systemically or
artificially. The most common source of inositols is myo-inositol, which does
occur in dietary
sources. Myo-inositol differs from D-chiro-inositol by inversion of the OH and
H at the position
indicated by the arrow in Figure I above. Methods of making D-chiro-inositol
are detailed in a
number of patents, among them, US 5,091,596; US 5,406,005; US 5,463,142; US
5,714,643, US
5,932,774; and US 6,660,891, all of which are incorporated herein by
reference. Phosphates
thereof for purposes of the present invention include those having one or more
of the hydroxyl
groups in formula I above phosphorylated. These include mono-, di-, tri-,
tetra-, penta-, hexa-
monophosphates, and heptaphosphates. For convenience, the phosphates of D-
chiroinositol will
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CA 02668580 2009-06-10
be referred to herein by the term D-chirolP,,, where x refers to the number of
phosphorylated
hydroxyl groups that are present. Where there is one or more numbers present
as in 1,2-D-
chiroIP2, the designation indicates the position of the phosphate(s) based on
the position
numbering in Figure I above. A designation such as 1,2-D-IP3 indicates that
positions 1 and 2
are phosphorylated and that another position is phosphorylated, but that it
can be at any other
position. The absence of any numerical designation before the "IP" indicates
that the phosphate
groups are not restricted to any particular position(s). The use of the term
"IP" without the
designation "D-chiro" shall mean that inositol phosphates more generally and
include
phosphorylated forms of any isomeric form of inositol. Specific mention of
particular isomeric
forms of inositol, such as myo-, or scyllo-, epi-, etc with the "IP,,"
designation shall refer only to
that particular inositol isomer phosphorylated in accordance with the numeric
prefix and "x"
designation in the foregoing convention. Thus, the present invention relates
to compositions and
methods of use of D-chiroinositol, its monophosphates (D-chirolPi),
diphosphates (D-chiroIP2),
triphosphates (D-chiroIP3), tetraphosphates (D-chirolP4), pentaphosphates (D-
chirolPs), and
hexaphosphate (D-chiroIP6). A compiound indicated as D-chirolP7 indicates a D-
choiroinositol
having 1 pyrophphosphate and 5 monophosphate groups or 2 pyrophosphate groups
and 3-
monophosphate groups, or 3-pyrophosphate groups and one monophosphate groups.
Higher
subscripts for the IP indicate similar multiple pyrophosphate and/or
monophosphate groups.
While polyphosphates of 3 or more linked phosphates (such as -O-P(O)(OH)-O-
P(O)(OH)-O-
P(O)(OH)-O-) are possible, they are more prone to rapid hydrolysis and
therefore less desirable.
D-chiroinositol has 6 distinct monophosphates, 15 distinct di(mono)phosphates,
20 distinct
tri(mono)phosphates, 15 distinct tetra(mono)phosphates, 6 distinct
penta(mono)phosphates, and
1 hexaphosphate, each of which are intended to be included within the scope of
the present
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CA 02668580 2009-06-10
invention (unless otherwise noted or the context compels otherwise). These are
1-D-chiroIPl, 2-
D-chiroIPl, 3-D-chirolP1, 4-D-chiroIPl, 5-D-chiroIPl, 6-D-chiroIPl, 1,2-D-
chirolP2, 1,3-D-
chirolP2, 1,4-D-chirolP2, 1,5-D-chiroIP2, 1,6-D-chirolP2, 2,3-D-chirolP2, 2,4-
D-chiroIP2, 2,5-D-
chiroIP2, 2,6-D-chiroIP2, 3,4-D-chiroIP2, 3,5-D-chiroIP2i 3,6-D-chiroIP2, 4,5-
D-chiroIP2, 4,6-D-
chiroIP2, 5,6-D-chiroIP2, 1,2,3-D-chirolP3, 1,2,4-D-chiroIP3, 1,2,5-D-
chiroIP3, 1,2,6-D-chirolP3,
1,3,4-D-chiroIP3, 1,3,5-D-chiroIP3, 1,3,6-D-chiroIP3, 1,4,5-D-chiroIP3, 1,4,6-
D-chiroIP3, 1,5,6-
D-chiroIP3, 2,3,5-D-chiroIP3, 2,3,6-D-chiroIP3, 2,4,5-D-chiroIP3, 2,4,6-D-
chiroIP3, 2,5,6-D-
chiroIP3, 3,4,5-D-chiroIP3, 3,4,6-D-chiroIP3, 3,5,6-D-chiroIP3, 4,5,6-D-
chiroIP3, 1,2,3,4-D-
chiroIP4, 1,2,3,5-D-chirolP4, 1,2,3,6-D-chiroIP4, 1,2,4,5-D-chirolP4, 1,2,4,6-
D-chirolP4, 1,2,5,6-
D-chiroIP4, 1,3,5,6-D-chiroIP4, 1,4,5,6,-D-chiroIP4, 2,3,4,5-D-chiroIP4,
2,3,4,6-D-chiroIP4,
2,3,5,6-D-chiroIP4, 2,4,5,6-D-chiroIP4, 1,2,3,4,5-D-chiroIP5, 1,2,3,4,6-D-
chiroIP5, 1,2,3,5,6-D-
chiroIP5, 1,2,4,5,6-D-chiroIP5, 1,3,4,5,6-D-chiroIP5, 2,3,4,5,6-D-chiroIP5,
and 1,2,3,4,5,6-D-
chiroIP6. In addition to these phosphates, the invention also includes the
corresponding
pyrophosphates where at least one of the hydroxyl groups is phosphorylated by
a pyrophosphate
rather than a monophosphate group, such as without limitation compounds such
as
OH OH
H 1 2 H
H H
6 3 OH OH
O5 4 H O-P'~O~ I /OH
OH I II
H OH O II
which would be 3-pyrophosphatidyl D-chiroinositol. Any of the 6 hydroxy
positions of the
inositol ring can be substituted by a pyrophosphate and some can be
unphosphorylated or
monophosphorylated with others pyrophosphorylated or phosphorylated with
higher phosphates.
CA 02668580 2009-06-10
Non-limiting examples of mixed mono- and pyro phyosphorylated D-chiroinositol
include: the
heptaphosphates such as 1,2,3,4,5-pentamonophosphatidyl-6-pyrophosphatidyl-D-
chiroinositol;
1,2,3,4,6-pentamonophosphatidyl-5-pyrophosphatidyl-D-chiroinositol; 1,2,3,5,6-
pentamonophosphatidyl-4-pyrophosphatidyl-D-chiroinositol; 1,2,4,5,6-
pentamonophosphatidyl-
3-pyrophosphatidyl-D-chiroinositol; 1, 3,4,5,6-pentamonophosphatidyl-2-
pyrophosphatidyl-D-
chiroinositol; and 2,3,4,5,6-pentamonophosphatidyl-l-pyrophosphatidyl-D-
chiroinositol; the
octaphosphates such as 1,2-dipyrophosphotidyl-3,4,5,6-tetramonophosphatidyl-D-
chiroinositol;
1,3-dipyrophosphotidyl-2,4,5,6-tetramonophosphatidyl-D-chiroinositol; 1,4-
dipyrophosphotidyl-
2,3,5,6-tetramonophosphatidyl-D-chiroinositol; 1,5-dipyrophosphotidyl-2,3,4,6-
tetramonophosphatidyl-D-chiroinositol; 1,6-dipyrophosphotidyl-2,3,4,5-
tetramonophosphatidyl-
D-chiroinositol; 2,3-dipyrophosphotidyl-1,4,5,6-tetramonophosphatidyl-D-
chiroinositol; 2,4-
dipyrophosphotidyl-1,3,5,6-tetramonophosphatidyl-D-chiroinositol; 2,5-
dipyrophosphotidyl-
1,3,4,6-tetramonophosphatidyl-D-chiroinositol; 2,6-dipyrophosphotidyl-1,3,4,5 -
tetramonophosphatidyl-D-chiroinositol; 3,4-dipyrophosphotidyl-1,2,4,5,6 -
tetramonophosphatidyl-D-chiroinositol; 3,5-dipyrophosphotidyl-1,2,4,6 -
tetramonophosphatidyl-
D-chiroinositol; 3,6-dipyrophosphotidyl-1,2,4,5 -tetramonophosphatidyl-D-
chiroinositol; 4,5-
dipyrophosphotidyl-1,2,3,6 -tetramonophosphatidyl-D-chiroinositol; and 5,6-
dipyrophosphotidyl-
1,2,3,4,-tetramonophosphatidyl-D-chiroinositol among others, for example
another mixed
octaphosphate, without limitation is 1-triphosphatidyl-3-pyrophosphatidyl-
4,5,6-
trimonophosphatidyl-D-chiroinositol. For simplicity, a pyrophosphatidyl group
will be indicated
as "PP", and longer phosphate chains will be designated as "Poly(y)P", where y
indicates the
number of phosphate groups in the chain, and y is generally not more than 4,
but typically 3.
Any of the free hydroxyl groups of the D-chiroinositol structure can be
phosphorylated with
76
CA 02668580 2009-06-10
either a single phosphate group, a pyrophosphate group or a longer
polyphosphate chain of 3 or
more phosphate groups and different hydroxyl groups in the same molecule can
be
phosphorylated with a variety of any of a mono, di, or poly phosphate. Thus,
for example,
without limitation, 1-monophosphatidyl-2-monopyrophosphatadiyl-D-chiroinositol
is also within
the scope of the invention, as is 1,2-di(monophosphatidyl)-3,4-diPP-5-Poly(3)P-
D-chiroinositol,
1,2,4,5,6-pentamonophosphatidyl-3-pyrophosphatidyl-D-chiroinositol, and
1,2,5,6-
tetramonophosphatidyl-3,4-dipyrophosphatidyl-D-chiroinositol along with the
corresponding
compounds having a different distribution of the mono and pyro phosphatate
groups around the
D-chiroinositol ring. When sterically possible, two hydroxyl groups of the D-
chiroinositol
structure (within the same molecule can be linked together through a single
phosphate group, PP,
or Poly(y)P group forming a ring structure or two or more D-chiroinositol
molecules can be
linked through such phosphate groups as in the non-limiting structure III:
OH OH
H H
iT
6 3 OH
OH H
4 O P O OH
OH 0
1 2 H H
H OH ::H
6 3
OH H H 5 4
H OH III
which exemplifies (but does not limit the invention to) a molecule in which
two D-chiroinositol
molecules are linked through a single phosphate group between position 3 of
one D-chiro-
inositol and position I of the other. The linking phosphate may be a single
phosphate, a PP, or
Poly(y)P group, and when two or more hydroxyl groups on the same D-
chiroinositol structure
77
CA 02668580 2009-06-10
are phosphorylated, longer chains of alternating D-chiroinositol and a
phosphate (single
phosphate, PP or Poly(y)P and mixtures thereof) are realized. Further, a
phosphate or a
pyrophosphate may link two hydroxyl groups as for example, without limitation,
in structure IV
below:
OH OH
H 1 2 H
H H
6 3 0
OH H
OH 5 4 O
/ P
O
H
OH IVa
OH OH OH OH
H 1 2 H H 1 2 H
H H H H
6 3 6 3
O H O OH H
H 411 5 4
O
5
H OH O
OH O\P/
P-OH
0 IVb HO O IVC
or in a more complex ring structure such as that of formula V below
78
CA 02668580 2009-06-10
OH OH
H 1 2 H
H H
6 3
OH H
OH 5 4 O
H i OH
HOOP O
O
O O
H 1 2 H
H H
6 3
OH H OH
OH 5 4
H OH `I
or the two remaining phosphate hydroxyl groups can be dehydrates to form a P-O-
P link as well.
In addition, any of the six hydroxy groups that are not phosphorylated can be
substituted or
replaced as indicated elsewhere in this specification and any of the hydrogen
atoms on the six
carbons or the inositol ring may be replaced as indicated elsewhere in this
specification. Each of
these more complex D-chiroinositol structures are also within the scope of the
present invention.
Manufacture of the compounds having PP or Poly(y)P as the phosphorylating
group (whether or
not linking multiple D-chiroinositol units together) can be prepared in an
analogous fashion to
the chemical synthesis of the phosphorylates that have only single phosphate
groups for any one
hydroxyl group by using pyrophosphate of Poly(y)P phosphate chains as the
phosphorylation
group source _With respect to the uses set forth herein other than in the
prevention and treatment
of spina bifida and cancer treatments, the invention compounds also
specifically include the
corresponding compounds, their phosphorylates and derivatives based on the
other inositol
isomers as well. For example, of particular interest to the the inventor is
the use or utility of the
inositol higher phosphates and/ or pyrophosphates to protect cells from alpha
tumor necrosis
79
CA 02668580 2009-06-10
factor (a-TNF) cell death as seen with autoimmune diseases and pathways
involved described
above. In analogous fashions to structures IV and V, the -P(=O)(OH)- can be
replaced by
-S(OZ)- or -S(=O)-.
[02081 Formulations in the literature containing chiro-inositol, inositol-
phosphates, etc.,
include, but are not limited to, those disclosed in US 5,124,360; US
5,614,510; US 5,760,222;
and US 6,784,209, all of which are incorporated herein by reference in their
entirety.
Formulations of the D-chiro inositols of the invention and their
phosphorylated, pyrophosphated,
and polyphosphated derivatives as indicated as being useful in the present
invention can be made
analogously.
[02091 In each of the above D-chiroinositol structures and D-chiroinositol
phosphate
structures, further derivatives of the invention can be made and utilized by
replacement of one or
more of the hydrogens on the D-chiroinositol ring or one or more of the
hydrogens of one or
more of the hydoxy groups on the D-chiroinositol ring or one or more of the
replaceable
hydrogens on one or more of the phosphate groups shown in structures I-V above
with a
substituent selected form those indicated earlier. In addition, one or more of
the hydroxyl groups
of the inositol ring can be completely replaced with the "substituents"
referred to earlier, except
that if such hydroxyl group is completely replaced by hydrogen, then the other
hydrogen of the
same inositol ring carbon must remain in place to be considered a D-
chiroinositol derivative of
the present invention (i.e. a deoxy variant), or both the hydrogen and the
hydroxyl of the same
carbon are replaced by a doubly bound oxygen (an oxo variant) or by a doubly
bound nitrogen
CA 02668580 2009-06-10
(an imino variant), while no such restriction generally exists if both the
hydrogen and the
hydroxyl of the same carbon atom are replaced by substituents which are
neither hydrogen nor
hydroxyl or derivitized hydroxy. For uses in which the base inositol structure
is not limited to
the D-chiro inositol configuration, no such limitation on the replacements of
the inositol ring
hydroxyl group exists, and all such allowable substituents within the above
substituent
definitions are possible. Other replacements of the hydroxyl group with
additionally
replacement of the other hydrogen of the same insitol ring carbon retains the
consideration of a
D-chiroinositol derivative for purposes of thepresent invention unless
specifically set forth
otherwise. Preferable, non-limiting groups for the replacement of one or more
of these
hydrogens on the D-chiroinositol ring carbons include aliphatic groups,
acylamino groups,
alkoxy alkylamino, alkylthio, amino, aralkyl, carbonyl, derivitized carbonyl,
thiocarbonyl,
derivitized thiocarbonyl, and aryl to name a few, all of which may be further
unsubstituted or
substituted in accordance with the aforementioned definitions of each of these
terms. Other than
the alkylthio and alkoxy, the same set of substituents is preferred for
choices to replace
replaceable hydrogen of either the D-chiroinositol hydroxy groups or the
replaceable hydrogens
of the phosphate groups. Also, in any of the foregoing compounds having a D-
chiroinositol ring
free hydroxy group, the hydroxyl group can be esterified or etherized and one
or more of the
remaining hydrogens of the (6) six ring position can be replaced by an
appropriate substituent as
substituent is defined above. Furthermore, One or more of the hydroxyl groups
of the inositol
ring can be completely replaced by an appropriate substituent as defined
above. Suitable
synthetic chemistry will be apparent to synthetic chemists once directed to a
particular D-
chiroinositol phosphate or derivative.
81
CA 02668580 2009-06-10
[02101 In other aspects of the invention, the present invention includes the
corresponding
phosphorylated and/or pyrophosphorylated and/or polyphosphorylated inositols
and/or
derivitized versions of such inositols or derivitized versions of such
phosphorylated,
pyrophosphorylated, or polyphosphorylated inositols (which are derivitized in
an analogous
manner as that set forth for D-chiroinositol above) where for purposes of this
paragraph,
"inositols" includes all of the inositol isomers for all of the foregoing
indications other than the
use of myoinositol and its phosphorylates with phosphoric acid (mono- to hexa-
monophosphates
so that any one to all six of the inositol -OH groups are replaced by a
monophosphate) for
prevention of spina bifida birth defect and prevention or amelioration of
breast cancer.
[02111 More specifically, and without limnitation to the foregoing, compounds
of the present
invention (subject to the limitations of certain aspects to those compounds
based on the D-
chiroinositol configuration and subject to exclusion of certain compounds as
set forth with
respect to their presence in the prior art as set forth earlier), compounds
for use in the present
invention include: (i)
R102 R103 R104
R101 R1 os
R112 R 106
R111 R1(
R109 R
R110 108
wherein each of R101, R103, Rios R107, R1o9, and R111 is independently
selected from H or a
substituent R201;
82
CA 02668580 2009-06-10
(a) each of R102, R104, R106 R108, R1lo, and R112 is independently selected
from OH, OR202,
OP(=O)(OR21 1)(OR212), OP(=O)(OR113)-OP(=O)(OR21 1)(OR212), OP(=O)(OR113)-
{OP(=O)(OR113)}a-OP(=O)(OR211)(0R212) (wherein a is 1-3);
or a substituent R203; or not more than 3 of R102, R104, R106 R108, R110, and
R112 is
independently H; or
(b) both of the respective R groups on the same carbon (that is independently
pair Rio, and
R102, pair R103 and R1o4, pair R105 and R106, pair R107 and R108, pair R109
and R110, and pair
R111 and R112) are together =0 or =N(R204); or
(ii)
R111 R R111 R
R111 112 R101 112 R101
R112 R1o1 R110 R
R11o R 102 Rico R
R1o9 C
R R1oz 109 B
109 A 1oz
R1o6R R103 R1o6R Rios
R10 R Rios 107 'l R1oa 107 R R1oa
107 R104 06 R105 106 R105
R106 R105
r
P
O
0 0 --P--O OH
O OH / . I
O ~P
OH P
OH P --O 0
1 s
O k
n
where each of the groups R111-R112, in each unit are
independently as set forth above except that one of such R groups in each of
the terminal
structures is a direct bond to the indicated oxygen instead of the foregoing,
and one of
83
CA 02668580 2009-06-10
such R groups in each intermediary structure is a direct bond to one of the
two indicated
oxygens instead of the above and a second of the R groups in each intermediary
structure
is a direct bond to the other indicated oxygen, p, r, and s are each 1, t and
k are each
independently an integer of from 0 to 2, and n is a an integer of from 0 to 8;
pharmaceutically acceptable salts thereof, and mixtures thereof; or in which
in the
structure above t=0 and any or all of the -P(O)(OH)- groups are independently
replaced
by -C(O)-,
-S(O)-, or -S(O)2-;
wherein R201, R202, R203, and R204 are each independently selected from
halogen (prefereably
fluorine, chlorine, bromine, or iodine, more preferably fluorine, or chlorine,
most preferably
fluorine); trihalomethyl (preferably trifluoromethyl), cyano, azido,
unsubstituted or substituted
aliphatic groups, unsubstituted or substituted aromatic groups, -C(O)-R205, -
C(O)-O-R206,
-S-C(O)-R207, -C(O)-S-R208, -C(S)-R205, -C(S)-O-R206, -S-C(S)-R207, -C(S)-S-
R208,
-SC(S)-S-R208, -C(NR209)-R205, -C(NR209)-O-R206, -S-C(NR209)-R207, -C(NR2o9)-S-
R208,
-S-R210, -S(O)R210, -S(O)OR210,-S(O)2R210, -S(0)2OR210, -NR,o9R21o, -N(R209)-
C(O)-O-R206,
-N(R2o9)-C(O)-S-R208, -N(R2o9)-QS)-R205, -N(R2o9)-C(S)-O-R206, -N(R2o9)-QS)-S-
R208,
-N(R2o9)-C(NR2o9)-R205, -N(R2o9)-C(NR2o9)-O-R206, -N(R2o9)-C(NR209)-S-R209, -S-
R210,
-N(R209)-S(O)R210, -N(R2o9)S(O)0R21o, -N(R2o9)S(O)20R21o, -N(R2o9)-S(O)2R21o,
and
-NR209R210, -P(O)(0R211)(0R212),
wherein each of R205 through R212 is itself independently selected from H, an
unsubstituted or
substituted aliphatic groups, or an unsubstituted or substituted aromatic
groups,
84
CA 02668580 2009-06-10
wherein the aliphatic groups are selected from straight chain and branched
carbon chains,
whether saturated or unsaturated, of up to 30 carbon atoms, prefereably of up
to 20 carbon
atoms, more preferably of up to 10 carbon atoms, more preferably of up to 7
carbon atoms. most
preferably of up to 5 carbons, especially methyl, ethyl, propyl, and
cycloaliphatic rings having
3-10 ring members such rings being carbocyclic or heterocyclic where the
heterocyclic rings
have one to four heteroatoms selected from oxygen, sulfur, and nitrogen; the
cycloaliphatic rings
being saturated or partially unsaturated, and the aromatic group having 6-8
ring members
selected from carbon, oxygen, sulfur, and nitrogen, the aliphatic and aromatic
groups further
containing up to four fused rings of either cycloaliphatic rings, aromatic
rings or both
cyclosaliphatic and aromatic rings, each of the aliphatic and aromatic groups
being further
unsubstituted or substituted by hydoxy, CI_7alkoxy, alkylthio, C1_20acyloxy,
phosphate, halogen
(prefereably fluorine, chlorine, bromine, or iodine, more preferably fluorine,
or chlorine, most
preferably fluorine); trihalomethyl (preferably trifluoromethyl), cyano, and
azido; each
substituent being mono or multiply present as valence permits; and further
provided that in the foregoing substitution patterns, no substitution pattern
results in a peroxy
group;
the R2õand /or the R212 of any -P(O)(OR211)(OR212) may be further joined to
any free hydroxy
group or to result in phosphate containing rings such as for example
CA 02668580 2009-06-10
\OR2,2
---O
O R103
R101 R105
R112 1R106
R111 R107
R110 R109 R108 (C)
\oR2,z
1 O
8103 8104P
*RR O O R212
05 R101 R105
R112 R112 R106 O
07 R111
R107
R
8110 R108 (D) R110 109 (E)
or the corresponding compounds to structures (C), (D), and (E) wherein (a) the
-P(OR212)(O)- group is replaced by -C(O)-, -S(O)-, or -S(O)2- or the
(OR212)together with any remaining group Rl 11 through R112 forms an -0- group
resulting in additional fused rings;
wherein each of R205 through R213 is itself independently selected from H,
unsubstituted
or substituted aliphatic groups, and unsubstituted or substituted aromatic
groups,
preferably,
86
CA 02668580 2009-06-10
OH OH
H 1 2 H
H H
6 3 O
OH H
4
OH O
P
O
H
OH IVa
OH OH
OH OH H 1
2 H
H H H
1 2 H 6 3
H H OH H
6 3 5 4
O H O
OH 5 q / OH O
H P-OH 0P/
OH\\ /\\
0 IVb HO 0 IVC
OH OH
H 1 2 H
H H
6 3
OH H
OH 5 4 O
H O / OH
HO-P;O \O
O O
H 1 2 H
H H
6 3
OH H OH
OH 5 4
H OH v
87
CA 02668580 2009-06-10
and/or, the R210 of any group -S(O)OR210 or -S(O)2R210 maybe joined to any
free hydroxy group
or to result in -S(O)- or -S(O)2- containing rings analogous to structures IV
and V except that
the -P(O)(OH)- group is replaced by -C(O)-, -S(O)- or -S(O)2- respectively;
or in which any of the foregoing compounds having a free hydroxy group is
esterified with an
acidic group of folic acid or an acidic group of a polysaccharide-folic acid
compound, or any of
the foregoing compounds having a free acidic group such as -P(=O)(OH),
-C(=O)(OH), -S(=O)(OH), or -S(=O)2(OH) is esterified with a free hydroxyl
group of folic acid
or of a polysaccharide-folic acid compound, or in which any of the foregoing
having a free
amine (-NHR (R being optionally H) or imine (=NH) forms together with the
acidic group of a
folic acid or of a polysaccharide-folic acid compound an amide (-C(=O)-NR-), a
sulfonamide
(-S(=O)2-NR-), or a group -S(=O)-NR-.
[02121 Of particular interest are the multiple phosphorylated insoitols having
3 or more
monophosphate groups per molecule without other derivatizations, the IPA, IP8,
and IP9 inositols
without other derivativzations; the derivatized inositols where one or more of
the ring hydrogens
is replaced with a halogen, especially fluorine, an amino group, an azido
group, or a cyano
group; the compounds where one or more of the inositol ring hydroxyl grousp is
replaced by a
hydroxymethyl; and those having the following substituents
C H3 O--alkyl or alkanoyl
O O
L>~'O
PN
HO O
88
CA 02668580 2009-06-10
CH 3
0 alkyl or alkanoyl
O 0
0
O
Hs
alkyl or alkanoyl
O O
0
I,O
S
0
CH3 alkyl or alkanoyl
O 0
O
1:~:--O
O'
O
where the alkyl is of 1-30 carbons and saturated or unsaturated, straight
chain or branched,
preferably octadecyloxy and the alkanoyl is up to 30 carbons and preferably
saturated or
unsaturated, straight chain or branched, preferably palmitoyl and the
substituent is bound through
the free oxygen atom to the inositol ring in place of one of the insoitol ring
hydroxyl groups or to
an inositol analog having a hydroxymethyl group in place of another inositol
ring hydroxyl group
as for example
89
CA 02668580 2009-06-10
OH OH
H H
H H CH 3
O O--palmitoyl or octadecyl
O
OH H
OH PiO
HO/ 0
OH OH
H H
H H CH
3
O palmitoyl or octedecyl
O 0
OH H
OH O
H 0
OH OH
H H
H H CHs
O O palmitoyl or octedecyl
OH H
OH SiO
H \O
OH OH
H H
H H CH
s
O 0 palmitoyl or octedecyl
OH H
OH
O'S i
H 0
CA 02668580 2009-06-10
OH
OH
H
HH H CH 3 palmitoyl or octadecyl
H
O O
OH H O
OH ~i0
H HO/ O
OH
OH
H H
H H CH3 /palmitoyl or octadecyl
O O
OH H O
OH O
H O
OH
OH
H H
H H CH3 palmitoyl or octadecyl
O O
NOH H
io H O
OH
OH
CH3 /palmitoyl or octadecyl
O 0
O
QH
L-O H O
91
CA 02668580 2009-06-10
with the particular position of the for the substituents on the inositol ring
and the particular
orientation of the various hydroxyl groups not being critical. In other words
the 2-methoxy-3-
(palmitoyloxy or octadecyloxy)-glycero phosphate substituent or the 2-methoxy-
3-(palmitoyloxy
or octadecyoxy)-glycerocarbonate substituent can be attached at any of the 6
ring positions and
the hydroxymethyl group can be at any of the inositol ring positions in place
of a hydroxyl at that
positon. Also of importance are the dipalmitoylglycerophosphate esters of the
various inositols
other than that of myoinositol, i.e., those having the following substituent
1almitoyl /palmitoyl
O
O
0
HOB Pi
O
bound through the free oxygen of the phosphate (shown at the left in the
structure above) to any
of the inositol ring positions ( and the corresponding molecules in which the -
P(O)(OH)- is
replaced by one of -S(O)-, or -S(O)2-) and those that have the
dipalmitoylglycerocarbonate
esters of the various inositols
almitoyl /palmitoyl
O O
O
O
O
Other important compounds of the invention include those having the group
92
CA 02668580 2009-06-10
C1_30alkyl or C1_30acyl
/C1_30alkyl or C1_30acyl
O
0
-P(=O)(OR211)-
0 or -C(=0) or
-S(=O)- or -S(0)2-
0 VI
bound through the free oxygen shown on the left of the above structure to any
of the inositol ring
positions. Where the compounds of structure VI has higher acyls (over 14
carbons) as the (C1_
3oalkyl or C1_30) groups and the linking group -P(=O)(OH)-, -C(=O)-, -S(O)-,
or -S(O)2- is
limited to -P(=O)(OH)-, and there is one such group bound to the inositol
ring, the compounds
are phosphatidylinositols. These phopsphatidylinositols being further
substituted having such
further substitution in patterns in accordance with the present invention
compounds are also
valuable for as inhibitors for PDK1 and its pathways.
[02131 While not including the unmodified, unphosphorylated, underivitized
inositol isomers
themselves in many embodiments, such isomers having been derivitized and/or
phosphorylated
are included and include scyllo-inositol, epi-inositol, cis-inositol, allo-
inositol, neo-inositol,
muco-inositol, dextro-inositol, levo-inositol, and L-chiro-inositol
phosphorylations/derivatizations. These materials may be used in amounts of
from below 2
mg/day to in excess of 8 grams/kg/day. Any of the above inositols may be used
in combination
for the inositol component of the invention and may be used with or without
other active agents
in an analogous fashion as described in more detail above concerning D-
chiroinositol. It should
be noted that the hedgehog, patched, and GL1,2,3 gain-of function or loss of
function can be
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CA 02668580 2009-06-10
determined for each of the above inositol based compounds with the screening
tests set forth in
the examples. Without being limited to theory, those that have hedgehog gain-
of-function or
smoothened loss-of-function can be used in an analogous manner to the D-
chiroinositol uses set
forth above and those that have hedgehog loss-of-function or smoothin gain-of-
function are not
for use in the prevention of birth defects purposes set forth above, but are
of use in a number of
the other indications of the present invention, especially in the prevention
and treatment of
various cancers and other indications referred to hereinabove. Some particular
compounds that
are beyond the D-chiro insoitol compounds described earlier are, without
limitation,
fluoroscylloinositol, fluoroepi-inositol, fluorocis-inositol, fluoroallo-
inositol, fluoroneo-inositol,
fluoromuco-inositol, fluorodextro-inositol, fluorolevo-inositol, fluoro D-
chiro-inositol,
deoxyscyllo-inositol, deoxyepi-inositol, deoxycis-inositol, deoxyallo-
inositol, deoxyneo-inositol,
deoxymuco-inositol, deoxydextro-inositol, deoxylevo-inositol, deoxyD-chiro-
inositol,
aminoscylloinositol, aminoepi-inositol, aminocis-inositol, aminoallo-inositol,
aminoneo-inositol,
aminomuco-inositol, aminodextro-inositol, aminolevo-inositol, aminoD-chiro-
inositol,
ketoscyllo-inositol, ketoepi-inositol, ketocis-inositol, ketoallo-inositol,
ketoneo-inositol,
ketomuco-inositol, ketodextro-inositol, ketolevo-inositol, ketoD-chiro-
inositol, sulfo
scylloinositol, sulfo epi-inositol, sulfo cis-inositol, sulfo allo-inositol,
sulfo neo-inositol, sulfo
muco-inositol, sulfo dextro-inositol, sulfo levo-inositol, sulfo D-chiro-
inositol and further sulfato
phosphorylates of an inositol isomer where the inositol isomer is selected
from the group
consisting of scyllo-inositol, epi-inositol, cis-inositol, allo-inositol, neo-
inositol, muco-inositol,
dextro-inositol, levo-inositol, and D-chiro-inositol; and the inositol
compound having at least one
phophsoryl group selected from monophosphoryl groups, pyrophosphoryl, groups,
and
polyphosphory groups and further having at least one sulfato group, which
phosphoryl group,
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CA 02668580 2009-06-10
and/or sulfato group and/or remaining free hydroxyl groups are each
independently unsubstituted
or substituted with a suitable substituent as hereinbefore described. Further
compounds for use
in the invention uses include D-chiro inositoll inositol derivative ofr
metabolite is selected from
the group of D-chiro-inositol phosphates, d-chiro-inositol esters, D-chiro-
inositol ethers, D-chiro
-inositol acetals, D-d-chiro-inositol ketals, polysaccharides containing D-
chiro-inositols, and D-
chiro-inositol phospholipids, which compounds are disclosed in US Patent
6486127,
incorporated herein in itsd entirety by reference.
102141 Inositol "salts" contemplated for use in the practice of all aspects of
the present
invention include any and all pharmaceutically acceptable salts, which may be
used in this aspect
of the invention as well as the D-chiroinositol more specific aspects set for
the earlier. Examples
are salts of therapeutically acceptable organic acids, such as acetic,
fumaric, lactic, maleic, citric,
malic, succinic, toluenesulfonic acid, and the like, salts of polymeric acids,
such as tannic acid,
alginic acid, carboxymethylcellulose, and the like, and salts of inorganic
acids, such as
hydrochloric acid, sulfuric acid, and the like. Others, however, may also be
utilized. Inositol
"derivatives" employed in the practice of this aspect of the present invention
( as well as those set
forth concerning the D-chiroinositol specific aspects set forth earlier)
include those which have
been modified as set forth earlier as well as those modified to vary the
hydrophilic or lipophilic
character of the inositol molecules. Such modifications may be desirable to
tailor the solubility
characteristics of the inositols to a particular mode or route of
administration. For example,
lipophilic side chains, including C1_20 hydrocarbon chains, which may be
saturated or unsaturated
and contain one or more non-hydrophilic substituents, may be added, as well as
conjugating the
inositols to a lipophilic molecule to enhance its lipid solubility.
Alternatively, the addition of
CA 02668580 2009-06-10
hydrophilic side chains or inositols conjugates to a hydrophilic molecule will
enhance the
hydrophilicity of the inositols, including C1_20 hydrocarbon chains which may
be unsaturated,
and may have hydrophilic substituents such as HO, HS, NH2, halo, keto, and the
like.
Exemplary inositol derivatives contemplated for use in the practice of one
aspect of the present
invention include amine-substituted, halogen-substituted, deoxy-, keto-, and
sulfo-inositol
analogues, and the like, as well as combinations of any two or more
substituents thereof.
Preferred inositol derivatives include substituted inositol derivatives,
including hydroxy, amino,
halo, e.g., fluoro, deoxy, keto, and sulfo inositol analogues, among other
hydrophilic
substituents, and combinations thereof, as well as the corresponding salts. In
some embodiments
of the invention, the further substituents set forth earlier in this
specification are useful as well.
102151 C. Exemplary Applications of Methods and Compositions
[02161 Folic acid (C19H19N7O6) and folates are well known in the art as are
various
formulations thereof. Any of the recognized folates or C19H19N7O6 is suitable
for use in the
present invention embodiments that include a folic acid and/or folate
component.
[02171 Women of child bearing age frequently are avoiding pregnancy by
utilizing birth
control pills. These are typically estrogenic substances that are administered
for a time period
and then either stopped for a short time, continued at altered dosage, and/or
supplemented or
replaced by progestogenic substances so as to induce menses. During the time
frame when the
estrogenic substance is reduced or stopped, it is possible for a woman to
become pregnant. On
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CA 02668580 2009-06-10
occasion, it is also possible that the intended "birth control" function of
the birth control pills
(even when containing a full complement of the estrogenic substance) may not
be totally
efficacious, such as when other medications or other substances are ingested
that interfere with
the proper workings of the birth control medication. In such situations a
pregnancy may result
despite being on such medications. Although there is a general awareness among
pregnant
women to have proper supplementation with folic acid, many women taking birth
control
medication do not take adequate supplements of folic acid or many other
nutrients that are
important to fetal development, simply because they believe that do not need
to be concerned
with a pregnancy at that time. Others are simply unaware of the need for
adequate
supplementation, and still others, even though educated about this either
neglect to take
appropriate supplements or still don't care. Others do not bother because of
economic reasons.
One aspect of the present invention is to include supplemental D-chiroinositol
(and/or one of its
phosphorylated derivatives) into birth control pills which may further have
folic acid (or other
appropriate folate source) incorporated into some or all of the pills in the
birth control pill
package so as to assure that the woman taken such birth control has adequate
stores of D-
chiroinositol (and folate, when folate is also incorporated) in the event that
she becomes pregnant
either while taking birth control pills or during the time period when she
initially stops the birth
control pill regimen. This is extremely important since both D-chiroinositol
and folate are most
effective against the various fetal defects that the present invention is
directed toward preventing
when these substances are administered pre-conception through the first
trimester of pregnancy.
The D-chiroinositol (and phosphorylated derivatives) and folic acid (and other
folates) can be
incorporated into just the tablets of the birth control pill package that have
either no other active
or have progestogenic but not estrogenic substances or have progesterins and
low levels of
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CA 02668580 2009-06-10
estrogens present, but preferably are incorporated into all of the tablets.
This is suitable because
generally the higher estrogenic substance tablets will prevent pregnancy and
the remaining
tablets will begin administering folic acid and D-chiroinositol (or their
counterparts) with the
first tablet after the estrogenic tablets. However, it is preferable to have
the compounds of the
invention in all of the tablets in case of a pregnancy that results from birth
control tablet failure
or due to interference with proper action of the estrogenic substance due to
drug interactions or
other dietary or environmental impacts that cause birth control failure.
[02181 Another aspect of the invention is a combination product having both D-
chiroinositol
(and/or a phosphorylated (either P, PP, and/or polyP) derivative thereof
and/or other derivative
thereof as set forth above) (hereinafter all such compounds collectively
referred to, whether as
single agents or combinations ofthese agents, as "the D-chiroinositol
compound"). and folic acid
(and/or other folate source) in a single composition as a supplement that is
especially suited for
women of child bearing age who are not yet pregnant (but generally intending
to become
pregnant), women who are not pregnant and not intentionally trying too become
pregnant, but
may be, and women who are pregnant. Such fixed combinations may be a
standalone product or
have other nutritional supplements (or other active agent) incorporated
therein. Such additional
supplements include vitamins and minerals as well as herbal products and are
well known (both
as to substances and their respective dosages) to those of ordinary skill in
the nutritional
supplement area. Without being held to theory, it is the inventors belief and
understanding that
co-therapy of C19H19N7O6 (and/or other folate sources) together with D-
chiroinositol (and/or
phosphorylated derivatives (P, PP and/or polyP) thereof and/or other
derivative thereof as set
forth above), whether simultaneously or sequentially, operate in a manner that
provides the best
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CA 02668580 2009-06-10
protective effects against fetal malformations beyond those achievable with
either component
alone, and further that such results are better than those achieved with each
alone or that would
have been predicted as additive effect. As such, such co-therapy is also
within the scope of the
present invention, whether such co-therapy is via a fixed combination
C19H19N706 (and/or other
folate) and D-chiroinositol (and/or phosphorylated derivates (P, PP and/or
polyP) thereof and/or
other derivative thereof as set forth above) or via separate administration of
these agents
generally within 12 hours of each other and generally on a daily basis.
Fractional dosing of
either or both components taken multiple times a day (i.e., for example 1/2
daily doses taken twice
daily or 1/3 daily dosing taken three times daily) is also within the scope of
the present invention.
Fractional dosing multiple times a day is particularly suitable when the
composition contains
only nutritional supplements as active agents and when the patient finds that
singe daily doing
upsets the stomach or the daily dose is large and not suitable for inclusion
into a single unit
dosage form.
[2191 An additional benefit of administering D-chiroinositol to women on
estrogenic
medications is downregulating the estrogen-receptor and/or, ErbB receptor
overexpressor
phenotypes and proliferation from estrogenic insult. For example, D-chiro
and/or its derivatives
are likely mediated by the production of second messenger lipids that elicit
transmemebrane
signal transduction cascades governing the activation and inhibition of
downstream effectors.
These views are also in line with the ideas on non-estrogen receptor
associated actions of the
compound by way of promoting binding sites that govern cellular proliferation.
Thus,
incorporation of D-chiroinositol (and/or its phosphorylated (P, PP and/or
polyp) derivatives
and/or other derivative thereof as set forth above) into fixed combinations
with estrogenic
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CA 02668580 2009-06-10
medications is a means to increase the safety of the use of estrogenic
substances. While D-
chiroinositol (and/or its phosphorylated (P, PP and/or polyP) derivatives
and/or other derivative
thereof as set forth above) can be used as separate medications or supplements
in co-therapy with
the estrogenic medication, it is highly preferred to have the D-chiroinositol
compound as a fixed
combination with the estrogenic substance as to assure patient compliance.
While estrogenic
sensitive breast tissue in men is rarer than in women, it does occur and co-
therapy in men having
estrogenic treatment is also within the scope of the present invention.
Furthermore, since
estrogenic insult is the result of excess estrogen from endogenous
overproduction of estrogen,
exogenous administration of estrogen, insufficient androgenic production, or
exogenous
administration of anti-androgens (androgen ablative therapy), the present
invention also includes
treating men or women with co-therapy of D-chiroinositol compound with anti-
androgens, which
co-therapy can be by separate administration of the compounds of the invention
with such anti-
androgens or via fixed combinations therewith. The invention still further
includes treating
patients with conditions that result in excess estrogen or conditions that
result in estrogenic-
receptor overexpression phenotypes (whether because of overproduction of
estrogen or
insufficient androgen production, or congential deformation in the breast
architecture
microenvimonment) with the D-chiroinositol compound as a means of decreasing
the the risk of
breast cancer from excess estrogenic insult. Finally, in this group of
treatments of the invention,
the invention further includes treating patients with a general overproduction
of hormonal
steroids (even though estrogenic/androgenic balance is maintained). A still
further benefit to
women who are or become pregnant while receiving the present invention
treatment is that of
reducing the incidents of gestational diabetes (or if they still do have such,
it is a milder case),
especially since there has been a connection between gestational diabetes and
some fetal
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CA 02668580 2009-06-10
malformations due poor maternal phosphoinositide turnover or derangement.
Specific active
agents which can be combined for co-therapy with D-chiroinositol compound
and/or derivatives
and optionally with addition folic acid (or other folate source) that are
within the invention
include, without limitation: antiprogestogens, androgens, antiandrogens,
estrogens, selective
estrogen receptor modulators, aromatase inhibitors, gonadotropins, ovulation
stimulators,
gonadotropin releasing hormone agonists, gonadotropin releasing hormone
antagonists, LHRH
agonists, progestins, and anti-progestins, to name a few. Many of these
classes are utilized in
opposing conditions but the co-therapy with the D-chiroinositol component of
the invention and
optionally the folate component of the invention is warranted in that in some
cases, the effect of
the D-chiroinositol component (and optional folate component) is complementary
to the other
active agent, while in other cases, the D-chiroinositol component (and
optional folate
component) are protective of one or more of the potential side effects of the
other active agent.
Specific compounds belonging to these classes of other active agents are
exemplified in the
following non-exclusive, non-limiting list, each agent of which is prepared in
its normal known
method and utilized in its known dosage, and include without limitation: 13-
cis-Retinoic Acid, 2-
CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil, 5-FU, 6-
Mercaptopurine, 6-MP,
6-TG, 6-Thioguanine, Abraxane, Accutane , Actinomycin-D, Adriamycin ,
Adrucil ,
Agrylin , Ala-Cort , Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-
AQ ,
Alkeran , All-transretinoic Acid, Alpha Interferon, Altretamine,
Amethopterin, Amifostine,
Aminoglutethimide, Anagrelide, Anandron , Anastrozole, Arabinosylcytosine,
Ara-C, Aranesp
, Aredia , Arimidex , Aromasin , Arranon Arsenic Trioxide, Asparaginase,
ATRA
Avastin , Azacitidine, BCG, BCNU, Bevacizumab, Bexarotene, BEXXAR ,
Bicalutamide,
BiCNU, Blenoxane , Bleomycin, Bortezomib, Busulfan, Busulfex , C225, Calcium
101
CA 02668580 2009-06-10
Leucovorin, Campath , Camptosar ,Camptothecin-11, Capecitabine
CaracTM,Carboplatin,
Carmustine, Carmustine, Wafer Casodex , CC-5013, CCNU, CDDP, CeeNU,
Cerubidine ,
Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone,
Cosmegen ,
CPT-11, Cyclophosphamide, Cytadren , Cytarabine, Cytarabine Liposomal Cytosar-
U,
Cytoxan , Dacarbazine, Dacogen, Dactinomycin, Darbepoetin Alfa, Dasatinib,
Daunomycin,
Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Liposomal, DaunoXome ,
Decadron,Decitabine, Delta-Cortef , Deltasone , Denileukin, diftitox,
DepoCyt TM,
Dexamethasone, Dexamethasone acetate, Dexamethasone Sodium Phosphate,
Dexasone,
Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil , Doxorubicin, Doxorubicin
liposomal,,
Droxia TM, DTIC, DTIC-Dome , Duralone , Efudex , Eligard TM, Ellence TM,
Eloxatin TM,
Elspar , Emcyt , Epirubicin, Epoetin alfa, Erbitux TM, Erlotinib, Erwinia,
Etanercept, L-
asparaginase, Estramustine, Ethyol, Etopophos , Etoposide, Etoposide
Phosphate, Eulexin ,
Evista , Exemestane,Fareston , Faslodex , Femara , Filgrastim,
Floxuridine, Fludara ,
Fludarabine, Fluoroplex , Fluorouracil, Fluorouracil (cream),
Fluoxymesterone, Flutamide,
Folinic Acid, FUDR , Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab,
ozogamicin,
Gemzar , Gleevec TM, Gliadel Wafer, GM-CSF, Goserelin, Granulocyte - Colony
Stimulating Factor, Granulocyte Macrophage Colony Stimulating Factor,
Halotestin ,
Herceptin , Hexadro, Hexalen , Hexamethylmelamine, HMM, Hycamtin , Hydrea
,
Hydrocort Acetate ,Hydrocortisone, Hydrocortisone Sodium Phosphate,
Hydrocortisone
Sodium Succinate, Hydrocortone Phosphate, Hydroxyurea, Ibritumomab,
Ibritumomab Tiuxetan,
Idamycin , Idarubicin, Ifex , IFN-alpha I fosfamide, IL-11, IL-2, Imatinib
mesylate,
Imidazole Carboxamide, Interferon alfa, Interferon Alfa-2b (PEG Conjugate),
Interleukin - 2,
Interleukin-11, Intron AS (interferon alfa-2b), Iressa , Irinotecan,
Isotretinoin, Kidrolase ,
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CA 02668580 2009-06-10
Lanacort , Lapatinib, L-asparaginase, LCR, Lenalidomide, Letrozole,
Leucovorin, Leukeran,
Leukine TM, Leuprolide, Leurocristine, Leustatin TM Liposomal, Ara-C Liquid
Pred ,
Lomustine, L-PAM, L-Sarcolysin, Lupron , Lupron Depot ,Matulane , Maxidex,
Mechlorethamine, Mechlorethamine Hydrochloride, Medralone , Medrol , Megace
,
Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna, Mesnex TM,
Methotrexate
,Methotrexate Sodium, Methylprednisolone, Meticorten , Mitomycin, Mitomycin-
C,
Mitoxantrone, M-Prednisol , MTC, MTX, Mustargen , Mustine, Mutamycin ,
Myleran ,
Mylocel TM, Mylotarg , Navelbine , Nelarabine, Neosar , Neulasta TM,
Neumega ,
Neupogen , Nexavar , Nilandron , Nilutamide, Nipent , Nitrogen Mustard,
Novaldex ,
Novantrone , Octreotide, Octreotide acetate, Oncospar , Oncovin , Ontak ,
Onxal TM,
Oprevelkin, Orapred , Orasone , Oxaliplatin, Paclitaxel, Paclitaxel Protein-
bound,
Pamidronate, Panitumumab, Panretin , Paraplatin , Pediapred , PEG
Interferon,
Pegaspargase, Pegfilgrastim, PEG-INTRON TM, PEG-L-asparaginase, PEMETREXED,
Pentostatin, Phenylalanine Mustard, Platinol , Platinol-AQ , Prednisolone,
Prednisone,
Prelone , Procarbazine, PROCRIT , Proleukin , Prolifeprospan 20 with
Carmustine
Implant, Purinethol , Raloxifene, Revlimid , Rheumatrex , Rituxan ,
Rituximab, Roferon-
A (Interferon Alfa-2a), Rubex , Rubidomycin hydrochloride, Sandostatin ,
Sandostatin
LAR , Sargramostim, Solu-Cortef , Solu-Medrol , Sorafenib, SPRYCEL TM, STI-
571,
Streptozocin, SU 11248, Sunitinib, Sutent , Tamoxifen, Tarceva , Targretin
, Taxol ,
Taxotere , Temodar , Temozolomide, Teniposide, TESPA, Thalidomide, Thalomid
,
TheraCys , Thioguanine,Thioguanine Tabloid , Thiophosphoamide, Thioplex ,
Thiotepa,
TICE ,Toposar , Topotecan, Toremifene, Tositumomab, Trastuzumab, Tretinoin,
Trexall TM,
Trisenox , TSPA, TYKERB , VCR, Vectibix TM, Velban , Velcade VePesid ,
Vesanoid
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CA 02668580 2009-06-10
Viadur TM, Vidaza , Vinblastine, Vinblastine Sulfate, Vincasar Pfs ,
Vincristine,
Vinorelbine, Vinorelbine tartrate, VLB, VM-26, Vorinostat, VP-16, Vumon ,
Xeloda ,
Zanosar , Zevalin TM, Zinecard , Zoladex , Zoledronic acid, Zolinza, Zometa
, etc.. A
preferred set includes, without limitation: abarelix,abraxane(paclitaxel),
adriamycin(doxorubicin), algestone, amadinone, aminoglutethimide, anagestrone,
anastrozole,
androisoxazole, androstanolone, androstenediol, 4-androstene-3,16,17-trione,
aredia(pamidronate
disodium), arimidex(anastrozole), aromasin(exemestane),bazedoxifene,
benorterone,
bicalutamide, bolandiol, bolasterone, bolazine, boldenone, bolenol,
bolmantalate, buserelin,
calusterone, chemotherapy regimens,(cyclophosphamide(cytoxan),
methotrexate(amethopetrin,
Mexate, folex, and flourorucil(fluorourcil, 5-fu, adrucil) (this therapy is
called CMF),
cyclophospamide, doxorubicin(adriamycin) and fluorouracil (this therapy is
called CAF),
doxorubicin(adriamycin) and cyclophosphamide(this therapy is called AC),
doxorubicin(adriamycin) and cyclophosphamide with paclitaxel(taxol),
doxorubicin(adriamycin)
followed by CMF, cyclophosphamide, eprubicin9ellence), and fluororacil,
chlorotrianisene,
chorionic gonadotropin, cioteronel, cingestol, clogestone, clomegestone,
clometherone,
clomifene, clostebol, conjugated estrogens, cyproterone,
cytoxan(cyclophasphamide), danazol,
delmadinone, deslorelin, desogestrel, detirelix, dienestrol,
diethylstilbestrol, dimethisterone,
dihydrogestrone, drospirenone, drostanolone, dydrogesterone,
ellence(epirubicin),epiestriol,
epimestrol, epitiostanol, epristeride, equilin, esterified estrogens,
estradiol, estrazinol, estriol,
estrofurate, estrone, estropipate, ethinylestradiol, ethisterone,
ethylestrenol, ethynerone,
ethynodiol, etonogestrel, evista(raloxifene), exemestane,
fareston(toremifene),
femara(letrozole),fenestrel, finasteride, fluoxymesterone, flurogestone,
flutamide, formebolone,
formestane, fosfestrol, fulvestrant, furazabol, ganirelin, gestaclone,
gestadienol, gestodene,
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CA 02668580 2009-06-10
gestonorone (especially gestonorone caproate), gestrinone, gonadorelin,
goserelin,
haloprogesterone, herceptin(trastuzumab),histrelin, 4-hydroxy-19-
nortestosterone,
hydroxyprogesterone, ibutamoren, idoxifene, infliamab,letrozole, leuprolide,
leuprorelin,
levonorgestrel, lutrelin, lynestrenol, mebolazine, medrogestone,
medroxyprogesterone,
megace(megestrol), melengestrel, menotropins (especially humegon, pergonal,
repronex),
mesabolone, mestranol, mesterolone, metandienone, metenolone, methandriol,
methenolone,
methestrol, methyltestosterone, methynodiol, metribolone, mibolerone,
mifepristone, nafarelin,
nafoxidine, nandrolone, nilutamide, nitromifene, norboletone, norbolethone,
norclostebol,
norelgestromin, norethandrolone, norethindrone, norethisterone, norethynodrel,
norgestimate,
norgestomet, norgestrel, norgestrienone, nylestriol, oxabolone, oxandrolone,
oxendolone,
oxogestone, oxymesterone, oxymetholone, polyestradiol (especially
polyestradiol phosphate),
pralmorelin, prasterone, progesterone, quinbolone, quinestrol, quinestradol,
quingestanol
(especially quingestanol acetate), quingestrone, raloxifene, rismorelin,
somalapor, somatrem,
somatropin, somenopor, somidobove, stanozolol, stenbolone, sumorelin,
tamoxifen,taxol(palitaxel), taxotere(docetaxel), testosterone, tibolone,
tigestrol, tiomesterone,
topterone, toremifene, trenbolone, trimegestone, trioxifene, triptorelin,
urofollitropin, vorozole,
xeloda(capecitabine),zanoterone, and zeranol,zoladex(goserelin),
zometa(zoledronic) among
others, each of which includes the pharmaceutically acceptable salts and
esters thereof. These
are all known compounds with known uses and are used in the normal course for
those known
indications. The co-therapy of the present invention adds the D-chiroinositol
compound and
optionally folic acid (and/or other folate source) thereto, with the amounts
of the D-chiroinositol
components and folic acid components being as set forth elsewhere herein. The
D-chiroinsoitol
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and optional folate can be separately administered with these other active
agents of combined in
fixed combinations therewith as may be convenient.
[02201 Turning to the fetal malformations of the present invention, fetal
development is a very
delicate and sensitive process and there are many points at which something
can go wrong,
resulting in a congenital defect. As such, no treatment will eliminate all
such fetal defects or
even all occurrences of any one type of fetal defect. Nonetheless, the
administration of D-
chiroinositol (and/or phosphorylated derivatives (P, PP and/or polyP) thereof)
alone or in
combination with folic acid (and/or other folate source) during the first
trimester of pregnancy,
preferably throughout the first trimester of pregnancy, even more preferably
from before
conception into the first trimester of pregnancy, and most preferably from
before conception
through at least the end of the first trimester of pregnancy will
significantly reduce the frequency
of a wide range of fetal defects, above those reported previously for those
patients who have not
been treated or those patients who have been treated with either of the D-
chiroinositol (and/or its
phosphorylated (P, PP and/or polyp) derivatives) or with folic acid (or other
folate source) alone
(where those treatments have been previously studied. The treatment of the
present invention
further reduces the frequency of these defects as compared to treatment with
other forms of
inositol (and/or phosphorylated derivatives thereof) where such treatment has
been previously
studied.
102211 The defects, the frequency of which the present invention is designed
to reduce,
include, but are not limited to wherein the defect is VATER/VACTERL
association (vertebral
[defects], [imperforate] anus, tracheoesophageal [fistula], radial and renal
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[dysplasia])rachischisis (aka spinal dysraphism) such as spina bifida
(including, but not limited
to spina bifida aperta (aka spinabifida cystica); spinabifida occulta; and
occult spinal disorder,
among others) and (b) craniorachischisis (aka cranial dysraphism) such as
cranium bifida (aka
encephalocele or craniocele) each of spina bifida and cranium bifida being of
any of the
following types meningocele, myelomeningocele, lipomeningocele, and
lipomyelomeningocele
among others; (c) anencephaly; and (d) chiari malformation types 0,12,3; (2)
caudal regression
syndrome, caudal dysplasia sequence, congenitalsacral agenesis;
sironmelia(mermaid syndrome),
sacral regression and the like; (3) crani-facial defects such as, without
limitation, facial cleft
(aka prosopoanoschisis, including without limitation cleft palate, cleft lip,
velopharyngeal
malformation (including without limitation bifid uvula), etc.); (4) anorectal
malformations
including, but not limited to (a) imperforate anus, (b) rectoperineal fistula,
(c) recto-bladder neck
fistula; (d) persistent urogenital sinus, (e) persistent cloaca, etc.; (5)
bucket-handle malformation;
among others. Biemond syndrome, Chiari malformation(, 0,1,2,3),Ectrodactyly-
ectoderma
dysplasia, cleft lip/palate, Ellis Van Creveld syndrome, Muir-Torre syndrome,
Cowden
syndrome, Carney complex, Birt-Hogg-Dube syndrome, Gorlin syndrome (ptc loss-
of-
function), Gorlin-Goltz syndrome, basal cell nevus syndrome, bifid-rib basal-
cell nevus
syndrome, basal cell cancer syndrome(shh gain of function), and multiple basal
cell nevi,
squamous cell carcinoma (increased ptc activity)Meckel Gruger syndrome,
McKusick-
Kaufmansyndrome, Mirror hand deformity(ulnar dimelia) Mohr syndrome, Oral-
facial-digital
syndrome, Pallister Hall syndrome, cephalopolysyndactyly, Post axial
polydactyly,
GreigRubinstein-Taybi syndrome, retinoblastoma, Cardiofaciocutaneous syndrome,
Noonan
syndrome,short rib polydactyly, extra deformed fingers and toes, Lowe syndrome
including
ocular and renal defects, Renal Colombo syndrome, retinoblastoma, retinitis
pigmentosa,
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holoprosencephaly, macular degeneration (whether it be due to a Shh
defects,age,or secondary
conditions like diabetes mellitus), mental retardation. All of these terms are
well known in the
art. However, for rapid reference, those unfamiliar with these terms are
referred (without
limitation to the Merck Manual, Eighteenth Edition 2006 and the PDR Medical
Dictionary,
Second Edition, 2000. The ultimate cause of these conditions can be genetic or
environmental, or
both. Nonetheless, it is also postulated here that certain cancers, and
possibly even breast cancer
in offspring are the result of signaling defects in utero and should be
considered a birth defect as
well since the results of the signaling defects in utero may not present until
much later in life
similar to that of other cancers when DNA damage and mutations accumulate
postnatally. For
example, our research suggests that in the microenvironment of mammary breast
architecture
comprise a population of epithelial cells and stem cells that continue to
communicate post natally
through converged signaling pathways. These mammary epithelial cells have the
ability for self-
renewal and harbor tumorigenic potential. Subsequent alterations in postnatal
signaling
mechanisms can result in a breast cancer as subsequent mammary development
proceeds
postnatally. Further studies also suggest that the stem-like, self-renewing
cells originate from the
progenitor fetal cells during early embryonic mammary development. These
breast cancer stem
cells have been identified as CD44+CD24 breast tumor cells. In certain mouse
mammary
experimental models stem/progenitor cells displayed sensitivity to altered or
hyper states of
signaling pathways and new mapping techniques have demonstrated that a
population of adult
neural stem cells that rarely divides responded to normal or hyperactive Shh
signaling even
though adult neural stem cells rarely divide. All of this this could result in
unintended
consequences in the formation of new malignancies. Nature 437, 894-897(6
October 2005). We
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propose to correct signaling defects in utero with the method compound and/or
its derivatives to
prevent breast cancer.
(02221 Without being bound to theory, the inventor believes that all of these
conditions are
related to failure of proper embryonic patterning (mapping sequences) during
the critical
embryonic first trimester. One embryonic patterning sequence that has been
identified is the
Sonic hedgehog (Shh) gene and some inositol phosphates (and kinases therefore)
(PI3K) have
been shown to be important in the proper expression of the Shh gene. It is the
present inventor's
belief that these two signaling pathways converge in order for sufficient gene
expression to
occur. Mutations in the human shh gene and genes that encode its downstream
intracellular
signaling pathway causes many clinical disorders including, but no way limited
to basal cell
carcinoma, nevoid basal cell carcinoma syndromes along with distinct
congential syndromes
syndromes as described above. Thus, insufficient D-chiroinositol levels
(and/or phosphates (P,
PP and/or polyp) thereof) interfere with or prevent the proper expression of
the Shh gene and the
result thereof is improper signaling of proper mapping of the embryonic
tissue. Thus, proper
supplementation with the D-chiroinositol compound will restore proper
signaling and mapping,
second messenger systems involved in embryonic patterning at that critical
period, especially, if
the embryo is one at risk of such improper signaling and mapping) so as to
substantially reduce
and/or eliminate the risk of the presentation of the above fetal
malformations. Since the risk of
some of the above conditions have been shown (but not statistically
significant) to benefit from
folate supplementation, co-therapy with both D-chiroinositol (and/or its
phosphorylated (P, PP
and/or polyp) derivatives) and folic acid (and/or another folate source) is
the preferred
embodiment of the invention. It is also believed that the D-chiroinositol
(plus one of its
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phosphorylated (P, PP and/or polyP) derivatives) is the natural active agent
involved in this
mechanism and that either other forms like myo-inositol have a weak (or
weaker) effect alone .
It is further believed that a significant number of the women having children
with these
malformations have (a) insufficient inositol intake and therefore cannot
convert a sufficient
amount to the D-chiro form or (b) simply cannot properly convert other
inositol forms to the D-
chiro variety and/or to the proper phosphorylated variety via
phosphoinositides. In this
subpopulation, supplementation with any of the D-chiroinositol and/or its
phosphorylated
derivatives will serve equally well. A small subpopulation however may have
defects in the
various kinases (for example, PKA or PKC isoforms) involved and thus, the best
supplementation would be with the particular phosphorylate that is after the
kinase defect. Since
finding the specific defect in a particular kinase may not be easily
identified in all cases, a
separate embodiment of the present invention is to use a mixture of D-
chiroinositol and a number
of its phosphorylated (P, PP and/or polyP) derivatives so as to be sure that
none of the
advantages of the present invention are missed in as many patients as
possible. A highly
preferred embodiment in this case is to use a mixture of D-chiroinositol and
at least one member
selected from D-chiroinositol-Phosphates(1_8). (In D-chiroinositol-
Phosphates(2_8), one or more of
the phosphates may be in the form of pyrophosphates, and in D-chiroinositol-
Phosphates(7_8) at
least one of the phosphates must be present as a pyrophosphate as there are
only 6 positions
which can be monophosphorylated).
[02231 Another aspect of the present invention relates to a method of
modulating a
differentiated state, survival, and/or proliferation of a cell, such as a
normal cell or a cell having a
ptc loss-of-function, hedgehog gain-of-function, or smoothened gain-of-
function, or by an
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aberrant P13K signaling pathway by contacting the cells with a compound as set
forth above
according to the subject method and as the circumstances may warrant as a way
to target,
manipulate, to optimize control of this pathway thereby mitigating its
contribution to oncogenic
and embryonic patterning activity.
[0224] Despite significant progress in reconstructive surgical techniques,
scarring can be an
important obstacle in regaining normal function and appearance of healed skin.
This is
particularly true when pathologic scarring such as keloids or hypertrophic
scars of the hands or
face causes functional disability or physical deformity. In the severest
circumstances, such
scarring may precipitate psychosocial distress and a life of economic
deprivation. Wound repair
includes the stages of hemostasis, inflammation, proliferation, and
remodeling. The proliferative
stage involves multiplication of fibroblasts and endothelial and epithelial
cells. Through the use
of the subject method, the rate of proliferation of epithelial cells in and
proximal to the wound
can be controlled in order to accelerate closure of the wound and/or minimize
the formation of
scar tissue". Fibroblasts can be stimulated Myo-ip6-SO4 (Stabilizers for
fibroblast growth
factors, Middaughet al, US 5348941) and play a critical role in wound healing.
Transplanted
fibroblasts can often retain positional memory of the location and tissue
context where they had
previously resided, at least over a few generations. Thus, the present
invention finds utility in
joints, hip, knee, cell, and tissue replacement. In one embodiment, embedding
a fibroblast with
the compound D-chiro-lp6- SO4 in a depot formulation, into the surgical site
to help wound
healing will promote the wound healing, especially in an elderly population
that does not heal
well due to age or people with diabetes mellitus, or immune system problems
etc.
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[02251 For instance, it is contemplated by the invention that, in light of the
findings of an
apparently broad involvement of P13K and hedgehog, ptc, and smoothened in the
formation of
ordered spatial arrangements of differentiated tissues in vertebrates, the
subject method is
suitable for use as part of a process for generating and/or maintaining an
array of different
vertebrate tissue both in vitro and in vivo. The compound, whether inductive
or anti-inductive
with respect proliferation or differentiation of a given tissue, can be, as
appropriate, any of the
preparations described above.
[02261 For example, the present method of using subject compounds is
applicable to cell
culture techniques wherein it is desirable to control the proliferation or
differentiation of the cell.
A subject compound may be employed in a method directed towards cells which
have a ptc loss-
of-function, hedgehog gain-of-function, or smoothened gain-of-function
phenotype. In vitro
neuronal culture systems have proved to be fundamental and indispensable tools
for the study of
neural development, as well as the identification of neurotrophic factors such
as nerve growth
factor (NGF), ciliary trophic factors (CNTF), and brain derived neurotrophic
factor (BDNF).
One use of the present method may be in cultures of neuronal stem cells, such
as in the use of
such cultures for the generation of new neurons and glia. In such embodiments
of the subject
method, the cultured cells can be contacted with a compound of the present
invention in order to
alter the rate of proliferation of neuronal stem cells in the culture and/or
alter the rate of
differentiation, or to maintain the integrity of a culture of certain
terminally differentiated
neuronal cells. In an exemplary embodiment, the subject method can be used to
culture, for
example, sensory neurons or, alternatively, motorneurons. Such neuronal
cultures can be used as
convenient assay systems as well as sources of implantable cells for
therapeutic treatments.
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[02271 According to the present invention, large numbers of non-tumorigenic
neural progenitor
cells can be perpetuated in vitro and their rate of proliferation and/or
differentiation can be
affected by contact with compounds of the present invention. Generally, a
method is provided
comprising the steps of isolating neural progenitor cells from an animal,
perpetuating these cells
in vitro or in vivo, preferably in the presence of growth factors, and
regulating the differentiation
of these cells into particular neural phenotypes, e.g., neurons and glia, by
contacting the cells
with a subject compound.
102281 Progenitor cells are thought to be under a tonic inhibitory influence
which maintains the
progenitors in a suppressed state until their differentiation is required.
However, recent
techniques have been provided which permit these cells to be proliferated, and
unlike neurons
which are terminally differentiated and therefore non-dividing, they can be
produced in
unlimited number and are highly suitable for transplantation into heterologous
and autologous
hosts with neurodegenerative diseases.
102291 By "progenitor" it is meant an oligopotent or multipotent stem cell
which is able to
divide without limit and, under specific conditions, can produce daughter
cells which terminally
differentiate such as into neurons and glia. These cells can be used for
transplantation into a
heterologous or autologous host. By heterologous is meant a host other than
the animal from
which the progenitor cells were originally derived. By autologous is meant the
identical host
from which the cells were originally derived.
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[02301 Cells can be obtained from embryonic, post-natal, juvenile or adult
neural tissue from
any animal. By any animal is meant any multicellular animal which contains
nervous tissue.
More particularly, is meant any fish, reptile, bird, amphibian or mammal and
the like. The most
preferable donors are mammals, especially mice and humans.
[02311 In the case of a heterologous donor animal, the animal may be
euthanized, and the brain
and specific area of interest removed using a sterile procedure. Brain areas
of particular interest
include any area from which progenitor cells can be obtained which will serve
to restore function
to a degenerated area of the host's brain. These regions include areas of the
central nervous
system (CNS) including the cerebral cortex, cerebellum, midbrain, brainstem,
spinal cord and
ventricular tissue, and areas of the peripheral nervous system (PNS) including
the carotid body
and the adrenal medulla. More particularly, these areas include regions in the
basal ganglia,
preferably the striatum which consists of the caudate and putamen, or various
cell groups such as
the globus pallidus, the subthalamic nucleus, the nucleus basalis which is
found to be
degenerated in Alzheimer's Disease patients, or the substantia nigra pars
compacta which is
found to be degenerated in Parkinson's Disease patients.
[02321 Human heterologous neural progenitor cells may be derived from fetal
tissue obtained
from elective abortion, or from a post-natal, juvenile or organ donor.
Autologous neural tissue
can be obtained by biopsy, or from patients undergoing neurosurgery in which
neural tissue is
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removed, in particular during epilepsy surgery, and more particularly during
temporal
lobectomies and hippocampalectomies.
[0233] Cells can be obtained from donor tissue by dissociation of individual
cells from the
connecting extracellular matrix of the tissue. Dissociation can be obtained
using any known
procedure, including treatment with enzymes such as trypsin, collagenase and
the like, or by
using physical methods of dissociation such as with a blunt instrument or by
mincing with a
scalpel to a allow outgrowth of specific cell types from a tissue.
Dissociation of fetal cells can
be carried out in tissue culture medium, while a preferable medium for
dissociation of juvenile
and adult cells is artificial cerebral spinal fluid (aCSF). Regular aCSF
contains 124 mM NaCl, 5
mM KCI, 1.3 mM MgC12, 2 mM CaC12, 26 mM NaHCO3, and 10 mM D-glucose. Low Ca2+
aCSF contains the same ingredients except for Mg C12 at a concentration of 3.2
mM and CaC12 at
a concentration of 0.1 mM.
[0234] Dissociated cells can be placed into any known culture medium capable
of supporting
cell growth, including MEM, DMEM, RPMI, F-12, and the like, containing
supplements which
are required for cellular metabolism such as glutamine and other amino acids,
vitamins, minerals
and useful proteins such as transferrin and the like. Medium may also contain
antibiotics to
prevent contamination with yeast, bacteria and fungi such as penicillin,
streptomycin, gentamicin
and the like. In some cases, the medium may contain serum derived from bovine,
equine,
chicken and the like. A particularly preferable medium for cells is a mixture
of DMEM and F-
12.
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[02351 Conditions for culturing should be close to physiological conditions.
The pH of the
culture media should be close to physiological pH, preferably between pH 6-8,
more preferably
close to pH 7, even more particularly about pH 7.4. Cells should be cultured
at a temperature
close to physiological temperature, preferably between 30 C.-40 C., more
preferably between
32 C.-38 C., and most preferably between 35 C.-37 C.
[02361 Cells can be grown in suspension or on a fixed substrate, but
proliferation of the
progenitors is preferably done in suspension to generate large numbers of
cells by formation of
"neurospheres" (see, for example, Reynolds et al. (1992) Science 255:1070-
1709; and PCT
Publications W093/01275, W094/09119, W094/10292, and W094/16718). In the case
of
propagating (or splitting) suspension cells, flasks are shaken well and the
neurospheres allowed
to settle on the bottom corner of the flask. The spheres are then transferred
to a 50 ml centrifuge
tube and centrifuged at low speed. The medium is aspirated, the cells
resuspended in a small
amount of medium with growth factor, and the cells mechanically dissociated
and resuspended in
separate aliquots of media.
[02371 Cell suspensions in culture medium are supplemented with any growth
factor which
allows for the proliferation of progenitor cells and seeded in any receptacle
capable of sustaining
cells, though as set out above, preferably in culture flasks or roller
bottles. Cells typically
proliferate within 34 days in a 37 C. incubator, and proliferation can be
reinitiated at any time
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after that by dissociation of the cells and resuspension in fresh medium
containing growth
factors.
[0238] In the absence of substrate, cells lift off the floor of the flask and
continue to proliferate
in suspension forming a hollow sphere of undifferentiated cells. After
approximately 3-10 days
in vitro, the proliferating clusters (neurospheres) are fed every 2-7 days,
and more particularly
every 2-4 days by gentle centrifugation and resuspension in medium containing
growth factor.
[0239] After 6-7 days in vitro, individual cells in the neurospheres can be
separated by physical
dissociation of the neurospheres with a blunt instrument, more particularly by
triturating the
neurospheres with a pipette. Single cells from the dissociated neurospheres
are suspended in
culture medium containing growth factors, and differentiation of the cells can
be control in
culture by plating (or resuspending) the cells in the presence of a subject
compound.
[0240] To further illustrate other uses of the subject compounds, it is noted
that intracerebral
grafting has emerged as an additional approach to central nervous system
therapies. For
example, one approach to repairing damaged brain tissues involves the
transplantation of cells
from fetal or neonatal animals into the adult brain (Dunnett et al. (1987) J
Exp Biol 123:265-289;
and Freund et al. (1985) J Neurosci 5:603-616). Fetal neurons from a variety
of brain regions
can be successfully incorporated into the adult brain, and such grafts can
alleviate behavioral
defects. For example, movement disorder induced by lesions of dopaminergic
projections to the
basal ganglia can be prevented by grafts of embryonic dopaminergic neurons.
Complex cognitive
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functions that are impaired after lesions of the neocortex can also be
partially restored by grafts
of embryonic cortical cells. The subject method can be used to regulate the
growth state in the
culture, or where fetal tissue is used, especially neuronal stem cells, can be
used to regulate the
rate of differentiation of the stem cells.
[02411 Stem cells useful in the present invention are generally known. For
example, several
neural crest cells have been identified, some of which are multipotent and
likely represent
uncommitted neural crest cells, and others of which can generate only one type
of cell, such as
sensory neurons, and likely represent committed progenitor cells. The role of
compounds
employed in the present method to culture such stem cells can be to regulate
differentiation of
the uncommitted progenitor, or to regulate further restriction of the
developmental fate of a
committed progenitor cell towards becoming a terminally differentiated
neuronal cell. For
example, the present method can be used in vitro to regulate the
differentiation of neural crest
cells into Glial cells, Schwann cells, Chromaffin cells, Cholinergic
sympathetic or
parasympathetic neurons, as well as peptidergic and serotonergic neurons. The
subject
compounds can be used alone, or can be used in combination with other
neurotrophic factors
which act to more particularly enhance a particular differentiation fate of
the neuronal progenitor
cell.
102421 In addition to the implantation of cells cultured in the presence of
the subject
compounds, yet another aspect of the present invention concerns the
therapeutic application of a
subject compound to regulate the growth state of neurons and other neuronal
cells in both the
central nervous system and the peripheral nervous system. The ability of ptc,
hedgehog, and
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smoothened to regulate neuronal differentiation during development of the
nervous system and
also presumably in the adult state indicates that, in certain instances, the
subject compounds can
be expected to facilitate control of adult neurons with regard to maintenance,
functional
performance, and aging of normal cells; repair and regeneration processes in
chemically or
mechanically lesioned cells; and treatment of degeneration in certain
pathological conditions. In
light of this understanding, the present invention specifically contemplates
applications of the
subject method to the treatment protocol of (prevention and/or reduction of
the severity of)
neurological conditions deriving from: (i) acute, subacute, or chronic injury
to the nervous
system, including traumatic injury, chemical injury, vascular injury and
deficits (such as the
ischemia resulting from stroke), together with infectious/inflammatory and
tumor-induced injury,
(ii) aging of the nervous system including Alzheimer's disease; (iii) chronic
neurodegenerative
diseases of the nervous system, including Parkinson's disease, Huntington's
chorea, amylotrophic
lateral sclerosis and the like, as well as spinocerebellar degenerations; and
(iv) chronic
immunological diseases of the nervous system or affecting the nervous system,
including
multiple sclerosis.
102431 As appropriate, the subject method can also be used in generating nerve
prostheses for
the repair of central and peripheral nerve damage. In particular, where a
crushed or severed axon
is intubulated by use of a prosthetic device, subject compounds can be added
to the prosthetic
device to regulate the rate of growth and regeneration of the dendritic
processes. Exemplary
nerve guidance channels are described in U.S. Pat. Nos. 5,092,871 and
4,955,892, incorporated
herein by reference.
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[0244] In another embodiment, the subject method can be used in the treatment
of neoplastic or
hyperplastic transformations such as may occur in the central nervous system.
For instance, the
subject compounds can be utilized to cause such transformed cells to become
either post-mitotic
or apoptotic. The present method may, therefore, be used as part of a
treatment for, e.g.,
malignant gliomas, meningiomas, medulloblastomas, neuroectodermal tumors, and
ependymomas, etc. In this connection, the invention still further relates to
inducing
antiangiogenesis in localized or distant metastasized tumors by affecting
cancer related vascular
cells.
[0245] In a preferred embodiment, the subject method can be used as part of a
treatment
regimen for malignant medulloblastoma and other primary CNS malignant
neuroectodermal
tumors.
[0246] In certain embodiments, the subject method is used as part of treatment
program for
medulloblastoma. Medulloblastoma, a primary brain tumor, is the most common
brain tumor in
children. A medulloblastoma is a primitive neuroectodermal tumor arising in
the posterior fossa.
They account for approximately 25% of all pediatric brain tumors (Miller).
Histologically, they
are small round cell tumors commonly arranged in true rosettes, but may
display some
differentiation to astrocytes, ependymal cells or neurons (Rorke; Kleihues).
PNETs may arise in
other areas of the brain including the pineal gland (pineoblastoma) and
cerebrum. Those arising
in the supratentorial region generally fare worse than their PF counterparts.
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[02471 Medulloblastoma/PNETs are known to recur anywhere in the CNS after
resection, and
can even metastasize to bone. Pretreatment evaluation should therefore include
an examination
of the spinal cord to exclude the possibility of "dropped metastases".
Gadolinium-enhanced MRI
has largely replaced myelography for this purpose, and CSF cytology is
obtained postoperatively
as a routine procedure.
[02481 In other embodiments, the subject method is used as part of treatment
program for
ependymomas. Ependymomas account for approximately 10% of the pediatric brain
tumors in
children. Grossly, they are tumors that arise from the ependymal lining of the
ventricles and
microscopically form rosettes, canals, and perivascular rosettes. Of the CHOP
series of 51
children reported with ependymomas, not all are malignant and approximately
2/3 arise from the
region of the 4th ventricle. One third presented in the supratentorial region.
Age at presentation
peaks between birth and 4 years, as demonstrated by SEER data as well as data
from CHOP.
The median age is about 5 years. Because so many children with this disease
are babies, they
often require multimodal therapy.
[02491 Yet another aspect of the present invention concerns the observation in
the art that ptc,
hedgehog, and/or smoothened are involved in morphogenic signals involved in
other vertebrate
organogenic pathways in addition to neuronal differentiation as described
above, having apparent
roles in other endodermal patterning, as well as both mesodermal and
endodermal differentiation
processes. Thus, it is contemplated by the invention that compositions
comprising one or more
of the subject compounds can also be utilized for both cell culture and
therapeutic methods
involving generation and maintenance of non-neuronal tissue.
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[02501 In one embodiment, the present invention makes use of the discovery
that ptc,
hedgehog, and smoothened are apparently involved in controlling the
development of stem cells
responsible for formation of the digestive tract, liver, lungs, and other
organs which derive from
the primitive gut. Shh serves as an inductive signal from the endoderm to the
mesoderm, which
is critical to gut morphogenesis. Therefore, for example, compounds of the
instant method can
be employed for regulating the development and maintenance of an artificial
liver which can
have multiple metabolic functions of a normal liver. In an exemplary
embodiment, the subject
method can be used to regulate the proliferation and differentiation of
digestive tube stem cells to
form hepatocyte cultures which can be used to populate extracellular matrices,
or which can be
encapsulated in biocompatible polymers, to form both implantable and
extracorporeal artificial
livers.
[02511 In another embodiment, therapeutic compositions of subject compounds
can be utilized
in conjunction with transplantation of such artificial livers, as well as
embryonic liver structures,
to regulate uptake of intraperitoneal implantation, vascularization, and in
vivo differentiation and
maintenance of the engrafted liver tissue.
[02521 In yet another embodiment, the subject method can be employed
therapeutically to
regulate such organs after physical, chemical or pathological insult. For
instance, therapeutic
compositions comprising subject compounds can be utilized in liver repair
subsequent to a
partial hepatectomy.
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[0253] The generation of the pancreas and small intestine from the embryonic
gut depends on
intercellular signalling between the endodermal and mesodermal cells of the
gut. In particular,
the differentiation of intestinal mesoderm into smooth muscle has been
suggested to depend on
signals from adjacent endodermal cells. One candidate mediator of endodermally
derived
signals in the embryonic hindgut is Sonic hedgehog. See, for example,
Apelqvist et al. (1997)
Curr Biol 7:801-4. The Shh gene is expressed throughout the embryonic gut
endoderm with the
exception of the pancreatic bud endoderm, which instead expresses high levels
of the
homeodomain protein Ipf l /Pdx 1 (insulin promoter factor 1 /pancreatic and
duodenal homeobox
1), an essential regulator of early pancreatic development. Apelqvist et al.,
supra, have examined
whether the differential expression of Shh in the embryonic gut tube controls
the differentiation
of the surrounding mesoderm into specialised mesoderm derivatives of the small
intestine and
pancreas. To test this, they used the promoter of the Ipfl/Pdx1 gene to
selectively express Shh in
the developing pancreatic epithelium. In Ipfl/Pdx1--Shh transgenic mice, the
pancreatic
mesoderm developed into smooth muscle and interstitial cells of Cajal,
characteristic of the
intestine, rather than into pancreatic mesenchyme and spleen. Also, pancreatic
explants exposed
to Shh underwent a similar program of intestinal differentiation. These
results provide evidence
that the differential expression of endodermally derived Shh controls the fate
of adjacent
mesoderm at different regions of the gut tube.
[0254] In the context of the present invention, it is contemplated therefore
that the subject
compounds can be used to control or regulate the proliferation and/or
differentiation of
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pancreatic tissue and intestinal tissue (see distal hindgut deformation
discussed elsewhere within
the present specification) both in vivo and in vitro.
102551 There are a wide variety of pathological cell proliferative and
differentiative conditions
for which the inhibitors of the present invention may provide therapeutic
benefits, with the
general strategy being, for example, the correction of abberrant insulin
expression, or modulation
of differentiation. More generally, however, the present invention relates to
a method of
inducing and/or maintaining a differentiated state, enhancing survival and/or
affecting
proliferation of pancreatic cells, by contacting the cells with the subject
inhibitors. For instance,
it is contemplated by the invention that, in light of the apparent involvement
of ptc, hedgehog,
and smoothened in the formation of ordered spatial arrangements of pancreatic
tissues, the
subject method is suitable for use as part of a technique to generate and/or
maintain such tissue
both in vitro and in vivo. For instance, modulation of the function of
hedgehog can be employed
in both cell culture and therapeutic methods involving generation and
maintenance a-cells and
possibly also for non-pancreatic tissue, such as in controlling the
development and maintenance
of tissue from the digestive tract, spleen, lungs, urogenital organs (e.g.,
bladder), and other
organs which derive from the primitive gut.
102561 In an exemplary embodiment, the present method can be used in the
treatment of
hyperplastic and neoplastic disorders effecting pancreatic tissue,
particularly those characterized
by aberrant proliferation of pancreatic cells. For instance, pancreatic
cancers are marked by
abnormal proliferation of pancreatic cells which can result in alterations of
insulin secretory
capacity of the pancreas. For instance, certain pancreatic hyperplasias, such
as pancreatic
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carcinomas, can result in hypoinsulinemia due to dysfunction of .beta.-cells
or decreased islet
cell mass. To the extent that one more more of aberrant P13K/hedgehog, ptc,
smoothened
signaling may be indicated in disease progression, the subject regulators can
be used to enhance
regeneration of the tissue after anti-tumor therapy.
[02571 Moreover, manipulation of P13K/hedgehog signaling properties at
different points may
be useful as part of a strategy for reshaping/repairing pancreatic tissue both
in vivo and in vitro.
In one embodiment, the present invention makes use of the apparent involvement
of ptc,
hedgehog, and smoothened in regulating the development of pancreatic tissue.
In general, the
subject method can be employed therapeutically to regulate the pancreas after
physical, chemical
or pathological insult. In yet another embodiment, the subject method can be
applied to cell
culture techniques, and in particular, may be employed to enhance the initial
generation of
prosthetic pancreatic tissue devices. Manipulation of proliferation and
differentiation of
pancreatic tissue, for example, by altering hedgehog activity, can provide a
means for more
carefully controlling the characteristics of a cultured tissue. In an
exemplary embodiment, the
subject method can be used to augment production of prosthetic devices which
require (3-islet
cells, such as may be used in the encapsulation devices described in, for
example, Aebischer et
al. U.S. Pat. No. 4,892,538, Aebischer et al. U.S. Pat. No. 5,106,627; Lim
U.S. Pat. No.
4,391,909, the Sefton U.S. Pat. No. 4,353,888, all incorporated herein by
reference. Early
progenitor cells to the pancreatic islets are multipotential, and apparently
coactivate all the islet-
specific genes from the time they first appear. As development proceeds,
expression of islet-
specific hormones, such as insulin, becomes restricted to the pattern of
expression characteristic
of mature islet cells. The phenotype of mature islet cells, however, is not
stable in culture, as
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reappearence of embryonal traits in mature .beta.-cells can be observed. By
utilizing the subject
compounds, the differentiation path or proliferative index of the cells can be
regulated.
[02581 Furthermore, manipulation of the differentiative state of pancreatic
tissue can be
utilized in conjunction with transplantation of artificial pancreas so as to
promote implantation,
vascularization, and in vivo differentiation and maintenance of the engrafted
tissue. For
instance, manipulation of hedgehog function to affect tissue differentiation
can be utilized as a
means of maintaining graft viability.
[02591 Bellusci et al. (1997) Development 124:53 report that Sonic hedgehog
regulates lung
mesenchymal cell proliferation in vivo. Accordingly, the present method can be
used to regulate
regeneration of lung tissue, e.g., in the treatment of emphysema.
[02601 Fujita et al. (1997) Biochem Biophys Res Commun 238:658 reported that
Sonic
hedgehog is expressed in human lung squamous carcinoma and adenocarcinoma
cells. The
expression of Sonic hedgehog was also detected in the human lung squamous
carcinoma tissues,
but not in the normal lung tissue of the same patient. They also observed that
Sonic hedgehog
stimulates the incorporation of BrdU into the carcinoma cells and stimulates
their cell growth,
while anti-Shh-N inhibited their cell growth. These results suggest that a
ptc, hedgehog, and/or
smoothened is involved in the cell growth of such transformed lung tissue and
therefore indicates
that the subject method can be used as part of a treatment of lung carcinoma
and
adenocarcinomas, and other proliferative disorders involving the lung
epithelia.
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102611 Numerous other tumors may (based on evidence such as involvement of the
hedgehog
pathway in these tumors, or detected expression of hedgehog or its receptor in
these tissues
during development) be affected by treatment with the subject compounds. Such
tumors include,
but are by no means limited to, tumors related to Gorlin's syndrome (e.g.,
basal cell carcinoma,
medulloblastoma, meningioma, etc.), tumors evidenced in pct knock-out mice
(e.g.,
hemangioma, rhabdomyosarcoma, etc.), tumors resulting from gli-1 amplification
(e.g.,
glioblastoma, sarcoma, etc.), tumors connected with TRC8, a ptc homolog (e.g.,
renal carcinoma,
thyroid carcinoma, etc.), Ext-l-related tumors (e.g., bone cancer, etc.), Shh-
induced tumors (e.g.,
lung cancer, chondrosarcomas, etc.), and other tumors (e.g., breast cancer,
urogenital cancer
(e.g., kidney, bladder, ureter, prostate, etc.), adrenal cancer,
gastrointestinal cancer (e.g.,
stomach, intestine, etc.), etc.).
[02621 In still another embodiment of the present invention, compositions
comprising one or
more of the subject compounds can be used in the in vitro generation of
skeletal tissue, such as
from skeletogenic stem cells, as well as the in vivo treatment of skeletal
tissue deficiencies. The
present invention particularly contemplates the use of subject compounds to
regulate the rate of
chondrogenesis and/or osteogenesis. By "skeletal tissue deficiency", it is
meant a deficiency in
bone or other skeletal connective tissue at any site where it is desired to
restore the bone or
connective tissue, no matter how the deficiency originated, e.g. whether as a
result of surgical
intervention, removal of tumor, ulceration, implant, fracture, or other
traumatic or degenerative
conditions.
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[02631 For example, the method of the present invention can be used as part of
a regimen for
restoring cartilage function to a connective tissue. Such methods are useful
in, for example, the
repair of defects or lesions in cartilage tissue which is the result of
degenerative wear such as that
which results in arthritis, as well as other mechanical derangements which may
be caused by
trauma to the tissue, such as a displacement of torn meniscus tissue,
meniscectomy, a Taxation of
a joint by a torn ligament, malignment of joints, bone fracture, or by
hereditary disease. The
present reparative method is also useful for remodeling cartilage matrix, such
as in plastic or
reconstructive surgery, as well as periodontal surgery. The present method may
also be applied
to improving a previous reparative procedure, for example, following surgical
repair of a
meniscus, ligament, or cartilage. Furthermore, it may prevent the onset or
exacerbation of
degenerative disease if applied early enough after trauma.
102641 In one embodiment of the present invention, the subject method
comprises treating the
afflicted connective tissue with a therapeutically sufficient amount of a
subject compound to
regulate a cartilage repair response in the connective tissue by managing the
rate of
differentiation and/or proliferation of chondrocytes embedded in the tissue.
Such connective
tissues as articular cartilage, interarticular cartilage (menisci), costal
cartilage (connecting the
true ribs and the sternum), ligaments, and tendons are particularly amenable
to treatment in
reconstructive and/or regenerative therapies using the subject method. As used
herein,
regenerative therapies include treatment of degenerative states which have
progressed to the
point of which impairment of the tissue is obviously manifest, as well as
preventive treatments of
tissue where degeneration is in its earliest stages or imminent.
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[0265] In an illustrative embodiment, the subject method can be used as part
of a therapeutic
intervention in the treatment of cartilage of a diarthroidal joint, such as a
knee, an ankle, an
elbow, a hip, a wrist, a knuckle of either a finger or toe, or a
tempomandibular joint. The
treatment can be directed to the meniscus of the joint, to the articular
cartilage of the joint, or
both. To further illustrate, the subject method can be used to treat a
degenerative disorder of a
knee, such as which might be the result of traumatic injury (e.g., a sports
injury or excessive
wear) or osteoarthritis. The subject regulators may be administered as an
injection into the joint
with, for instance, an arthroscopic needle. In some instances, the injected
agent can be in the
form of a hydrogel or other slow release vehicle described above in order to
permit a more
extended and regular contact of the agent with the treated tissue.
[0266] The present invention further contemplates the use of the subject
method in the field of
cartilage transplantation and prosthetic device therapies. However, problems
arise, for instance,
because the characteristics of cartilage and fibrocartilage varies between
different tissue: such as
between articular, meniscal cartilage, ligaments, and tendons, between the two
ends of the same
ligament or tendon, and between the superficial and deep parts of the tissue.
The zonal
arrangement of these tissues may reflect a gradual change in mechanical
properties, and failure
occurs when implanted tissue, which has not differentiated under those
conditions, lacks the
ability to appropriately respond. For instance, when meniscal cartilage is
used to repair anterior
cruciate ligaments, the tissue undergoes a metaplasia to pure fibrous tissue.
By regulating the
rate of chondrogenesis, the subject method can be used to particularly address
this problem, by
helping to adaptively control the implanted cells in the new environment and
effectively
resemble hypertrophic chondrocytes of an earlier developmental stage of the
tissue.
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[0267] In similar fashion, the subject method can be applied to enhancing both
the generation
of prosthetic cartilage devices and to their implantation. The need for
improved treatment has
motivated research aimed at creating new cartilage that is based on collagen-
glycosaminoglycan
templates (Stone et al. (1990) Clin Orthop Relat Red 252:129), isolated
chondrocytes (Grande et
al. (1989) J Orthop Res 7:208; and Takigawa et al. (1987) Bone Miner 2:449),
and chondrocytes
attached to natural or synthetic polymers (Walitani et al. (1989) J Bone Jt
Surg 71B:74; Vacanti
et al. (1991) Plast Reconstr Surg 88:753; von Schroeder et al. (1991) J Biomed
Mater Res
25:329; Freed et al. (1993) J Biomed Mater Res 27:11; and the Vacanti et al.
U.S. Pat. No.
5,041,138). For example, chondrocytes can be grown in culture on
biodegradable,
biocompatible highly porous scaffolds formed from polymers such as
polyglycolic acid,
polylactic acid, agarose gel, or other polymers which degrade over time as
function of hydrolysis
of the polymer backbone into innocuous monomers. The matrices are designed to
allow
adequate nutrient and gas exchange to the cells until engraftment occurs. The
cells can be
cultured in vitro until adequate cell volume and density has developed for the
cells to be
implanted. One advantage of the matrices is that they can be cast or molded
into a desired shape
on an individual basis, so that the final product closely resembles the
patient's own ear or nose
(by way of example), or flexible matrices can be used which allow for
manipulation at the time
of implantation, as in a joint.
[0268] In one embodiment of the subject method, the implants are contacted
with a subject
compound during certain stages of the culturing process in order to manage the
rate of
differentiation of chondrocytes and the formation of hypertrophic
chrondrocytes in the culture.
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[0269] In another embodiment, the implanted device is treated with a subject
compound in
order to actively remodel the implanted matrix and to make it more suitable
for its intended
function. As set out above with respect to tissue transplants, the artificial
transplants suffer from
the same deficiency of not being derived in a setting which is comparable to
the actual
mechanical environment in which the matrix is implanted. The ability to
regulate the
chondrocytes in the matrix by the subject method can allow the implant to
acquire characteristics
similar to the tissue for which it is intended to replace.
[0270] In yet another embodiment, the subject method is used to enhance
attachment of
prosthetic devices. To illustrate, the subject method can be used in the
implantation of a
periodontal prosthesis, wherein the treatment of the surrounding connective
tissue stimulates
formation of periodontal ligament about the prosthesis.
[0271] In still further embodiments, the subject method can be employed as
part of a regimen
for the generation of bone (osteogenesis) at a site in the animal where such
skeletal tissue is
deficient Indian hedgehog is particularly associated with the hypertrophic
chondrocytes that are
ultimately replaced by osteoblasts. For instance, administration of a compound
of the present
invention can be employed as part of a method for regulating the rate of bone
loss in a subject.
For example, preparations comprising subject compounds can be employed, for
example, to
control endochondral ossification in the formation of a "model" for
ossification.
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[02721 In yet another embodiment of the present invention, a subject compound
can be used to
regulate spermatogenesis. The hedgehog proteins, particularly Dhh, have been
shown to be
involved in the differentiation and/or proliferation and maintenance of
testicular germ cells. Dhh
expression is initiated in Sertoli cell precursors shortly after the
activation of Sry (testicular
determining gene) and persists in the testis into the adult. Azospermic and
oligospermic males
are viable but infertile, owing to a complete absence of mature sperm.
Examination of the
developing testis in different genetic backgrounds suggests that Dhh regulates
both early and late
stages of spermatogenesis. Bitgood et al. (1996) Curr Biol 6:298. In a
preferred embodiment,
the subject compound can be used as a contraceptive. In a similar fashion,
compounds of the
subject method are potentially useful for modulating abnormal ovarian
function, and at the same
time, offering protective effects against the use of ovulation inductors for
treating infertility in
phenotypes with receptor loss of function (non-limiting drug list incorporated
herein).
[02731 In yet another embodiment of the present invention, a subject compound
can be used to
regulate ovulation as describe in the preceeding paragraph. The hedgehog
proteins, particularly
Dhh, have been shown to be involved induced expression of the hedgehog target
genes Ptchl and
Glil, in the surrounding pre-theca cell compartment. Cyclopamine, a highly
specific hedgehog
signaling antagonist, inhibits this induced expression of target genes in
cultured neonatal mouse
ovaries. The theca cell compartment remains a target of hedgehog signaling
throughout follicle
development, showing induced expression of the hedgehog target genes Ptch1,
Ptch2, Hip], and
Glil. In periovulatory follicles, a dynamic synchrony between loss of hedgehog
expression and
loss of induced target gene expression is observed. Oocytes are unable to
respond to hedgehog
because they lack expression of the essential signal transducer Smo
(smoothened). The present
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results point to a prominent role of hedgehog signaling in the communication
between granulosa
cells and developing theca cells (Endocrinology Vol. 146, No. 8 3558-3566,
2005).
[02741 The subject method also has wide applicability to the treatment or
prophylaxis of
disorders afflicting epithelial tissue, as well as in cosmetic uses. In
general, the method can be
characterized as including a step of administering to an animal an amount of a
subject compound
effective to alter the growth state of a treated epithelial tissue. The mode
of administration and
dosage regimens will vary depending on the epithelial tissue(s) which is to be
treated. For
example, topical formulations will be preferred where the treated tissue is
epidermal tissue, such
as dermal or mucosal tissues.
[02751 A method which "promotes the healing of a wound" results in the wound
healing more
quickly as a result of the treatment than a similar wound heals in the absence
of the treatment.
"Promotion of wound healing" can also mean that the method regulates the
proliferation and/or
growth of, inter alia, keratinocytes, or that the wound heals with less
scarring, less wound
contraction, less collagen deposition and more superficial surface area. In
certain instances,
"promotion of wound healing" can also mean that certain methods of wound
healing have
improved success rates, (e.g., the take rates of skin grafts) when used
together with the method of
the present invention. (See the earlier discussion concening fibroblasts and
wound healing
above.)
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[0276] Despite significant progress in reconstructive surgical techniques,
scarring can be an
important obstacle in regaining normal function and appearance of healed skin.
This is
particularly true when pathologic scarring such as keloids or hypertrophic
scars of the hands or
face causes functional disability or physical deformity. In the severest
circumstances, such
scarring may precipitate psychosocial distress and a life of economic
deprivation. Wound repair
includes the stages of hemostasis, inflammation, proliferation, and
remodeling. The proliferative
stage involves multiplication of fibroblasts and endothelial and epithelial
cells. Through the use
of the subject method, the rate of proliferation of epithelial cells in and
proximal to the wound
can be controlled in order to accelerate closure of the wound and/or minimize
the formation of
scar tissue.
[0277] The subject method can also be used in the treatment of corneopathies
marked by
corneal epithelial cell proliferation, as for example in ocular epithelial
disorders such as
epithelial downgrowth or squamous cell carcinomas of the ocular surface. Also,
for example, the
subject method and compounds can be used to treat degenerative diseases of the
retina.
[0278] Levine et al. (1997) J Neurosci 17:6277 show that hedgehog proteins can
regulate
mitogenesis and photoreceptor differentiation in the vertebrate retina, and
Ihh is a candidate
factor from the pigmented epithelium to promote retinal progenitor
proliferation and
photoreceptor differentiation. Likewise, Jensen et al. (1997) Development
124:363 demonstrated
that treatment of cultures of perinatal mouse retinal cells with the amino-
terminal fragment of
Sonic hedgehog results in an increase in the proportion of cells that
incorporate
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bromodeoxuridine, in total cell numbers, and in rod photoreceptors, amacrine
cells and Muller
glial cells, suggesting that Sonic hedgehog promotes the proliferation of
retinal precursor cells.
Thus, the subject method can be used in the treatment of proliferative
diseases of retinal cells and
regulate photoreceptor differentiation. Furthermore, Bennett, Jeffrey L
:Journal of Neuro-
Ophthalmology: Volume 22(4) December 2002pp 286-296 cites recent studies that
have
identified several factors important for the determination and function of the
optic disc: sonic
hedgehog (Shh), Pax Gli3 transcription factors. Deficient Shh expression in
zebrafish leads to
cyclopia, whereas ectopic expression results in small optic cups and enlarged
optic stalks. Pax2
null mutant mice fail to form optic discs, resulting in medial extension of
retinal pigment
epithelial cells into the optic stalk, failure of axons to cross at the optic
chiasm, and optic nerve
coloboma.The murine Gli3 mutant, 'extra-toes,' also has optic nerve coloboma).
Mutations in
the human Shh and Pax2 genes are known to result in holoprosencephaly and the
renal-coloboma
syndrome, and possibly retinoblastoma. Retinitis Pigmentosa is a photoreceptor
degenerative
disease leading to blindness in adulthood. Ala Moshiri et al, The Journal of
Neuroscience, January 7, 2004, 24(1):229-237; doi: 10. 1 523/JNEUROSCI.2980-
03.2004 also
postulates that the hedgehog signaling pathway is a key regulator of neural
development,
affecting both proliferation and differentiation of neural progenitors. Sonic
hedgehog (Shh) is a
mitogenic factor for retinal progenitors in vitro. They wanted to determine
whether this signaling
system is important in vivo for regulating retinal progenitor proliferation,
they analyzed mice
with a single functional allele of the Shh receptor patched (ptc). They found
that ptc+/- mice had
increased numbers of neural progenitors at every stage of retinal development
that they
examined. In addition, these mice had persistent progenitors at the retinal
margin for up to 3
months of age, reminiscent of the ciliary marginal zone of lower vertebrates.
To test whether the
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progenitors at the retinal margin of ptc+/- mice could be induced to
regenerate retinal neurons in
response to damage, they bred ptc+/- mice onto a retinal degeneration
background (pro23his
rhodopsin transgenic) and labeled newly generated cells with combined
immunohistochemistry
for bromodeoxyuridine and retinal neuron and photoreceptor-specific markers.
Ala Moshiri et al,
(2004) found newly generated neurons and photoreceptors at the retinal margin
in ptc+/-;
pro23his mice. They propose that the Shh pathway may act as a regulator of
both prenatal and
postnatal retinal growth. Through the use of the subject method and compounds,
there is hope in
treating diseases associated with degeneration of the photorecptors and for
treating other
childhood cancers like retinoblastoma related to deficiencies in Shh pathways.
[02791 The subject method and compositions can also be used to treat wounds
resulting from
dermatological diseases, such as lesions resulting from autoimmune disorders
such as psoriasis.
Atopic dermititis refers to skin trauma resulting from allergies associated
with an immune
response caused by allergens such as pollens, foods, dander, insect venoms and
plant toxins.
[02801 In another aspect of the invention, the subject method can be used to
induce
differentiation and/or inhibit proliferation of epithelially derived tissue.
Such forms of these
molecules can provide a basis for differentiation therapy for the treatment of
hyperplastic and/or
neoplastic conditions involving epithelial tissue. For example, such
preparations can be used for
the treatment of cutaneous diseases in which there is abnormal proliferation
or growth of cells of
the skin.
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102811 Yet another aspect of the present invention relates to the use of the
subject method to
control hair growth. Hair is basically composed of keratin, a tough and
insoluble protein; its
chief strength lies in its disulphide bond of cystine. Each individual hair
comprises a cylindrical
shaft and a root, and is contained in a follicle, a flask-like depression in
the skin. The bottom of
the follicle contains a finger-like projection termed the papilla, which
consists of connective
tissue from which hair grows, and through which blood vessels supply the cells
with
nourishment The shaft is the part that extends outwards from the skin surface,
whilst the root has
been described as the buried part of the hair. The base of the root expands
into the hair bulb,
which rests upon the papilla. Cells from which the hair is produced grow in
the bulb of the
follicle; they are extruded in the form of fibers as the cells proliferate in
the follicle. Hair
"growth" refers to the formation and elongation of the hair fiber by the
dividing cells.
[02821 As is well known in the art, the common hair cycle is divided into
three stages: anagen,
catagen and telogen. During the active phase (anagen), the epidermal stem
cells of the dermal
papilla divide rapidly. Daughter cells move upward and differentiate to form
the concentric
layers of the hair itself. The transitional stage, catagen, is marked by the
cessation of mitosis of
the stem cells in the follicle. The resting stage is known as telogen, where
the hair is retained
within the scalp for several weeks before an emerging new hair developing
below it dislodges
the telogen-phase shaft from its follicle. From this model it has become clear
that the larger the
pool of dividing stem cells that differentiate into hair cells, the more hair
growth occurs.
Accordingly, methods for increasing or reducing hair growth can be carried out
by potentiating
or inhibiting, respectively, the proliferation of these stem cells.
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[02831 In certain embodiments, the subject method can be employed as a way of
reducing the
growth of human hair as opposed to its conventional removal by cutting,
shaving, or depilation.
For instance, the present method can be used in the treatment of trichosis
characterized by
abnormally rapid or dense growth of hair, e.g. hypertrichosis. In an exemplary
embodiment,
subject compounds can be used to manage hirsutism, a disorder marked by
abnormal hairiness.
The subject method can also provide a process for extending the duration of
depilation.
[02841 Moreover, because a subject compound will often be cytostatic to
epithelial cells, rather
than cytotoxic, such agents can be used to protect hair follicle cells from
cytotoxic agents which
require progression into S-phase of the cell-cycle for efficacy, e.g.
radiation-induced death.
Treatment by the subject method can provide protection by causing the hair
follicle cells to
become quiescent, e.g., by inhibiting the cells from entering S phase, and
thereby preventing the
follicle cells from undergoing mitotic catastrophe or programmed cell death.
For instance,
subject compounds can be used for patients undergoing chemo- or radiation-
therapies which
ordinarily result in hair loss. By inhibiting cell-cycle progression during
such therapies, the
subject treatment can protect hair follicle cells from death which might
otherwise result from
activation of cell death programs. After the therapy has concluded, the
instant method can also
be removed with concommitant relief of the inhibition of follicle cell
proliferation.
[02851 The subject method can also be used in the treatment of folliculitis,
such as folliculitis
decalvans, folliculitis ulerythematosa reticulata or keloid folliculitis. For
example, a cosmetic
prepration of a subject compound can be applied topically in the treatment of
pseudofolliculitis, a
chronic disorder occurring most often in the submandibular region of the neck
and associated
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with shaving, the characteristic lesions of which are erythematous papules and
pustules
containing buried hairs.
[02861 In another aspect of the invention, the subject method can be used to
induce
differentiation and/or inhibit proliferation of epithelially derived tissue.
Such forms of these
molecules can provide a basis for differentiation therapy for the treatment of
hyperplastic and/or
neoplastic conditions involving epithelial tissue. For example, such
preparations can be used for
the treatment of cutaneous diseases in which there is abnormal proliferation
or growth of cells of
the skin.
[02871 For instance, the pharmaceutical preparations of the invention are
intended for the
treatment of hyperplastic epidermal conditions, such as keratosis, as well as
for the treatment of
neoplastic epidermal conditions such as those characterized by a high
proliferation rate for
various skin cancers, as for example basal cell carcinoma or squamous cell
carcinoma. The
subject method can also be used in the treatment of autoimmune diseases
affecting the skin, in
particular, of dermatological diseases involving morbid proliferation and/or
keratinization of the
epidermis, as for example, caused by psoriasis or atopic dermatosis.
[02881 Many common diseases of the skin, such as psoriasis, squamous cell
carcinoma,
keratoacanthoma and actinic keratosis are characterized by localized abnormal
proliferation and
growth. For example, in psoriasis, which is characterized by scaly, red,
elevated plaques on the
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skin, the keratinocytes are known to proliferate much more rapidly than normal
and to
differentiate less completely.
102891 In one embodiment, the preparations of the present invention are
suitable for the
treatment of dermatological ailments linked to keratinization disorders
causing abnormal
proliferation of skin cells, which disorders may be marked by either
inflammatory or non-
inflammatory components. To illustrate, therapeutic preparations of a subject
compound, e.g.,
which promotes quiescense or differentiation, can be used to treat varying
forms of psoriasis, be
they cutaneous, mucosal or ungual. Psoriasis, as described above, is typically
characterized by
epidermal keratinocytes which display marked proliferative activation and
differentiation along a
"regenerative" pathway. Treatment with an antiproliferative embodiment of the
subject method
can be used to reverse the pathological epidermal activiation and can provide
a basis for
sustained remission of the disease.
[02901 A variety of other keratotic lesions are also candidates for treatment
with the subject
method. Actinic keratoses, for example, are superficial inflammatory
premalignant tumors
arising on sun-exposed and irradiated skin. The lesions are erythematous to
brown with variable
scaling. Current therapies include excisional and cryosurgery. These
treatments are painful,
however, and often produce cosmetically unacceptable scarring. Accordingly,
treatment of
keratosis, such as actinic keratosis, can include application, preferably
topical, of a subject
compound composition in amounts sufficient to inhibit hyperproliferation of
epidermal/epidermoid cells of the lesion.
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[02911 Acne represents yet another dermatologic ailment which maybe treated by
the subject
method. Acne vulgaris, for instance, is a multifactorial disease most commonly
occurring in
teenagers and young adults, and is characterized by the appearance of
inflammatory and
noninflammatory lesions on the face and upper trunk. The basic defect which
gives rise to acne
vulgaris is hypercornification of the duct of a hyperactive sebaceous gland.
Hypercornification
blocks the normal mobility of skin and follicle microorganisms, and in so
doing, stimulates the
release of lipases by Propinobacterium acnes and Staphylococcus epidermidis
bacteria and
Pitrosporum ovale, a yeast. Treatment with an antiproliferative subject
compound, particularly
topical preparations, may be useful for preventing the transitional features
of the ducts, e.g.
hypercornification, which lead to lesion formation. The subject treatment may
further include,
for example, antibiotics, retinoids and antiandrogens.
102921 The present invention also provides a method for treating various forms
of dermatitis.
Dermatitis is a descriptive term referring to poorly demarcated lesions which
are either pruritic,
erythematous, scaly, blistered, weeping, fissured or crusted. These lesions
arise from any of a
wide variety of causes. The most common types of dermatitis are atopic,
contact and diaper
dermatitis. For instance, seborrheic dermatitis is a chronic, usually
pruritic, dermatitis with
erythema, dry, moist, or greasy scaling, and yellow crusted patches on various
areas, especially
the scalp, with exfoliation of an excessive amount of dry scales. The subject
method can also be
used in the treatment of stasis dermatitis, an often chronic, usually
eczematous dermatitis.
Actinic dermatitis is dermatitis that due to exposure to actinic radiation
such as that from the sun,
ultraviolet waves or x- or gamma-radiation. According to the present
invention, the subject
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method can be used in the treatment and/or prevention of certain symptoms of
dermatitis caused
by unwanted proliferation of epithelial cells. Such therapies for these
various forms of dermatitis
can also include topical and systemic corticosteroids, antipuritics, and
antibiotics.
[0293] Ailments which may be treated by the subject method are disorders
specific to non-
humans, such as mange as well as any of the disease states or conditions
occurring in animals
corresponding to the conditions and disease states in humans described above
or below.
[0294] In still another embodiment, the subject method can be used in the
treatment of human
cancers, particularly basal cell carcinomas and other tumors of epithelial
tissues such as the skin.
For example, subject compounds can be employed, in the subject method, as
part, of a treatment
for basal cell nevus syndrome (BCNS), and other other human carcinomas,
adenocarcinomas,
sarcomas and the like.
[0295] In a preferred embodiment, the subject method is used as part of a
treatment of
prophylaxis regimen for treating (or preventing) basal cell carcinoma. The
deregulation of the
hedgehog signaling pathway may be a general feature of basal cell carcinomas
caused by ptc
mutations. Consistent overexpression of human ptc mRNA has been described in
tumors of
familial and sporadic BCCs, determined by in situ hybridization. Mutations
that inactivate ptc
may be expected to result in overexpression of mutant Ptc, because ptc
displays negative
autoregulation. Prior research demonstrates that overexpression of hedgehog
proteins can also
lead to tumorigenesis. That sonic hedgehog (Shh) has a role in tumorigenesis
in the mouse has
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been suggested by research in which transgenic mice overexpressing Shh in the
skin developed
features of BCNS, including multiple BCC-like epidermal proliferations over
the entire skin
surface, after only a few days of skin development. A mutation in the Shh
human gene from a
BCC was also described; it was suggested that Shh or other Hh genes in humans
could act as
dominant oncogenes in humans. Sporadic ptc mutations have also been observed
in BCCs from
otherwise normal individuals, some of which are UV-signature mutations. In one
recent study of
sporadic BCCs, five UV-signature type mutations, either CT or CCTT changes,
were found out
of fifteen tumors determined to contain ptc mutations. Another recent analysis
of sporadic ptc
mutations in BCCs and neuroectodermal tumors revealed one CT change in one of
three ptc
mutations found in the BCCs. See, for example, Goodrich et al. (1997) Science
277:1109-13;
Xie et al. (1997) Cancer Res 57:2369-72; Oro et al. (1997) Science 276:817-21;
Xie et al. (1997)
Genes Chromosomes Cancer 18:305-9; Stone et al. (1996) Nature 384:129-34; and
Johnson et al.
(1996) Science 272:1668-71.
[02961 The subject method can also be used to treat patients with BCNS, e.g.,
to prevent BCC
or other effects of the disease which may be the result of ptc loss-of-
function, hedgehog gain-of-
function, or smoothened gain-of-function. Basal cell nevus syndrome is a rare
autosomal
dominant disorder characterized by multiple BCCs that appear at a young age.
BCNS patients
are very susceptible to the development of these tumors; in the second decade
of life, large
numbers appear, mainly on sun-exposed areas of the skin. This disease also
causes a number of
developmental abnormalities, including rib, head and face alterations, and
sometimes
polydactyly, syndactyly, and spina bifida. They also develop a number of tumor
types in
addition to BCCs: fibromas of the ovaries and heart, cysts of the skin and
jaws, and in the central
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nervous system, medulloblastomas and meningiomas. The subject method can be
used to
prevent or treat such tumor types in BCNS and non-BCNS patients. Studies of
BCNS patients
show that they have both genomic and sporadic, mutations in the ptc gene,
suggesting that these
mutations are the ultimate cause of this disease.
[0297] In another aspect, the present invention provides pharmaceutical
preparations and
methods for controlling the formation of megakaryocyte-derived cells and/or
controlling the
functional performance of megakaryocyte-derived cells. For instance, certain
of the
compositions disclosed herein may be applied to the treatment or prevention of
a variety
hyperplastic or neoplastic conditions affecting platelets.
[0298] In certain embodiments, the invention compound can be chosen on the
basis of
selectivity for the hedgehog pathway. This selectivity can be for the hedgehog
pathway vs other
mediated pathways that are used with the compound as well as selectivity for
particular
hedgehog pathways, e.g., which isotype specific for hedgehog (e.g., Shh, Ihh,
Dhh) or the
patched receptor (e.g., ptc-1, ptc-2). For instance, the subject method may
employ different
compounds with different phosphates which do not interfere with the biological
activity of
compounds used in birth control, chemotherapeutic agents, or other ablative
therapies (without
limitation incorporated herein): Aldosterone, androstane, androstene,
androstenedione,
androsterone, cholecalciferol, cholestane, cholic acid, corticosterone,
cortisol, cortisol acetate,
cortisone, cortisone acetate, deoxycorticosterone, digitoxigenin,
ergocalciferol, ergosterol,
estradiol-l7-.alpha., estradiol-l7-.beta., estriol, estrane, estrone,
hydrocortisone, lanosterol,
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lithocholic acid, mestranol, .beta.-methasone, prednisone, pregnane,
pregnenolone, progesterone,
spironolactone(as used in lybrel ), testosterone, triamcinolone and their
derivatives.
[02991 In this manner, untoward side effects which may be associated with
certain members of
these steroidal alkaloids, cancer therpeutics, and other class of drugs
described above and
incorporated herein can be reduced by using the D-chiroinositol compound. For
example, some
methods and compostions may be employed as a means of reducing such unwanted
negative side
effects to certain drug regimes during chemotherapy or infertility treatments.
These side effects
include hirsuitism (excess hair growth due to hormones), shortened life spans,
cardiovascular
diseases (with the use chemotherapeutic agents like tamoxifen and herceptin)
and vascular
occlusion (stroke risk with hormonal/birthcontrol use), organ toxicity,
hyperglycemia and
diabetes exacerbation (with hormonal/birthcontrol use), steroidal glaucoma,
hypertension (from
birth control use or hormone use), and increased susceptibility to infections
(from steroid
akaloids and chemotherapeutics agents) or other types of cancers. In this
manner, unwanted
side effects which may be associated with certain members of steroidal
aklyloids can be reduced
with the method compounds. For example, using the drug screening assays
described herein,
and the application of combinationatorial and medicinal chemistry techniques,
provides a means
for identifiacation of individual agents best suited for reducing the unwanted
negative side
effects of other actives that is part of this application.
[03001 Dosages of C19H19N706 can vary from about 100 g to about 2 mg per day,
preferably at
least about 200 g per day, more preferably at least 400 g per day and should
preferably be no
more than about 1.6 mg per day, more preferably not more than about 1.2 mg per
day. Specific
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pre-natal dosages of folic acid are well known and any of the literature
dosages of this
component will be suitable, especially 0.4 mg, 0.6 mg, 0.8 mg, 1.0 mg, 1.2 mg,
and 1.4 mg for
example. Other folate sources beyond C19H19N7O can be used with or instead of
C19H19N7O6 in
amounts that appropriate to result in the same C19H19N7O6 delivery as the
aforementioned folic
acid. Combinations of C19H19N7O6 and other folate sources are administered in
appropriate
amounts so that the total is equivalent to a C19H19N7O6 dose within the above
limitations.
[03011 D-Chiroinositol doses (and the various derivatives thereof calculated
on the basis of
unphosphorylated D-chiroinositol) range from about 0.05 mg/day to about 60
grams per day,
preferably about 0.05 mg/day to about 30 grams per day, preferably about 0.1
mg to about 25
grams/day, more preferably, about 1 mg to about 20 grams/day, still more
preferably about 5 mg
to about 10 grams per day, even more preferably about 10 mg to about 5 grams
per day, yet more
preferably about 25 mg to about 2 grams/day, still even more preferably about
20 mg to about
1.8 grams/day. Highly preferred dosages of D-chiroinositol (and its P, PP, and
PolyP
derivatives) further include, about 10 mg/kg/day to about 500 mg/kg/day; about
100 mg to about
1 gram/day; about 1.2 gram to about 1.8 gram/day; about 500 mg/day; about 500
to about 700
mg/day; about 25 mg/kg/day to about 100 mg/kg/day. Particular daily doses
(based on
unphosphorylated D-chiroinositol) include: about 0.1 mg, about 0.2 mg, about
0.5 mg, about .8
mg, about 1 mg, about 1.25 mg, about 1.5 mg, about 2 mg, about 2.5 mg, about 3
mg, about 5
mg, about 10 mg, about 12.5 mg, about 15 mg, about 20 mg, about 25 mg, about
40 mg, about 50
mg, about 75 mg, about 100 mg, about 125 mg, about 150 mg, about 200 mg, about
250 mg,
about 300 mg, about 350 mg, about 400 mg, about 500 mg, about 750 mg, about
800 mg, about 1
g, about 1.2 g, about 1.4 g, about 1.6 g, about 1.8 g, about 2 g, about 2.4 g,
about 2.5 g, about
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2.75 g, about 3 g, about 3.5 g, about 4 g, about 5 g, about 6 g, about 8 g,
about 10 g, about 12 g,
about 15 g, about 18 g, about 20 g, about 22.5 g, about 25 g, about 30 g,
about 40 g, about 50 g
and about 60 g. These, particularly the larger doses, may be administered in
fractional doses, all
at a single time or spread out over the day as may be convenient.
[03021 In another aspect, the present invention provides pharmaceutical
preparations
comprising the subject compounds. The compounds for use in the subject method
may be
conveniently formulated for administration with a biologically acceptable
and/or sterile medium,
such as water, buffered saline, polyol (for example, glycerol, propylene
glycol, liquid
polyethylene glycol and the like) or suitable mixtures thereof. The optimum
concentration of the
active ingredient(s) in the chosen medium can be determined empirically,
according to
procedures well known to medicinal chemists. As used herein, "biologically
acceptable
medium" includes any and all solvents, dispersion media, and the like which
may be appropriate
for the desired route of administration of the pharmaceutical preparation. The
use of such media
for pharmaceutically active substances is known in the art. Except insofar as
any conventional
media or agent is incompatible with the activity of the subject compounds, its
use in the
pharmaceutical preparation of the invention is contemplated. Suitable vehicles
and their
formulation inclusive of other proteins are described, for example, in
Remington's
Pharmaceutical Sciences (Mack Publishing Company, Easton, Pa., USA 1985).
These vehicles
include injectable "deposit formulations".
[03031 The composition which is applicable to all aspects of the present
invention is provided
in various forms and formulations, and includes without limitation, as an
implant, a topical and
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transdermal formulation, as a slow release formulation, as an inhalable and
vaporizable
composition, and in injectable form among others. The composition may be part
of a kit, along
with instructions for the administration of the therapeutic agent, and
optionally syringe(s) and
needles, an inhalant device, a transdermal device, and the like.
[03041 In addition to forms set out earlier, the agent may be administered in
the form of a solid,
such as tablets, dragees, capsules, powders, suppositories, etc., and as a
solution, suspension, or
emulsion in a carrier. Particularly desirable are formulations for systemic
and topical
administration, e.g., oral, injectable, topical, transdermal, including those
for iontophoretical
delivery, implantable, and vaginal, rectal, intranasal, intrapulmonary, and
other types of
formulations, which may be prepared by methods known in the art. Solid and
liquid carriers are
suitable, and are known in the art. Liquid carriers typically used in
preparing solutions,
suspensions, and emulsions, which are contemplated for use in the practice of
the present
invention include water, salt solutions, such as saline, pharmaceutically
acceptable organic
solvent(s) and their mixtures, pharmaceutically acceptable oils or fats, and
mixtures of any and
all of the above. The carrier may contain other therapeutic agents and
suitable pharmaceutically
acceptable additives such as solubilizers, emulsifiers, nutrients, buffers,
preservatives,
suspending agents, thickening agents, viscosity regulators, and stabilizers,
among others.
Suitable organic solvents include, for example, monohydric alcohols, such as
ethanol, and
polyhydric alcohols, such as glycols. Suitable oils include, for example,
soybean oil, coconut oil,
olive oil, safflower oil, cottonseed oil, and the like. For parenteral
administration, the carrier
may also be an oily ester such as ethyl oleate, isopropyl myristate, and the
like. Other
pharmaceutically acceptable forms include microparticles, microcapsules,
liposomal
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encapsulates, and the like, as well as their combinations of the agent of the
invention, alone or
with one or more other therapeutic agents may be formulated into sustained
release
microparticles or microcapsules. Materials suitable for the microparticle
matrix include
materials such as starch, polyvinyl alcohol, polyvinylpyrrolidinone,
polyacrylic acid, and the
like, as well as combinations of any two or more thereof. Biodegradable
polymers suitable for
use as a microparticle or microcapsule matrix include, without limitation, for
example, poly-l-
lactide, poly-dl-lactide, polyglycolide, poly(glycolide-co-dl-lactide),
polyanhydrides,
polyorthoesters, poly(alpha-hydroxybutyric acid), poly-p-dioxinone, and block
polymers of
polyglycolide, trimethylene carbonate, polyethylene oxide, proteins,
polysaccharides, and
derivatives and mixtures thereof. The microparticles and microcapsules
containing the agent
may be prepared employing methods which are well known in the art, e.g., by
solvent
evaporation, phase separation, and interphase reaction methods, spray drying,
physical methods,
and the like. As already indicated, the present agent may also be encapsulated
into liposomes,
and the microparticles, microcapsules, and/or liposomes loaded with the agent
may then be
suspended or emulsified in a suitable liquid carrier.
[03051 In addition to the manners set forth above, any aspect of the present
agent may be
administered in a variety of ways, including topical, enteral, and parenteral
routes of
administration. For example, without limnitation, suitable modes of
administration include
subcutaneous, transdermal, transmucosal, including iontophoretic, intravenous,
subcutaneous,
transnasal, intrapulmonary, transdermal, oral, rectal, vaginal, implantable
and the like, as well as
their combinations. The particular pharmaceutically acceptable form of the
therapeutic agent
employed will depend on the route of administration selected. The agent may
be, for example,
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administered in a form that enhances its bioavailability when compared with
standard oral
formulations. Suitable forms include a lipid carrier system that promotes the
oral absorption of
compounds through the intestinal epithelium. Examples of these systems are oil-
in-water, and
water-in-oil emulsions. Exemplary oils that are contemplated for use in oil-in-
water and water-
in-oil based systems include castor oil, olive oil, soybean oil, safflower
oil, coconut oil,
cottonseed oil, their combinations, and the like. Other suitable forms that
enhance the
bioavailability of the orally administered agent of this invention include
single surfactant, and
mixed micelle systems. The agent may, for example, be orally administered in
the form of a
mixed micelle system containing linoleic acid and polyoxyethylene-hardened
castor oil. Suitable
surfactants contemplated for use in single and mixed micelle systems include
polyoxyethylene
ether, polyoxypropylene ether, polyoxyethylene lauryl, cetyl and cholesteryl
ethers,
polyoxyethylene derivatives of lanolin alcohols, and the like, as well as
their mixtures.
[0306] When intravenous or subcutaneous administration is contemplated, the
use of a solution
of the therapeutic agent is preferred. For transdermal administration by
iontophoresis, the agent
is preferably administered in charged form, such as in the form of a salt. The
salt may be in
solution or in a gel reservoir. Therapeutic agent-containing gels may be used
as a drug reservoir
for many routes of administration. An agent containing gel may be prepared by
blending the
inositol based compound with a hydrogel-forming polymer such as polyvinyl
alcohol,
polyacrylamides, copolymers of propylene oxide and ethylene oxide, e.g.,
Pluronic®,
polyvinylpyrrolidinone, gelatin, polymers and copolymers of maleic anhydride,
polyacrylic acid
and salts and derivatives thereof, polysaccharides, and salts and derivatives
thereof, cellulosic
polymers, and salts and derivatives thereof, polycarboxylic acids, and the
like, as well as their
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mixtures. The agent may also be administered transdermally through the use of
a skin patch,
with a carrier. Suitable carriers are typically inert to the agent, non-toxic
to the skin, and allow
the delivery of the agent for systemic absorption into the blood stream via
the skin. Carriers for
transdermal absorption may include pastes, such as absorptive powders
dispersed in petroleum or
hydrophilic petroleum with the agent, with or without a carrier, or a matrix
containing inositol.
Preparations of agent-based compounds may also be administered topically as a
solution, cream,
lotion, or gel, formulated with pharmaceutically acceptable vehicles
containing the agent. The
agent may also be administered intra or transnasally or intrapulmonarily as an
aerosol spray of a
solution, suspension or emulsion, or as microparticles, microcapsules, or
liposomes containing
the agent. Also contemplated are formulations of the agent of this invention
with
pharmaceutically acceptable excipients. Suitable excipients contemplated for
use as processing
aids and drug delivery modifiers and enhancers include calcium phosphate,
magnesium stearate,
talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methyl
cellulose, sodium
carboxymethyl cellulose, dextrose, polyvinylpyrrolidinone, low melting waxes,
ion exchange
resins, and the like, as well as their combinations. Typically, such
formulations are prepared as
tablets or capsules. Other formulations are, however, also within those
contemplated in this
patent.
[03071 The agent may be administered as a single dose or in multiple doses.
Multiple doses
may be administered either continuously, in intervals, or a combination of
both. The agent, for
example, may be administered as a single dose, optionally coupled with a
follow-up dose. The
follow-up dose may be administered by the same or different route of
administration as a single
or sustained dose. Accordingly, the present composition is presented in unit
dosage form or in
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multiple dosage form, as well as in the form of a kit, which may be for self
administration, along
with instructions for the use of the therapeutic agent, and optionally a
syringe(s) and needle(s),
an inhaler or vaporizer, a transdermal patch, optionally for iontophoresis,
and the like. The
composition is also provided as a cream or gel for topical application, and as
an implant. The
manufacture of implants is known in the art and commercially available.
[03081 One particular form of implantable version of the present invention is
the use of a
dendrimeric type of carrier, which may have the inositol based compound
attached thereto by an
ionic, covalent, or hydrogen bonding. Alternatively, the inositol component of
the invention
bound to a fibroblast bound dendrimer (which fibroblast will anchor the
delivery system in
place) is also contemplated. Dendritic molecules have multiple protrusions
which may also be
used to attach the dendrimer to a particular site within the body, to
particular cells, or can be
implanted for migration within the body for attachment to particular cites at
some other point in
time. (See for example Dendrimers Improve Cancer Drug Uptake and Antitumor
Activity, Drug
Delivery, Boston University, NCI Alliance for Nanotechnology in Cancer -
NanoTechWire_com
- The online resource for Nano Technology And Research, 1/15/2007 available at
http://nanotechwire.com/news.asp?nid=4213. Alternatively, the protrusions
themselves may
have the inositol component bound thereto or coated thereon.
[03091 Another implantable version is the use of nanobots or nanorobots for
the delivery of the
acitive agent. While nanorobotics is a rather recent development, those of
ordinary skill will
appreciate the advantages of such a delivery, which can be achieved in manners
set forth in for
example (a) Shanthi, et al; Prospects for medical Robots; AZojono Journal of
Nanotechnology
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Online, posted 13-November-2007;
http//www.azonano.com/Details.asp?ArticleID=2035; (b)
Adriano Cavalcanti, Bijan Shirinzadeh, Tad Hogg, Julian A. Smith, "Hardware
Architecture for
Nanorobot Application in Cancer Therapy", IEEE-RAS ICAR Int'l Con on Advanced
Robotics, Jeju, Korea, pp. 200-205, August 2007; (c) Hede et al, "Nano": the
new nemesis of
Cancer, J Can Res Ther [serial online] 2006 [cited 2007 Dec 11]; 2:186-195
available from
http://www.cancerjournal.net/text.asp?2006/2/4/186/29829 and (d) Nanorobotics
Control Design
and 3D Simulation, available at hllp://www.nanorobotdesipn.co m/ (2007). A
preferred version
of the nanorobotic delivery in thepresent invention is designed to be
implanted in or around the
site of specific delivery such as a cancerous lesion excision site or into an
inoperable tumor and
which delivers the active agent or active agent precursor on a single
prolonged or multiple
release schedule which can be pre-programmedfor delivery over short or
extended periods
extending for as much as multiple years. As may be desired, the nanorobotic
delivery system
can be one which can mirate or be fixed in position by virtue of specific
adherence mechanisms
including fibroblasts, monoclonal antibodies, charged particle portions,
antisence DNA, etc.
103101 Yet another implantable or injectable and migratable delivery system
utilizes
monoclonal antibodies that are specific to cancer cell receptors or other
cancer cell specific
proteins. These antibodies having the active agent linked thereto migrate to
the specific cancer
cells and deliver the active agent directly to the cell on which it is to act.
103111 Pharmaceutical formulations of the present invention can also include
veterinary
compositions, e.g., pharmaceutical preparations of the subject compounds
suitable for veterinary
uses, e.g., for the treatment of live stock or domestic animals, e.g., dogs.
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103121 Compositions of the present invention may be single active agent
entities that are
merely co-administered as described herein or fixed combinations as indicated
above. The other
components beyond the folic acid (and/or other folate) and the inositol
compounds (and/or P, PP,
and/or PolyP derivative thereof, and the other derivatives thereof discussed
further herein) can be
selected from a wide variety of compounds. Additional active agents that may
be included in or
merely co-administered with the above components include those estrogenic and
progestogenic
substances used in birth control pills, hormone replacement therapy, androgen
ablative therapy,
etc. (including, but not limited to conjugated estrogens, ethinyl estradiol,
levonorgestrel,
norgestrel, norgestimate, norethidrone, norethidrone acetate, mestranol,
ethynodiol diacetate,
norelgestromin, etonogestrel, desogestrel, etc). These hormones are currently
marketed under
the following (non-limiting) trade names: ALESSE, ANGELIQ, DIANE, LEVLEN, LO-
OVRAL, LYBREL,TRICYCLEN, ORTHOCEPT, ORTHOEVRA, MIRENA, MENOSTAR,
NUVA RING, OVRAL, TRI-LEVLEN, TRIPHASIL, BREVICON, FEMHRT, LOESTRIN,
LoOGESTREL, MICROGESTIN, YAZMIN, among others. Where the birth control or
hormone
replacement therapy dosage form is other than an oral dosage form (such as,
for example, a
transdermal patch (in the case of currently marketed norelgestromin) or a
vaginal ring (in the
case of currently marketed etonogestrel estradiol), ORTHOEVRA marketed as
transdermal
birthcontrol patch (recently linked to higher than average acute
thromboembolitic events in
female users). The invention compound is designed to eliminate the inherent
risk of this type of
hormonal contraception. Also transdermal patches for hormonal replacement
incuding but not
limited to Vivelle and Vivelle-DotTM, Estradot , combination
estrogen/progestin transdermal
delivery systems (including CombiPatchTM, licensed to Aventis, and Estalis
,Testoderm . The
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invention objectives are achieved with a co-therapy of a suitable dosage form
with or without the
folic acid (and/or other folate source) and D-chiroinositol (and/or P, PP,
and/or Poly P
derivatives thereof). Andogen ablative therapies for which the instant
invention can be used
include treatment with for example, without limitation, finasteride as well as
other known
androgen ablative drugs. Other active compounds for use in combination with or
in cotherapy
with the inositol compounds of the invention include, especially in the fetal
alcohol syndrome
prevention aspect, those used in cholesterol reduction regimens, especially
the statins.
[03131 Compositions of the present invention in which the D-chiroinositol or
other inositol
component and or the folic acid component are the only active agents can be
prepared as in or
analogously to those set forth in the patents indicated above as being
incorporated herein by
reference. For compositions that are disclosed therein that have an inositol
component, the D-
chiroinositol or other inositolcomponent (and/or P, PP, and/or PolyP
derivative thereof or other
derivative thereof discussed further herein) can be used in direct replacement
of the such inositol
component indicated in such reference. The folic acid (and/or other folate
source) can be
incorporated therein by merely replacing a small portion of filler or merely
adding the folic acid
(and/or other folate source) thereto. Where the referenced formulation is a
folic acid formulation
and the dose selected for the inositol (and/or P, PP, and/or PolyP derivative
thereof or other
derivative thereof discussed further herein) is sufficiently small, the
inositol (and/or derivative
thereof) can be used in place of a portion or all of the filler used in the
referenced formulation, or
added to it. If larger amounts are needed, then the filler used in the
referenced formulation is
replaced with the inositol (and/or P, PP, and/or PolyP derivative thereof or
other derivative
thereof) component if the resulting tablet size is not of concern. If the size
of the dosage form is
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insufficient to accommodate the full dose of the inositol component, then
either a separate
dosage form is used or multiple dosage forms having a fraction of the daily
dose is used and the
patient will need to take more than 1 dosage form to achieve the daily dosages
set forth.
103141 In preferred dosage forms of one embodiment of the invention, the D-
chiroinositol
(and/or P, PP, and/or PolyP derivatives thereof or other derivatives thereof)
is substantially free
of the other isomers of inositol. In highly preferred formulations of this
embodiment, the D-
chiroinositol (and/or the P, PP, and/or PolyP derivatives thereof or other
derivatives thereof) and
the dosage forms thereof are completely free of the other isomers of inositol
as well as their
corresponding phosphorylated derivatives. For purposes of the present
invention, "substantially
free" means not more than about 5% based on the combined D-chiro forms
present, more
preferably not more than about 2.5%, still more preferably not more than about
1%, most
preferably not more than 0.5%. For purposes of the present invention,
"completely free of " or
"free of' means below the limit of detection of said non-D-chiro forms
respectively in common
analytical techniques used in common pharmaceutical quality control of bulk
materials as of the
date of the invention herein. Similarly with respect to formulations of other
embodiments of the
invention in which other inositol isomers, their respective phosphorylates (of
varying size) or
other derivatives thereof oas described further herein, presferred are those
that are substantially
free of other isomeric forms of inositol than the one being primarily present,
and more preferably
completely freee of such other isomeric forms.
103151 Actual dosage levels of the active ingredients in the pharmaceutical
compositions of
this invention may be varied so as to obtain an amount of the active
ingredient which is effective
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to achieve the desired therapeutic response for a particular patient,
composition, and mode of
administration, without being toxic to the patient.
103161 The selected dosage level will depend upon a variety of factors
including the activity of
the particular compound of the present invention employed, or the ester, salt
or amide thereof,
the route of administration, the time of administration, the rate of excretion
of the particular
compound being employed, the duration of the treatment, other drugs, compounds
and/or
materials used in combination with the particular compound employed, the age,
sex, weight,
condition, general health and prior medical history of the patient being
treated, and like factors
well known in the medical arts.
[03171 The therapeutic agent of this aspect of the invention (the non-D-
chiroinositol based
inositol compounds), is typically administered to a subject at a dose of about
20 to about 8,000
mg/kg/day, and preferably at about 30 to about 5,000 mg/kg/day. Other amounts,
however, may
also be administered. Higher or lower doses of these agents, however, may also
be administered.
103181 US 5,998,485 (incorporated herein by reference) reports that unmodified
scyllo inositol
is more potent than other unmodified inositol isomers tested there in
modulating a subject's
immune response in a dose dependent manner. When the agent is administered at
relatively low
doses, it effectively enhances a mammal's immune response and, when
administered at relatively
high doses, inositol effectively inhibits or suppresses the mammal's immune
response. Thus, the
gain-of-function or loss-of-function activity of the inositols may vary
depending on dose and the
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CA 02668580 2009-06-10
above screenings should be undertaken at multiple dosing schedules to
determine whether an
analogous result is present with resepect to other inositol based compounds
within the present
invention, and if so, the compound may be used in the appropriate indication
above at the
appropriate dose NOTWITHSTANDING prior direction to not use any particular
active agent
in such indication.
103191 The dose response range for gain-of-function as opposed to loss-of-
function for any
particular pathway may vary somewhat depending on the form of the agent
employed, such as,
the particular stereoisomer, derivative, or salt employed. One of ordinary
skill in the art,
however, may readily determine the range of response-enhancing doses of the
therapeutic agent
by the above screening tests and by other means known in the art, e.g., by
generating a dose-
response curve for any particular form of therapeutic agent. The dose of the
therapeutic agent
administered for any particular activity will, of course, also vary with
factors such as the
pharmacodynamic characteristics of the agent employed, its mode and route of
administration,
the age, health, and weight of the recipient, the nature and extent of the
symptoms, the kind of
concurrent treatment(s), the frequency of treatment, the effect desired, and
the like. Modulation
of the doses within the ranges set forth herein are within the skill of the
ordinary skilled clinician
and can be adjusted by such persons appropriately.
103201 A physician or veterinarian having ordinary skill in the art can
readily determine and
prescribe the effective amount of the pharmaceutical composition required. For
example, the
physician or veterinarian could start doses of the compounds of the invention
employed in the
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pharmaceutical composition at levels lower than that required in order to
achieve the desired
therapeutic effect and gradually increase the dosage until the desired effect
is achieved.
[03211 In general, a suitable daily dose of a compound of the invention will
be that amount of
the compound which is the lowest dose effective to produce a therapeutic
effect. Such an
effective dose will generally depend upon the factors described above.
Generally, intravenous,
intracerebroventricular and subcutaneous doses of the compounds of this
invention for a patient
will range from about 0.0001 to about 100 mg per kilogram of body weight per
day.
[0322] If desired, the effective daily dose of the active compound may be
administered as two,
three, four, five, six or more sub-doses administered separately at
appropriate intervals
throughout the day, optionally, in unit dosage forms.
[03231 Methods of introduction may also be provided by rechargeable or
biodegradable
devices. Various slow release polymeric devices have been developed and tested
in vivo in
recent years for the controlled delivery of drugs, including proteinacious
biopharmaceuticals. A
variety of polymers (including hydrogels), can be used to form an implant for
the sustained
release of a subject compound at a particular target site. Other methods of
drug delivery may
also be provided by nanotechnology utilizing such nanomaterials,
nanostructures, nanofibers,
nanowires, nanoparticles, quantum dot, nanotube, dendrimer, nanocystal, or
nanobot. (See
HIGH-PRESSURE POLYMERIZATION OF SINGLE WALL CARBON NANOTUBES; M.
Popov, M. Kyotani and Y. Koga Joint Research Consortium of Frontier Carbon
Technology,
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CA 02668580 2009-06-10
JFCC, c/o NIMC, Higashi, Tsukuba, Ibaraki, 305-8565, Japan and R. J. Nemanich
Department
of Physics, North Carolina State University, 408A Cox, Box 8202, Raleigh, NC,
27695-8202,
USA; http://www.eng.auburn.edu/department/ee/ADC-
FCT2001/ADCFCTabstract/101.htm)
[03241 Other methods of entry may also be provided by a battery. For example,
a battery
operated transdermal drug delivery device (iontophoresis) utilizing a current
distribution member
for delivering a pulsed direct current sufficient to iontophoretically deliver
composition across a
stratum corneum layer of the epidermis. The current distribution member
comprises an
electrochemically active component in electrical connection with a battery, a
voltage pulse
generator and a precision resistor.
103251 The preparations of the present invention may be given orally,
parenterally, topically, or
rectally. They are of course given by forms suitable for each administration
route. For example,
they are administered in tablets or capsule form, by implant, injection,
inhalation, eye lotion,
ointment, drops, suppository, controlled release patch, etc. administration by
injection, infusion
or inhalation; topical by lotion or ointment; and rectal by suppositories.
Oral and topical
administrations are preferred.
103261 The phrases "parenteral administration" and "administered parenterally"
as used herein
means modes of administration other than enteral and topical administration,
usually by
injection, and includes, without limitation, intravenous, intramuscular,
intraarterial, intrathecal,
intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal, subcutaneous,
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subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and
intrasternal injection and
infusion.
[03271 The phrases "systemic administration," "administered systemically,"
"peripheral
administration" and "administered peripherally" as used herein mean the
administration of a
compound, drug or other material other than directly into the central nervous
system, such that it
enters the patient's system and, thus, is subject to metabolism and other like
processes, for
example, subcutaneous administration.
[03281 These compounds may be administered to humans and other animals for
therapy by any
suitable route of administration, including orally, nasally, as by, for
example, a spray, rectally,
intravaginally, parenterally, intrasystemically, and topically, as by powders,
ointments or drops,
including buccally and sublingually.
[03291 Regardless of the route of administration selected, the compounds of
the present
invention, which may be used in a suitable hydrated form, and/or the
pharmaceutical
compositions of the present invention, are formulated into pharmaceutically
acceptable dosage
forms such as described below or by other conventional methods known to those
of skill in the
art.
103301 The term "treatment" is intended to encompass prophylaxis, therapy and
cure, unless
specifically indicated otherwise or the context requires otherwise.
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103311 The patient receiving this treatment is any animal in need, including
primates, in
particular humans, and other mammals such as equines, cattle, swine and sheep;
and poultry and
pets in general.
[03321 The compound of the invention can be administered as such or in
admixtures with
pharmaceutically acceptable and/or sterile carriers and can also be
administered in conjunction
with other antimicrobial agents such as penicillin, cephalosporins,
aminoglycosides and
glycopeptides. Conjunctive therapy, thus includes sequential, simultaneous and
separate
administration of the active compound in a way that the therapeutical effects
of the first
administered one is not entirely disappeared when the subsequent is
administered.
[03331 V. Pharmaceutical Compositions
[0334] While it is possible for a compound of the present invention to be
administered alone, it
is preferable to administer the compound as a pharmaceutical formulation
(composition). The
subject compounds according to the invention may be formulated for
administration in any
convenient way for use in human or veterinary medicine. In certain
embodiments, the compound
included in the pharmaceutical preparation may be active itself, or may be a
prodrug, e.g.,
capable of being converted to an active compound in a physiological setting.
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[0335] Thus, another aspect of the present invention provides pharmaceutically
acceptable
compositions comprising a therapeutically effective amount of one or more of
the compounds
described herein, formulated together with one or more pharmaceutically
acceptable carriers
(additives) and/or diluents. As described in detail below, the pharmaceutical
compositions of the
present invention may be specially formulated for administration in solid or
liquid form,
including those adapted for the following: (1) oral administration, for
example, drenches
(aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders,
granules, pastes
for application to the tongue; (2) parenteral administration, for example, by
subcutaneous,
intramuscular or intravenous injection as, for example, a sterile solution or
suspension; (3)
topical application, for example, as a cream, ointment or spray applied to the
skin; or (4)
intravaginally or intrarectally, for example, as a pessary, cream or foam.
However, in certain
embodiments the subject compounds may be simply dissolved or suspended in
sterile water. In
certain embodiments, the pharmaceutical preparation is non-pyrogenic, i.e.,
does not elevate the
body temperature of a patient.
[0336] The phrase "therapeutically effective amount" as used herein means that
amount of a
compound, material, or composition comprising a compound of the present
invention which is
effective for producing some desired therapeutic effect, e.g., with respect to
D-chiroinositol and
its derivatives by overcoming a ptc loss-of-function, hedgehog gain-of-
function, or smoothened
gain-of-function, in at least a sub-population of cells in an animal and
thereby blocking the
biological consequences of that pathway in the treated cells, at a reasonable
benefit/risk ratio
applicable to any medical treatment.
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103371 The phrase pharmaceutically acceptable" is employed herein to refer to
those
compounds, materials, compositions, and/or dosage forms which are, within the
scope of sound
medical judgment, suitable for use in contact with the tissues of human beings
and animals
without excessive toxicity, irritation, allergic response, or other problem or
complication,
commensurate with a reasonable benefit/risk ratio. Such materials will be
known to those of
ordinary skill in the pharmaceutical formulation art.
[03381 The phrase "pharmaceutically acceptable carrier" as used herein means a
pharmaceutically acceptable material, composition or vehicle, such as a liquid
or solid filler,
diluent, excipient, solvent or encapsulating material, involved in carrying or
transporting the
subject regulators from one organ, or portion of the body, to another organ,
or portion of the
body. Each carrier must be "acceptable" in the sense of being compatible with
the other
ingredients of the formulation and not injurious to the patient Some examples
of materials which
can serve as pharmaceutically acceptable carriers includewithout limitation:
(1) sugars, such as
lactose, glucose and sucrose; (2) starches, such as corn starch and potato
starch; (3) cellulose,
and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose
and cellulose
acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)
excipients, such as cocoa
butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil,
safflower oil, sesame
oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene
glycol; (11) polyols, such
as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as
ethyl oleate and ethyl
laurate; (13) agar, (14) buffering agents, such as magnesium hydroxide and
aluminum hydroxide;
(15) alginic acid; (16) pyrogen-free water, (17) isotonic saline; (18)
Ringer's solution; (19) ethyl
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alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible
substances
employed in pharmaceutical formulations.
[0339] As set out above, certain embodiments of the present compounds may
contain a basic
functional group, such as amino or alkylamino, and are, thus, capable of
forming
pharmaceutically acceptable salts with pharmaceutically acceptable acids. The
term
"pharmaceutically acceptable salts" in this respect, refers to the relatively
non-toxic, inorganic
and organic acid addition salts of compounds of the present invention. These
salts can be
prepared in situ during the final isolation and purification of the compounds
of the invention, or
by separately reacting a purified compound of the invention in its free base
form with a suitable
organic or inorganic acid, and isolating the salt thus formed. Representative
salts include the
hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate,
valerate, oleate,
palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate,
maleate, fumarate,
succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionate, and
laurylsulphonate
salts and the like. (See, for example, Berge et al. (1977) "Pharmaceutical
Salts", J. Pharm. Sci.
66:1-19)
103401 The pharmaceutically acceptable salts of the subject compounds include
the
conventional nontoxic salts or quaternary ammonium salts of the compounds,
e.g., from non-
toxic organic or inorganic acids. For example, such conventional nontoxic
salts include those
derived from inorganic acids such as hydrochloride, hydrobromic, sulfuric,
sulfamic, phosphoric,
nitric, and the like; and the salts prepared from organic acids such as
acetic, propionic, succinic,
glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic,
maleic, hydroxymaleic,
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phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic,
fumaric,
toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isothionic, and
the like.
103411 As described above, where the compounds used in the invention are
capable of salt
formation, the reference to the compound includes the pharmaceutically
acceptable salts thereof.
These salts can be prepared in situ during the final isolation and
purification of the compounds of
the invention, or by separately reacting a purified compound of the invention
in its free form
with a suitable organic or inorganic acid or base as appropriate, and
isolating the salt thus
formed, or reacting a salt of the compound with an appropriate organic or
inorganic acid or base
to result in a different salt formation. Representative pharmaceutically
acceptable acid addition
salts include, without limitation, the hydrobromide, hydrochloride, sulfate,
bisulfate, phosphate,
nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,
lactate, phosphate, tosylate,
citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate,
glucoheptonate,
lactobionate, and laurylsulphonate salts and the like. (See, for example,
Berge et al. (1977)
"Pharmaceutical Salts", J. Pharm. Sci. 66:1-19).
103421 In other cases, the compounds of the present invention may contain one
or more acidic
functional groups and, thus, are capable of forming pharmaceutically
acceptable salts with
pharmaceutically acceptable bases. The term "pharmaceutically acceptable
salts" in these
instances refers to the relatively non-toxic, inorganic and organic base
addition salts of
compounds of the present invention. These salts can likewise be prepared in
situ during the final
isolation and purification of the compounds, or by separately reacting the
purified compound in
its free acid form with a suitable base, such as the hydroxide, carbonate or
bicarbonate of a
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pharmaceutically acceptable metal cation, with ammonia, or with a
pharmaceutically acceptable
organic primary, secondary or tertiary amine. Representative alkali or
alkaline earth salts
include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts
and the like.
Representative organic amines useful for the formation of base addition salts
include ethylamine,
diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and
the like. (See, for
example, Berge et al., supra).
[03431 Wetting agents, emulsifiers and lubricants, such as sodium lauryl
sulfate and
magnesium stearate, stearic acid, and talc, as well as coloring agents,
release agents, coating
agents, sweetening, flavoring and perfuming agents, preservatives and
antioxidants can also be
present in the compositions.
103441 Examples of pharmaceutically acceptable antioxidants include: (1) water
soluble
antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate,
sodium
metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such
as ascorbyl palmitate,
butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin,
propyl gallate,
alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric
acid,
ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric
acid, and the like.
[03451 Formulations of the present invention include those suitable for oral
or topical
(including buccal and sublingual), rectal, vaginal, and/or parenteral,
transdermal, iontophoresis,
nano particle delivery (without limitation), and various polypeptide vectors
"carrier"
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administration from one organ to another as necessary. The formulations may
conveniently be
presented in unit dosage form and may be prepared by any methods well known in
the art of
pharmacy. The amount of active ingredient which can be combined with a carrier
material to
produce a single dosage form will vary depending upon the host being treated,
the particular
mode of administration. The amount of active ingredient which can be combined
with a carrier
material to produce a single dosage form will generally be that amount of the
compound which
produces a therapeutic effect.
[03461 Methods of preparing these formulations or compositions include the
step of bringing
into association a compound of the present invention with the carrier and,
optionally, one or
more accessory ingredients. In general, the formulations are prepared by
uniformly and
intimately bringing into association a compound of the present invention with
liquid carriers, or
finely divided solid carriers, or both, and then, if necessary, shaping the
product. Alternative
methods will be appreciated by those of ordinary skill in the pharmaceutical
formulating art.
[03471 Formulations of the invention suitable for oral administration may be
in the form of
capsules, cachets, pills, tablets, lozenges (usually using a flavored basis,
usually sucrose and
acacia or tragacanth), powders, granules, or as a solution or a suspension in
an aqueous or non-
aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as
an elixir or syrup, or as
pastilles (using an inert base, such as gelatin and glycerin, or sucrose and
acacia) and/or as mouth
washes and the like, each containing a predetermined amount of a compound of
the present
invention as an active ingredient. A compound of the present invention may
also be
administered as a bolus, electuary or paste.
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[03481 In solid dosage forms of the invention for oral administration
(capsules, tablets, pills,
dragees, powders, granules and the like), the active ingredient is mixed with
one or more
pharmaceutically acceptable carriers, such as, without limitation, sodium
citrate or dicalcium
phosphate, and/or any of the following: (1) fillers or extenders, such as
starches, lactose, sucrose,
glucose, mannitol, and/or silicic acid; (2) binders, such as, for example,
carboxymethylcellulose,
alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3)
humectants, such as glycerol;
(4) disintegrating agents, such as agar-agar, calcium carbonate, potato or
tapioca starch, alginic
acid, certain silicates, and sodium carbonate; (5) solution retarding agents,
such as paraffin; (6)
absorption accelerators, such as quaternary ammonium compounds; (7) wetting
agents, such as,
for example, cetyl alcohol and glycerol monostearate; (8) absorbents, such as
kaolin and
bentonite clay, (9) lubricants, such a talc, calcium stearate, magnesium
stearate, solid
polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10)
coloring agents. In
the case of capsules, tablets and pills, the pharmaceutical compositions may
also comprise
buffering agents. Solid compositions of a similar type may also be employed as
fillers in soft
and hard-filled gelatin capsules using such excipients as lactose or milk
sugars, as well as high
molecular weight polyethylene glycols and the like.
103491 A tablet may be made by compression or molding, optionally with one or
more
accessory ingredients. Compressed tablets may be prepared using binder (for
example, gelatin or
hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,
disintegrant (for example,
sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),
surface-active or
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dispersing agent, etc. Molded tablets may be made by molding in a suitable
machine a mixture of
the powdered compound moistened with an inert liquid diluent.
[03501 The tablets, and other solid dosage forms of the pharmaceutical
compositions of the
present invention, such as dragees, capsules, pills and granules, may
optionally be scored or
prepared with coatings and shells, such as enteric coatings and other coatings
well known in the
pharmaceutical-formulating art. They may also be formulated so as to provide
slow or
controlled release of the active ingredient therein using, for example,
hydroxypropylmethyl
cellulose in varying proportions to provide the desired release profile, other
polymer matrices,
liposomes and/or microspheres. They may be sterilized by, for example,
filtration through a
bacteria-retaining filter, or by incorporating sterilizing agents in the form
of sterile solid
compositions which can be dissolved in sterile water, or some other sterile
injectable medium
immediately before use. These compositions may also optionally contain
opacifying agents and
may be of a composition that they release the active ingredient(s) only, or
preferentially, in a
certain portion of the gastrointestinal tract, optionally, in a delayed
manner. Examples of
embedding compositions which can be used include polymeric substances and
waxes. The
active ingredient can also be in micro-encapsulated form, if appropriate, with
one or more of the
above-described excipients.
[03511 Liquid dosage forms for oral administration of the compounds of the
invention include
pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions,
syrups and
elixirs. In addition to the active ingredient, the liquid dosage forms may
contain inert diluents
commonly used in the art, such as, for example, water or other solvents,
solubilizing agents and
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emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl
acetate, benzyl
alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in
particular, cottonseed,
groundnut, corn, germ, olive, castor and sesame oils), glycerol,
tetrahydrofuryl alcohol,
polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
103521 Besides inert diluents, the oral compositions can also include
adjuvants such as wetting
agents, emulsifying and suspending agents, sweetening, flavoring, coloring,
perfuming and
preservative agents.
[03531 Suspensions, in addition to the active compounds, may contain
suspending agents as,
for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and
sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and
tragacanth, and
mixtures thereof.
[03541 Formulations of the pharmaceutical compositions of the invention for
rectal or vaginal
administration may be presented as a suppository, which may be prepared by
mixing one or more
compounds of the invention with one or more suitable nonirritating excipients
or carriers
comprising, for example, cocoa butter, polyethylene glycol, a suppository wax
or a salicylate,
and which is solid at room temperature, but liquid at body temperature and,
therefore, will melt
in the rectum or vaginal cavity and release the active compound.
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[03551 Formulations of the present invention which are suitable for vaginal
administration also
include pessaries, tampons, creams, gels, pastes, foams or spray formulations
containing such
carriers as are known in the art to be appropriate.
103561 Dosage forms for the topical or transdermal administration of a
compound of this
invention include powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches
and inhalants. The active compound may be mixed under sterile conditions with
a
pharmaceutically acceptable carrier, and with any preservatives, buffers, or
propellants which
may be required.
[03571 The ointments, pastes, creams and gels may contain, in addition to an
active compound
of this invention, excipients, such as animal and vegetable fats, oils, waxes,
paraffins, starch,
tragacanth, cellulose derivatives, polyethylene glycols, silicones,
bentonites, silicic acid, talc and
zinc oxide, or mixtures thereof.
[03581 Powders and sprays can contain, in addition to a compound of this
invention, excipients
such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and
polyamide powder,
or mixtures of these substances. Sprays can additionally contain customary
propellants, such as
chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as
butane and propane.
[03591 Transdermal patches have the added advantage of providing controlled
delivery of a
compound of the present invention to the body. Such dosage forms can be made
by dissolving or
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dispersing the subject compounds in the proper medium. Absorption enhancers
can also be used
to increase the flux of the compound across the skin. The rate of such flux
can be controlled by
either providing a rate controlling membrane or dispersing the compound in a
polymer matrix or
gel or microneedle technology. Battery operated trandermal drug delivery
utilizing a current is
another example of controlling rate of flux (Iontophoretic transdermal
delivery)
103601 Ophthalmic formulations,ophthalmalic eye implant for medication
delivery, eye
ointments, drug eluting contact lenses, powders, solutions and the like, are
also contemplated as
being within the scope of this invention.
[03611 Pharmaceutical compositions of this invention suitable for parenteral
administration
comprise one or more compounds of the invention in combination with one or
more
pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions,
dispersions,
suspensions or emulsions, or sterile powders which may be reconstituted into
sterile injectable
solutions or dispersions just prior to use, which may contain antioxidants,
buffers, bacteriostats,
solutes which render the formulation isotonic with the blood of the intended
recipient or
suspending or thickening agents.
[03621 Examples of suitable aqueous and nonaqueous carriers which may be
employed in the
pharmaceutical compositions of the invention include water, ethanol, polyols
(such as glycerol,
propylene glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable
oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
Proper fluidity can be
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maintained, for example, by the use of coating materials, such as lecithin, by
the maintenance of
the required particle size in the case of dispersions, and by the use of
surfactants.
103631 These compositions may also contain adjuvants such as preservatives,
wetting agents,
emulsifying agents and dispersing agents. Prevention of the action of
microorganisms may be
ensured by the inclusion of various antibacterial and antifungal agents, for
example, paraben,
chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to
include isotonic
agents, such as sugars, sodium chloride, and the like into the compositions.
In addition,
prolonged absorption of the injectable pharmaceutical form may be brought
about by the
inclusion of agents that delay absorption such as aluminum monostearate and
gelatin.
[03641 In some cases, in order to prolong the effect of a drug, it is
desirable to slow the
absorption of the drug from subcutaneous or intramuscular injection. This may
be accomplished
by the use of a liquid suspension of crystalline, or nanocrystal, or amorphous
material having
poor water solubility. The rate of absorption of the drug then depends upon
its rate of dissolution
which, in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed
absorption of a parenterally administered drug form is accomplished by
dissolving or suspending
the drug in an oil vehicle.
[03651 Injectable depot forms are made by forming microencapsule matrices of
the subject
compounds in polymers such as, without limitation, polylactide-polyglycolide.
Depending on
the ratio of drug to polymer, and the nature of the particular polymer
employed, the rate of drug
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release can be controlled. Examples of other polymers include poly
(orthoesters) and
poly(anhydrides). Depot injectable formulations are also prepared by
entrapping the drug in
liposomes or microemulsions which are compatible with body tissue.
[03661 When the compounds of the present invention are administered as
pharmaceuticals, to
humans and animals, they can be given per se or as a pharmaceutical
composition containing, for
example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in
combination with a
pharmaceutically acceptable carrier.
[03671 The addition of the active compound of the invention to animal feed is
preferably
accomplished by preparing an appropriate feed premix containing the active
compound in an
effective amount and incorporating the premix into the complete ration.
[03681 Alternatively, an intermediate concentrate or feed supplement
containing the active
ingredient can be blended into the feed. The way in which such feed premixes
and complete
rations can be prepared and administered are described in reference books
(such as "Applied
Animal Nutrition", W.H. Freedman and CO., San Francisco, U.S.A., 1969 or
"Livestock Feeds
and Feeding" 0 and B books, Corvallis, Ore., U.S.A., 1977).
[03691 Synthetic schemes using coupling reactions to identify active
regulators of genes
or gene expression
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[0370] The subject compounds, and derivatives thereof, can be prepared readily
by employing
synthetic methodolody well-known in the art. Additional compounds may be
synthesized and
tested in a combinatorial fashion, to facilitate the identification of
additional compounds which
may be employed in the subject method.
[0371] a. Combinatorial Libraries
[0372] The compounds of the present invention, particularly libraries of
variants having
various representative classes of substituents, are amenable to combinatorial
chemistry and other
parallel synthesis schemes (see, for example, PCT WO 94/0805 1). The result is
that large
libraries of related compounds, e.g. a variegated library of compounds
represented above, can be
screened rapidly in high throughput assays in order to identify potential
hedgehog regulator lead
compounds, as well as to refine the specificity, toxicity, and/or cytotoxic-
kinetic profile of a lead
compound. For instance, ptc, hedgehog, or smoothened bioactivity assays, such
as may be
developed using cells with either a ptc loss-of-function, hedgehog gain-of-
function, or
smoothened gain-of-function, can be used to screen a library of the subject
compounds for those
having agonist activity toward ptc or antagonist activity towards hedgehog or
smoothened.
Alternatively, bioactivity assays using cells with either a ptc gain-of-
function, hedgehog loss-of-
function, or smoothened loss-of-function, can be used to screen a library of
the subject
compounds for those having antagonist activity toward ptc or agonist activity
towards hedgehog
or smoothened.
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103731 Simply for illustration, a combinatorial library for the purposes of
the present invention
is a mixture of chemically related compounds which may be screened together
for a desired
property. The preparation of many related compounds in a single reaction
greatly reduces and
simplifies the number of screening processes which need to be carried out.
Screening for the
appropriate physical properties can be done by conventional methods.
[03741 Diversity in the library can be created at a variety of different
levels. For instance, the
substrate aryl groups used in the combinatorial reactions can be diverse in
terms of the core aryl
moiety, e.g., a variegation in terms of the ring structure, and/or can be
varied with respect to the
other substituents.
103751 A variety of techniques are available in the art for generating
combinatorial libraries of
small organic molecules such as the subject compounds. See, for example,
Blondelle et al.
(1995) Trends Anal. Chem. 14:83; the Affymax U.S. Pat. Nos. 5,359,115 and
5,362,899: the
Ellman U.S. Pat. No. 5,288,514: the Still et al. PCT publication WO 94/08051;
the ArQule U.S.
Pat. Nos. 5,736,412 and 5,712,171; Chen et al. (1994) JACS 116:2661: Kerr et
al. (1993) JACS
115:252; PCT publications W092/10092, W093/09668 and W091/07087; and the
Lerner et al.
PCT publication W093/20242). Accordingly, a variety of libraries on the order
of about 100 to
1,000,000 or more diversomers of the subject compounds can be synthesized and
screened for
particular activity or property.
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[0376] Combinatorial library development and screening can be conducted, for
example in
analogy to the methods and procedures set forth in Beachy et al US 7,291,626,
incorporated
herein by reference in its entirety. In an exemplary embodiment, a library of
candidate
compound diversomers can be synthesized utilizing a scheme adapted to the
techniques
described in the Still et al. PCT publication WO 94/08051, e.g., being linked
to a polymer bead
by a hydrolyzable or photolyzable group, optionally located at one of the
positions of the
candidate regulators or a substituent of a synthetic intermediate. According
to the Still et al.
technique, the library is synthesized on a set of beads, each bead including a
set of tags
identifying the particular diversomer on that bead. The bead library can then
be "plated" with,
for example, ptc loss-of-function, hedgehog gain-of-function, or smoothened
gain-of-function
cells for which a hedgehog antagonist is sought. The diversomers can be
released from the bead,
e.g. by hydrolysis.
[0377] Many variations on the above and related pathways permit the synthesis
of widely
diverse libraries of compounds which may be tested as regulators of hedgehog
function.
[0378] b. Screening Assays
[0379] There are a variety of assays available for determining the ability of
a compound such
as a hedgehog regulator to regulate ptc, smoothened, or hedgehog function,
many of which can
be disposed in high-throughput formats. In many drug screening programs which
test libraries of
compounds and natural extracts, high throughput assays are desirable in order
to maximize the
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number of compounds surveyed in a given period of time. Thus, libraries of
synthetic and natural
products can be sampled for other compounds which are hedgehog regulators.
Such assays can
be conducted by analogy described below by Beachy et al US 7,291,626, (2007).
[0380] In addition to cell-free assays, test compounds can also be tested in
cell-based assays.
In one embodiment, cells which have a ptc loss-of-function, hedgehog gain-of-
function, loss of
kinase activity, smoothened gain-of-function phenotype can be contacted with a
test agent of
interest, with the assay scoring for, e.g., inhibition of proliferation of the
cell in the presence of
the test agent.
[0381] A number of gene products have been implicated in receptor-mediated
signal
transductions, including patched, GLI, GL2, GL3 family of transcription the
serine/threonine
kinase fused (fu) and smoothned, and patched and the induction of cells by
hedgehog proteins
sets in motion a cascade involving the activation and inhibition of downstream
effectors, the
ultimate consequence of which is, in some instances, a detectable change in
the transcription or
translation of a gene. Potential transcriptional targets of hedgehog-mediated
signaling are
described.
[0382] Reporter gene based assays described in the invention by Beachy et al,
2007 can be
utilized by analogy for the subject compositions in the same way. These gene
based assays
appear to measure the end stage of the above described cascade of events,
e.g., transcriptional
modulation.
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103831 Accordingly, in practicing one embodiment of the assay, a reporter gene
construct is
inserted into the reagent cell in order to generate a detection signal
dependent on ptc loss-of-
function, hedgehog gain-of-function, smoothened gain-of-function, or
stimulation by Shh itself
or inhibitions of protein kinases. The amount of transcription from the
reporter gene may be
measured using any method known to those of skill in the art to be suitable.
For example,
mRNA expression from the reporter gene may be detected using RNAse protection
or RNA-
based PCR, or the protein product of the reporter gene may be identified by a
characteristic stain
or an intrinsic biological activity. The amount of expression from the
reporter gene is then
compared to the amount of expression in either the same cell in the absence of
the test compound
or it may be compared with the amount of transcription in a substantially
identical cell that lacks
the target receptor protein. Any statistically or otherwise significant
decrease in the amount of
transcription indicates that the test compound has in some manner agonized the
normal ptc signal
(or modulated, antagonized the gain-of-function hedgehog or smoothened
signal), e.g., the test
compound is a potential hedgehog antagonist.
103841 Exemplification
[03851 The invention now being generally described, it will be more readily
understood by
reference to the following examples which are included merely for purposes of
illustration of
certain aspects and embodiments of the present invention, and are not intended
to limit the
invention.
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[03861 Examples
[03871 The following non-limiting examples are presented only to exemplify
various
embodiments of the invention and do not limit it in any fashion.
[03881 Example 1
[03891 In Jenkins, D. at al Anat 2007Sep 11, it states that studies of mouse
mutants have
demonstrated that Sonic hedgehog (SHH) signalling has a functional role in
morphogenesis and
differentiation at multiple sites within the forming urinary tract, and
urinary tract malformations
have been reported in humans with mutations that disrupt SHH signalling.
However, there is
only strikingly sparse and fragmentary information about the expression of SHH
and associated
signalling genes in normal human urinary tract development. (Jenkins,2007)
used
immunohistochemistry to demonstrate that SHH protein was localised in distinct
urinary tract
epithelia in developing normal humans, in the urothelium of the nascent
bladder and in kidney
medullary collecting ducts. The expression patterns of the SHH-transducing
proteins Patched
(PTCH) and Smoothened (SMO) were consistent with long-range paracrine
signalling associated
with detrusor smooth muscle differentiation in the urogenital sinus. In the
developing kidney,
SHH and PTCH were expressed in epithelia of the collecting system between 16-
26 weeks -
surprisingly, SMO was not detected. Analysis of cell proliferation and Cyclin
BI
immunohistochemistry at 26 weeks, as compared with a 28 week sample in which
SHH
expression was down-regulated, was consistent with the idea that SHH and PTCH
might
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influence medullary collecting duct growth by regulating the subcellular
localisation of Cyclin
B 1 independently of SMO.
[0390] Collectively, this descriptive experimental result is similar to that
of Beachy et al, 2007.
These results, along with the inventors own literature research, certainly
generates new
hypotheses and evidence regarding SHH signal transduction in human urinary
tract development
as well as other anomalies described in the patent that help to explain the
varied urinary tract
malformations associated with anorectal malformation phenotypes noted in
individuals with
mutations in the SHH pathway
[0391] Example 2
[0392] Females determined to be at risk of fetal malformations and who are
seeking a further
pregnancy are split into no treatment, folate treatment, D-chiroinositol
treatment, and Folate + D-
chiroinositol treatment arms. The respective regimens are administered once
daily from before
conception through the end of the first trimester. Relative to the untreated
controls, the
frequency of fetal malformations is reduced in each of the non-control arms.
However, the
reduction in frequency of fetal malformations in the co-therapy of the present
invention is
significantly better than in either of the other treatment arms.
[0393] Example 3
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[0394] Females beginning birth control medication are assigned to similar
treatment and
control groups as in Example 1. Treatment is begun at the time of initiation
of birth control
medication, and continued until after a pregnancy occurs and for the following
first trimester of
pregnancy. Similar reductions as reported in Example 2 are seen. In addition,
follow up of these
females shows a lower level of breast cancer development than expected.
[0395] Example 4
[0396] Men preparing to initiate androgen ablative therapy are initiated on a
course of D-
chiroinositol prior to and throughout the treatment with the androgen ablative
therapeutic. The
frequency of male breast cancer found in these patients is substantially
reduced as compared to
controls not receiving the D-chiroinositol therapy.
[0397] Example 5
[0398] In order to demonstrate an effect of D-chiroinositol plus folate on Shh
signaling during
embryogenesis, curly tail mice (38) are chosen because of the genetic
propensity for spinal
defects and associated genitourinary(GU) defects. These embryos are exposed to
the present
invention compound D-chiroinositol vs. myoinositol and internal examination is
conducted. The
severityof these defects among treated embryos treated with myoinositol are
severe compared to
those embryos treated with D-chiroinositol .
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[03991 Example 6
[04001 To determine whether the downstream targets of the phosphatidyl
inositol(PI3K)
signaling pathway could be effected by an inhibitor, we can use a vehicle to
inhibit the effect of
inositol on neural tube closure. The curly tail mice embryos are cultured from
E9.5 to El0.5,
and the length of unclosed neural folds at the pnp are measured to indicate
predisposition to
spina bifida.
[04011 Embryos which are exposed to the kinase inhibitor alone have enlarged
PNP's,
reflecting the in vivo development of spinal NTD's by 50%-60% of curly tail
embryos. Hence,
inhibtion of the down stream targets of the phosptidylinositol signaling
pathway blocks the
protective effect of the inositol. Therefore, D-chiroinositol normalizes PNP
closure in vitro.
[04021 D-chiroinositol, a phosphate derivative, plus folic acid could
represent a possible novel
adjunct therapy to prevent NTD's because the activation of certain isoforms by
the
phostidylinositol pathway is essential for prevention of these defects.
104031 It is well-known that cancer remains a major health problem in the
United States and in
other developed countries. It is our effort here to reduce the burden of
cancer on a physical,
social, and psychological level. There is a constant search for more effective
cancer treatments.
While, it it well-known that many cancer specialists have won the battle for
detecting and
treating many cancers, however, there is still one problem that remains; the
concept of
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prevention. This is a promising approach to control cancer. Also, the
discovery of convergent
signaling pathways in these contexts are something that cannot be overlooked
as well because
these signaling pathways control many cellular processes including cellular
proliferation,
survival, growth, and motility, all of which are critical processes for
tumorigenesis. Without
being bound to theory, it is the inventor's belief that the alteration of
these converged pathways
occurs in many cancerous states, many embryonic dysmorphic states, as well as
many conditions
that are the result of abberant signaling, but otherwise unrelated to each
other.
[04041 Based on the foregoing and the results described below by Beachy et al,
2007, the
inventor suggests that the inclusion of the inositol compound, alone or in
combination with other
compounds as set forth more fully herein, is warranted as a treatment or
prevention for a wide
range of conditions related to aberrant growth (such as for cancers) birth
defects due to pattern
formation dysregulation during gestation, as well as other chronic diseases.
[0405] The goal is to identify combinations of the invention that target the
tumor at vulnerable
sites and interrupt specific pathways suspected in carcinogenesis. From the
behavior and
characteristics of malignant cells, several principal pathways of malignancy
have been
established. They include: Cell proliferation, cell cycle progression,
metastases and invasion,
angiogenesis, and apopotosis. Interestingly, we believe that at least one of
the D-
chiroinositolphosphates targets and acts on all of them. MDA-MB 231 human
breast cancer
cells are highly invasive tumor cells. These, and most other, tumors cells
emit substances known
as matrix metalloproteinases that allow metastastic cells to pass into blood
vessels.
myoinositolhexaphosphate significantly inhibits secretion of MMP-9 from MDA-MB
231 cells.
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104061 Introduction
[04071 Beachy, (2007) goes on to describe an essential role for Shh during
Hair Follicle
Morphogenesis. The hair follicle is a source of epithelial stem cells and site
of origin for several
types of skin tumors. While it is clear that follicles arise by way of a
series of inductive tissue
interactions, identification of the signaling molecules driving this process
remains a major
challenge in skin biology. Hair germs comprising epidermal placodes and
associated dermal
condensates were detected in both control and Shh -/- embryos, but progression
through
subsequent stages of follicle development was blocked in mutant skin. The
expression of Gli 1
and Ptc 1 was reduced in Shh -/- dermal condensates and they failed to evolve
into hair follicle
papillae, suggesting that the adjacent mesenchyme is a critical target for
placode-derived Shh.
Despite the profound inhibition of hair follicle morphogenesis, late-stage
follicle differentiation
markers were detected in Shh -/- skin grafts, as well as cultured vibrissa
explants treated with
cyclopamine to block Shh signaling. These findings are stated as revealing an
essential role for
Shh during hair follicle morphogenesis, where it is required for normal
advancement beyond the
hair germ stage of development.
104081 Early stages of organogenesis are marked by the appearance of
mesenchymal
condensates and focal cellular aggregates, or placodes, in adjacent epithelia.
This process is
driven to completion by a series of inductive signals traveling between
epithelial and
mesenchymal cell populations which ultimately give rise to the adult structure
(reviewed in
Gurdon, 1992; Thesleff et al., 1995). In skin appendages such as vibrissae and
hair follicles,
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detailed analysis of tissue recombinants has revealed the existence of at
least three
morphogenetic signals: the embryonic dermis instructs overlying ectoderm to
initiate placode
formation; the placode transmits a signal generating a dermal condensate with
hair follicle-
inductive properties; and the condensate in turn sends a signal to nascent
follicle keratinocytes
stimulating their proliferation, downgrowth into the developing dermis, and
reorganization to
form the mature follicle (reviewed in Sengel, 1976; Hardy, 1992). The
epithelial and
mesenchymal components of the follicle remain in close proximity in mature
hair bulbs, where
the dermal papilla is surrounded by matrix cells giving rise to at least six
phenotypically distinct
epithelial cell types in the hair shaft and inner root sheath of the hair
follicle. After birth the
follicle epithelium cycles through periods of active growth (anagen), followed
by regression
(catagen) and inactivity (telogen) (reviewed in Cotsarelis, 1997). The
morphogenetic program
that accompanies the transition from telogen to anagen bears similarities to
follicle development
during embryogenesis, making this structure a unique model for studying
certain aspects of
organogenesis in the adult animal. Although a, large number of genes have been
implicated at
various stages of hair follicle development and cycling (reviewed in
Rosenquist and Martin,
1996; Sterm et al, 1996; Widelitz et al, 1997; Millar, 1997), the molecular
nature of the inductive
signals that underlie the formation of the follicle is largely unknown.
[04091 In situ localization of transcripts encoding potential morphogens has
revealed focal
expression of Sunic hedgehog (Shh) in placodes of the epidermis and several
other epithelia at
early stages of development, with Ptc 1 transcripts encoding a putative Shh
receptor also present
in adjacent mesenchymal cells (Bitgood and McMahon, 1995; Iseki et al., 1996;
Oro et al., 1997;
Motoyama et al., 1998). These findings, coupled with the accumulating evidence
demonstrating
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CA 02668580 2009-06-10
a pivotal role for secreted Hedgehog proteins in a variety of developmental
processes (reviewed
in Hammerschmidt et aL, 1997). Since the follicle is a source of cutaneous
stem cells and a
likely, site of origin for certain epithelial skin cancers (Cotsarelis et al.,
1990; Lavker et al.,
1993; Rochat et al., 1994; Hansen and Tennant, 1994), understanding the
developmental biology
of this organ is likely to provide insights relevant to normal skin function
as well as wound-
healing and neoplasia, and may shed light on fundamental aspects of
organogenesis involving
other structures as well.
[04101 To summarize, they concluded an obligatory role for Shh in the
progression of hair
follicle morphogenesis past the hair germ stage of development. The reduced
expression of Ptcl
and Gli 1 in Shh -/- dermal condensates, coupled with their failure to evolve
into recognizable
dermal papillae, argue that Shh is involved in regulating development of the
mesenchymal
component of the hair follicle, although a requirement for Shh signaling in
the epithelial
component of the follicle cannot be excluded. In the absence of dermal
papillae normal hair
follicle morphogenesis does not proceed, underscoring the critical influence
these cells have on
growth and remodeling of developing follicle epithelium (Jahoda et al., 1984;
Weinberg et al.,
1993). Interestingly, biochemical differentiation of the follicle can take
place in the absence of
normal morphogenesis, implying that these two processes are regulated
independently in this
organ. According to Beachy et al, (2007), additional experiments will be
required to formally
define which component of the developing follicle is functionally impaired in
Shh -/- embryos,
and to determine whether Shh has additional roles at later stages of follicle
development or
during hair cycling. (Johnson et al., 1996; Hahn et al., 1996; Oro et al.,
1997; Fan et al., 1997;
Xie et al., 1998).
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The experiments detailed below by Beachy et al (2007) are believed to be
equally applicable to
the D-chiroinositol compounds (phosphates thereof and other derivatives
thereof) used in the
present invention.
[04111 Methods
[04121 Animals and Skin Transplantation
[0413) The generation and identification of Shh mutant mice was performed as
described
(Chiang et al., 1996). Embryonic skin was grafted onto the dorsal fascia of
nude mice beneath a
protective silicone chamber using a modification of a previously-described
technique (Dlugosz et
al., 1995). The chamber was removed 11-12 days after grafting and tissue
harvested for analysis
after an additional one to four weeks. Animals were handled in accordance with
NIH guidelines.
[04141 Immunohistochemistry
[04151 Tissue is fixed overnight in Carnoy's or Bouin's solution for detecting
keratins (KI,
K10, K5, K14, and K 17), loricrin, and filaggrin; fixation with neutral-
buffered formalin is used
for tissues immunostained with Lef-1, Ki67, and hair keratin (AE13)
antibodies. Samples are
embedded in paraffin and 8 m sections cut for immunostaining. Immunoreactivity
of antigens in
formalin-fixed sections is restored by immersing slides in a boiling 0.01 M
citrate buffer, pH 6,
for 10 minutes. The following primary antibodies are used at the indicated
dilutions for
immunostaining: rabbit anti-keratins K 1, K 10, K5 and K 14 (1:500) (Roop et
al., 1984), loricrin
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and filaggrin (1:500) (Roop et al., 1987); rabbit anti-K17 (1:1000) (McGowan
and Coulombe,
1998),; rabbit anti-Lef-1 (1:200) (Travis et al., 1991),; rabbit anti-Ki67,
NCL-Ki67p (Novocastra
Laboratories, Ltd., Newcastle upon Tyne, UK) (1:200); and mouse monoclonal AE
13
hybridoma supernatant, which recognize type I hair keratins (1:5) (Lynch et
al., 1986), as
described in Beachy et al (2007). Tissue sections are incubated with primary
antibodies diluted
in tris-buffered saline containing I% bovine serum albumin, typically for 1-2
hours at room
temperature. Subsequent immunostaining procedures are performed using
peroxidase Vectastain
ABC kits (Vector Laboratories, Inc., Burlingame, Calif.) and
3,3'diaminobenzidine (Sigma, St.
Louis, Mo.) as a substrate, according to the manufacturers' recommendations.
Sections are
counterstained with hernatoxylin and mounted using Permount (Fisher
Scientific, Pittsburgh,
Pa.).
104161 In Situ Hybridization
[04171 Non-radioactive RNA in situ hybridization is performed on 5 m sections
essentially as
described (Groves et al., 1995), using previously described sequences for Gli
1 (Walterhouse et
al., 1993), Ptcl (Goodrich et al., 1996), and BMP-4 (Jones et al., 1991).
104181 Vibrissa Follicle Explants
[04191 Vibrissa follicle explants are established using CD-I mouse embryos at
13.5 days of
gestation according to a previously described protocol (Hirai et al., 1989),
with minor
midifications. Vibrissa pads are transferred onto Nuclepore filters (13 mm, 8
m pores), and
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floated on, 2 ml of medium [DMEM (Life Technologies, Gaithersburg, Md.)+Ham's
F12
medium (Life Technologies) (1:1), with 1% FCS (Intergen, Purchase, N.Y.),
penicillin (50
units/ml) and streptomycin (50 gg/ml) (Life Technologies)] in 6-well plates.
Similar results are
obtained using a DMEM-based medium, without the addition of Ham's F 12.
Explants are fed
fresh medium every two days. Microdissection is performed with the aid of a
Nikon SMZ-2T
stereomicroscope and photomicrographs were taken using an Olympus OM-4 camera.
Cyclopamine is stored at -20 as a 10 mM stock in 95% EtOH.
[0420] RNA Isolation and RT-PCR
[04211 RNA is obtained by solubilizing individual explants in TriZol (Life
Sciences) and
isolating as recommended by the manufacturer. cDNA is synthesized using
SuperScript 11 Rnase
H reverse transcriptase with random primers (Life Technologies), and RT-PCR
performed using
the primers set forth in Beachy et al's, (2007) (Walterhouse et al., 1993).
The following PCR
conditions are used for MHKA1, Hacl-1, and actin: 95×3 min "hot start";
95×50 sec,
58×30 sec, and 72×60 sec for 25 (actin) or 35 cycles (MHKA I and
Hacl-1);
72×7 min. PCR conditions for profilaggrin primers were as previously
described
(Bickenbach et al., 1995). Reaction products are run through 1.5% agarose gels
and visualized
with ethidiurn bromide.
[0422] Early stages of hair follicle development appear similar in control and
Shh -/- embryos.
Hair germs, consisting of clusters of columnar basal keratinocytes protruding
into the developing
dermis with associated dermal condensates, are detected in the skin of both
mutant and control
191
CA 02668580 2009-06-10
embryos at 15.5 days of gestation. Despite the similar morphology of control
and Shh-deficient
hair germs, a dramatic difference in gene expression patterns is revealed by
in situ hybridization.
The level of Glil mRNA is markedly reduced in both the epithelial and
mesenchymal
components of Shh -/- primary hair germs. In addition, expression of Ptc 1 is
reduced in Shh
mutant hair germs, although some placodes contain levels slightly above
background. These
findings are consistent with previous reports identifying Shh as a positive
regulator of both Glil
and Ptcl (Mango and Tabin, 1996; Mango et al., 1996; Lee et al., 1997; Sasaki
et al., 1997), and
suggest that Shh is signaling in both the epithelial and mesenchymal cells of
the developing
follicle. In contrast to Gli1 and Ptc1, BMP-4 mRNA is clearly detectable in
condensates of
mutant and control embryos, arguing against a requirement for Shh in the
induction of BMP-4
expression. Thus, although Shh is not required for the initiation of hair
follicle development,
primary hair germs that arise in Shh mutant skin are deficient in the
expression of at least some
Shh target genes.
[04231 In control embryos, the interval between E15.5 and E17.5 is marked by
rapid
proliferation and downgrowth of the follicle into the developing dermis,
accompanied by a
several-fold increase in the mass of the follicle epithelium and
reorganization into distinct
cellular compartments. In the most mature follicles, keratinocytes in the most
peripheral cell
layer, which give rise to the outer root sheath in the mature follicle, assum
a columnar
arrangement perpendicular to the long axis of the developing follicle; cells
located centrally are
without a definite orientation at this stage but will eventually be replaced
by the three concentric
layers of inner root sheath cells and the three cell types comprising the hair
shaft; and the
epithelial cells of the deepest portion of the follicle, the future hair bulb,
have surrounded what.
192
CA 02668580 2009-06-10
is at this stage a well-defined cluster of mesenchymal cells, the dermal
papilla. Even the less
mature follicles exhibit an organized "cap" of mesenchymal cells at their
invaginating tips. In
striking contrast, hair follicles in skin from mutant embryos at E 17.5 fail
to develop past the hair
germ stage seen at E 15.5 . Although the follicle epithelium is most obviously
affected due to its
lack of growth, organizing dermal condensates and dermal papillae are
conspicuously absent in
mutant skin. These results are consistent with the idea that epidermis-derived
Shh (Bitgood and
McMahon, 1995; Iseki et al., 1996; Oro et al., 1997; Motoyama et al., 1998)
functions as a
paracrine signal regulating development of the mesenchymal component of the
hair follicle.
Inhibition of follicle formation is not likely to be due to a general
disruption of skin development
since epidermal morphogenesis, marked by the appearance of granular and
cornified cell layers,
took place by E 17.5 in both control and mutant embryos.
[04241 Additional studies are performed to determine whether Shh influenced
the expression of
epithelial differentiation markers in embryonic skin. Keratinocytes in
developing hair follicles
can be distinguished by a relative deficiency of K5 and K14, keratins that are
abundant in
surrounding epidermal basal cells (Kopan and Fuchs, 1989; Byrne et al., 1994).
Immunohistochemical staining of E 17.5 embryos reveals greatly reduced or
undetectable levels
of K14 in a sub-population of cells comprising the normal follicles in control
embryos as well as
the primordial follicles seen in Shh -/- embryos. Moreover, KI 7, which is
normally not detected
in interfollicular epidermis but is expressed in developing and mature hair
follicles (Panteleyev
et al., 1997; McGowan and Coulombe, 1998), is localized to the follicular
epithelium in both
control and mutant skin. Thus, although morphogenesis of hair follicles in Shh
-/- skin fails to
progress past the hair germ stage, these structures contain epithelial cells
that have initiated a
193
CA 02668580 2009-06-10
terminal differentiation program characteristic of developing follicle
keratinocytes. Consistent
with these morphological findings, the expression level of epidermal-specific
differentiation
markers (keratins 1 and 10, loricrin, and filaggrin) in Shh -/- skin is
similar to or greater than in
control epidermis, based on immunchistochemical staining.
[04251 Since Shh -/- mice are not viable, post-natal analysis of mutant skin
is performed
following grafting onto nude mice. Whereas skin from control mice produced
abundant
pigmented hairs, transplanted Shh -/skin failed to generate detectable hairs
but exhibited a
pigmented graft site, consistent with the strain of donor skin. The histology
of control skin grafts
reveals the typical structures seen in normal mouse skin, including numerous
hair follicles and
sebaceous glands. In striking contrast, mutant skin failed to produce normal-
appearing follicles,
hair shafts, or sebaceous glands, but in some cases exhibit a thickened
epidermis with focal areas
of hyperkeratosis. Conspicuous aggregates of basophilic cells with scant
cytoplasm are detected
at the dermal-epidermal junction in these mutant grafts. Interestingly, the
morphology of cells in
the Shh-deficient keratinocyte aggregates is reminiscent of cells in control
hair bulbs, and
additional analyses revealed biochemical similarities. Cells in these
aggregates are unreactive
with K5 antibodies, exhibit abundant nuclear Lef- 1 expression (Zhou et al.,
1995), and contain a
high proportion of proliferating cells detected by Ki67 immunostaining.
Interestingly, short
columnar structures resembling abortive hair shafts are associated with some
of the Shh mutant
keratinocyte aggregates. Moreover, these structures express hair-specific
keratin, indicating that
an advanced stage in the follicle differentiation program is achieved despite
a dramatic
disruption of normal morphogenesis. Rarely, a small cluster of mesenchymal
cells is seen
associated with the base of a keratinocyte aggregate, where these cells
immunostain with Lef- 1
194
CA 02668580 2009-06-10
antibody. These findings suggest that a rudimentary dermal papilla is present
in at least some of
the hair germs seen in Shh mutant grafts.
[04261 To better define the temporal requirement for Shh during follicle
development, tissue
culture studies are performed using cyclopamine (GaTield and Keeler, 1996),
which has recently
been shown to block Shh signaling in neural plate explants (Cooper et al.,
1998; Incardona et al.,
1998). Explants are established using vibrissa pads from mice at 13.5 days of
gestation (Hirai et
al., 1989). When grown for six to eight days in culture, explants undergo
robust morphogenesis
resulting in the formation of elongated, grossly normal-appearing vibrissa
follicles . These
follicles contain hair shafts and express genes encoding mouse hair keratin A
I (MHKA 1)
(Kaytes et al., 1991) and a hair cortex-specific marker Hacl-1 (Huh et al.,
1994), detected by RT-
PCR (FIG. 11B). Treatment of explants with cyclopamine results in striking
inhibition of
morphogenesis, indicating that Shh signaling is required during or shortly
after the hair germ
stage of vibrissa. follicle development. In keeping with results using Shh
mutant skin, hair-
specific transcripts are detected in cyclopamine-treated grafts despite their
altered development,
providing further support for the notion that biochemical differentiation of
the follicle is not
necessarily coupled to its morphogenesis. Both control and cyclopamine-treated
explants
accumulate profilaggrin mRNA, indicating that disruption of Shh signaling does
not inhibit
epidermal differentiation.
Fetal Alcohol Syndrome Examples
104271 Example 7
195
CA 02668580 2009-06-10
104281 Female rats are administered alcohol for 2 weeks and then mated. The
alcohol
administration continues through out pregnancy and the rate of fetal alcohol
syndrome related
defects in the offspring are noted. A second set of rats from the same strain
are mated without
having the alcohol treatment as a control and the rate of defects of the same
type as noted in the
first arm are noted. The control arm does not display any significant number
of the defects that
are noticed in the alcohol treatment arm. A third arm of the study includes
rats that are
administered D-chiroinoitol or a phosphorylate thereof before and during the
alcohol treatment
and are otherwise treated in the same manner as in the alcohol treatment
above. The rate of fetal
alcohol syndrome defects noted in the inositol treatment group is
substantially less than the rate
of defects seen in the inositol free alcohol treated group.
[04291 Example 8
[04301 Example 7 is repeated except that a cholesterol reducing medication is
used instead of
the alcohol treatment. Similar results to that in Example 1 are obtained.
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[0481] All of the references cited above and throught the specification are
hereby incorporated
by reference herein.
[0482] Those skilled in the art will recognize, or be able to ascertain using
no more than
routine experimentation, many equivalents to the specific embodiments of the
invention
described herein. Such equivalents are intended to be encompassed by the
following claims.
[0483] In general inositol (underivitzed) can be recovered in the myo form
from plant sources.
Chemical synthesis of the underivitized inositol enantiomers is known in the
art and many are
available, although many in small quantities not readily convenient for
commercial batch
production. Pinitol is another known compound which is a monomethyl ester of
an inositol
isomer. Conversion of these known compounds to the deoxo versions can
generally be
accomplished by virtue of the Barton Mccombie deoxygenation reaction,
exemplified with
respect to nucleosides in US 6,822,089, incorporated herein by reference.
Esterification of one
or more of the hydroxy groups of the inositol structure with phosphoric acid,
pyrophosphoric
acid, carboxylic acids, carbonic acids, sulfonic acid, sulfouous acid and
further esters of the other
205
CA 02668580 2009-06-10
hdyroxy groups of these acidic compounds can be achieved by techniques
available to the
synthetic chemist. Techniques for the introduction of other substituents such
as halogens, oxo
groups, and substituents having a carbon atom bound to the inositol ring will
also be known to
those of ordinary skill in the synthetic chemistry art. Phosphorylation may
also be obtained in a
variety of substitution patterns by complete phosphorylation of the inositol
and then exposure to
specific enzymes or bacterial for specific dephosphorylation of particular
positions, leaving
different phsophoryaltion patterns. Once prepared, the various phosphorylates
can be separated
from each other by commonly known analytical and separation techniques such as
HPLC etc.
[04841 Compositions of the invention include those of an inositol, an inositol
derivative, an
inositol metabolite, polysaccharides containing inositol, and inositol
containing phospholipid and
an additional active agent which is not an inositol. Where the additional
active agent is a folate,
it is administered in an amount equivalent to an amount of folic acid selected
from about 200 g,
about 250 g, about 300 g, about 350 g, about 400 g, about 450 g, about
500 g, about 600
g, about 650 g, about 700 g, about 750 g, about 800 g, about 850 g, about
900 g, about
950 g, about 1 mg, about 1.05 mg, about 1.1 mg, about 1.15 mg, about 1.2 mg,
about 1.25 mg,
about 1.3 mg, about 1.35 mg, about 1.4 mg, about 1.45 mg, about 1.5 mg, about
1.55 mg, and
about 1.6mg per day. Birth defects within the invention uses include those
where the defect is
VATERNACTERL association (vertebral [defects], [imperforate] anus,
tracheoesophageal
[fistula], radial and renal [dysplasia]), rachischisis, (aka spinal
dysraphism) such as spina bifida
(including, but not limited to spina bifida aperta (aka spinabifida cystica);
spinabifida occulta;
and occult spinal disorder, among others) and (b) craniorachischisis (aka
cranial dysraphism)
such as cranium bifida (aka encephalocele or craniocele) each of spina bifida
and cranium bifida
206
CA 02668580 2009-06-10
being of any of the following types meningocele, myelomeningocele,
lipomeningocele, and
lipomyelomeningocele among others; (c) anencephaly; and (d) chiari
malformation; (2) caudal
regression syndrome, caudal dysplasia sequence, congenitalsacral agenesis;
sironmelia(mermaid
syndrome), sacral regression and the like; (3) cranio-facial defects such as,
without limitation,
facial cleft (aka prosopoanoschisis, including without limitation cleft
palate, cleft lip,
velopharyngeal malformation (including without limitation bifid uvula), etc.);
(4) anorectal
malformations including, but not limited to (a) imperforate anus, (b)
rectoperineal fistula, (c)
recto-bladder neck fistula; (d) persistent urogenital sinus, (e) persistent
cloaca, etc.; (5) bucket-
handle malformation; among others, acrocallosal syndrome, Basal cell nevus
syndrome, bardet-
Biedl syndrome, Biemond syndrome, Ectrodactyly-ectoderma dysplasia, cleft
lip/palate, Ellis
Van Creveld syndrome, meckel Gruger syndrome McKusick-kaufman syndrome, Mirror
hand
deformity(ulnar dimelia) Mohr syndrome, oral-facial-digital syndrome,
Pallister Hall syndrome,
Greig cephalopolysyndactyly,Post axial polydactyly, GreigRubinstein-Taybi
syndrome,
Cardiofaciocutaneous syndrome, noonan syndrome,short rib polydactyly, extra
deformed fingers
and toes, Lowe syndrome including ocular and renal defects, mental
retardation. This inositol
type compound, whether inositol or an inositol derivative, metabolite,
polysaccharides
containing inositol, and inositol containing phospholipid is administered in
an amount that is
equivalent to an amount of D-chiroinositol selected from the group consisting
of about 0.1 mg,
about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about
7 mg, about 8
mg, about 9 mg, about 10 mg, about 15 mg, about 20 mg, about 25 mg, about 50
mg, about 75
mg, about 100 mg, about 125 mg, about 150 mg, about 200 mg, about 250 mg,
about 300 mg,
about 350 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about
750 mg, about
800 mg, about 900 mg, about 950 mg, about 1 g per day, about 1.2 g per day,
about 1.8 g per
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CA 02668580 2009-06-10
day, about 2 g per day, about 2.5 g per day, about 3 g per day, about 5 g per
day, about 10 g per
day, about 12 g per day, about 18 g per day, about 24 g per day, about 30 g
per day, about 45 g
per day, and about 60 g per day. Certain methods include administration to
patients where the
patient is is a female receiving at least one treatment selected from birth
control, hormonal
replacement therapy, or antiandrogenic therapy; or said patient is a male
receiving at least one
treatment selected from estrogenic treatment and hormonal ablative therapy; or
said patient is a
male to female trans-sexual receiving at least one therapy selected from
estrogenic treatment and
antiandrogenic treatment, more specifically in some patients, the estrogenic
insult is from a
medicinal source and said medicinal source is selected from the group
consisting of estrogenic,
progestogenic, or antiandrogenic therapy. In some embodiments, the treatment
is of a cancer
selected from basal cell carcinoma (shh gain of function), Multiple basal cell
nevi, squamous cell
carcinoma(ptc activity) medulloblastoma, primitive neuroectodermal
tumor(PNET), Gorlin
syndrome, nevoid basal cell carcinoma syndrome, harmartomas,blue rubber-bleb
nevus
syndrome, Turcot syndrome, glioma polyposis syndrome, Rubinstein-Taybi
syndrome, Cowden
tumor syndrome,rabdomyosarcoma(RMS), alveolar rhabdomyosarcoma, botryoid
rhabdomyosarcoma, embryonal rhabdomyosarcoma, spindle cell rhabdomyosarcoma,
pleomorphic rhabdomyosarcoma, soft tissue sarcoma, rhabdomyoblasts, pediatric
sarcoma, cell
carcinoma, carcinosarcoma, adenocystic carcinoma, epidermoid carcinoma,
nasopharyngeal
carcinoma, bladder carcinoma,renal cell carcinoma, papilloma, or an
epidermoidoma. In other
embodiments, the birth defect is VATER/VACTERL association (vertebral
[defects],
[imperforate] anus, tracheoesophageal [fistula], radial and renal
[dysplasia])rachischisis (aka
spinal dysraphism) such as spina bifida (including, but not limited to spina
bifida aperta (aka
spinabifida cystica); spinabifida occulta; and occult spinal disorder, among
others) and (b)
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CA 02668580 2009-06-10
craniorachischisis (aka cranial dysraphism) such as cranium bifida (aka
encephalocele or
craniocele) each of spina bifida and cranium bifida being of any of the
following types
meningocele, myelomeningocele, lipomeningocele, and lipomyelomeningocele among
others;
(c) anencephaly; and (d) chiari malformation; (2) caudal regression syndrome,
caudal dysplasia
sequence, congenitalsacral agenesis; sironmelia(mermaid syndrome), sacral
regression and the
like; (3) cranio-facial defects such as, without limitation, facial cleft (aka
prosopoanoschisis,
including without limitation cleft palate, cleft lip, velopharyngeal
malformation (including
without limitation bifid uvula), etc.); (4) anorectal malformations including,
but not limited to (a)
imperforate anus, (b) rectoperineal fistula, (c) recto-bladder neck fistula;
(d) persistent urogenital
sinus, (e) persistent cloaca, etc.; (5) bucket-handle malformation; among
others. Biemond
syndrome, Ectrodactyly-ectoderma dysplasia, cleft lip/palate, Ellis Van
Creveld syndrome,
Muir-Torre syndrome, Cowden syndrome, Carney complex, Birt-Hogg-Dube syndrome,
Gorlin
syndrome (ptc loss-of-function), Gorlin-Goltz syndrome, basal cell nevus
syndrome, bifid-rib
basal-cell nevus syndrome, multiple basal cell nevi, Meckel Gruger syndrome,
McKusick-
Kaufmansyndrome, Mirror hand deformity(ulnar dimelia) Mohr syndrome, Oral-
facial-digital
syndrome, Pallister Hall syndrome, cephalopolysyndactyly,Post axial
polydactyly,
GreigRubinstein-Taybi syndrome, retinoblastoma, Cardiofaciocutaneous syndrome,
Noonan
syndrome,short rib polydactyly, extra deformed fingers and toes, Lowe syndrome
(including
ocular and renal defects), Renal Colombo syndrome, Retinitis pigmentosa. In
some
embodiments cotherapy of an oinositol or derivative thereof or metabiolite
thereof or of a
polysacharride having inositol components or an inositol phospholipid is
administered as a
cotherwith other active agents to a patient selected from the group selected
from (a) those in need
of preventing of inhibiting of proliferation, growth, and/or metastases of
tissues or conditions
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CA 02668580 2009-06-10
selected from the group consisting of breast tissue, prostate tissue, cervical
cancer caused by
human papiloma virus subtypes(HPV), Kaposis sarcoma, lung cancer,
adenocarcinoma, gut
derived tumors, colon cancers due to adenocarcinomas, and human
erythroleukemia and (b)
those in need of tissue regulation selected from the group consisting of
regulation of neural
tissues, regulation of bone and cartilage formation and repair, regulation of
ovulation, regulation
of spermatogenesis, regulation of smooth muscle, regulation of lung, liver,
intestines, colon,
rectum and other organs arising from the primitive gut as well as the distal
hindgut, regulation of
hematopoietic function, hemopoietic stem cells, and regulation of skin and
hair growth, and
modulation of cell proliferation which includes, without limitation,
inhibition of angiogenesis.
The modulation of cell proliferation also includes a basal cell carcinoma,
medulloblastoma,
primitive neuroectodermal tumor, PNET, Gorlin syndrome, nevoid basal cell
carcinoma
syndrome, harmartomas, blue rubber-bleb nevus syndrome, Turcot syndrome,
glioma polyposis
syndrome, Rubinstein-Taybi syndrome, Cowden tumor syndrome, rabdomyosarcoma,
RMS,
alveolar rhabdomyosarcoma, botryoid rhabdomyosarcoma, embryonal
rhabdomyosarcoma,
spindle cell rhabdomyosarcoma, pleomorphic rhabdomyosarcoma, soft tissue
sarcoma,
rhabdomyoblasts, pediatric sarcoma, sarcoma squamous cell carcinoma,
carcinosarcoma,
adenocystic carcinoma, epidermoid carcinoma, nasopharyngeal carcinoma, bladder
carcinoma,
renal cell carcinoma, papilloma, karposi's sarcoma, and an epidermoidoma. The
inihibiton of
cell proliferation also includes preventing or inhibiting the proliferation,
growth, and/or
metastases of one or more cancers selected from the group consisting of breast
cancer, prostate
cancer, especially prostatic androgen dependent PCA-LNA-p cells cervical
cancer, caused by
human papiloma virus subtypes(HPV), Kaposis sarcoma, lung cancer, (in
particular, small cell
lung cancer, adenocarcinoma; gut derived tumors (including but not limited to
cancer of the
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CA 02668580 2009-06-10
esophagus, stomach, pancreas, biliary duct system, intestinal(gastric) system,
colon cancers due
to adenocarcinomas, and human erythroleukemia.
104851 The instant compounds may also be co-administered with other well known
therapeutic
agents that are selected for their particular usefulness against the condition
that is being treated.
[04861 For example, instant compounds are useful in combination with known
anti-cancer
agents. Combinations of the presently disclosed compounds with other anti-
cancer or
chemotherapeutic agents are within the scope of the invention. Examples of
such agents can be
found in Cancer Principles and Practice of Oncology by V. T. Devita and S.
Hellman (editors),
8th edition (April, 2008), Lippincott Williams & Wilkins Publishers. A person
of ordinary skill
in the art would be able to discern which combinations of agents would be
useful based on the
particular characteristics of the drugs and the cancer involved. Such anti-
cancer agents include
the following: estrogen receptor modulators, androgen receptor modulators,
retinoid receptor
modulators, cytotoxic/cytostatic agents, antiproliferative agents, prenyl-
protein transferase
inhibitors, HMG-CoA reductase inhibitors and other angiogenesis inhibitors and
agents that
interfere with cell cycle checkpoints. The instant compounds are particularly
useful when co-
administered if needed with radiation therapy.
[0487] "Estrogen receptor modulators" refers to compounds that interfere with
or inhibit the
binding of estrogen to the receptor, regardless of mechanism. Examples of
estrogen receptor
modulators include, but are not limited to, tamoxifen, raloxifene, idoxifene,
LY353381,
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CA 02668580 2009-06-10
LY 117081, toremifene, fulvestrant, 4-[7-(2,2-dimethyl-l-oxopropoxy-4-methyl-2-
[4-[2-(1-
piperidinyl)ethoxy]ph-enyl]-2H-1-benzopyran-3-yl]-phenyl-2,2-
dimethylpropanoate,4,4'-
dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone,and SH646.
104881 "Androgen receptor modulators" refers to compounds which interfere or
inhibit the
binding of androgens to the receptor, regardless of mechanism. Examples of
androgen receptor
modulators include finasterideandother5.alpha.-reductaseinhibitors,
nilutamide, flutamide,
bicalutamide, liarozole, and abiraoacetate.
[0489] "Retinoid receptor modulators" refers to compounds which interfere or
inhibit the
binding of retinoids to the receptor, regardless of mechanism. Examples of
such retinoid receptor
modulators include bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoic
acid,alpha-
difluromethylomithine,ILX23-7553,trans-N-(4'-hydroxyphenyl)retinamide, N-4-
carboxyphenyl,retinamide.
[04901 "Cytotoxic/cytostatic agents" refer to compounds which cause cell death
or inhibit cell
proliferation primarily by interfering directly with the cell's functioning or
inhibit or interfere
with cell myosis, including alkylating agents, tumor necrosis factors,
intercalators, hypoxia
activatable compounds, microtubule inhibitors/microtubule-stabilizing agents,
inhibitors of
mitotic kinesins, anti-metabolites; biological response modifiers;
hormonal/anti-hormonal
therapeutic agents, haematopoietic growth factors, monoclonal antibody
targeted therapeutic
agents and topoisomerase inhibitors.
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CA 02668580 2009-06-10
[04911 Examples of cytotoxic agents include, but are not limited to, sertenef,
cachectin,
ifosfamide, tasonermin, lonidamine, carboplatin, altretamine, prednimustine,
dibromodulcitol,
ranimustine, fotemustine, nedaplatin, oxaliplatin, temozolomide, heptaplatin,
estramustine,
improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride,
pumitepa, lobaplatin,
satraplatin, profiromycin, cisplatin, irofulven, dexifosfamide,cis-
aminedichloro(2-methyl-
pyridine)platinum,benzylguanine, glufosfamide, GPX100, (trans,trans,trans)-bis-
mu-(hexane-
1,6-diamine)-mu-[diamine-platinum(II)]-
bis[diamine(chloro)platinum(II)]tetrachloride,
diarizidinylspermine, arsenic trioxide, 1-(11-dodecylamino-l0-hydroxyundecyl)-
3,7-
dimethylxanthine, zorubicin, idarubicin, daunorubicin, bisantrene,
mitoxantrone, pirarubicin,
pinafide, valrubicin, amrubicin, antineoplaston, 3'-deamino-3'-morpholino-13-
deoxo-10-
hydroxycarminomycin, annamycin, galarubicin, elinafide, MEN10755, and 4-
demethoxy-3-
deamino-3-aziridinyl-4-methylsulphonyl-daunorubicin (see WO 00/50032). An
example of of a
hypoxia activatable compound for cancer is tirapazmine.
[04921 Examples of microtubule inhibitors/microtubule-stabilizing agents
include paclitaxel,
vindesine sulfate, 3',4'-didehydro-4'-deoxy-8'-norvincaleukoblastine,
docetaxol, rhizoxin,
dolastatin, mivobulin isethionate, auristatin, cemadotin, RPR109881, BMS
184476, vinflunine,
cryptophycin, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene
sulfonamide,
anhydrovinblastine, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-
proline-t-
butyla- mide, TDX258, the epothilones (see for example U.S. Pat. Numbers
6,284,781 and
6,288,237, and BMS 188797
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CA 02668580 2009-06-10
[0493] Some examples of topoisomerase inhibitors are topotecan, hycaptamine,
iminotecan,
rubitecan, 6-ethoxypropionyl-3',4'-O-exo-benzylidene-chartreusin,9-methoxy-N,N-
dimethyl-5-
nitropyrazolo[3,4,5-kl] acridine-2-(6H)propanamine, l-amino-9-ethyl-5-fluoro-
2,3-dihydro-9-
hydroxy-4-methyl-1 H,12H-benzo [de]pyrano[3',4':b,7]-indolizino[
1,2b]quinoline-
10,13(9H,15H)dione,lurtotecan,7-[2-(N-isopropylaminoamino)ethyl]-
(20S)camptothecin,BNP 13 50,BNPI 1100,BN80915,
BN80942,etoposidephosphate,teniposide,sobuzoxane,2'-dimethylamino-2'-deoxy-
etoposide,
GL331, N-[2-(dimethylamino)ethyl]-9-hydroxy-5,6-dimethyl-6H-pyrido[4,3-
b]carbazo-le-1-
carboxamide, asulacrine, (5 a, 5 aB, 8 aa, 9b)-9- [2- [N- [2-dimethyl amino)
ethyl] -N-
methylamino] ethyl] -5- [-4-hydroxy-3, 5 -dimethoxyphenyl] -5,5 a, 6, 8, 8 a,
9-
hexohydrofuro (3', 4':6, 7)naphtho(2, 3 -d)-1, 3 -dioxol-6-one,2, 3 -(methyl
enedioxy)-5 -methyl-7-
hydroxy-8-methoxybenzo [c]-phenanthridiniu-m,6,9-bis[(2-
aminoethyl) amino]benzo [g] i soguinoline-5,10-dione, 5 -(3 -aminopropylamino)-
7,10-dihydroxy-2-
(2-hydroxyethylaminomethyl)-6H-pyrazolo[4,5,1-de]acridin-6-one, N-[ 1-
[2(diethylamino)ethylamino]-7-methoxy-9-oxo-9H-thioxanthen-4-ylmethy-
1]formamide,N-(2-
(dimethylamino)ethyl)acridine-4-carboxamide,6-[ [2-(dimethylamino)ethyl]
amino] -3-hydroxy-
7H-indeno[2,1-c]quinolin-7-on-e,and dimesna.
[04941 Examples of inhibitors of mitotic kinesins, and in particular the human
mitotic kinesin
KSP, are described in PCT Publications WO 01/30768 and WO 01/98278, and
pending U.S. Ser.
Nos. 60/338,779 (filed Dec. 6, 2001), 60/338,344 (filed Dec. 6, 2001),
60/338,383 (filed Dec. 6,
2001), 60/338,380 (filed Dec. 6, 2001), 60/338,379 (filed Dec.
6,2001)and60/344,453 (filedNov.7,2001).
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[0495] "Antiproliferative agents" includes antisense RNA and DNA
oligonucleotides such as
G3139, ODN698, RVASKRAS, GEM231, and INX3001, and antimetabolites such as
enocitabine, carmofur, tegafur, pentostatin, doxifluridine, trimetrexate,
fludarabine, capecitabine,
galocitabine, cytarabine ocfosfate, fosteabine sodium hydrate, raltitrexed,
paltitrexid, emitefur,
tiazofurin, decitabine, nolatrexed, pemetrexed, nelzarabine, 2'-deoxy-2'-
methylidenecytidine, 2'-
fluoromethylene-2'-deoxycytidine, N-[5-(2,3-dihydro-benzofuryl)sulfonyl]-N'-
(3,4-
dichlorophenyl)urea,N6-[4-deoxy-4-[N2- [2(E),4(E)-tetradecadienoyl]
glycylamino]-L-glycero-B-
L--manno-heptopyranosyl]adenine, aplidine, ecteinascidin,troxacitabine,4-[2-
amino-4-oxo-
4,6,7,8-tetrahydro-3H-pyrimidino[5,4-b] [ 1,4]thiazin-6-yl- -(S)-ethyl]-2,5-
thienoyl-L-glutamic
acid, aminopterin, 5-flurouracil, alanosine, l l -acetyl-8-
(carbamoyloxymethyl)-4-formyl-6-
methoxy-l4-oxa-1,11-diazatetr- acyclo(7.4.1Ø0)-tetradeca-2,4,6-trien-9-yl
acetic acid ester,
swainsonine, lometrexol, dexrazoxane, methioninase, 2'-cyano-2'-deoxy-N-4-
palmitoyl- l -B-D-
arabino furanosylcytosine,3-aminopyridine-2-carboxaldehydethiosemicarbazone
and
trastuzamab.
[0496] Examples of monoclonal antibody targeted therapeutic agents include
those therapeutic
agents which have cytotoxic agents or radioisotopes attached to a cancer cell
specific or target
cell specific monoclonal antibody. One particular example is Bexxar.
[0497] "HMG-CoA reductase inhibitors" refers to inhibitors of 3-hydroxy-3-
methylglutaryl-
CoA reductase. Compounds which have inhibitory activity for HMG-CoA reductase
can be
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CA 02668580 2009-06-10
readily identified by using assays well-known in the art. For example, see the
assays described or
cited in U.S. Pat. No. 4,231,938 at col. 6, and WO 84/02131 at pp. 30-33. The
terms "HMG-CoA
reductase inhibitor" and "inhibitor of HMG-CoA reductase" have the same
meaning when used
herin.
[04981 Examples of HMG-CoA reductase inhibitors that may be used include but
are not
limited to lovastatin (MEVACOR®; see U.S. Pat. Nos. 4,231,938, 4,294,926
and
4,319,039), simvastatin (ZOCOR®; see U.S. Pat. Nos. 4,444,784, 4,820,850
and
4,916,239), pravastatin (PRAVACHOL®; see U.S. Pat. Nos. 4,346,227,
4,537,859,
4,410,629, 5,030,447 and 5,180,589), fluvastatin (LESCOL®; see U.S. Pat.
Nos. 5,354,772,
4,911,165, 4,929,437, 5,189,164, 5,118,853, 5,290,946 and 5,356,896),
atorvastatin
(LIPITOR®; see U.S. Pat. Nos. 5,273,995, 4,681,893, 5,489,691 and
5,342,952) and
cerivastatin (also known as rivastatin and BAYCHOL®; see U.S. Pat. No.
5,177,080). The
structural formulas of these and additional HMG-CoA reductase inhibitors that
may be used in
the instant methods are described at page 87 of M. Yalpani, "Cholesterol
Lowering Drugs",
Chemistry & Industry, pp. 85-89 (5 Feb. 1996) and U.S. Pat. Nos. 4,782,084 and
4,885,314. The
term HMG-COA reductase inhibitor as used herein includes all pharmaceutically
acceptable
lactone and open-acid forms (i.e., where the lactone ring is opened to form
the free acid) as well
as salt and ester forms of compounds which have HMG-CoA reductase inhibitory
activity, and
therefor the use of such salts, esters, open-acid and lactone forms is
included within the scope of
this invention.
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[04991 In HMG-CoA reductase inhibitors where an open-acid form can exist, salt
and ester
forms may preferably be formed from the open-acid, and all such forms are
included within the
meaning of the term "HMG-CoA reductase inhibitor" as used herein. Preferably,
the HMG-CoA
reductase inhibitor is selected from lovastatin and simvastatin, and most
preferably simvastatin.
Herein, the term "pharmaceutically acceptable salts" with respect to the HMG-
CoA reductase
inhibitor shall mean non-toxic salts of the compounds employed in this
invention which are
generally prepared by reacting the free acid with a suitable organic or
inorganic base, particularly
those formed from cations such as sodium, potassium, aluminum, calcium,
lithium, magnesium,
zinc and tetramethylammonium, as well as those salts formed from amines such
as ammonia,
ethylenediamine, N-methylglucamine, lysine, arginine, ornithine, choline, N,N'-
dibenzylethylenediamine,chloroprocaine,diethanolamine,procaine,N-
benzylphenethylamine, l -p-
chlorobenzyl-2-pyrrolidine-1'-yl-methylbenzimidazole, diethylamine,
piperazine, and
tris(hydroxymethyl)aminomethane. Further examples of salt forms of HMG-CoA
reductase
inhibitors may include, but are not limited to, acetate, benzenesulfonate,
benzoate, bicarbonate,
bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate,
chloride,
clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate,
fumarate, gluceptate,
gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine,
hydrobromide,
hydrochloride, hydroxynapthoate, iodide, isothionate, lactate, lactobionate,
laurate, malate,
maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate,
oleate, oxalate, pamaote,
palmitate, panthothenate, phosphate/diphosphate, polygalacturonate,
salicylate, stearate,
subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide,
and valerate.
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105001 Ester derivatives of the described HMG-CoA reductase inhibitor
compounds may act as
prodrugs which, when absorbed into the bloodstream of a warm-blooded animal,
may cleave in
such a manner as to release the drug form and permit the drug to afford
improved therapeutic
efficacy.
[0501] "Prenyl-protein transferase inhibitor" refers to a compound which
inhibits any one or
any combination of the prenyl-protein transferase enzymes, including farnesyl-
protein
transferase (FPTase), geranylgeranyl-protein transferase type I (GGPTase-I),
and
geranylgeranyl-protein transferase type-II (GGPTase-II, also, called Rab
GGPTase). Examples
of prenyl-protein transferase inhibiting compounds include (±)-6- [amino(4-
chlorophenyl)(1-
methyl-1 H-imidazol-5-yl)methyl]-4-(3-ch-lorophenyl)-1-methyl-2 (1 H)-
quinolinone,(-)-6-
[amino(4-chlorophenyl)(1-methyl-1 H-imidazol-5-yl)methyl]-4-(3-chlor- ophenyl)-
1-methyl-2
(1H)-quinolinone, (+)-6-[amino(4-chlorophenyl)(1-methyl-IH-imidazol-5-
yl)methyl]-4-(3-chlor-
ophenyl)-1-methyl-2 (1 H)-quinolinone, 5(S)-n-butyl- l -(2,3-dimethylphenyl)-4-
[ 1-(4-
cyanobenzyl)-5-imidazolylmeth-yl]-2-iperazinone, (S)-1-(3-chlorophenyl)4-[ 1-
(4-cyanobenzyl)-
5-imidazolylmethyl]-5-[2-ethanesulfonyl)methyl)-2-p-iperazinone,5(S)-n-Butyl-1-
(2-
methylphenyl)-4-[ 1-(4-cyanobenzyl)-5-imidazolylmethyl]-2-piperazinone, l -(3-
chlorophenyl)4-
[ 1-(4-cyanobenzyl)-2-methyl-5-midazolylmethyl]-2-piperazinone, 1-(2,2-
diphenylethyl)-3-[N-
(1-(4-cyanobenzyl)-1 H-imidazol-5-ylethyl)carba-moyl]piperidine,4- { 5-[4-
hydroxymethyl-4-(4-
chloropyridin-2-ylmethyl)-piperidine- l -ylmethy-l]-2-methylimidazol- l -
ylmethyl} benzonitrile,4-
{ 5-[4-hydroxymethyl-4-(3-chlorobenzyl)-piperidine-1-ylmethyl]-2-methyli-
midazol- l -
ylmethyl} benzonitrile, 4- {3-[4-(2-oxo-2H-pyridin- l -yl)benzyl]-3H-imidazol-
4-
ylmethyl} benzonitril-e,4- {3-[4-(5-chloro-2-oxo-2H-[ 1,2']bipyridin-5'-
ylmethyl]-3H-imidazol-4-
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CA 02668580 2009-06-10
y- lmethyl}benzonitrile, 4-{3-[4-(2-oxo-2H-[1,2']bipyridin-5'-ylmethyl]-3H-
imidazol-4-
ylmethyl}ben-zonitrile,4-[3-(2-oxo-l-phenyl-1,2-dihydropyridin-4-ylmethyl)-3H-
imidazol- -4-
ylmethyl} benzonitrile, 18,19-dihydro-19-oxo-5H,17H-6,10:12,16-dimetheno-1 H-
imidazo[4,3-
c] [1,11,4]- dioxaazacyclononadecine-9-carbonitrile, (±)-19,20-dihydro-l9-
oxo-5H-18,21-
ethano-12,14-etheno-6,10-metheno-22H--benzo[d]imidazo[4,3-k] [
1,6,9,12]oxatriaza-
cyclooctadecine-9-carbonitrile, 19,20-dihydro-19-oxo-5H,17H-18,21-ethano-
6,10:12,16-
dimetheno-22H-imidazo-[3,4-h][1,8,11,14]oxatriazacycloeicosine-9-
carbonitrile,and (+)-19,20-
dihydro-3-methyl- l 9-oxo-5H-18,21-ethano-12,14-etheno-6,10-methe-no-22H-
benzo[d-
]imidazo[4,3-k] [ 1,6,9,12] oxa-triazacyclooctadecine-9-carbonitrile.
105021 Other examples of prenyl-protein transferase inhibitors can be found in
the following
publications and patents: WO 96/30343, WO 97/18813, WO 97/21701, WO 97/23478,
WO
97/38665, WO 98/28980, WO 98/29119, WO 95/32987, U.S. Pat. No. 5,420,245, U.S.
Pat. No.
5,523,430, U.S. Pat. No. 5,532,359, U.S. Pat. No. 5,510,510, U.S. Pat. No.
5,589,485, U.S. Pat.
No. 5,602,098, European Patent Publ. 0 618 221, European Patent Publ. 0 675
112, European
Patent Publ. 0 604 181, European Patent Publ. 0 696 593, WO 94/19357, WO
95/08542, WO
95/11917, WO 95/12612, WO 95/12572, WO 95/10514, U.S. Pat. No. 5,661,152, WO
95110515, WO 95/10516, WO 95/24612, WO 95134535, WO 95/25086, WO 96/05529, WO
96/06138, WO 96/06193, WO 96/16443, WO 96/21701, WO 96/21456, WO 96/22278, WO
96/24611, WO 96/24612, WO 96/05168, WO 96/05169, WO 96/00736, U.S. Pat. No.
5,571,792,
WO 96/17861, WO 96/33159, WO 96/34850, WO 96/34851, WO 96/30017, WO 96/30018,
WO
96/30362, WO 96/30363, WO 96/31111, WO 96/31477, WO 96/31478, WO 96/31501, WO
97/00252, WO 97/03047, WO 97/03050, WO 97/04785, WO 97/02920, WO 97/17070, WO
219
CA 02668580 2009-06-10
97/23478, WO 97/26246, WO 97/30053, WO 97/44350, WO 98/02436, and U.S. Pat.
No.
5,532,359. For an example of the role of a prenyl-protein transferase
inhibitor on angiogenesis
see European J. of Cancer, Vol. 35, No. 9, pp. 1394-1401 (1999).
[05031 "Angiogenesis inhibitors" refers to compounds that inhibit the
formation of new blood
vessels, regardless of mechanism. Examples of angiogenesis inhibitors include,
but are not
limited to, tyrosine kinase inhibitors, such as inhibitors of the tyrosine
kinase receptors Flt-1
(VEGFRI) and Flk-1/KDR (VEGFR2), inhibitors of epidermal-derived, fibroblast-
derived, or
platelet derived growth factors, MMP (matrix metalloprotease) inhibitors,
integrin blockers,
interferon-.alpha., interleukin- 12, pentosan polysulfate, cyclooxygenase
inhibitors, including
nonsteroidal anti-inflammatories (NSAIDs) like aspirin and ibuprofen as well
as selective
cyclooxygenase-2 inhibitors like celecoxib and rofecoxib (PNAS, Vol. 89, p.
7384 (1992); JNCI,
Vol. 69, p. 475 (1982); Arch. Opthalmol., Vol. 108, p. 573 (1990); Anat. Rec.,
Vol. 238, p. 68
(1994); FEBS Letters, Vol. 372, p. 83 (1995); Clin, Orthop. Vol. 313, p. 76
(1995); J. Mol.
Endocrinol., Vol. 16, p. 107 (1996); Jpn. J. Pharmacol., Vol. 75, p. 105
(1997); Cancer Res., Vol.
57, p. 1625 (1997); Cell, Vol. 93, p. 705 (1998); Intl. J. Mol. Med., Vol. 2,
p. 715 (1998); J. Biol.
Chem., Vol. 274, p. 9116 (1999)), steroidal anti-inflammatories (such as
corticosteroids,
mineralocorticoids, dexamethasone, prednisone, prednisolone, methylpred,
betamethasone),
carboxyamidotriazole, combretastatin A-4, squalamine, 6-O-chloroacetyl-
carbonyl)-fumagillol,
thalidomide, angiostatin, troponin- 1, angiotensin II antagonists (see
Fernandez et al., J. Lab.
Clin. Med. 105:141-145 (1985)), and antibodies to VEGF (see, Nature
Biotechnology, Vol. 17,
pp. 963-968 (October 1999); Kim et al., Nature, 362, 841-844 (1993); WO
00/44777; and WO
00/61186).
220
CA 02668580 2009-06-10
[0504] Other therapeutic agents that modulate or inhibit angiogenesis and may
also be used in
combination with the compounds of the instant invention include agents that
modulate or inhibit
the coagulation and fibrinolysis systems (see review in Clin. Chem. La. Med.
38:679-692
(2000)). Examples of such agents that modulate or inhibit the coagulation and
fibrinolysis
pathways include, but are not limited to, heparin (see Thromb. Haemost. 80:10-
23 (1998)), low
molecular weight heparins and carboxypeptidase U inhibitors (also known as
inhibitors of active
thrombin activatable fibrinolysis inhibitor [TAFIa]) (see Thrombosis Res.
101:329-354 (2001)).
TAFIa inhibitors have been described in U.S. Ser. Nos. 60/310,927 (filed Aug.
8, 2001) and
60/349,925 (filed Jan. 18, 2002).
[05051 "Agents that interfere with cell cycle checkpoints" refer to compounds
that inhibit
protein kinases that transduce cell cycle checkpoint signals, thereby
sensitizing the cancer cell to
DNA damaging agents. Such agents include inhibitors of ATR, ATM, the Chkl and
Chk2
kinases and cdk and cdc kinase inhibitors and are specifically exemplified by
7-
hydroxystaurosporin,flavopiridol,CYC202(Cyclacel)andB MS-3 87032.
105061 As described above, the combinations with NSAID's are directed to the
use of NSAID's
which are potent COX-2 inhibiting agents. For purposes of this specification
an NSAID is potent
if it possesses an IC50 for the inhibition of COX-2 of 1 mu.M or less as
measured by cell or
micrsosomal assays.
221
CA 02668580 2009-06-10
105071 The invention also encompasses combinations with NSAID's which are
selective COX-
2 inhibitors. For purposes of this specification NSAID's which are selective
inhibitors of COX-2
are defined as those which possess a specificity for inhibiting COX-2 over COX-
1 of at least 100
fold as measured by the ratio of IC50 for COX-2 over IC50 for COX-1
evaluated by
cell or microsomal assays. Such compounds include, but are not limited to
those disclosed in
U.S. Pat. No. 5,474,995, issued Dec. 12, 1995, U.S. Pat. No. 5,861,419, issued
Jan. 19, 1999,
U.S. Pat. No. 6,001,843, issued Dec. 14, 1999, U.S. Pat. No. 6,020,343, issued
Feb. 1, 2000, U.S.
Pat. No. 5,409,944, issued Apr. 25, 1995, U.S. Pat. No. 5,436,265, issued Jul.
25, 1995, U.S. Pat.
No. 5,536,752, issued Jul. 16, 1996, U.S. Pat. No. 5,550,142, issued Aug. 27,
1996, U.S. Pat. No.
5,604,260, issued Feb. 18, 1997, U.S. Pat. No. 5,698,584, issued Dec. 16,
1997, U.S. Pat. No.
5,710,140, issued Jan. 20, 1998, WO 94/15932, published Jul. 21, 1994, U.S.
Pat. No. 5,344,991,
issued Jun. 6, 1994, U.S. Pat. No. 5,134,142, issued Jul. 28, 1992, U.S. Pat.
No. 5,380,738,
issued Jan. 10, 1995, U.S. Pat. No. 5,393,790, issued Feb. 20, 1995, U.S. Pat.
No. 5,466,823,
issued Nov. 14, 1995, U.S. Pat. No. 5,633,272, issued May 27, 1997, and U.S.
Pat. No.
5,932,598, issued Aug. 3, 1999, all of which are hereby incorporated by
reference.
[05081 General and specific synthetic procedures for the preparation of the
COX-2 inhibitor
compounds described above are found in U.S. Pat. No. 5,474,995, issued Dec.
12, 1995, U.S.
Pat. No. 5,861,419, issued Jan. 19, 1999, and U.S. Pat. No. 6,001,843, issued
Dec. 14, 1999, all
of which are herein incorporated by reference.
[05091 Other examples of angiogenesis inhibitors include, but are not limited
to, endostatin,
ukrain, ranpirnase, IM862, 5-methoxy-4-[2-methyl-3-(3-methyl-2-
butenyl)oxiranyl]-1-
222
CA 02668580 2009-06-10
oxaspiro[2,5]oct--6-yl(chloroacetyl)carbamate,acetyldinanaline,5-amino- l -
[[3,5-ichloro-4-(4-
chlorobenzoyl)phenyl]methyl]-1H-1,2,3-triaz-ole-4-carboxamide,CM101,
qualamine,
combretastatin, RPI4610, NX31838, sulfated mannopentaose phosphate, 7,7-
(carbonyl-
bis[imino-N-methyl-4,2-pyrrolocarbonylimino[N-methyl-4,2-py-rrole]-
carbonylimino]-bis-(1,3-
naphthalenedisulfonate), and3-[(2,4-dimethylpyrrol-5-ylmethylene]-
2indolinone(SU5416).
[0510] As used above, "integrin blockers" refers to compounds which
selectively antagonize,
inhibit or counteract binding of a physiological ligand to the
.alpha.v.beta.3 integrin, to
compounds which selectively antagonize, inhibit or counter-act binding of a
physiological ligand
to the .alpha.v.beta.5 integrin, to compounds which antagonize, inhibit or
counteract binding of a
physiological ligand to both the .alpha.v.beta.3 integrin and the
.alpha.v.beta.5
integrin, and to compounds which antagonize, inhibit or counteract the
activity of the particular
integrin(s) expressed on capillary endothelial cells. The term also refers to
antagonists of
the.alpha.. sub. v.beta.. sub. 6,. alpha.. sub.v.beta.. sub. 8,. alpha.. sub.
l .beta.1,
alpha.. sub.2.beta.. sub. 1,. alpha.. sub. 5.beta.. sub. 1,. alpha.. sub.
6.beta.. sub. I and
.alpha.. sub.6.beta.4 integrins. The term also refers to antagonists of
any combination
of.alpha.v.beta.. sub. 3,. alpha.. sub.v.beta. 5,.alpha..
sub.v.beta.. sub. 6,
.alpha.. sub. v.beta.. sub. 8,. alpha.. sub. l .beta.. sub.1,. alpha..
sub.2.beta.. sub. 1,
.alpha.. sub. 5.beta.. sub. 1, .alpha. 6.beta. 1 and
.alpha.6.beta.4 integrins.
[0511] Some specific examples of tyrosine kinase inhibitors include N-
(trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide, 3-[(2,4-dimethylpyrrol-
5 -
yl)methylidenyl)indolin-2-one, 17-(allylamino)-17-demethoxygeldanamycin, 4-(3-
chloro-4-
223
CA 02668580 2009-06-10
fluorophenylamino)-7-methoxy-6-[3 -(4-morpholinyl)propoxyl ] q-uinazoline,N-(3
-
ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-
quinazolinamine,BIBX1382,2,3,9,10,11,12-
hexahydro-10-(hydroxymethyl)-10-hydroxy-9-methyl-9,12-epox-y-I H-diindolo[ 1
,2, 3-fg:3',2',1'-
kl]pyrrolo[3,4-i][1,6]benzodiazocin-l-one, SH268, genistein, ST1571, CEP2563,
4-(3-
chlorophenylamino)-5,6-dimethyl-7H-pyrrolo [2,3-d]pyrimidinemethane sulfonate,
4-(3-bromo-
4-hydroxyphenyl)amino-6,7-dimethoxyquinazoline, 4-(4'-hydroxyphenyl)amino-6,7-
dimethoxyquinazoline, SU6668, STI571 A, N-4-chlorophenyl-4-(4-pyridylmethyl)-1-
phthalazinamine,andEMD 121974.
105121 Combinations with compounds other than anti-cancer compounds are also
encompassed
in the instant methods. For example, combinations of the instantly claimed
compounds with
PPAR-.gamma. (i.e., PPAR-gamma) agonists and PPAR-.delta. (i.e., PPAR-delta)
agonists are
useful in the treatment of certain malingnancies. PPAR-.gamma. and PPAR-
.delta. are the
nuclear peroxisome proliferator-activated receptors gamma. and delta. The
expression of
PPAR-.gamma. on endothelial cells and its involvement in angiogenesis has been
reported in the
literature (see J. Cardiovasc. Pharmacol. 1998; 31:909-913; J. Biol. Chem.
1999;274:9116-9121;
Invest. Ophthalmol Vis. Sci. 2000; 41:2309-2317). More recently, PPAR-.gamma.
agonists have
been shown to inhibit the angiogenic response to VEGF in vitro; both
troglitazone and
rosiglitazone maleate inhibit the development of retinal neovascularization in
mice. (Arch.
Ophthamol. 2001; 119:709-717). Examples of PPAR-.gamma. agonists and PPAR-
.gamma./.alpha. agonists include, but are not limited to, thiazolidinediones
(such as DRF2725,
CS-011, troglitazone, rosiglitazone, and pioglitazone), fenofibrate,
gemfibrozil, clofibrate,
GW2570, S13219994, AR-H039242, JTT-501, MCC-555, GW2331, GW409544, NN2344,
224
CA 02668580 2009-06-10
KRP297, NP0110, DRF4158, NN622, G1262570, PNU182716, DRF552926, 2-[(5,7-
dipropyl-3-
trifluoromethyl-1,2-benzisoxazol-6-yl)oxy]-2-methylpro- pionic acid (disclosed
in U.S. Ser. No.
09/782,856), and 2(R)-7-(3-(2-chloro-4-(4-fluorophenoxy)phenoxy)propoxy)-2-
ethylchromane-
2- -carboxylic acid (disclosed in U.S.Ser.No.60/235,708and60/244,697).
[0513] Another embodiment of the instant invention is the use of the presently
disclosed
compounds in combination with gene therapy for the treatment of cancer. For an
overview of
genetic strategies to treating cancer see Hall et al (Am J Hum Genet 61:785-
789, 1997) and Kufe
et al (Cancer Medicine, 5th Ed, pp 876-889, BC Decker, Hamilton 2000). Gene
therapy can be
used to deliver any tumor suppressing gene. Examples of such genes include,
but are not limited
to, p53, which can be delivered via recombinant virus-mediated gene transfer
(see U.S. Pat. No.
6,069,134, for example), a uPA/uPAR antagonist ("Adenovirus-Mediated Delivery
of a
uPA/uPAR Antagonist Suppresses Angiogenesis-Dependent Tumor Growth and
Dissemination
in Mice," Gene Therapy, August 1998;5(8):1105-13), and interferon gamma (J
Immunol
2000;164:217-222).
[0514] The compounds of the instant invention may also be administered in
combination with
an inhibitor of inherent multidrug resistance (MDR), in particular MDR
associated with high
levels of expression of transporter proteins. Such MDR inhibitors include
inhibitors of p-
glycoprotein (P-gp), such as LY335979, XR9576, OC144-093, R101922, VX853
andP SC833 (valspodar).
225
CA 02668580 2009-06-10
[05151 A compound of the present invention may be employed in conjunction with
anti-emetic
agents to treat nausea or emesis, including acute, delayed, late-phase, and
anticipatory emesis,
which may result from the use of a compound of the present invention, alone or
with radiation
therapy. For the prevention or treatment of emesis, a compound of the present
invention may be
used in conjunction with other anti-emetic agents, especially neurokinin-1
receptor antagonists,
5HT3 receptor antagonists, such as ondansetron, granisetron, tropisetron, and
zatisetron,
GABAB receptor agonists, such as baclofen, a corticosteroid such as Decadron
(dexamethasone),
Kenalog, Aristocort, Nasalide, Preferid, Benecorten or others such as
disclosed in U.S. Pat. Nos.
2,789,118, 2,990,401, 3,048,581, 3,126,375, 3,929,768, 3,996,359, 3,928,326
and 3,749,712, an
antidopaminergic, such as the phenothiazines (for example prochlorperazine,
fluphenazine,
thioridazine and mesoridazine), metoclopramide or dronabinol. For the
treatment or prevention
of emesis that may result upon administration of the instant compounds,
conjunctive therapy
with an anti-emesis agent selected from a neurokinin-1 receptor antagonist, a
5HT3 receptor
antagonist and a corticosteroid is preferred.
105161 Neurokinin-1 receptor antagonists of use in conjunction with the
compounds of the
present invention are fully described, for example, in U.S. Pat. Nos.
5,162,339, 5,232,929,
5,242,930, 5,373,003, 5,387,595, 5,459,270, 5,494,926, 5,496,833, 5,637,699,
5,719,147;
European Patent Publication Nos. EP 0 360 390, 0 394 989, 0 428 434, 0 429
366, 0 430 771, 0
436 334, 0 443 132, 0 482 539, 0 498 069, 0 499 313, 0 512 901, 0 512 902, 0
514 273, 0 514
274, 0 514 275, 0 514 276, 0 515 681, 0 517 589, 0 520 555, 0 522 808, 0 528
495, 0 532 456, 0
533 280, 0 536 817, 0 545 478, 0 558 156, 0 577 394, 0 585 913, 0 590 152, 0
599 538, 0 610
793, 0 634 402, 0 686 629, 0 693 489, 0 694 535, 0 699 655, 0 699 674, 0 707
006, 0 708 101, 0
226
CA 02668580 2009-06-10
709 375, 0 709 376, 0 714 891, 0 723 959, 0 733 632 and 0 776 893; PCT
International Patent
Publication Nos. WO 90/05525, 90/05729, 91/09844, 91/18899, 92/01688,
92/06079, 92/1215 1,
92/15585, 92/17449, 92/20661, 92/20676, 92/21677, 92/22569, 93/00330,
93/00331, 93/01159,
93/01165, 93/01169, 93/01170, 93/06099, 93/09116, 93/10073, 93/14084,
93/14113, 93/18023,
93/19064, 93/21155, 93/21181, 93/23380, 93/24465, 94/00440, 94/01402,
94/02461, 94/02595,
94/03429, 94/03445, 94/04494, 94/04496, 94/05625, 94/07843, 94/08997,
94/10165, 94/10167,
94/10168, 94/10170, 94/11368, 94/13639, 94/13663, 94/14767, 94/15903,
94/19320, 94/19323,
94/20500, 94/26735, 94/26740, 94/29309, 95/02595, 95/04040, 95/04042,
95/06645, 95/07886,
95/07908, 95/08549, 95/11880, 95/14017, 95/15311, 95/16679, 95/17382,
95/18124, 95/18129,
95/19344, 95/20575, 95/21819, 95/22525, 95/23798, 95/26338, 95/28418,
95/30674, 95/30687,
95/33744, 96/05181, 96105193, 96/05203, 96/06094, 96/07649, 96/10562,
96/16939, 96/18643,
96/20197, 96/21661, 96/29304, 96/29317, 96/29326, 96/29328, 96/31214,
96/32385, 96/37489,
97/01553, 97/01554, 97/03066, 97/08144, 97/14671, 97/17362, 97/18206,
97/19084, 97/19942
and 97/21702; and in British Patent Publication Nos. 2 266 529, 2 268 931, 2
269 170, 2 269
590, 2 271 774, 2 292 144, 2 293 168, 2 293 169, and 2 302 689. The
preparation of such
compounds is fully described in the aforementioned patents and publications,
which are
incorporatedhereinbyreference.
[05171 A neurokinin-1 receptor antagonist for use in conjunction with the
compounds of the
presentinventionis2-(R)-(1-(R)-(3,5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-
(4-fluoropheny-l)-
4-(3-(5-oxo-1H,4H-1,2,4-triazolo)methyl)morpholine or a pharmaceutically
acceptable salt whicj
is described in US pat No 5,719,147.
227
CA 02668580 2009-06-10
[05181 A compound of the instant invention may also be administered with an
agent useful in
the treatment of anemia. Such an anemia treatment agent is, for example, a
continuous
erythropoiesis receptor activator, such as apoetin alfa.
[05191 A compound of the instant invention may also be administered with an
agent useful in
the treatment of neutropenia. Such a neutropenia treatment agent is, for
example, a hematopoietic
growth factor which regulates the production and function of neutrophils such
as a human
granulocyte colony stimulating factor, (G-CSF). Examples of a G-CSF include
filgrastin.
[05201 A compound of the instant invention may also be administered with an
immunologic-
enhancing drug such as levamisole, Isoprinosine, and zadaxin.
[05211 Thus, the scope of the instant invention encompasses the use of the
instantly claimed
compounds in combination with a second compound selected from: 1)PI3k
inhibitor, 2)Akt
inhibitor, 3)TNF inhibitor such as Inflixamab, Humira, Etanercept 4) an
estrogen receptor
modulator, 5) an androgen receptor modulator, 6) retinoid receptor modulator,
7) a cytotoxic
agent, 8) an antiproliferative agent, 9) a prenyl-protein transferase
inhibitor, 10) an HMG-CoA
reductase inhibitor, 11) an HIV protease inhibitor, 12) a reverse
transcriptase inhibitor, 13) an
angiogenesis inhibitor, 14) an inhibitor of inherent multidrug resistance, 15)
an anti-emetic
agent, 16) an agent useful in the treatment of anemia, 17) agent useful in the
treatment of
neutropenia, and 18) an immunologic-enhancing drug in certain cases.
228
CA 02668580 2009-06-10
105221 Also included in the scope of the claims is a method of treating cancer
that comprises
administering a therapeutically effective amount of one or more inositol
compounds of the
invention or derivative thereof described above in combination with radiation
therapy and/or in
combination with a compound selected from: 1)PI3k inhibitor, 2)Akt inhibor,
3)TNF inhibitor
such as Inflixamab, Humira, Etanercept 4) an estrogen receptor modulator, 5)
an androgen
receptor modulator, 6) retinoid receptor modulator, 7) a cytotoxic agent, 8)
an antiproliferative
agent, 9) a prenyl-protein transferase inhibitor, 10) an HMG-CoA reductase
inhibitor, 11) an
HIV protease inhibitor, 12) a reverse transcriptase inhibitor, 13) an
angiogenesis inhibitor, 14) an
inhibitor of inherent multidrug resistance, 15) an anti-emetic agent, 16) an
agent useful in the
treatment of anemia, 17) agent useful in the treatment of neutropenia, and 18)
an immunologic-
enhancing drug.
105231 When a composition according to this invention is administered into a
human subject,
the daily dosage will normally be determined by the prescribing physician with
the dosage
generally varying according to the age, weight, and response of the individual
patient, as well as
the severity of the patients symptom's
[05241 In one exemplary application, in addition to amounts set forth above, a
suitable amount
of an inhibitor of Akt/PKB is administered to a mammal undergoing treatment
for cancer.
Administration occurs in an amount of inhibitor of between about 0.1 mg/kg of
body weight to
about 60 mg/kg of body weight per day, preferably of between 0.5 mg/kg of body
weight to
about 40 mg/kg of body weight per day. A particular therapeutic dosage that
comprises the
229
CA 02668580 2009-06-10
instant composition includes from about 0.01 mg to about 1000 mg of inhibitor
of Akt/PKB.
Preferably, the dosage comprises from about 1 mg to about 1000 mg of inhibitor
of Akt/PKB.
[05251 In addition to the above, the compounds of the invention can be used in
combinations
with monoclonal antibodies for autoimmune condition treatments such as
Adalimumab and
certolizumab. Still further the compounds of the invention can be used in
combinations with
various active agents currently in use for chronic, active hepatitis, in
particular, without
limitation, pegylated interferon, Ribavarin (copegus, reberol), rebetron,
Inteferons, epivir-HBV,
inteferon alpha/alpha 2 plus ribavarin combination.
[05261 All patents, publications and pending patent applications identified
are hereby
incorporated by reference.
105271 The compounds of the invention can be also be used in the treatment of
or as a
synergistic inhibitor of autoimmune diseases mediated by defective or
overactive signaling
pathways: More particularly, the compounds of the invention can be used in
treating patients
with Achlorhydra Autoimmune Active Chronic Hepatitis, Addison's Disease,
Alopecia, Areata,
Amyotrophic Lateral Sclerosis (ALS, Lou Gehrig's Disease), Ankylosing
Spondylitis, Anti-
GBM Nephritis or anti-TBM Nephritis, Antiphospholipid Syndrome, Aplastic
Anemia,
Rheumatoid Arthritis, Asthma, Atopic Allergy, Atopic Dermatitis, Autoimmune
Inner Ear
Disease (AIED), Autoimmune Lymphoproliferative Syndrome (ALPS), Balo Disease,
Behcet's
Disease, Berger's Disease, (IgA Nephropathy), Bullous Pemphigoid,
cardiomyopathy, Celiac
Disease, Chronic Fatigue Immune Dysfunction Syndrome (CFIDS), Churg Strauss
Syndrome,
230
CA 02668580 2009-06-10
Cicatricial Pemphigoid, Cogan's Syndrome, Cold Agglutunin Disease, Colitis,
Cranial Arteritis,
CREST Syndrome, Crohn's Disease, Cushing's Syndrome, Dego's Disease,
Dermatitis,
Dermatomyositis, Devic Disease, Type 1 Diabetes, Type 2 Diabetes, Dressler's
Syndrome,
Discoid Lupus, Eczema, Essential Mixed cryoglobulinemia, Eosinophilic,
Fasciitis,
Epidermolysis Bullosa Acquisita, Evan's Syndrome, Fibromyalgia, Fibromyositis,
Fibrosing
Alveolitis, Gastritis, Giant Cell Artertis, Glomerulonephritis, Goodpasture's
Disease, Grave's
Disease, Guillian-Barre Syndrome, Hashimoto's Thyroiditis, Hemolytic Anemia,
Henoch-
Schonlein Purpura, Hepatitis, Hughes Syndrome, Idiopathic Adrenal Atrophy,
Idiopathic
Pulmonary Fibrosis, Idiopathic Thrombocytopenia Purpura, Inflammatory
Demylinating
Polyneuropathy, Irritable Bowel Syndrome, Kawasaki's Disease, Lichen Planus,
Lou Gehrig's
Disease, Lupoid Hepatitis, Lupus, Lyme Disease, Meniere's Disease, Mixed
Connective Tissue
Disease, Multiple Myeloma, Multiple Sclerosis, Myasthenia Gravis, Myositis,
Ocular Cicatricial
Pemphigoid, Osteoporosis, Pars Planitis, Pemphigus Vulgaris, Polyglandular
Autoimmune
Syndromes, Polymyalgia Rheumatica (PMR), Polymyositis, Primary Biliary
Cirrhois, Primary
Sclerosing Cholangitis, Psoriasis, Raynaud's Phenomenon, Reiter's Syndrome,
Rheumatic Fever,
Rheumatoid Arthritis, Sarcoidosis, Scleritis, Scleroderma, Sjogren's Syndrome,
Sticky Blood
Syndrome, Still's Disease, Stiff Man Syndrome, Sydenham Chorea, Systemic Lupus
Erythmatosis (SLE), Takayasu's Arteritis, Temporal Arteritis, Ulcerative
Colitis, Vasculitis,
Vitiligo, Wegener's Granulomatosis, and Wilson'sSyndrome.
231
