Note: Descriptions are shown in the official language in which they were submitted.
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BETA-ALETHINE USE IN CELL CULTURE AND THERAPY
BACKGROUND OF THE INVENTION
1. Field of the Invention
The invention relates to the use of [3-alethine and/or its corresponding
monosulfide,(3-aletheine,for the inducement of cell differentiation,
adaptation
of cells to culture, and enhancement of cell phenotypic expression, vitality,
longevity, and production. In particular, the invention relates to the use of
(3-alethine for inducing the differentiation of precursor cells into
specialized
cells, for normalizing function of malfunctioning cells, and for eliminating
intractable cells (cells which are "resistant to cure, relief, or control",
Dorland's Illustrated Medical Dictionary, 26tr' Edition, 1974, W.B. Saunders,
Philadelphia, PA, USA).
Cellular differentiation is a well-known phenomenon which broadly refers
to processes by which precursor cells (commonly termed "stem cells") develop
into specialized cells. Differentiation compounds, i.e., those factors which
induce cell multiplication, in the literature (see, e.g., "Growth,
Differentiation, and the Reversal of Malignancy", Scientific American, pp. 40-
47,
January 1986, and the publications cited therein) and the implications of each
with respect to therapeutic use in the treatment of disease or disorders of
the
body are of much current interest. The present application relates to the
identification of (3-alethine as a non-cell-lineage-dependent differentiation
compound, and the use of(3-alethine to induce differentiation and/or
normalization
of the function of a variety of cells, particularly for therapeutic benefits.
"Phenotypic cell expression" is defined herein as the manifestation of
an entire range of physical, biochemical, and physiological characteristics of
an individual cell as determined both genetically and environmentally,
incontrast
to "genotypic cell expression", which in the art solely refers to the
expression
of the cell chromosomal sequence. [See, for example, Dorland' s Illustrated
Medical
Dictionary, 26th Edition, 1974, W. B. Saunders, Philadelphia]. Biological
activity
of the compounds of the invention thus includes modulation of the expression
of genetic material of cells in culture as influenced by the condition and
environment of each cell, including the age of the cell, the culture or
conditions
employed, and the presence of optionally added biological effectors.
The invention further provides a method for treating neoplasias with
3-alethine. In particular, the invention provides methods for treating a
variety
of neoplasias which reduce tumor burden, inhibit tumor growth, and inhibit
tumor
intravascularization, for example from metastasizing tumors. 3-alethine has
been
identified as a compound inducing cell differentiation and modulating cell
growth,
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CA 02087883 2010-05-28
phenotypic expression (including bioproduction and function), vitality, and
longevity ; a correspondence between cell differentiation and reversal of
malignancy has been suggested; see, for example, "Growth, Differentiation, and
the Reversal of Malignancy", Scientific American, pp. 40-47, January 1986, and
the publications cited therein.
The present application particularly relates to the identification of
R-alethine as a non-cell-lineage-dependent anti-tumor compound, and the use of
P-alethine to induce normalization of the function of a variety of neoplastic
cells, particularly for therapeutic benefits.
2. Discussion of Related Art
3-alethine is an endogenous thiol known to be produced in vivo as a byproduct
of metabolic pathways. It is related via these pathways to pantothenic acid,
which is a vitamin having known nutritional benefits (see, e.g., J. Reprod.
Fert. ,
57:505-510 , 1979), and related compounds have been suggested for use in
conjunction with radiotherapy as radioprotectors (J. Med. Chem., 29:2217-2225,
1986; WO 35/00157, 17 Jan. 1985) . No other relevant asserted biological
functions
of this compound are known to be described in the prior art. The compound is
primarily well-known as a starting material for the chemical synthesis of
related
compounds (see, e.g., Japanese patent applications (83) 198461; (83) 46063A2)
(81) 156256A2; (81) 104861A2; (80) 124755; (75) 62932; (80) 07222; andU.S.
Patents
2,835,704 and 4,552,765; for examples of the preparation of (3-alethine,
p-aletheine, pantetheine, and its derivatives or intermediates, and also for
Coenzyme A and Coenzyme A derivatives or intermediates).
It is well-known that endogenous thiols and disulfides are critical to
the function of a multitude of thiol- and disulfide-dependent branch-point
enzymes
controlling access to major metabolic pathways. Glutathione (GSH, gamma-
glutamyl
cysteinylglycine, an acid tripeptide thiol) is the most abundant thiol in
mammalian
cells, and an entire regulatory and regenerating system ensures an adequate
supply
of this reducing agent (3,4,5), which maintains and buffers cell thiol/disulf
ide
ratios. Coenzyme A (CoA) and lipoic acid are prevalent in mammalian systems
and
also regulate dependent enzyme activity. Xenobiotic thiols such as
dithiothreitol
(DTT, Cleland's reagent) or dithioerythritol are routinely used experimentally
to regulate activity of thiol-dependent enzymes.
In response to demand, thiols such as GSH, CoA, and lipoic acid can, for
example, activate thiol-activatable enzyme by reducing inactive oxidized
(disulfide) enzyme to the corresponding thiol with a concomitant oxidation of
the activating thiol to its corresponding disulfide (GSSG in the case of
glutathione-GSH) according to the following scheme, wherein P is protein:
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CA 02087883 2010-05-28
p p~ SH SH p pf
+ < H~ \SH +
HO OH HO OH
Inactive Active
(Active) DTT (Inactive) Oxidized DTT
Activity of thiol-dependent enzymes is a function of the availability of the
thiols involved as expressed by the thiol/disulfide ratios of their
thiol/disulfide redox buffers (upper arrow); interaction is complex, however,
and activity is further dependent on additional factors such as substrate,
ambient
ions, and type of reducing thiol (membrane-bound enzymes, for example, are
resistant to reduction by glutathione). Similarly, activity of
disulfide-dependent enzymes (in parenthesis) is a function of the availability
of disulfides, as expressed by the thiol/disulfide ratios of their redox pairs
(lower arrow).
By the above mechanisms, endogenous thiol/disulfide redox buffers such
as GSH/GSSG systems control the activity of many critical enzymes; thyroxine
monodeiodinase is exemplary of thiol-dependent enzymes. Regulation of the
activity of this enzyme by thiol/disulfide buffer controls the induction of a
host of important enzymes, including HMG-CoA reductase, the branch-point
enzyme
for the isoprenoid pathway, which in turn regulates the production of
essential
isoprenoids such as steroid hormones, dolichol, cholesterol, and ubiquinone
and
the isoprenylation of proteins . Glycolysis is also controlled by thiol-
dependent
and disulfide-dependent enzyme systems; phospho- fructokinase, for example, is
inactivated by disulfides, whereas fructose-1,6-bis-phosphatase with the
reverse
enzyme activity is activated by certain disulfides. Thiol-dependent enzymes
also
directly and/or indirectly control isoprenoid and oligosaccharide biosynthesis
and the synthesis and utilization of thyroid hormones.
One mechanism postulated to participate in the in vivo regulation of
thiol/disulfide equilibria is the oxidation of thiol to disulfide catalyzed by
microsomal flavin-containing mixed function monooxygenase (herein referred to
as "monooxygenase"). This monooxygenase catalyzes, for example, the oxidation
of cysteamine to its corresponding disulfide, cystamine. A comparable
oxygenase
activity thus appears to be critical to the regulation of at least some thiol-
and disulfide-dependent enzymes in vivo.
Certain other thiols (glutathione or cysteine or N-acetyl-cysteine) have
been demonstrated in vivo to inhibit neoplasia (Am. J. Med., 91 (3C) :1225-
1305,
1991); to inhibit replication of HIV in cell cultures (Proc. Natl. Acad. Sci.
USA, 87(12):4884-8, 1990); to be markedly elevated in preneoplastic/neoplastic
hepatocytes (Mol. Carcin., 2(3):144-9, 1989); to influence the proliferation
of human peripheral blood lymphocytes (HPBL) and T-cells (Am. J. Med.,
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CA 02087883 2010-05-28
91(3C):1405-1445, 1991); to reverse inhibition of lymphocyte DNA synthesis by
glutamate in cells from HIV-infected patients (Int. Immunol. , 1 (4) :367-72,
1989);
to reduce infectivity of herpes virus in vitro (Acta.Virol.Praha. , 11(6):559-
61,
1967); to suppress HIV expression in monocytes (Proc. Natl. Acad. Sci. USA,
88: 986-990, 1991); and to be systemically deficient in HIV-infected
individuals
(Biol. Chem. Hoppe Seyler, 370:101-08, 1989 and The Lancet, 11:1294-97, 1989).
Regulation of HMG-CoA reductase activity by thiols and disulfides is well-
known;
as noted above, thyroxine monodeiodinase is a thiol-dependent enzyme, and this
enzyme controls the induction of HMG-CoA raductase (Eur. J. Biochem., 4:273-
278,
1968) . Hypercholesteremia and atherosclerosis, leading factors in heart
disease,
are now clearly linked to HMG-CoA reductase activity, and treatment of these
conditions with various regulators of HMG-CoA reductase is known. HMG-CoA
reductase activity is also linked to neoplasia, most recently by evidence of
its role in the transformation of cells by activation of Ras protein which
regulates
oncogene expression (Adv. Enzymol., 38:373-412, 1973; Biochem. Soc. Trans.,
17:875-876, 1989; Science, 245:379-385, 1989; 3-Hydroxy-3-Methylglutaryl
Coenzyme A Reductase, Sabine ed. CRC Press Inc., Boca Raton, FL, USA, pp. 245-
257,
1983).
SUMMARY OF THE INVENTION
The invention accordingly provides methods for inducing cell
differentiation and normalization at cell function, and for enhancing cell
phenotypic expression, vitality, longevity, and production employing(3-
alethine
as differentiation compound. The invention has particular application in the
differentiation of mammalian cells, including human cells, both in vitro and
in vivo, most especially for normalizing cell development with respect to both
cell maturation and differentiation-dependent cell growth; however, the use of
R-alethine as differentiation compound for other cells, such as reptilian,
avian,
plant, insect, arachnid, rickettsial, bacterial, yeast, mold, protozoan, and
fungus cells is also contemplated. Exemplary applications include the
following:
normalization of immnunodeficient and autoimmune cell function (including the
therapeutic use of (3-alethine in the treatment of immunodeficiency and
autoimmune
diseases and disorders, particularly in mammals, and especially in humans) ;
delay
of cell senescence (including the therapeutic use at (3-alethine in the
treatment
of diseases or disorders characterized by presenescent or prematurely
senescing
cells, particularly in mammals, and especially in humans); enhancement of
cellular
phenotypic expression, production, and vitality, and the therapeutic benefits
derived therefrom; and adaptation of resistant cells to culture and the
diagnostic
and therapeutic uses derived therefrom.
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CA 02087883 2010-05-28
The invention in further particular provides methods far the recognition,
normalization, and elimination of neoplastic cells particularly for the
treatment
of cancer. Within the scope of the present invention, (3-aletheine, the
reduced
form of (3-alethine, is to be considered the biological equivalent of (3-
alethine
for purposes of practicing the invention, as (3-alethine is readily reduced to
P-aletheine in vivo, for example by abundant intracellular thiol compounds,
such
as glutathione in mammals, including humans. Both compounds have the advantage
of having inherent antioxidative properties; however, R-alethine is chemically
more resistant to autoxidation than (3-aletheine, and the use of (3-alethine
in
the present invention is generally preferred for this reason.
BRIEF DESCRIPTION OP THE DRAWINGS
FIG. 1 illustrates data from a series of experiments designed to test the
effect of (3-alethine on the maximum population doubling level (PDL) of IMR-90
human fetal lung fibroblasts;
FIG. 2 illustrates data from a series of experiments designed to study
the effect of (3-alethine on non-antigen specific immunoglobulin synthesis and
secretion by human peripheral blood leukocytes (HPBLs) in vitro;
FIG. 3 illustrates data from a series of experiments designed to study
the effect of (3-alethine on murine splenocyte production of non-specific
immunoglobulin;
FIG. 4 illustrates data from a series of experiments designed to study
the effect of 3-alethine on murine splenocyte IgG production;
FIG. 5 illustrates data from an experiment designed to study the effect
of (3-alethine on the ability to adapt cells taken from in vivo for in vitro
growth;
FIGS. 6-8 illustrate data demonstrating the effect of R-alethine at varying
dosages on mice inoculated with NS-1 myeloma cells;
FIG. 9 is a three-dimensional composite of the data of FIGS. 6-8;
FIG. 10 is an 85 clockwise rotation of the data of FIG. 9; and
FIG. 11 illustrates modulation of tumor in two mice, each with differing
amounts of 3-alethine.
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DETAILED DESCRIPTION OF THE INVENTION
As indicated above, (3-alethine is a known compound
[(H2NCH2CH,(C=O)NHCH2CH2S)2 and Figure 1, following] commonly produced by
oxidation of the correspondingmonosulfide, (3-aletheine [H2NCH2CH2 (C=0)
NHCH2CH,SH
and Figure II, following], which is unstable in air and aqueous solutions (The
Merck Index, 9th edition (#221), Merck & Co., Rahway, NJ, USA):
H iH
j -CH2-CH2-C-NH-CH2-CH2 S-S-CH2CH2-NH-C-CH2-CHZ (1)
H
H 0
~ 41
IT-CH2-CH2-C-NH-CH2-CH2-SH ( I I )
H
Both compounds are stabilized as their acid salts, particularly their
hydrogen halide salts, and especially their hydrochloride salts. Various
techniques for the synthesis of (3-alethine based on deblocking of
(N,N'-bis-carbobenzoxy)-blocked (3-alethine are described in the literature
(carbobenzoxy is often abbreviated as CBZ); however, most of the known
procedures
result in an unsatisfactory yield, purity of product, or both. Accordingly, it
is preferred that P-alethine for use in the process of the invention be
prepared
by processes which ensure purity of product and preferably also maximize
yield,
for example by the process of the invention comprising coupling N-CBZ-blocked
[i-alanine to N-hydroxysuccinimide to produce the corresponding active ester,
which is then coupled to cystamine prepared by oxidation of cysteamine with
hydrogen
peroxide; the product, bis-CBZ-blocked R-alethine, is then recovered and
deblocked. The process is described in detail in the Examples, and provides a
high-yield, high-purity product suitable for pharmaceutical use.
According to the invention, (3-alethine appears to regulate a set of generic
differentiation mechanisms that are not cell-lineage specific and that are
common
to cells regardless of phenotypic specialization. Consistent with this
premise,
the use of (3-alethine according to the invention is not significantly
dosage-dependent with respect to cell lineage, phenotype, or point of
intervention
in the cell cycle, except as noted below. Broadly, dosages starting from about
approximately 10 pg/ml culture are useful for cellular differentiation. For in
vivo applications, from about 10 pg/kg of body weight are recommended,
particularly amounts from about 10 pg/kg up to about 200 }ig/kg, and more
particularly, up to about 100 pg/kg, which may be administered by any
customary
route in the presence of conventional carriers (such as physiological saline
for non-oral routes including parenteral, or with suitable enterocoating in
oral
routes) preferably on a daily or alternate-daily regimen as described more
fully
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below, until the desired results are achieved, although other regimens, such
as weekly or biweekly regimens may suffice, particularly when results are
apparent; i.e., decreases in dosages as normalization progresses maybe
suitable.
Use of amounts of (3-alethine substantially in excess of those required to
obtain
differentiation, normalization of cell function, or other results noted herein
are not recommended, as excessive dosages may be counterproductive or at least
ineffective. For in vitro applications, dosages starting from about 10 pg/ml
culture are suggested, with replenishment as described below.
It is contemplated that (3-alethine is useful for the differentiation of
cells of living organisms in general, including mammalian (especially human),
reptilian, avian, plant, insect, arachnid, rickettsial, bacterial, yeast,
mold,
protozoan, and fungus cells, owing to the commonality of results obtained with
corresponding dosages observed in both experiments reported herein and
unreported
experiments and the ubiquitous presence of precursors to )3-alethine in living
systems. Further, (3-alethine is useful for adapting to culture cells which
are
generally not regarded as so adaptable (herein referred to as culture-
resistant
cells), such as hepatic cells.
According to one aspect of the invention, R-alethine comprises a
differentiation compound which normalizes cell function (i.e., increases
insufficient function, or decreases excess function); promotes cell longevity
and/or bioproductivity; and/or diversifies cell function (i. e, expands
phenotypic
cellular expression) . R-alethine specifically functions to (1) adapt
resistant
cells to culture; (2) delay senescence of cells in vitro, wherein senescence
is broadly defined as the cell's increasing inability to reproduce itself in
culture, typically characterized by markedly increasing cell generation times
(Tg) at specific population doubling levels (PDL) ; or the time required for
one
complete round ofcell celldivision (see, e.g., HayfR.,Exp.Cell.Res.,37:614-636
(1965), for a discussion of markers of senescence including T. and PDL); and
(3) normalize or improves function, such as immunological surveillance (the
recognition and elimination of intractable cells), and/or production of cells,
especially those of the immune system (immunocytes). Particularly for delay
of senescence in cell cultures, it is preferable to expose cells to be treated
to R-alethine prior to significant cell malfunction (in this case, onset of
senescence), as it has been found difficult, for example, to reverse cellular
senescence once it has begun; thus treatment with (3-alethine according to the
invention includes prevention of cellular differentiative malfunction as
described herein, as well as therapeutic treatment of existing differentiative
malfunction, such as that associated with various diseases or disorders.
Markers
of abnormal cellular differentiative function denoting incipient diseases or
disorders, including markers of approaching cellular senescence, are well-
known
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CA 02087883 2010-05-28
or developing in the art, and practitioners are referred to the literature for
methods for assessing such markers.
In order to normalize the life cycle of cells in culture, i.e., optimize
growth and maturation of cells with respect to senescence and death, and adapt
resistant cells to culture, it is preferred that the cells be exposed to R-
alethine
before the onset of senescence. Since cellular aging is a gradual procedure,
senescence may to some degree be arrested even if the cells are exposed to the
compound at a later stage in the life of the cells, depending upon the
particular
cell type, culture conditions and other factors. However, senescent cells are
less viable and productive by definition, so maintaining them at this late
stage
of the lifespan is counterproductive for most aspects of the invention,
unless,
for example, study of senescent cells is of concern. Consequently, for optimum
results in most instances (e.g., when optimization of cell life and function
is desired) it is preferable to expose cells to the compound as early in their
life-cycle as is convenient.
Culture-resistant cells (i.e., cells which have a brief lifespan under
conventional culture conditions, for example of two weeks or less; or those
which
do not express normal biofunctions in culture, such as those wherein normal
production of hormones, enzymes, or other bioproducts is suppressed in vitro)
are adaptable to culture by early exposure to adaptive amounts of the
compound,
preferably by combining the compound with the culture medium before
introducing
the cells. Exemplary resistant cells include lymphoid, hepatic, pancreatic,
neural, thyroid, and thymus mammalian cells.
R-alethine maybe added to any known culture medium, optionally supplemented
with protein components such as serum, e.g., fetal or new-born calf serum, to
obtain the results of the invention; the media employed do not form a part of
this invention. Exemplary media include Eagle's Basal Medium; Eagle's Minimal
Essential Medium; Dulbecco's Modified Eagle's Medium; Ham's Media, e.g. F10
Medium or F12 Medium; Puck's N15 Medium; Puck's N16 Medium; Waymoth's MB 5421
Medium; McCoy's 5AMedium; RPMI Media 1603, 1534, and 1640; Leibovitz' s L15
Medium;
ATCC (American Type Culture Collection) CRCM 30; MCDB Media 101, 102, 103,
104;
CMRL Media 1066, 1415; and Hank's or Earl's Balanced Salt Solution. The basal
medium employed, as known in the art, contains nutrients essential for
supporting
growth of the cell under culture, commonly including essential amino acids,
fatty
acids, and carbohydrates. The media typically include additional essential
ingredients such as vitamins, cofactors, trace elements, and salts in
assimilable
quantities. Other factors which promote the growth and maintenance of cells,
including compounds and factors necessary for the survival, function,
production,
and/or proliferation of the cells, such as hormones, for example peptidyl or
steroidal hormones, growth factors, and antibiotics are also typically
included.
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The media also generally include buffers, pH adjusters, pH indicators and the
like.
Media containing the modulators of the invention are applicable to a variety
of cells, especially eukaryotic cells. The media of the invention are suitable
for culturing animal, especially mammalian cells including human cells; plant
cells, insect cells; microorganisms such as bacteria, fungi, molds, protozoa,
and rickettsia, especially antibiotic-producing cells. R-alethine is broadly
useful for promoting viability of living cells in a broad spectrum of so-
called
tissue culture media adapted for the culture of such cells. Exemplary
applications
include the culture of cloned cells, such as hybridoma cell lines; of cells,
mammalian cells in particular and especially human cells, for the production
of cell products, particularly proteins and peptides such as hormones,
enzymes,
and immunofactars; of virally-infected cells for the production of vaccines;
of plant cells in, for example, meristem or callus culture; of epithelial
cells
to provide tissue for wound healing; of resistant cells for medical and
diagnostic
use; and in media adapted far the production and preservation of biological
organs
and implant tissue.
Specific cell types useful for culture in the processes of the invention
accordingly include: cells derived from mammalian tissues, organs, and glands
such as the brain, heart, lung, stomach, intestines, thyroid, adrenal, thymus,
parathyroid, testes, liver, kidney, bladder, spleen, pancreas, gall bladder,
ovaries, uterus, prostate, and skin; reproductive cells (sperm and ova); lymph
nodes, bone, cartilage, and interstitial cells; blood cells including
immunocytes,
cytophages such as macrophages, lymphocytes, leukocytes, erythrocytes, and
platelets. Additional cell types include stem, leaf, pollen, and ovarian cells
of plants; microorganisms and viruses as described above; and cells derived
from
insect tissues, organs, and glands.
Culture techniques useful in conjunction with the modulators of the
invention include the use of solid supports, (especially for anchorage-
dependent
cells in, for example, monolayer or suspension culture) such as glass, carbon,
cellulose, hollow fiber membranes, suspendable particulate membranes, and
solid
substrate forms, such as agarose gels, wherein the compound is caged within
the
bead, trapped with the matrix, or covalently attached, i.e. as a mixed
disulfide.
13-alethine is useful in primary cultures; serial cultures; subcultures;
preservation of cultures, such as frozen or dried cultures; and encapsulated
cells; cultures also may be transferred from conventional media to media
containing the compound by known transfer techniques.
According to the practice of this one aspect of the invention, cells are
treated with (3-alethine in an amount effective to promote culture of these
cells
in vitro, as measured, for example, by significant increase in cell viability
or lifespan, increase in cell biomass, or increase in cell bioproductivity, as
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CA 02087883 2010-05-28
compared to untreated cells. For in vitro applications, from about 10 pg/mi of
cell culture (based on a density of from about 105 to about 107 cells/ml) are
suggested. The culture should be replenished with the compound as necessary,
generally on a daily basis; again, treatment on an alternate-day or biweekly
basis nay suffice, depending upon the desired results.
In immunological applications, immunocytes are exposed to (3-alethine to
promote and/or diversify the function of immunocytes such as leukocytes,
lymphocytes, splenocytes, T-cells, B-cells, natural killer (NK) cells, and
cytophages such as macrophages. In particular, (3-alethine is useful in vivo
or
in vitro to diversify or improve splenocyte or antibody systems; to diversify
or improve immunoglobulin production; to generally promote normal
immunofunction
of immunocytes; and to treat diseases or disorders, especially those of the
immune
system, by treating the organism, mammals in particular and especially humans,
with effective dosages of 3-alethine (optionally including an agent that
promotes
the growth and maintenance of cells described above) either directly, or by
removing the affected cells, treating them in vitro, and reinjecting them into
the affected organism. Human diseases contemplated to respond to these
therapies
include autoimmune diseases, hypogammaglobulinemia, and AIDS (acquired immune
deficiency syndrome).
It is contemplated that 13-alethine is useful for the treatment of neoplasias
of cells of living organisms in general, including mammalian, especially
human,
reptilian, avian, and plant cells, owing to the commonality of results
obtained
with corresponding dosages observed in experiments reported herein and
unreported
experiments. According to the invention, (3-alethine comprises an anti-tumor
compound which normalizes cell function (i.e., increases insufficient function
or decreases excess function) .(-alethine specifically functions to (1)
inhibit
tumor growth, especially that of malignant tumors; (2) regress tumors,
especially
malignant tumors; (3) inhibit tumor metastasis; and (4) normalize growth
characteristics of neoplastic cells; and/or (5) improve recognition and/or
elimination of neoplastic cells.
In cancer applications, neoplastic cells or immunocytes, or both neoplastic
cells and immunocytes, are exposed to (3-alethine to promote differentiation
of
the cells and normalize the cell cycle. Treatment of tumor cells is
effectively
segregated from treatment of the immunocytes by removing immunocytes from the
afflicted mammal, including humans. The immunocytes are then treated in
culture
with (3-alethine, or with a combination of (3-alethine and tumor cells derived
from the afflicted mammal, until either the immunocytes are activated, or the
tumor cells are completely attenuated for health reasons, respectively. The
activated immunocytes preferably devoid of metastatic tumor cells are then
reinjected into the mammal. R-alethine is useful in vivo for reducing soft
(hematolymphoid) tumor burden, particularly in mammals, especially in humans,
CA 02087883 2010-05-28
and inhibiting intravascularization of tumor cells, especially cells of
metastasizing tumors. The compounds are thus useful for reducing tumor burden,
by inhibiting tumor growth or by inhibiting tumor metastasis, or both. In
particular, (3-alethine is contemplated to be useful in the treatment of
numerous
soft and lymphoid malignant tumors, such as lymphomas; leukemias;
hepatocellular
tumors; liver tumors; and Hodgkin's disease; especially tumors such as
myelomas.
R-alethine is contemplated inter alia as useful in the treatment of neoplasia
1) prophylactically; 2) as a primary therapy for inhibiting tumor growth,
particularly that of slowly-growing tumors; and 3) as a supplemental therapy
pursuant to surgical intervention f or removal or debulking of tumors,
particularly
virulent or primary tumors. Treatment with R-alethine has been found to
regress
tumors, reduce tumor mass, inhibit tumor growth, inhibit tumor metastasis, and
inhibit tumor ascites production.
It is recommended that anti-tumor therapy commence at the earliest tumor
stage possible, particularly to avoid peripheral physiological complications
caused by the presence or metastasis of large tumors. (3-alethine for tumor
therapy
is administered by any convenient route as noted above, for example i.v. or
i.p.,
in a suitable conventional carrier such as physiological saline of at least
therapeutic threshold amounts; from about 1 ng/kg body weight up to about 100
pg/kg are particularly suitable, depending upon the stage of the tumor.
Dosages
toward the higher end of the therapeutic range are recommended for Stage II
tumors
and above, whereas dosages toward the lower end of the range are suitable for
Stage I or incipient tumors. For cancer prophylaxis, dosages ranging from
about
pg/kg body weight, preferably from about 1 ng/kg up to about 100 pg/kg body
weight are contemplated. Therapeutic regimens of alternate days for the
dosages
noted above for cancer treatment are suitable, and appear to be preferable,
based
on the observation that in vivo induction of biochemicals such as enzymes
thought
to be responsive to (3-alethine therapy appear to follow chemical stimulation
by about 48 hrs. Prophylactic regimens may be followed on a daily basis.
At least at the dosage levels indicated, 3-alethine appears to be a
substantially non-toxic compound in healthy mice, with no observed adverse
side-effect.
For in vitro applications, immunocytes and optionally tumor cells removed
by methods described in the prior art are treated by exposing the cells to an
appropriate amount of (3-alethine prior to reinjection of the immunocytes into
the afflicted mammal according to methods described in the prior art.
Techniques
for removing and maintaining cells in culture (immunocytes in particular),
treating these calls with immunopotentiating agents in cell culture, and
reinjecting these immunopotentiated cells into mammals are described in the
prior
art, for example, Immune Responses to Metastases (volumes I and II, 1987, CRC
Press, Boca Raton, Florida, USA), Ann. Surg. (201: 158-163, 1985), New England
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CA 02087883 2010-05-28
J. Med. (319:1676-1680, 1988), Cancer Immunol. Immunother. (26:1-10, 1988),
Surgery (pp. 262- 272, August 1986), and Current Protocols in Immunology
(volumes
1 and 2, Green Publishing Associates and Wiley-Interscience, New York, New
York,
USA) . Broadly suitable techniques for removing cells from the afflicted
mammal
include needle or core biopsies or surgical removal of organs, tumors, or
humoral
fluids. Outgrowth from needle biopsies, or treatment of core biopsies or
tissues
with collagenase or sieving through appropriately sized sterile meshes to
disrupt
organization of solid tissues, comprise exemplary methods for establishing
cultures of cells, in vitro. According to the present invention, similarly
removed
cells are treated with (3-alethine for an appropriate amount of time in cell
culture,
and similarly reinjected into the afflicted mammal, the improvement in
technique
being the treatment of the cells with R-alethine. In certain instances, it may
be desirable to remove R-alethine prior to reinjection of the immunocytes into
the afflicted mammal by washing the cells as described in the prior art. For
in vitro applications according to the invention, exposure of the cells to
from
about 10 pg (3-alethine included per ml of cell culture media (based on a
density
of from about 105 to about 107 cells/ml) for an appropriate amount of time,
for
example from about 30 minutes to four weeks or until the desired
immunopotention
is evident as described in the prior art, is suggested. In particular, in
vitro
stimulation of antibody production by human peripheral blood leukocytes or
murine
splenocytes is most efficacious between 10 pg and 1 pg (3-alethine/ml cell
culture
after about four to six days of exposure (see, e.g., Examples V and VI). The
culture should be replenished with the compound as necessary, generally on a
daily basis; treatment on an alternate day or biweekly basis may suffice,
depending
upon the desired results. During this period, exposure of the cells to
immunogenic
substances or cells other than extracted tumor cells, such as allogeneic serum
proteins in the cell culture media, should be minimized to preclude diversion
of the immunological response to non-tumor targets.
EXAMPLES
I. Preparation of P-alethine
)3-alethine was produced by deblocking N,N'-bis-carbobenzoxy (CBZ) blocked
(3-alethine produced as follows:
A. Preparation of N,N'-bis-(CBZ)-(3-alethine or
5,5-Bisis[N-carbobenzoxy-3-alanyl)-2-aminoethyl) disulfide
A solution of dicyclohexylcarbodiimide (23.3g) was added to a solution
of N-CBZ-[3-alanine (24.84g) and N-hydroxy-succinimide (12. 92g) in a total
volume
of about 500 ml of dry 10% acetonitrile in dichloromethane. Dicyclohexylurea
(24.51g) precipitated as a by-product upon formation of the active ester. The
12
CA 02087883 2010-05-28
active ester was dried to an oil and triturated with anhydrous ethyl ether.
The
precipitate was resuspended in dichloromethane and additional dicyclohexylurea
was allowed to precipitate. The resulting dichloromethane solution of active
ester was filtered and added to a previously prepared solution of cystamine
(8. 5g) .
The desired product, N,N'-bis-(CBZ)-(3-alethine precipitated from this
mixture.
The mother liquor, anhydrous ether, dichloromethane extracts of the product,
and the anhydrous ether extract of the active ester recovered above were dried
and recombined to augment the yield of product. The product was substantially
insoluble in water, hot (above about 70 C) ethyl acetate, and hot (above about
30 C) ether, and these can be used to further extract impurities. The product
can also be recrystallized from dimethyl sulfoxide with acetonitrile or water,
and again rinsed with ethyl acetate and ether. The later process results in a
1 C increase in product melting point, from 180 to 181 C (uncorrected) .
Yields
of N, N' -bis- (CBZ) -(3-alethine of up to theoretical yields are
contemplated; yields
of 85-90% of theory have been routinely obtained. When dried over P2051 in
vacuo,
the product appears to retain one mole equivalent of water, and was analyzed
accordingly as the monohydrate.
Anal. Calcd. for C26H34N406S2 H20: C, 53.78; H, 6.25; N, 9.65.
Found: C, 54.23; H, 6.56; N, 9.66. Sample analyzed by Ruby Ju, Department of
Chemistry, University of New Mexico, Albuquerque, New Mexico.
B. Deblocking of CBZ-blocked 3-alethine obtained from I.A., above
[Preparation of R-alethine 2HC1; or N,N'-bis-p-alanyl)-cystamine; or
N,N'-bis-(3-alanyl-2-aminoethyl) disulfide]
Complete removal of the carbobenzoxy group was accomplished according to
procedures described in J. Am. Chem. Soc., 86:1202-1206 (1964). After
deblocking
with four equivalents of hydrogen bromide in glacial acetic acid per mole of
the N,N'-bis-(CBZ)-(3-alethine for 15 hours, the (3-alethine was purified by
precipitation with acetonitrile, rinsing with anhydrous ethyl ether,
resuspension
in water and filtering, and precipitating the mixed salt product with
acetonitrile.
Initial yields were in excess of 80% of theoretical maximum yields. G3-
alethine
was converted to the hydrochloride salt by passing the preparation over a 30
ml X 15 cm long column of Dowex AG lX8 (chloride form) (Dow Chemical Corp.,
Midland,
MI, USA) which had been previously prepared by eluting with 1M KC1 and rinsing
thoroughly with DI (deionized) water. Neutralization with Ca(OH)2 and
recrystallization of the [3-alethine HC1 from water with acetonitrile resulted
in fine needles which melted at 224-225 C (uncorrected).
Anal. Calcd. for C10H22N402S2 2HC1: C, 32.69; H, 6.59; N, 15.25.
Found: C, 32.52; H, 6.69; N, 15.32. Sample analyzed by Ruby Ju, Department of
Chemistry, University of New Mexico, Albuquerque, New Mexico.
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CA 02087883 2010-05-28
C. Characterization of 3-alethine: [13C]-NMR; ['H]-NMR; and IR spectra of
(3-alethine
a b c d e f
S-CH2 CH2-N H-N-C=O O-C-CH2 CHZ-N -~I-H
=__-----------> H/ + H
L"C1-NXR
$-alethine 37.59 39.04 172.79 32.9 36.71 ---
E'H 1-NMR
0-alethine 2.524 3.094 2.694 3.367 ---
______--____=__________-_______ H-N-C=O
bis-(CBZ)- b-R
-3-alethine
(DMSO) 2.740 3.309 8.085 2.254 3.192 7.24
a b c d e f
IR (cm')
a b c d e f
H + H
/3-alethine 660w 3250w --- 3270v
1555w-s 2970s-w
1286= 1462s
1620s 1620s
1128s
aaa=ce~~a===~aomsmsacam~aom=~~casa=a=s~a~o~asa=x=~> H-N-C=O
bis- (CBZ) - O-R
-f3-alethine ---- 3345s --- 3345s
1545= 1535s
1640s 1270=
1682s
a b c d e f
(R is a benzyl moiety in this table.)
(3-alethine is unusual in that changes in pH (neutralization with Ca(OH)2)
cause pronounced shifts in the positions and intensities of IR bands.
Peaks (HC1 salt) : 3270s, 3170s, 2970s, 2700w, 2550w, 2020w, 1657s, 1595m,
1560s,
1450s, 1409m, 1390w, 1354w, 1325m, 1300w, shoulder/1252m/shoulder, 1188m,
1129m,
1097m, 1079w, 1030w, 950w, 905w, 829m.
Peaks (neutralized) 3250W,3180w,2940m/broad,2375s,2230s,2157s, 1936w, 1620s,
1555w, 1462s, 1432 shoulder, 1400m, 1342m, 1286m, 1217m, 1188=, 1128s, 1020m,
810w, 719m, 660w.
Bis-(CBZ)-(3-alethine displays only a few of the resonances present in
(3-alethine.
Peaks: 3345s, 3310s, 1682s, 1640s, 1545m shoulder, 1535s, 1450w, 1427w, 1375w,
1332m, 1270m, 1231=, 1178w, 1120w, 1030m/broad.
14
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II. Delay of Cellular Senescence of Fibroblasts with 0-alethine
Figure 1 shows data from a series of experiments designed to test the effect
of (3-alethine on the maximum population doubling level (PDL) of IMR-90 human
fetal lung fibroblasts . This cell line is available from the American Type
Culture
Collection (ATCC, Bethesda, MD, USA) and is used as a standard for in vitro
cellular
senescence studies. Cellular senescence is loosely defined as those cellular
process(es) that together result in the cell's inability to replenish itself
in culture. This model represents aging processes in vivo, and cell lines
developed
from humans of different ages have PDL's in vitro which are inversely related
to the chronological age of the donor. The IMR-90 cells depicted in Figure 1
were grown under ideal conditions in McCoy's 5A synthetic medium supplemented
with HEPES buffer at 10 mM; 100 units penicillin G/ml and 100 Pg
streptomycin/ml
at standard concentrations; new born calf serum (NBCS) at 20% (V/V); and
L-glutamine at 2 mM. The treated cell cultures were augmented with different
concentrations of (3-alethine (as indicated) dissolved in phosphate-buffered
saline (PBS) - always in a standard volume of 30 microliters per culture. The
P-alethine was added to the cultures at PDL 35 which is considered Phase II or
midlife of these cells in chronological terms, at the time the cells were
passaged.
The culture process comprised removing the adherent cells from their substrate
by treating the cells with 0.25% trypsin/EDTA (ethylenediamine tetreacetic
acid)
solution for two to five minutes. The cells were then washed twice with the
complete
medium and counted in a double Neubauer hemacytometer; 2 million cells were
aliquoted into a fresh tissue culture flask (T-75, polystyrenebyLUX,
FlowGeneral,
McClean, MD, USA) with 20 ml of fresh complete medium. This process was
repeated
every 48 to 72 hours when the cells reached approximately 80% surface
confluency.
Thus the cells were maintained under conditions which facilitate logarithmic
growth, i.e., between 30 and 80% surface conf luency. Under these conditions
IMR-90
cells senesce at approximately PDL 45-47 (first bar on graph). Cells treated
with (3-alethine continued to grow well beyond this point and finally senesced
in a dose-dependent manner from PDL67to PDL 101. 5. During the(3-alethine-
dependent
growth extension the cells were observed to have phenotypes similar to Phase
II fibroblasts. At the point of eventual senescence their phenotype was
similar
to that of the PDL 47 untreated control. The augmentation of growth
represented
a doubling of the life expectancy of the cells and in absolute terms
represented
an increase in cell number (biomass) by a factor of 2 raised to the power 55,
or 3.6 X 1016 fold. It was concluded that this was a differentiative
phenomenon,
based in part on the observation that the treated cells have similar
generation
times (the time required for one complete round of division) before and after
treatment with (3-alethine, beyond the normal senescence point.
CA 02087883 2010-05-28
III. Differentiation of a Peripheral Lymphoid Organ with ¾-alethine
Figure 2 illustrates data from a series of experiments designed to assess
the effect of (3-alethine on non-antigen-specific immunoglobulin synthesis and
secretion in vitro by human peripheral blood leukocytes (HPBLs), generally
characterized asa peripheral lymphoid organ primarily populated by medium-
sized,
mature lymphocytes. In these experiments, blood was taken from healthy male
humans; the blood was then defibrinated on glass beads, and the leukocytes
separated
by centrifugation (huffy coat technique). Residual red blood cells were lysed
with a brief treatment with 0.85% ammonium chloride. The leukocytes were
counted
and dispensed into 24 well tissue culture trays (LUX, Flow General, McClean,
MD, USA) in 1 ml of RPMI 1640 basal medium supplemented with penicillin,
streptomycin, 10% fetal calf serum, and L-glutamine. (3-alethine was added to
test cultures at various concentrations in 30 microliter doses. The cells were
harvested as indicated in FIG. 2 at various times between 72 and 144 hours of
culture and tested for antibody production using a conventional protein-A
facilitated plaque assay (A-PFC or Ig-PFC). In this assay, protein-A was
covalently conjugated to washed sheep red blood cells (SRBC's) using chromium
chloride in saline (6 mg/100ml) and used as target in the plaque assay. In
addition,
aliquots of cells were also tested for proliferation status by treating them
with 0. 5 pCi of tritiatedthymidine (6-9 Ci/mole) followed by assessing the
level
of incorporation of radioactivity into newly synthesized DNA. Figure 2 shows
that (3-alethine stimulated the HPBLs to produce immunoglobulin in a dose-
dependent
manner at approximately 60 times the untreated control levels. The optimal
concentration was about 5 nanograms /ml culture. The proliferation index
indicated
a low level of increased thymidine incorporation at 5 ng/ml doses. This 2- to
3-fold increase over background has miner significance as compared to truly
proliferative stimulants such as LPS (lipopoly-saccharide) of PHA
(phytohemagglutinin), which under similar conditions result in the
incorporation
of about 200,000 cpm of radioactivity, approximately 100- to 200-fold that of
control levels. It was concluded that the level of proliferation observed in
the experiment was attributable to differentiation-dependent proliferation
rather than to independent proliferative processes stimulated by P-alethine,
or to an increase in both the survival of cells and the retentive capacity for
deoxyribonucleic acids associated with viable cells.
IV. Differentiation of a Central Lymphoid Organ with P-alethine
Figure 3 shows data from a series of experiments designed to study the
effect of (3-alethine on murine splenocyte production of non-specific
immunoglobulin. The assays and culture techniques were the same as those
described
above (Example III) for the HPBL model. The animals, 4 to 6 week old female
BALBc/J
mice, were sacrificed by cervical dislocation, and their spleens aseptically
16
CA 02087883 2010-05-28
removed and pressed through 90 mesh stainless steel screens. After several
washes
with the complete medium the cells were counted and dispensed into the 24 well
trays and test cultures were dosed with the (3-alethine. These cultures were
also
harvested over various culture times of from 72 to 144 hours of culture, and
tested for antibody production and proliferation, as shown in Figure 3. Figure
3 illustrates that 3-alethine markedly stimulated the murine splenocytes to
produce immunoqlobulin in a dose-dependent manner, with an illustrated optimum
at approximately 10 ng/ml dosages. In this case there was no signif icant
stimulation
of proliferation, based on the thymidine assay described in Example III.
V. Differentiation of a Central Lymphoid Organ with P-alethine
Figure 4 shows data from a series of experiments designed to study the
effect of P-alethine on murine splenocyte production of specific types of
immunoglobulin. The assays and culture techniques were the same as those
described
in Examples III and IV for the murine splenocyte model, with the exception
that
rabbit antibodies which are specific for the gamma chain of mouse antibodies
is used to detect murine splenocytes producing IgG. The plaque assay is
described
in detail in the prior art by N.K. Jerne et al, in Transplant. Rev., 18:130-
191
(1974). The animals, 4 to 6 week old female BALBc/J mice, were sacrificed by
cervical dislocation, and their spleens aseptically removed and pressed
through
90 mesh stainless steel screens. After several washes with the complete medium
the cells were counted and dispensed into the 24 well trays (LUX, Flow
General,
McClean, MD, USA) in 1 ml of RPMI 1640 basal medium supplemented with
penicillin,
streptomycin, 10% fetal calf serum, and L-glutamine. 3-alethine was added to
test cultures at various concentrations in 30 microliter doses. The cells were
harvested as indicated in Figure 4 at various times between 72 and 144 hours
of culture and tested for antibody production using a conventional protein-A
facilitated plaque assay (A-PFC orIg-PFC).In this assay, protein-A was
covalent ly
conjugated to washed sheep red blood cells (SRBC's) using chromium chloride in
saline (6 mg/100ml), and the conjugate was used as target in the plaque assay.
Figure 4 illustrates that (3-alethine markedly stimulates the murine
splenocytes
to produce IgG in a dose-dependent manner, with an illustrated optimum at
approximately 10 ng/ml dosages and about four days into the study.
VI. Adaptation of Culture-Resistant Cells (Hepatocytes) to Culture with
P-alethine
Figure 5 illustrates data from a single experiment designed to study the
use of (3-alethine for adapting cells to culture. The culture-resistant cells
employed (hepatocytes) were taken from in vivo to in vitro growth. Murine
hepatocytes were chosen owing to their being especially difficult to adapt to
culture. It has been suggested that difficulty in adapting cells to culture is
17
CA 02087883 2010-05-28
related to the relative degree of differentiation of the selected cells, i.e.,
the more highly the cells are differentiated, the more difficult it is to
culture
them; accordingly, the hepatocyte mixed population of cell types were selected
for this experiment on the basis that these cells are highly differentiated.
In this experiment a single liver lobe from one of the BALBc/J mice was
excised,
pressed through a 90 mesh stainless steel screen, washed, and placed in T-25
tissue culture flasks (10 for LUX, Flow General, McClean, MD, USA) . All
cultures
were maintained in 10 ml of the same medium as described in Example III (RPMI-
1640
plus fetal calf serum plus pen/strep plus L-glutamine) and the test cultures
exposed to various concentrations of R-alethine as indicated in FIG. 5. Figure
shows that no colonies of hepatocytes were found in the control culture, while
approximately 50 colonies were observed in cultures treated with 10 ng/ml of
(3-alethine. It was possible to obtain some viable colonies from the control
cultures,
but only if 10- to 20-fold higher initial cell concentrations were used;
therefore
the (3-alethine was between 500- and 1000-fold more efficient in adapting
murine
hepatocytes to in vitro culture than control, i.e., saline-treated medium. In
addition, the (3-alethine-treated cells were stable in culture, in contrast to
the controlled cells which are notoriously unstable in long-term culture. The
effect is again dose-dependent, as illustrated.
VII. Inoculation of Mice with NS-1 Myeloma Cells
NS-lmyeloma cells (ATCCTIB18,P3/NSl/1-Ag4-1) were employed asinoculant;
these cells have proven to be about 90% effective in establishing myelomas in
mice according to the exemplified procedure, and the untreated myelomas are
substantially fatal within about two weeks.
The cells were grown for several passages (preferably one week) in a sterile
environment consisting of RPMI 1640 (Whittaker M.A. Bioproducts, Walkersville,
MD, USA) containing 10% fetal calf serum (Hyclone Laboratories, Logan, UT,
USA),
2mM L-glutamine, 5,000 units of penicillin, and 5 mg streptomycin in 75 cm2
polystyrene tissue-culture flasks (Corning Glassworks, Corning, NY, USA) in a
humidified chamber at 37 C and under 6% C02. To assure NS-1 propagation in
vivo
it is essential to remove DMSO (the cryostatic agent dimethyl sulfoxide)
through
several medium changes and dilutions; this also serves to maintain the cells
in log-phases growth. Female BALBc/J mice were injected i.p. with 104 cells in
0.1 ml of standard phosphate-buffered saline as soon as possible after
weaning,
transport, and indexing, as it has been found that the NS-1 cell line employed
does not generally perform optimally in animals which are mature or which have
equilibrated with their environment. The mice were maintained with Wayne
Rodent
Blox (Wayne Research Animal Diets, Chicago, IL, USA) ad lib and tap water.
18
CA 02087883 2010-05-28
VIII. Treatment of Inoculated Mice (Example VII) with A-alethine (Early
Intervention)
A. Concentrations of (3-alethine as obtained above (Example I) of 1 ng/kg,
1 pg/kg, 10 pg/kg and 100 pg/kg (based on the body weight of the inoculated
mice)
were injected i.p. in 0.1 ml physiological saline starting the second day
after
tumor inoculation (day 2), and continuing every Monday, Wednesday and Friday
through day 47. This regimen was predicated on the observation that enzymes
thought
responsive to these compounds and which may play a role in the reported
results
are induced 48 hours after chemical stimulation. The inoculated mice were
compared
to a) untreated controls and b) carrier-injected (saline-injected) controls.
B. Conclusions
(3-alethine is of fective for preventing the onset of NS-1 myeloma in, BALBc/J
mice over the concentration range from 10 pg (3-alethine/kg mouse to 100 pg/kg
mouse. Without treatment, 75% of the mice in the experiment either had to be
euthanized or died as the result of tumor development. At doses of (3-alethine
below the effective threshold (i.e., below about 10 pg/kg, or at about 10
pg/kg
or 1 ng/kg (data not illustrated for the latter) one-third to two-thirds of
the
animals ultimately contracted tumor. At dosages approaching the maximal
effective
dose (i.e., above about 10 pg/kg, or at about 10 pg/kg or 100 pg/kg), only one
mouse developed a palpable tumor, which persisted for 20 days but eventually
regressed. Figures 6-8 illustrate early and late tumor development (based on
weight of mouse not attributable to normal weight gain) in mice treated with
decreasing concentrations of P-alethine (100pg, lOpg, and 10 pg per kg mouse,
respectively). In FIG. 8, the biphasic curve in the center illustrates early
and late tumor development in these mice and corresponds to two deaths at this
dosage (10 pg/kg mouse) of (3-alethine which is the therapeutic minimal
threshold
for this compound in this model (Figure 6) . Normal weight gain of the mice is
slightly inhibited at 100 pg/kg (FIG. 6) , but not significantly at 10 ug/kg
(FIG.
7). At 10 pg/kg (FIG. 7) one mouse developed a palpable tumor which persisted
for 20 days but eventually regressed. At effective dosages of the
antineoplastic
compound 3-alethine (from about 10 pg/kg body weight to about 10 ug/kg body
weight), there is a striking difference between the weights of the nice
(reflecting
tumor burden) in the untreated control group (the vehicle-injected control
group),
compared with the mice in the treatment group.
The effectiveness of (3-alethine in the treatment of NS-1 myeloma is further
illustrated in FIG. 9, comprising a three-dimensional representation of the
study;
and in FIG. 10, comprising a 85 clockwise rotation of the illustration of
FIG.
9, with higher doses of the drug in the front to no doses of the drug in the
back. The controls inoculated with tumor (far left, FIG. 9) gain tumor and
therefore
weight (Z-axis) at an accelerated rate with respect to the day of the study (Y-
axis) .
Mice receiving high doses of (3-alethine (far right) develop at a near-normal
19
CA 02087883 2010-05-28
rate and show no signs of chronic tumor. Ridges or increases in elevation
illustrate
tumor development at the lower concentrations (farther left on the X-axis)
which
are coded for the morphology of the developing tumor (Ta = ascites and Ts =
solid
tumors) and for deaths resulting from either ascites or solid tumors (Da or Ds
respectively). Complete regression is indicated by R at the point at which the
tumor is no longer palpable. When normalized to the initial weights of the
mice
in the control group and plotted (analyzed as in FIGS. 6-10), control mice
(physiological saline injections only) displayed normal growth and development
approximating the growth and development of the mice receiving tumor and lOpg
(i-alethine/kg mouse (FIGS. 7 and 9) . This is further illustrated in FIG. 10
in
a mouse in which a tumor is modulated with different concentrations of (3-
alethine
(below). The only departure from the normal growth curve coincides with the
appearance and disappearance of a palpable tumor.
A tumor appeared and regressed without any signs of malaise in a mouse
undergoing a therapy of l0 pg/kg (3-alethine (FIG. 7) . Tumors developing in
untreated
controls with some exceptions typically promoted ascites development, while
tumors developing in mice undergoing therapy with (3-alethine were with few
exceptions solid masses; since it thus appears that P-alethine promotes
consolidation of tumor into discrete masses, (3-alethine is proj ected to be
valuable
in the design of treatment regimens involving surgical debulking.
IX. Treatment of Inoculated Mice (Example VII) with P-alethine
A single mouse developing a tumor late in the experiment (at 4 on the X-axis,
FIG. 9, approximately 40 days after the 10 pg (3-alethine/kg mouse treatment
was
discontinued) was treated with 100 }.ig (3-alethine/kg mouse to determine the
effect
of late therapeutic intervention on the treatment of the
myeloma(FIG.11).Massive
log-phase growth of tumor persisted along the right side of the mouse from
shoulder
to hip and in the abdomen for 10 to 14 days after treatment with the higher
dose
was begun, indicating pronounced infiltration of the tumor into
extraperitoneal
tissues, and a considerable lag phase before the treatment became effective.
The growth then ceased, and was followed by rapid reduction of both the tumor
mass and the tumor-dependent weight of the mouse. There was a brief period in
which the malaise subsided and the mouse's rough coat improved. This coincided
with a temporary stabilization (approximately 1 week) of the mouse's weight
suggesting that the tumor also stabilized during this time. This was followed
by another precipitous drop in the mouse's weight and obvious decrease in the
tumor masses. The mouse was euthanized when the weight returned to normal even
though a palpable mass remained in the abdomen. At the time the mouse was
euthanized,
phlebitis was evident in the extremities, possibly resulting from the
processing
of the tumor equivalent of roughly 10% of the body weight per day. After
correcting
for necrosis evident histologically, it was estimated that between 85 and 90%
CA 02087883 2010-05-28
of the original tumor was either necrotic or resorbed at the time of
euthanasia.
Considering the rate of resorption, there would have been complete regression
of the tumor if the therapy had been maintained for the full month.
Wasting of the tumor and not the mouse proper was confirmed by weighing
the debulked carcass. In this mouse, unlike untreated controls, there was no
gross evidence of infiltration of organs by the tumor, and the remaining tumor
appeared necrotic (yellowish-green and granular like an old sponge).
Histological
examination of the tissues indicated remaining tumor cells in the skeletal
muscle
adjacent to the abdominal wall, the subcutaneous tissue, and a mammary gland.
Hepatic, Urogenital, and gastrointestinal tumors, as well as a variety of
other
tumors, have been consistently observed in untreated mice, indicating the
highly
invasive and metastatic nature of the NS-1 cell line; however, in the treated
mouse, none of the remaining organs contained tumor cells. An apparent
pathological
bone fracture was observed in this mouse, but no tumor cells were evident in
the marrow of this bone. It was thus tentatively determined that the bone
demineralized due to rapid growth of the tumor resulting in the fracture, a
process
which requires calcium and phosphate, and which would also explain the
subsequent
rapid extraskeletal deposition of calcium phosphate as the tumor was resorbed.
Based on this and other studies, it is recommended that in some instances
(particularly when treating large inoperable tumors)(3-alethine initial
dosages
of about 100 pg/kg used in late intervention therapy be gradually reduced to
slow the resorptive process and permit the organism to adjust to the therapy.
Gradual decreases of dosages (on an alternating 48 hour regimen) from about
100
pg/kg down to about 1 ng/kg are suggested as the tumor responds to the
therapy.
21
