Note: Descriptions are shown in the official language in which they were submitted.
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CYTOTOXINS AND DIAGNOSTIC IMAGING
AGENTS COMPRISING HSP90 LIGANDS
RELATED APPLICATIONS
This application claims priority to Kamal et al., PCT/US02/39993, filed
December 12, 2002, entitled ASSAYS AND IMPLEMENTS FOR DETERMINING
AND MODULATING HSP90 BINDING ACTIVITY, which claims priority to
United States Provisional Patent Application Ser. No. 60/340,762, Eled
December 12,
2001, and entitled ASSAYS FOR DETERMINING HSP90 BINDING ACTIVITY,
each of which applications is herein incorporated by reference in its
entirety.
FIELD OF INVENTION
The invention relates generally to assays for assessing ligand binding and
binding affinity, and more specifically to heat shock protein 90 ("HSP90")
binding
assays.
BACKGROUND
The following description includes information that may be useful in
understanding the present invention. It is not an admission that any of the
information
provided herein is prior art or relevant to the presently claimed inventions,
or that any
publication specifically or implicitly referenced is prior art.
17-allylamino-geldanamycin (17-AAG) is a synthetic analog of geldanamycin
(GDM). Both molecules belong to a broad class of antibiotic molecules known as
ansamycins. GDM, as first isolated from the microorganism Streptomyces
hygroscopicus, was originally identified as a potent inhibitor of certain
kinases, and
was later shown to act by stimulating kinase degradation, specifically by
targeting
"molecular chaperones," e.g., heat shock protein 90s (HSP90s). Subsequently,
various
other ansamycins have demonstrated more or less such activity, with 17-AAG
being
among the most promising and the subject of intensive clinical studies
currently being
conducted by the National Cancer Institute (NCI). See, e.g., Federal Register,
66(129): 35443-35444; Erlichman et al., Proc. AACR (2001),42, abstract 4474.
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HSP90s are ubiquitous chaperone proteins that are involved in folding,
activation and assembly of a wide range of proteins, including key proteins
involved
in signal transduction, cell cycle control and transcriptional regulation.
Researchers
have reported that HSP90 chaperone proteins are associated with important
signaling
proteins, such as steroid hornone receptors and protein kinases, including,
e.g., Raf 1,
EGFR, v-Src family kinases, Cdk4, and ErbB-2 (Buchner J., 1999, TIBBS, 24:136-
141; Stepanova et al., 1996, Genes Dev. 10:1491-502; Dai, K. et al., 1996, J
Biol.
Chem, 271:22030-4).
Studies further indicate that certain co-chaperones, e.g., Hsp70,
p60/Hop/Stil,
Hip, Bagl, HSP40/Hdj2/Hsj 1, immunophilins, p23, and p50, may assist HSP90 in
its
function. See, e.g., Caplan, A., 1999, Trends in Cell Biol., 9: 262-68.
Ansamycin antibiotics, e.g., herbimycin A (HA), geldanamycin (GM), and 17-
AAG are thought to exert their anticancerous effects by tight binding of the N-
terminus pocket ofHSP90 (Stebbins, C. et al., 1997, Cell, 89:239-250). This
pocket is
highly conserved and has weak homology to the ATP-binding site of DNA gyrase
(Stebbins, C. et al., supra; Grenert, J.P. et al., 1997, J. Biol. Chem.,
272:23843-50).
Further, ATP and ADP have both been shown to bind this pocket with low
affinity
and to have weak ATPase activity (Proromou, C. et al., 1997, Cell, 90: 65-75;
Panaretou, B. et al., 1998, EMBO J., 17: 4829-36). In vitro and in vivo
studies have
demonstrated that occupancy of this N-terminal pocket by ansarnycins and other
HSP90 inhibitors alters HSP90 function and inhibits protein folding. At high
concentrations, ansamycins and other HSP90 inhibitors have been shown to
prevent
binding of protein substrates to HSP90 (Scheibel, T., H. et al., 1999, Proc.
Natl. Acad.
Sci. USA 96:1297-302; Schulte, T. W. et al., 1995, J. Bioi. Chern. 270:24585-
8;
Whitesell, L., et al., 1994, Proc. Natl. Acad. Sci. USA, 91:8324-8328).
Ansamycins
have also been demonstrated to inhibit the ATP-dependent release of chaperone-
associated protein substrates (Schneider, c., L. et aL, 1996, Proc. Natl.
Acad. Sci.
USA, 93:14536-41; Sepp-Lorenzino et al., 1995, J Biol. Chem. 270:16580-16587).
In
either event, the substrates are degraded by a ubiquitin-dependent process in
the
proteasome (Schneider, C., L., supra; Sepp-Lorenzino, L., et al., 1995, J.
Biol. Chem.,
270:16580-16587; Whitesell, L. et al., 1994, Proc. Natl. Acad. Sci. USA, 91:
8324-
8328).
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This substrate destabilization occurs in tumor and non-transformed cells alike
and has been shown to be especially effective on a subset of signaling
regulators, e.g.,
Raf (Schulte, T. W. et ai., 1997, Biochem. Biophys. Res. Commun. 239:655-9;
Schulte, T. W., et al., 1995, J. Biol. Chem. 270:24585-8), nuclear steroid
receptors
(Segnitz, B:, arid U. Gehring. 1997, J. Biol. Chem, 272:18694-18701; Srnith,
D. F. et
al., 1995, Mol. Cell. Biol. 15:6804-12), v-src (Whitesell, L., et al., 1994,
Proc. Natl.
Acad. Sci. USA, 91:8324-8328) and certain transmembrane tyrosine kinases (Sepp-
Lorenzino, L. et al., 1995, J. Biol. Chem. 270:16580-16587) such as EGF
receptor
(EGFR) and Her2/Neu (Hartmann, F., et al., 1997, Int. J. Cancer 70:221-9;
Miller, P.
et al., 1994, Cancer Res. 54:2724-2730; Mimnaugh, E. G., et al., 1996, J.
Biol. Chem.
271:22796-801; Schnur, R. et al., 1995, J. Med. Chem. 38:3806-3812), CDK4, and
mutant p53. Erlichman et al., Proc. AACR (2001), 42, abstract 4474. The
ansamycin-
induced loss of these proteins leads to the selective disruption of certain
regulatory
pathways and results in growth arrest at specific phases of the cell cycle
(Muise-
Heimericks, R. C. et al., 1998, J Biol. Chem. 273 :29864-72), and apoptosis,
and/or
differentiation of cells so treated (Vasilevskaya, A. et al., 1999, Cancer
Res., 59:3935-
40). Ansamycins and HSP90 ligands in general thus hold great promise for the
treatment and/or prevention of many types of cancers and proliferative
disorders.
In addition to anti-cancer and antitumorgenic activity, IiSP90 inhibitors have
also been implicated in a wide variety of other utilities, including use as
anti-
inflammation agents, anti-infectious disease agents, agents for treating
autoimmunity,
agents for treating ischernia, and agents useful in promoting nerve
regeneration (See,
e.g., Rosen et al., WO 02109696; PCT/USOI/23640; Degranco et al., WO 99/51223;
PCT/IJS99/07242; Gold, U.S. Patent 6,210,974 Bl). There are reports in the
literature
that fibrogenetic disorders including but not limited to scleroderma,
polymyositis,
systemic lupus, rheumatoid arthritis, liver cirrhosis, keloid formation,
interstitial
nephritis, and pulmonary fibrosis may be treatable. (Strehlow, WO 02/02123;
PCT/LTSOI/20578). Still further HSP90 modulation, modulators, and uses thereof
are
reported in PCT/LTS03/04283, PCT/US02/35938, PCT/LTS02/16287,
PCT/US02/06518, PCT/US98/09805, PCT/US00/09512, PCT/USO1/09512,
PCT/LTSOl/23640, PCT/LTSO1/46303, PCT/LTSOl/46304, PCT/LTS02/06518,
PCT/LTS02/29715, PCT/US02/35069, PCT/LTS02/35938, PCT/US02/39993,
PCTlUS03/10533, and PCT/US03/02686.
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Recently, Nicchitta et aL, WO 01/72779 (PCT/LTSOI/09512), demonstrated
that HSP90 can assume a different conformation upon heat shock and/or binding
by
the fluorophore bis-ANS. Specifically, Nicchitta et al. demonstrated that this
induced
conformation exhibits a higher affinity for certain HSP90 ligands than another
conformation that predominates in normal cells.
A fundamental step in identifying and evaluating HSP90 ligands is to be able
to conveniently assay their binding affinity for HSP90. A variety of
nonisotopic
procedures, e.g., colorimetric, enzymatic, and densitometric, afford
sufficient
sensitivity in other contexts where they are preferred over isotopic
procedures for
health and disposal reasons, and Chiosis et al., Chemistry and Biology, 8:289-
299
(2001), recently described a procedure for evaluating HSP90 ligand ability.
The
Chiosis procedure, however, is cumbersome and time-consuming from the
standpoint
of requiring gels to be run, blotted, and probed with antibody. The Chiosis
assay is
further limited in its ability to conveniently support high-throughput
screening.
Further, it appears that Chiosis employed a standard form of HSP90 that is
characteristic of normal, healthy cells.
Needed are new diagnostic agents and therapies based on selective targeting of
the HSP90 conformation that typifies cancerous, tumorous and infected cells.
SUMMARY OF THE INVENTION
The aspects and embodiments of the present invention build on those featured
in PCT/US02/39993, which reported the identification of high-affinity
conformations
of HSP90 in cancer cells and uses thereof in HSP90 binding assays and HSP90
inhibitor identification. This application features the use of such high-
affinity agents
in vitro and/or in vivo to selectively target cells containing such high
affinity forms
for, on the one hand, diagnostic and prognostic assessment, and on the other
hand for
selective therapeutics and/or prophylactic protection against diseases and
afflictions
mediated and/or characterized by high affinity forms of HSP90. Applicants'
report
herein of the selective accumulation and persistence of HSP90 inhibitors in
tumor and
cancer cells relative to normal cells suggests that these agents will find
excellent use
as both diagnostic and therapeutic agents.
Thus, in a first aspect the invention features a diagnostic or cytotoxic
composition having an HSP90 inhibitor functionalized with an additional moiety
that
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affords imaging and/or cytotoxin properties. The HSP90 ligand preferably
includes a
member selected from the group consisting of purines, ansamycins, radicicol,
zearalanols, ATP analogs, indoles, chalcones, and benzimidazoles. In some
preferred
embodiments, the inhibitor is a benzoquinoid ansamycin, preferably derivatized
at
position 17 with the cytotoxic and/or imaging agent/moiety. One embodiment for
such a compound may be generically illustrated by the following formula,
wherein X comprises the functional moiety, and wherein positions 4 and 5 are
optionally both hydroxylated.
In some preferred embodiments, the functional moiety comprises a
radioisotope/radionuclide, e.g., one selected from the group consisting of
Iodenetzs,
Iodenet3l, Technitium99m , Indiumltt, Rheniumt88, Gallium6~, Copper6~,
Yttrium9o,
and Astatine2l, ~aF, t tC, t3N, iz3h tz4h izsh t3lh zi3Bi and 150.; it is
expected that
these will find use not only as cytotoxic agents but also as imaging agents.
In other
1 S preferred embodiments, the functional moiety comprises a muslin or mustard-
type
molecule. Illustrative species of these two embodiments include compounds of
formulas
p I
0 ~
HN
HN ~ , ~zs ~HN I % ~ s NCI N
1 ~I~a 33~
Radionuclide moieties I I
Radjonuclide treatment ~
H HN
p ' N~N~CI ~ ~ ~ NCI
HN
/ ~~i F~a CI I
Radionuclide scanning (Positron Emission Tomography or PET) nitrogen mustard
or "muslin"
Common isotopes are: 18F, 11C, 13N, 1231, 124) and 150 moieties
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In addition or as an alternative to the foregoing, the functional moieties of
the
invention may also include a member selected from the group consisting of
antibodies, recombinant products, small molecules, antineoplastic agents,
herceptin,
taxol, taxanes and taxane derivatives, gleevec, alkylating agents, anti-
rnetabilites;
epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor;
procarbazine;
mitoxantrone; platinum coordination complexes; biological response
modifiers/growth inhibitors; hormonal/anti-hormonal therapeutic agents and
haematopoietic growth factors, anthracycline drugs, vinca drugs, mitomycins,
bleomycins, cytotoxic nucleosides, tepothilones, discodermolide, pteridine
drugs,
diynenes, podophyllotoxins, carminomycin, daunorubicin, aminopterin,
methotrexate,
methopterin, dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil,
6-
mercaptopurine, gemcitabine, cytosine arabinoside, podophyllotoxin, podo-
phyllotoxin derivatives, etoposide, etoposide phosphate or teniposide,
melphalan,
vinblastine, vincristine, leurosidine, vindesine, leurosine, paclitaxel,
estramustine,
carboplatin, cyclophosphamide, bleomycin, gemcitibine, ifosamide, melphalan,
hexamethyl melamine, thiotepa, cytarabin, idatrexate, trimetrexate,
dacarbazine, L-
asparaginase, camptothecin, CPT -11, topotecan, ara-C, bicalutamide,
flutamide,
leuprolide, pyridobenzoindole derivatives, interferons and interleukins.
In another aspect, the invention features a method of treating or preventing
an
HSP90-mediated disease by administering to a subject a pharmaceutically
effective
amount of a composition according to the preceding aspect and embodiments. The
disease can be a neoplastic disease or one established of fostered by a
pathogen, e.g.,
a virus, bacteria, or fungus. In some preferred embodiments, the disease is a
cancer or
tumor, preferably one selected from the group consisting of melanoma, breast,
lung,
or prostate cancer or tumor. In some embodiments, the diseased cells express
supra-
normal levels of Her-2 transcript, protein, or HSP90 client proteins. The
treatment
may be administered, e.g., orally, topically, subcutaneously, intramuscularly,
intratumorally, parenterally or intravenously.
In some embodiments, the administration is made to removed tissues or cells
of an organism (ex vivo). In other embodiments, the administration is made to
the
living organism itself (in vivo), which may entail an in situ administration
directly to
the site of a tumor and/or a general systemic administration. Preferably the
subject is a
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mammal, more preferably a human. In some embodiments, the treatment is part of
a
chemotherapy regimen.
In another aspect, the invention features a method of diagnosing or monitoring
the progression or regression of an HSP90-mediated disease by administering to
the
cells of a subject having or suspected of having an HSP90-mediated disease a
composition according to the first aspect, and then evaluating those cells for
the
presence of underlying compound. Because the compound selectively accumulates
and persists in diseased cells, it is easy to image such cells using, e.g.,
the well-known
procedure of positron emission tornography (PET) or equivalent imaging
technology.
The images created can, e.g., assist surgeons in identifying more precisely
the location
of turnorous or cancerous cells so as to excise or biopsy them. Alternatively,
the
imaging can be used to help diagnose disease or monitor its progression,
regression,
or the effect of treatment thereon. In the case of infectious diseases, normal
cells and
infected cells or tissues can be compared for the relative presence and/or
concentration of the compounds of the invention as a measure of the disease
state, or
as a measure of therapy progress.
Additional advantages, aspects, and embodiments of the invention will be
apparent from the figures, the detailed description, and claims to follow.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows competitive binding of geldanamycin and biotinylated
geldanamycin for HSP90.
Figure 2 shows competitive binding of 17-allyl amino geldanamycin (17-
AAG) and biotinylated geldanamycin for HSP90.
Figure 3 shows competitive binding of free geldanamycin, 17-AAG, and
biotinylated geldanamycin for HSP90 .
Figure 4 shows that 17-AAG (CF7) has a higher apparent binding affinity for
HSP90 from tumor cells (BT474) than normal cells (fibroblasts, RPTEC) or
purified
HSP90 alone, as determined using methods described herein.
Figure 5 shows that 17-AAG (CF7) has a higher apparent binding affinity fox
HSP90 from the specific high Her2 expressing cells, SKOY-3, SKBR-3, and N87,
than from normal cells, heat-shocked HSP90, or bis-ANS treated HSP90.
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Figure 6 shows the results of various test compounds used in certain assay
embodiments of the invention. The cell line used was MCF7. Synthesis and use
of the
modulators shown is described in PCT/US02129715.
Figure 7 shows the pharmacokinetic proftle of CF237 in a subcutaneous
human tumor xenograft and in the liver and intestine of the host mice.
Figure 8 shows the pharmacokinetic proftle of CF237 in a subcutaneous
human tumor xenograft and in the blood of the host mice.
Figure 9 shows the in vitro cellular drug uptake kinetics of CF237 in a human
tumor breast tumor cell line and in normal human epithelial and endothelial
cells.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
Compounds of the invention may embrace pharmaceutical salts of the
underlying compounds. When a compound is named or displayed, it will be
understood that pharmaceutically acceptable salts of such compound are also
subsumed within the deftnition or structure.
A "pharmaceutically acceptable salt" may be prepared for any compound of
the appropriate aspect of the invention having a functionality capable of
forming a
salt, for example an acid or base functionality. Pharmaceutically acceptable
salts may
be derived from organic or inorganic acids and bases. Compounds of the
invention
that contain one or more basic functional groups, e.g., amino or alkylamino,
are
capable of forming pharmaceutically acceptable salts with pharmaceutically
acceptable organic and inorganic acids. These salts can be prepared in situ
during the
Enal 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. Examples of
suitable
acids include hydrochloric, hydrobromic, sulfuric, nitric, perchloric,
fumaric, malefic,
phosphoric, glycolic, gluconic, lactic, salicylic, succinic, toluene-p-
sulfonic, tartaric,
acetic, citric, methanesulfonic, formic, benzoic, malonic, naphthalene-2-
sulfonic,
benzenesulfonic, 1,2 ethanesulfonic acid (edisylate), galactosyl-d-gluconic
acid, and
the like. Other acids, such as oxalic acid, while not themselves
pharmaceutically
acceptable, may be employed in the preparation of pharmaceutically acceptable
acid
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addition salts. See, e.g., Berge et al. "Pharmaceutical Salts", J. Pharm. Sci.
66:1-19
(1977).
Compounds of the present invention that contain one or more acidic functional
groups 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 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.
Illustrative
examples of some of the bases that can be used include sodium hydroxide,
potassium
hydroxide, choline hydroxide, sodium carbonate, and the like. Representative
organic
amines useful for the formation of base addition salts include ethyl amine,
diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and
the
like. See, for example, Berge et al., supra.
A "pharmacological composition" refers to a mixture of one or more of the
compounds described herein or pharmaceutically acceptable salts thereof, with
other
chemical components, such as pharmaceutically acceptable carriers and/or
excipients.
The purpose of a pharmacological composition is to facilitate administration
of a
compound to an oxganism.
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 agent 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 include: (1) sugars, such as lactose, glucose and sucrose; (2)
starches, such as
com starch and potato starch; (3) cellulose, and its derivatives, such as
sodium
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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, com oil and soybean oil; (10) glycols, such as propylene
glycol; (11)
polyols, such as glycerin, sorbitol, mannitol arid polyethylene glycol; (12)
esters, such
as ethyl oleate and ethyllaurate; (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 alcohol; (20) phosphate
buffer
solutions; and (21) other non-toxic compatible substances employed in
pharmaceutical formulations. A physiologically acceptable carrier should not
cause
significant irritation to an organism and does not abrogate the biological
activity and
properties of the administered compound.
An "excipient" refers to an inert substance added to a pharmacological
composition to further facilitate administration of a compound. Examples of
excipients include but are not limited to calcium carbonate, calcium
phosphate,
various sugars and types of starch, cellulose derivatives, gelatin, vegetable
oils and
polyethylene glycols.
A "pharmaceutically effective amount" means an amount which is capable of
providing a therapeutic and/or prophylactic effect. The specific dose of
compound
administered according to this invention to obtain therapeutic and/or
prophylactic
effect will, of course, be determined by the particular circumstances
surrounding the
case, including, for example, the specific compound administered, the route of
administration, the condition being treated, and the individual being treated.
A typical
daily dose (administered in single or divided doses) will contain a dosage
level of
from about 0.01 mg/kg to about 50-100 mg/kg of body weight of an active
compound
of the invention. Preferred daily doses generally will be from about 0.05
mg/kg to
about 20 rng/kg and ideally from about 0.1 rnglkg to about 10 mg/kg. Factors
such as
clearance rate and half life and maximum tolerated dose (MTD) have yet to be
determined but one of ordinary skill in the art can determine these using
standard
procedures.
In some method embodiments, the preferred therapeutic effect is the
inhibition, to some extent, of the growth of cells characteristic of a
proliferative
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disorder, e.g., breast cancer. A therapeutic effect will also normally, but
need not,
relieve to some extent one or more of the symptoms other than cell growth or
size of
cell mass. A therapeutic effect may include, for example, one or more of 1) a
reduction in the number of cells; 2) a reduction in cell size; 3) inhibition
(i.e., slowing
to some extent, preferably stopping) of cell infiltration into peripheral
organs, e.g., in
the instance of cancer metastasis; 3) inhibition (i.e., slowing to some
extent,
preferably stopping) of tumor metastasis; 4) inhibition, to some extent, of
cell growth;
and/or 5) relieving to some extent one or more of the symptoms associated with
the
disorder.
In some preferred embodiments of the invention, the "IC50" value of a
compound of the invention is greater for normal cells than for cells
exhibiting a
proliferative disorder, e.g., breast cancer cells. The value depends on the
assay used.
By a "standard" is meant a positive or negative control. A negative control in
the context ofHER-2 expression levels is, e.g., a sample possessing an amount
ofHER-2 protein that correlates with a normal cell. A negative control may
also
include a sample that contains no HER-2 protein. By contrast, a positive
control does
contain HER-2 protein, preferably of an amount that correlates with over-
expression
as found in proliferative disorders, e.g., breast cancers.. The controls may
be from cell
or tissue samples, or else contain purified ligand (or absent ligand),
immobilized or
otherwise. In some embodiments, one or more of the controls may be in the form
of a
diagnostic "dipstick."
By "selectively targeting" is meant affecting one type of cell to a greater
extent
than another, e.g., in the case of cells with high as opposed to relatively
low or normal
Her-2 levels.
HSP90 Li~ands
HSP90 Iigands useful in the compositions and methods of the invention
include, e.g., purines, ansamycins, radicicol, zearalanols, ATP analogs,
indoles,
chalcones, and benzimidazoles, which compounds are well-known in the art.
Preferred are ansamycins, which are known to inhibit HSP90 function. The term
"ansamycin" as used herein is well-known in the art and refers to a broad
class of
structures characterized by aliphatic rings of various length and constitution
bridging
opposite ends of aromatic ring structures and their reduced equivalents.
Subsumed
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within this broad class is the sub-class, benzoquinone ansamycins. A
"benzoquinone
ansamycinin" as used herein possesses a benzoquinone as the aromatic ring
structure
and includes any benzoquinone ansamycin known in the art having an alkoxy
moiety
on the benzoquinone portion of the molecule, preferably a methoxy, and
preferably at
the 17 position, that can be replaced by a nucleophile. The result of the
reaction is the
formation of a "benzoquinone ansamycin derivative." Ansarnycins further
include
dimers and pharmaceutically acceptable salts of the ansamyin compound.
Ansamycins may be synthetic, naturally-occurring, or a combination of the two,
i.e.,
"semi-synthetic." Exemplary ansamycins useful in the processes of the
invention and
their methods of preparation include but are not limited to those described,
e.g., in
U.S. Patents 3,595,955 (describing the preparation of geldanamycin),
4,261,989,
5,387,584, and 5,932,566. Geldanamycin is also commercially available, e.g.,
from
CN Biosciences, an affiliate of Merck headquartered in San Diego, California,
USA
(cat. no. 345805). The biochemical purification of 4,5-Dihydrogeldanamycin and
its
hydroquinone from cultures of Streptomyces hygroscopicus (ATCC 55256) are
described Cullen et al. in International Application Number PCT/US92/10189 (WO
93/14215), assigned to Pfizer Inc. An alternative method of synthesis for 4,5-
Dihydrogeldanamycin by catalytic hydrogenation of geldanamycin is also known.
See, e.g., Progress in the Chemistry of Organic Natural Products, Chemistry
a/the
Ansamycin Antibiotics, 33 1976, p. 278. Applicants recently described the
preparation of numerous other ansamycin compounds in commonly owned co-
pending applications PCT/LJS03/04283 and PCT/LJS02/29715, including the dimer
CF237 which appears in one of the examples below.
HSP90s
HSP90 proteins are ubiquitous cellular proteins that are highly conserved in
nature. The term "an HSP90" or "an HSP90 member" as claimed herein includes
but
is not limited to the following: NCBI accession #'s P07900 and XM 004515
(human a
and ~3 HSP90 respectively), P11499 (mouse), AAB2369 (rat), P46633 (chinese
hamster), JC1468 (chicken), AAF69019 (flesh fly), AAC21566 (zebrafish),
AAD30275 (salmon), 002075 (pig), NP 015084 (yeast), and CAC29071 (frog).
Further included in the definition are any variations of such proteins that
may exist in
nature or that are man- made. All are expected to have more or less utility in
connection with the methods, assays, and ligands of the invention, e.g., in
identifying
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and/or quantifying binding affinities of various HSP90 ligands, and thereby
identifying andlor prioritizing new drug candidates for clinical trials. One
aspect of
the invention exploits Applicants' finding that cancer and tumor cells possess
a more
sensitive, high affinity, form of HSP90s than do normal cells. Ligands bind
HSP90s
found in cancer or tumor cells much more avidly despite the protein itself
having an
identical amino acid constituency. Without being limiting of the invention,
this is
thought to be a consequence of a different tertiary or quarternary
configuration of the
protein that is present in such cells, possibly being afforded by co-
chaperone proteins
or client proteins that are bound to the HSP90 to make it behave as such.
The following discussion sections concerning labeling and solid supports and
high throughput screening are borrowed largely from United States Patents
6,203,989,
6,153,442, 6,096,508, 5,846,537, and 5,585,241. The procedures described
therein
and below can be assimilated, adapted, and/or otherwise implemented in
furtherance
of the novel and unobvious features of the present invention.
Labels and Labeling
The following discussion sections concerning labeling and solid supports and
high throughput screening are borrowed largely from United States Patents
6,203,989,
6,153,442, 6,096,508, 5,846,537, and 5,585,241. The procedures described
therein
and below can be assimilated, adapted, and/or otherwise implemented in
furtherance
of the novel and unobvious features of various aspects and embodiments of the
present invention.
Biotin:(Strept)Avidin Labels. Some preferred embodiments of the invention
exploit the natural high affinity of streptavidin for biotin. Streptavidin is
related to
avidin, a 67 kilodalton (kD) glycoprotein found in e~g whites and which has an
exceptionally high binding affinity (K<sub>d</sub> =l0<sup>-15</sup> M) for biotin. Avidin
consists of four subunits, each capable of binding one biotin molecule.
Streptavidin is
produced by Streptomyces avidihii and shares significant conformation and
amino
acid composition with avidin, as well as high affinity for biotin and
stability.
Streptavidin is also not glycosylated and reportedly exhibits less non-
specific binding
than avidin, making it the superior choice of the two for most biotin-based
applications.
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Biotin, a member of the B-complex vitamins, is found naturally and is
essential for amino acid and fatty acid degradation, gluconeogenesis, and
fatty acid
synthesis. The binding interactions between biotin and the biotin binding site
of
avidin and streptavidin are the result of non covalent hydrogen bonding and
van der
Waals interactions between biotin and avidin, together with the ordering of
surface
polypeptide loops that bury the biotin in the protein interior. Biotin has
previously
been chemically or enzyrnatically coupled extensively to probe biomolecules in
ways
that minimize interference with target recognition, and the results described
herein for
geldanamycin provide further examples of how this may be done.
Reagents for labeling streptavidin or avidin with a fluorescent tag are
commercially available. For example, the reagents, 5(6)-Carboxyfluorescein-N-
hydroxysuccinirnide ester (FLUOS), 7-amino-4-methyl-coumarin-3-acetic acid-N'-
hydroxysuccinimide ester (AMCA, activated) and fluorescein isothiocyanate
(FITC)
are available from Boehringer Mannheim, Indianapolis, Ind., USA. Methods for
fluorescently labeling proteins with fluorescent labels, and methods for
detection of
the fluorescent labels, are described in Howard, G., Labeling Proteins with
Fluorochromes, in "Methods in Nonradioactive Detection,", G, Howard; Ed.,
Appleton and Lange, Norwalk, Conn, USA. 1993, pp. 39-6~, the disclosure of
which
is incorporated herein. Additionally, there are a variety of commercially
available
streptavidin- and avidin-labeled molecules. Examples include streptavidin-
gold,
streptavidin-fluorochrorne, streptavidin-AMCA, streptavidin- fluorescein,
streptavidin-phycoerythrin (STPE), streptavidin-sulforhodamine 101, avidin-
FITC
and avidin-Texas red.RTM:, which are commercially available from Boehringer
Mannheim, Indianapolis, Ind., USA. A working example of the use of
streptavidin-
phycoerythrin in the methods and reagents of some aspects and embodiments of
the
invention is described below.
In addition to the biotin-steptavidin system described above, other labels or
label complexes can also be used to generate a detectable signal to relate the
amount
of bound and/or unbound label. The label can be any molecule that produces or
can be
induced to produce a signal, and may be, for example, a fluoresces, radio-
label,
enzyme, dense element such as gold, chemiluminescer or photosensitizes. Thus,
the
signal, depending on the label embodiment, can be detected and/or measured by
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detecting enzyme activity, luminescence, light absorbance or radioactivity as
the case
may be. In some applications, nonradioactive applications are preferred.
Specific labels that can be used illustratively include, e.g., enzymes such as
alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH") and
horseradish peroxidase;.dyes; fluorescers such as fluorescein, isothiocyanate,
rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and
fluorescamine; chemiluminescers such as isoluminol; sensitizers; coenzymes;
enzyme
substrates; radiolabels such as <sup>125</sup> I, <sup>l31</sup> I, <sup>l4</sup> C, <sup>3</sup> H,
<sup>57</sup> Co
and <sup>75</sup> Se; panicles such as latex, or carbon particles; metals;
crystallite;
liposomes; cells, photoactivatable compounds etc., which may be further
labeled with
a dye, catalyst or other detectable group. Other suitable enzymes and
coenzymes are
disclosed in Litman, et al., U.S. Pat. No. 4,275,149, columns 19-28, and
Boguslaski,
et al., U.S. Pat. No. 4,318,980, columns 10-14. Suitable fluorescers and
chemiluminescers are disclosed in Litman, et al., U.S. Pat. No. 4,275,149, at
columns
30 and 31. Photoactivatable compounds include but are not limited to those
containing reactive enediyne and terminal diazo groups, e.g., the natural
products
Dynemicin, Kinamycin C, and Prekinamycin, as disclosed at
http~//www Indiana edu/~zale~rt~/overview acs.html. Others include, e.g.,
UVADEX
and Methoxypsoralen (8-MOP) that are reported efficacious in extracorporeal
photochemotherapy (ECP), as well as TER286 [g-glutarnyl-a-amino-b(2-ethyl-
N,N,N',N'-tetrakis (2-chloroethyl) phos-phorodiamidate)-sulfonyl)-propionyl-
(R)-(-)
phenylglycine] (a nitrogen mustard prodrug) and 35S]-azidophenacyl-glutathione
([35S]APA-SG), chlorins, porphyrins, and phthalocyanines. See, e.g.,
htt~:l/www.ibmtindy.com./faq/photopheresis.htm;
htty//www fccc.edu/research/reports/current/tew.repoutframe.html
http ~//www.info.med.yale.eduldermatolo~/clinical/nhoto.html;
http~/lsearch msn.com/results.asp?FORM=sCPN&RS=C:HECKED&un=doc~v=1&~
~hotoactivatable°1o20dru~; Qualls et al., "Synergistic Phototoxicity of
Chloroaluminum Phthalocyanine Tetrasulfonate Delivered via Acid-Labile
Diplasmenylcholine-Folate Liposomes", International Journal of Cancer 2001,
93,
384-392. Each of the preceding is herein incorporated by reference.
The label used may directly produce a signal. Alternatively, the label may
indirectly produce a signal and therefore require additional reagents and/or
physical
CA 02549463 2006-06-12
WO 2004/054624 PCT/US2003/018776
stimulation, e.g., bombardment with electromagnetic energy or the addition of
a
chemical substrate or co-factor. In the instance of fluorescers, for example,
these are
able to absorb ultraviolet and visible light where the light absorption
transfers energy
to these molecules and elevates them to an excited energy state, which
absorbed
energy is then dissipated by emission of light at a second wavelength. By
contrast,
labels that directly produce a signal include, e.g., radioactive isotopes and
dyes.
Examples of labels that need other reagent components to produce a Signal
include, e.g., substrates and coenzymes (for enzyme labels), substances that
react with
enzymic products, catalysts, activators, cofactors, inhibitors, scavengers,
metal ions,
and a specific binding substance required for binding of signal generating
substances.
Additional discussion of suitable labeling systems can be found, e.g., in
Ullrnan, et al.,
U.S. Pat. 5,155,243, columns 11-13, the disclosure of which is herein
incorporated by
reference.
The assay methods of the invention may be conveniently performed on a solid
support, such as in multi-well plates for an ELISA or on any solid support for
high
density or chip array analysis, for example, in an ELISA type format, the
ligand or
receptor molecule is adsorbed to a solid support such as the wells of a 96-
well plate.
The corresponding complement (receptor or ligand, whichever the case may be),
is/are added to the wells and incubated. Alternatively, the complexes may
first be
formed and then adhered to the solid support, to be competed with later by
another
ligand or compound of interest. In yet another embodiment, multiple ligands,
at least
one of which is known and labeled, are mixed together in the presence of an
HSP90
and, by whichever of a broad variety of means known in the art, noncomplexed
and
nonadhered species (in solid support embodiments) are removed. The amount of
label
is then assessed using a detection device. Removal of noncomplexed and
nonadhered
species can be done by wash steps, and in some embodiments, centrifugation.
Unbound ligand/receptor is washed away and the presence of labeled complex is
then
detected. Many variations are possible.
Detection hardware devices that can be used in connection with various
embodiments of the present invention are well known in the art and include but
are
not limited to, e.g., densitometers, mass spectrometers, fluorometers,
scintillation
counters, spectrophotometers, nrnr devices, positron emission tomography (PET)
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devices, luminometers, cameras, and other imaging or detection devices. These
devices and their use are all well known in the art. For example, PET is
described in
Mandelkern et al., Positron enaission tonaography in cancer research and
treatment.
Technologies in Cancer Research and Treatment (2002) 1(6):423-39, and Zimny et
al., Positron emission tomography scanning in gynecologic and breast cancers,
Current Opinion in Obstetrics and Gynecology. (2003) 15(1):69-75.
Assays to Determine HSP90 Binding and Downstream Effect .
In addition to the innovations described herein, a variety of in vitro and in
vivo
assays are available to test the effect of the compounds of the invention on
HSP90.
HSP90 competitive binding assays and functional assays can be performed as
known
in the art substituting in the compounds of the invention. Chiosis et al.,
Chemistry &
Biology 8:289-299 (2001 ) describe some of the known ways in which this can be
done. For example, competition binding assays using, e.g., geldanamycin or 17-
AAG
as a competitive binding inhibitor ofHSP90 can be used to determine relative
HSP90
affinity of the compounds of the invention by immobilizing the compound of
interest
or other competitive inhibitor on a gel or solid matrix, preincubating HSP90
with the
other inhibitor, passing the preincubated mix over the gel or matrix, and then
measuring the amount of HSP90 that sticks or does not stick to the gel or
matrix.
Downstream effects can also be evaluated based on the known effect ofHSP90
inhibition on function and stability of various steroid receptors and
signaling proteins
including, e.g., Rafl and Her2. Compounds of the present invention induce dose-
dependent degradation of these molecules, which can be measured using standard
techniques. Inhibition of HSP90 also results in up-regulation of HSP90 and
related
chaperone proteins that can similarly be measured. Antiproliferative activity
on
various cancer cell lines can also be measured, as can morphological and
functional
differentiation related to HSP90 inhibition.
Many different types of methods are known in the art for determining protein
concentrations and measuring or predicting the level of proteins within cells
and in
fluid samples. Indirect techniques include nucleic acid hybridization and
amplification
using, e.g., polymerase chain reaction (PCR). These techniques are known to
the
person of skill and are discussed, e.g., in Sarnbrook, Fritsch & Maniatis,
Molecular
Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor
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Laboratory Press, Cold Spring Harbor, N.Y., Ausubel, et al., Current Protocols
in
Molecular Biology, John Wiley & Sons, NY, 1994, and, as specifically applied
to the
quantification, detection, and relative activity of Her-2/neu in patient
samples, e.g., in
U.S. Patents 4,699,877, 4,918,162, 4,968,603; and 5,846,749. A brief
discussion of
two generic techniques that can be used follows.
The determination of whether cells over-express or contain elevated levels of
HER- 2 can be determined using well known antibody techniques such as
immunoblotting, radioimmunoassays, western blotting, iminunoprecipitation,
enzyme-linked immunosorbant assays (ELISA), and derivative techniques that
make
use of antibodies directed against HER-2. As an example, HER-2 expression in
breast
cancer cells can be determined with the use of an immunohistochernical assay,
such as
the Dako HercepTM test (Dako Corp., Carpinteria, CA). The HercepTM test is an
antibody staining assay designed to detect HER-2 over-expression in tumor
tissue
specimens. This particular assay grades HER-2 expression into four levels: 0,
l, 2,
and 3, with level 3 representing the highest level of HER-2 expression.
Accurate
quantitation can be enhanced by employing an Automated Cellular Imaging System
(ACIS) as described, e.g., by Press, M, et al. , 2000, Modern Pathology,
13:225A.
Antibodies, polyclonal or monoclonal, can be purchased from a variety of
commercial suppliers, or may be manufactured using well-known methods, e.g.,
as
described in Harlow et al" Antibodies: A Laboratory Manual, 2nd Ed; Cold
Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988).
HER-2 over-expression can also be determined at the nucleic acid level since
there is a reported high correlation between over-expression of the HER-2
protein and
amplification of the gene that codes for it. One way to test this is by using
RT - PCR.
The genomic and cDNA sequences for HER-2 are known. Specific DNA primers can
be generated using standard, well-known techniques, and can then be used to
amplify
template already present in the cell. An example of this is described in
Kurokawa, H.
et al., Cancer Res. 60: 5887-5894 (2000). PCR can be standardized such that
quantitative differences are observed as between normal and abnormal cells,
e.g.,
cancerous and noncancerous cells. Well known methods employing, e.g.,
densitometry, can be used to quantitate and/or compare nucleic acid levels
amplified
using PCR.
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Similarly, fluorescent in situ hybridization (FISH) assays and other assays
can
be used, e.g., Northern and/or Southern blotting. These rely on nucleic acid
hybridization between the HER-2 gene or mRNA and a corresponding nucleic acid
probe that can be designed in the same or a similar way as for PCR primers,
above.
See, e.g., Mitchell, MS, and Press, MF, 1999, Semin. Oncol., Suppl. 12:108-16.
For
FISH, this nucleic acid probe can be conjugated to a fluorescent molecule,
e.g.,
fluorescein andlor rhodamine, that preferably does not interfere with
hybridization,
and which fluorescence can later be measured following hybridization. See,
e.g.,
Kurokawa, H et al., Cancer Res. 60: 5887- 5894 (2000) (describing a specific
nucleic
acid probe having sequence 5'-FAM- NucleicAcid-TAMRA-p-3' sequence). ACIS-
based approaches as described above can be employed to make the assay more
quantitative (de la Torre-Bueno, J, et al , 2000, Modem Pathology 13:221A).
Immuno and nucleic acid detection can also be directed against proteins other
than HSP90 and Her-2, which proteins are nevertheless affected in response to
HSP90
inhibition.
Pharmaceutical Compositions, Dosa~in~ and Modes of Administration
Compounds identified as promising using the assays of the invention can in
turn be formulated into pharmaceutical compositions and then administered to
subj ects.
Those of ordinary skill in the art are familiar with formulation and
administration techniques that can be employed with the compounds and methods
of
the invention, e.g., as discussed in Goodman and Gilman, The Pharmacological
Basis
of Therapeutics, current edition; Pergamon Press; and Remington Pharmaceutical
Sciences (current edition.), Mack Publishing Co., Easton, Pa., USA.
The compounds utilized in the methods of the instant invention may be
administered either alone or in combination with pharmaceutically acceptable
carriers,
excipients or diluents, in a pharmaceutical composition, according to standard
pharniaceutical practice. The compounds can be administered orally or
parenterally,
are nevertheless affected in response to HSP90 including the intravenous,
intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of
administration.
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For example, the therapeutic or pharmaceutical compositions of the invention
can be administered locally to the area in need of treatment. This may be
achieved by,
for example, but not limited to, local infusion during surgery, topical
application, e.g.,
cream, ointment, injection, catheter, or implant, said implant made, e.g., out
of a
porous, non- porous, or gelatinous material, including membranes, such as
sialastic
membranes, or fibers. The administration can also be by direct injection at
the site (or
former site) of a tumor or neoplastic or pre-neoplastic tissue.
Still further, the compounds or compositions of the invention can be delivered
in a vesicle, e.g., a liposome (see, for example, Langer, 1990, Science,
249:1527-
1533; Treat et al., 1989, Liposomes in the Therapy of Infectious Disease and
Cancer,
Lopez-Bernstein and Fidler (Eds.), Liss, N.Y., USA, pp. 353-365).
The compounds and pharmaceutical compositions used in the methods of the
present invention can also be delivered in a controlled release system. In one
embodiment, a pump may be used (see, Sefton, 1987, CRC Grit. Ref. Biomed. Eng.
14:201; Buchwald et al., 1980, Surgery, 88:507; Saudek et al., 1989, N. Eng.
J. Med.,
321:574). Additionally, a controlled release system can be placed in proximity
of the
therapeutic target (see, e.g., Goodson, 1984, Medical Applications of
Controlled
Release, Vol. 2, pp. 115-138).
The pharmaceutical compositions used in the methods of the instant invention
can also contain the active ingredient in a form suitable for oral use, for
example, as
tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders
or
granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions
intended
for oral use may be prepared according-to any method known to the art for the
manufacture of pharmaceutical compositions, and such compositions may contain
one
or more agents selected from the group consisting of sweetening agents,
flavoring
agents, coloring agents and preserving agents in order to provide
pharmaceutically
elegant and palatable preparations. Tablets contain the active ingredient in
admixture
with non-toxic pharmaceutically acceptable excipients which are suitable for
the
manufacture of tablets. These excipients may be, for example, inert diluents,
such as
calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium
phosphate; granulating and disintegrating agents, such as microcrystalline
cellulose;
sodium crosscarmellose, corn starch, or alginic acid; binding agents, for
example
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starch, gelatin, polyvinyl-pyrrolidone or acacia, and lubricating agents, for
example,
magnesium stearate, stearic acid or talc. The tablets may be un-coated or
coated by
known techniques to mask the taste of the drug or delay disintegration and
absorption
in the gastrointestinal tract and thereby provide a sustained action over a
longer
period. For example, a water soluble taste masking material such as
hydroxypropylrnethyl-cellulose or hydroxypropylcellulose, or a time delay
material
such as ethyl cellulose, or cellulose acetate butyrate may be employed as
appropriate.
Formulations for oral use may also be presented as hard gelatin capsules
wherein the active ingredient is mixed with an inert solid diluent, for
example,
calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules
wherein
the active ingredient is mixed with water soluble carrier such as
polyethyleneglycol or
an oil medium, for example peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions contain the active material in admixture with excipients
suitable for the manufacture of aqueous suspensions. Such excipients are
suspending
agents, for example sodium carboxymethyl cellulose, methylcellulose,
hydroxypropylrnethyl-cellulose, sodium alginate, polyvinyl-pyrrolidone, gum
tragacanth and gum acacia; dispersing or wetting agents may be a naturally-
occurring
phosphatide, for example lecithin, or condensation products of an alkylene
oxide with
fatty acids, for example polyoxyethylene stearate, or condensation products of
ethylene oxide with long chain aliphatic alcohols, for example
heptadecaethylene-
oxycetanol, or condensation products of ethylene oxide with partial esters
derived
from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or
condensation products of ethylene oxide with partial esters derived from fatty
acids
and hexitol aziliydrides, for example polyethylene sorbitan monooleate. The
aqueous
suspensions may also contain one or more preservatives, for example ethyl, or
n-
propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring
agents,
and one or more sweetening agents, such as sucrose, saccharin or aspartame.
Oily suspensions may be formulated by suspending the active ingredient in a
vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil,
or in
mineral oil such as liquid paraffin. The oily suspensions may contain a
thickening
agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents
such as
those set forth above, and flavoring agents may be added to provide a
palatable oral
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preparation. These compositions may be preserved by the addition of an anti-
oxidant
such as butylated hydroxyanisol or alpha-tocopherol.
Dispersible powders and granules suitable for preparation of an aqueous
suspension by the addition of water provide the active ingredient in admixture
with a
dispersing or wetting agent, suspending agent and one or more preservatives.
Suitable
dispersing or wetting agents and suspending agents are exemplified by those
already
mentioned above. Additional excipients, for example sweetening, flavoring and
coloring agents, may also be present. These compositions may be preserved by
the
addition of an anti=oxidant such as ascorbic acid.
The compounds and pharmaceutical compositions used in the methods of the
instant invention may also be in the form of oil-in-water emulsions. The oily
phase
may be a vegetable oil, for example olive oil or arachis oil, or a mineral
oil, for
example liquid paraffin or mixtures of these. Suitable emulsifying agents may
be
natural ly-occurnng phosphatides, for example soy bean lecithin, and esters or
partial
esters derived from fatty acids and hexitol anhydrides, for example sorbitan
monooleate, and condensation products of the said partial esters with ethylene
oxide,
for example polyoxyethylene sorbitan monooleate. The emulsions may also
contain
sweetening agents, flavoring agents, preservatives and antioxidants.
Syrups and elixirs may be formulated with sweetening agents, e.g., glycerol,
propylene glycol: sorbitol or sucrose. Such formulations may also contain a
demulcent, a preservative, flavoring and coloring agents and antioxidant.
The pharmaceutical compositions may be in the form of sterile injectable
aqueous solutions. Acceptable vehicles and solvents that may include, e.g.,
water,
Ringer's solution and isotonic sodium chloride solution.
The sterile injectable preparation may also be a sterile injectable oil-in-
water
microemulsion where the active ingredient is dissolved in the oily phase. For
example, the active ingredient may be first dissolved in a mixture of soybean
oil and
lecithin. The oil solution is then introduced into a water and glycerol
mixture and
processed to form a micro emulsion.
The injectable solutions or micro emulsions may be introduced into a patient's
blood-stream by local bolus injection. Alternatively, it may be advantageous
to
administer the solution or micro emulsion in such a way as to maintain a
constant
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circulating concentration of the instant compound. In order to maintain such a
constant concentration, a continuous intravenous delivery device may be
utilized. An
example of such a device is the Deltec CADD-PLUSTM model 5400 intravenous
pump.
The pharmaceutical compositions may be in the form of a sterile injectable
aqueous or oleagenous suspension for intramuscular and subcutaneous
administration.
This suspension may be formulated according to the known art using those
suitable
dispersing or wetting agents and suspending agents which have been mentioned
above. The sterile injectable preparation may also be a sterile injectable
solution or
suspension in a non-toxic parenterally-acceptable diluent or solvent, for
example as a
solution in 1,3- butane dial. In addition, sterile, fixed oils are
conventionally
employed as a solvent or suspending medium. For this purpose any bland fixed
oil
may be employed including synthetic mono- or diglycerides. In addition, fatty
acids
such as oleic acid find use in the preparation of injectables.
The Compounds of the present invention used in the methods of the present
invention may also be administered in the form of suppositories for rectal
administration of the drug. These compositions can be prepared by mixing the
inhibitors with a suitable non-irritating excipient which is solid at ordinary
temperatures but liquid at the rectal temperature and will therefore melt in
the rectum
to release the drug. Such materials include cocoa butter, glycerinated
gelatin,
hydrogenated vegetable oils, mixtures of polyethylene glycols of various
molecular
weights and fatty acid esters of polyethylene glycol.
For topical use, creams, ointments, jellies, solutions or suspensions, etc.,
containing a compound or composition of the invention can be used. As used
herein,
topical application can include, e.g., mouth washes and gargles.
The compounds used in the methods of the present invention can be
administered in intianasal form via topical use of suitable intranasal
vehicles and
delivery devices, or via trans dermal routes, using those forms of transdermal
skin
patches well known to those of ordinary skill in the art. To be administered
in the
form of a transdermal delivery
system, the dosage administration will, of course, be continuous rather than
intermittent throughout the dosage regimen.
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The methods, compounds and compositions of the instant invention may also
be used in conjunction with other well known therapeutic agents that are
selected for
their particular usefulness against the condition that is being treated. For
example, the
instant compounds may be useful in combination with known anti-cancer and
cytotoxic agents. Further, the instant methods and compounds may also be
useful in
combination with other inhibitors of parts of the signaling pathway that links
cell
surface growth factor receptors to nuclear signals initiating cellular
proliferation.
The methods of the present invention may also be useful with other agents that
inhibit angiogenesis and thereby inhibit the growth and invasiveness of tumor
cells,
including, but not limited to VEGF receptor inhibitors, including ribozymes
and
antisense targeted to VEGF receptors. angiostatin and endostatin.
Examples of antineoplastic agents that can be used in combination with the
compounds and methods of the present invention include, in general, and as
appropriate, alkylating agents, anti-metabolites; epidophyllotoxin; an
antineoplastic
enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinum
coordination complexes; biological response modifiers and growth inhibitors;
hormonallanti-hormonal therapeutic agents and haematopoietic growth factors.
Exemplary classes of antineoplastics include theanthracycline family of drugs,
the
vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the
epothilones, discodermolide, the pteridine family of drugs, diynenes and the
podophyllotoxins. Particularly useful members of those classes include, for
example,
carminomycin,. daunorubicin, aminopterin, methotrexate, methopterin,
dichloromethotrexate, mitomycin C, porfiromycin, 5-fluorouracil, 6-
mercaptopurine,
gemcitabine, cytosine arabinoside, podophyllotoxin or podo-phyllotoxin
derivatives
such as etoposide, etoposide phosphate or teniposide, melphalan, vinblastine,
vincristine, leurosidine, vindesine, leurosine, paclitaxel and the like. Other
useful
antineoplastic agents include estramustine, carboplatin, cyclophosphamide,
bleomycin, gemcitibine, ifosamide, melphalan, hexamethyl melamine, thiotepa,
cytarabin, idatrexate, trimetrexate, dacarbazine, L-asparaginase,
camptothecin, CPT-
II, topotecan, ara-C, bicalutamide, flutamide, leuprolide, pyridobenzoindole
derivatives, interferons and interleukins.
24
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When a compound or composition of the 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 patient's
symptoms.
In one exemplary application, a suitable amount of compound is administered
to a mammal undergoing treatment for cancer, for example, breast cancer.
Administration typically occurs in an amount of between about 0.01 mg/kg of
body
weight to about 100 mg/kg of body weight per day (administered in single or
divided
doses), more preferably at least about 0.1 mg/kg of body weight per day. A
particular
therapeutic dosage can include, e.g., from about 0.01 mg to about 1000 mg of
compound, and preferably includes, e.g., from about 1 mg to about 1000 mg. The
quantity of active compound in a unit of preparation may be varied or adjusted
from
about 0.1 mg to 1000 mg, preferably from about 1 mg to 300 mg, more preferably
10
mg to 200 mg, according to the particular application. The amount administered
will
vary depending on the particular IC50 value of the compound used and the
judgment
of the attending clinician taking into consideration factors such as health,
weight, and
age. In combinational applications in which the compound is not the sole
active
ingredient, it may be possible to administer lesser amounts of compound and
still have
therapeutic or prophylactic effect.
Preferably, the pharmaceutical preparation is in unit dosage form. In such
form, the preparation is subdivided into unit doses containing appropriate
quantities of
the active component, e.g., an effective amount to achieve the desired
purpose.
The actual dosage employed may be varied depending upon the requirements
of the patient and the severity of the condition being treated. Determination
of the
proper dosage for a particular situation is within the skill of the art.
Generally,
treatment is initiated with smaller dosages which are less than the optimum
dose of
the compound. Thereafter, the dosage is increased by small amounts until the
optimum effect under the circumstances is reached. For convenience, the total
daily
dosage may be divided and administered in portions during the day if desired.
The amount and frequency of administration of the compounds and
compositions of the present invention used in the methods of the present
invention,
and if applicable other chemotherapeutic agents and/or radiation therapy, will
be
CA 02549463 2006-06-12
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regulated according to the judgment of the attending clinician (physician)
considering
such factors as age, condition and size of the patient as well as severity of
the disease
being treated.
The chemotherapeutic agent and/or radiation therapy can be administered
according to therapeutic protocols well known in the art. It will be apparent
to those
skilled in the art that the administration of the chemotherapeutic agent
andlor
radiation therapy can be varied depending on the disease being treated and the
known
effects of the chemotherapeutic agent andlor radiation therapy on that
disease. Also,
in accordance with the knowledge of the skilled clinician, the therapeutic
protocols
(e.g., dosage amounts and times of administration) can be varied in view of
the
observed effects of the administered therapeutic agents (i.e., antineoplastic
agent or
radiation) on the patient, and in view of the observed responses of the
disease to the
administered therapeutic agents.
Also, in general, the compounds of the invention need not be administered in
the same pharmaceutical composition as a chemotherapeutic agent, and may,
because
of different physical and chemical characteristics, be administered by a
different
route. For example, the compounds/compositions may be administered orally to
generate and maintain good blood levels thereof, while the chemotherapeutic
agent
may be administered intravenously. The determination of the mode of
administration
and the advisability of administration, where possible, in the same
pharmaceutical
composition, is well within the knowledge of the skilled clinician. The
initial
administration can be made according to established protocols known in the
art, and
then, based upon the observed.
The particular choice of compound (and where appropriate, chemotherapeutic
agent and/or radiation) will depend upon the diagnosis of the attending
physicians and
their judgment of the condition of the patient and the appropriate treatment
protocol.
The compounds/compositions of the invention (and where appropriate
chemotherapeutic agent andlor radiation) may be administered concurrently
(e.g.,
simultaneously, essentially simultaneously or within the same treatment
protocol) or
sequentially, depending upon the nature of the proliferative disease, the
condition of
the patient, and the actual choice of chemothexapeutic agent and/or radiation
to be
26
CA 02549463 2006-06-12
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administered in conjunction (i.e., within a single treatment protocol) with
the
compound/cornposition.
In combinational applications and uses, the compound/composition and the
chemotherapeutic agent and/or radiation axe not administered simultaneously or
essentially simultaneously, then the initial order of administration of the
compound/composition, and the chemotherapeutic agent and/or radiation, may not
be
important. Thus, the compounds/compositions of the invention may be
administered
first followed by the administration of the chemotherapeutic agent and/or
radiation; or
the chernotherapeutic agent and/or radiation may be administered first
followed by the
administration of the compounds/compositions of the invention. This alternate
administration may be repeated during a single treatment protocol. The
determination
of the order of administration, and the number of repetitions of
administration of each
therapeutic agent during a treatment protocol, is well within the knowledge of
the
skilled physician after evaluation of the disease being treated and the
condition of the
patient. For example, the chemotherapeutic agent andlor radiation may be
administered first, especially if it is a cytotoxic agent, and then the
treatment
continued with the administration of the compounds/cornpositions of the
invention
followed, where determined advantageous, by the administration of the
chemotherapeutic agent and/or radiation, and so on until the treatment
protocol is
complete.
Thus, in accordance with experience and knowledge, the practicing physician
can modify each protocol for the administration of a eompound/composition for
treatment according to the individual patient's needs, as the treatment
proceeds.
The attending clinician, in judging whether treatment is effective at the
dosage
administered, will consider the general well-being of the patient as well as
more
definite signs such as relief of disease-related symptoms, inhibition of tumor
growth,
actual shrinkage of the tumor, or inhibition of metastasis. Size of the tumor
can be
measured by standard methods such as radiological studies, e.g., CAT or MRI
scan,
and successive measurements can be used to judge whether or not growth of the
tumor has been retarded or even reversed. Relief of disease-related symptoms
such as
pain, and improvement in overall condition can also be used to help evaluate
treatment efficacy.
27
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EXAMPLES
The following examples are offered by way of illustration only and not by way
of limitation. Examples 1-3 illustrate alternative methods ofproducing a
biotinylated
geldanamycin, of formulalcompound 5. Example 4 illustrates that such a
compound is
useful in competitive binding assays with other HSP90 ligands, e.g., other
ansamycins
such as geldanamycin and 17-AAG. Example 5 illustrates how HSP90s taken from
tumor cell lines more avidly bind known HSP90 ligands. Example 6 demonstrates
the
selective accumulation and persistence of CF237 (an ansamycin dimer) in mouse
and
human tumors. Example 7 illustrates prophetic examples of preparing
radionuclide-
functionalized or cytotoxin-functionalized geldanamycin derivatives.
Example 1: Synthesis of biotinylated ansamycins useful for competition binding
studies with HSP90.
This example follows the scheme:
0
NON /v I I N
O °H ~O
S N'/~0~~O'/~'N
O ~ _
2
0
NON
O
N~ ~O~
THF S o ° NH I I °
--~ ~ ~N -~' I
RT '~~'' ° off ~o
3 o w o
-
wherein the compound numbers are denoted beneath the corresponding structures;
and wherein the details of the synthesis are as follows.
To 50 mg (0.134 mmol) of (+)-biotinyl-3,6-dioxaoctanediamine 1 in 3 mL of
15:1 THF-H20 was added 29.9 (0.053 mmol) of geldanamycin 2 at room
temperature.
The reaction was stirred overnight, quenched with water (50 mL) and extracted
with 2
x 50 mL of EtOAc. The EtOAc extracts were combined, washed with 2 x 50 mL of
28
CA 02549463 2006-06-12
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H20, 1 x 50 mL of brine, dried (MgS04) and purified by silica gel flash
chromatography to give 88 mg (0.095 mmol) in 71 %yield of 3. MP 113-117
°C: MS
926 (M + Na).
Example 2: Synthesis of biotinylated ansamycins useful for competition binding
studies with HSP90.
This example follows the scheme:
0
,o
o I I o
NON N i V
N~NH2 ~ ~ O OH 'O
S O ~ O
O I
Z H~o
0
N~'N
H O
THF _~S~ o NON I I o
~'N ~~I
RT .~~'' o off 'o
o '. o
I
- NH~O
5
wherein the compound numbers are denoted beneath the corresponding structures,
and wherein the details of the synthesis are as follows.
To 50 mg (0.152 mmol) of 5-(biotinamido)pentylamine 4 in 3 mL of 15:1
THF-H20 was added 28 mg (0.050 mmol) of geldanamycin 2 at room temperature.
The reaction was stirred overnight, quenched with water (50 mL) and extracted
with 2
x 50 mL of EtOAc. The EtOAc extracts were combined, washed with 2 x 50 mL of
H20, 1 x 50 mL of brine, dried (MgS04) and purified by silica gel flash
chromatography to give 100 rng (0.114 mmol) of 5 in 75 % yield. MP 143-147
°C.
MS 880 (M + Na).
Example 3: Synthesis of biotinylated ansamycins useful for competition binding
studies with HSP90.
This example follows the scheme:
29
CA 02549463 2006-06-12
WO 2004/054624 PCT/US2003/018776
0
0
o I I
NON N ~ I
~~~"~ O OHH ~~
-~N~..O~O~O~NHz
O . ~ O
O t
6 2 = Hz o
TNF
RT
wherein the compound numbers are denoted beneath the corresponding structures
and
the details of the synthesis are as follows.
To 50 mg (0.119 rnmol) of (+)-biotinyl-3,6,9-trioxaundecanediamine 6 in 3
mL of 15:1 THF-H20 was added 26.8 mg (0.048 mmol) of geldanamycin 2 at room
temperature. The reaction was stirred overnight, quenched with water (50 mL)
and
extracted with 2 x 50 mL of EtOAc. The EtOAc extracts were combined, washed
with
2 x 50 mL of H20, 1 x 50 mL of brine, dried (MgS04) and purified by silica gel
flash
chromatography to give 84 mg (0.087 mmol) of 7 in 73°!° yield.
MP 103-104 °C. MS
970 (M + Na).
Example 4: HSP90 Binding Assay Utilizing A Biotinylated Ansamycin
Purified native HSP90 protein was coated onto 96-well plates by incubating
for 1 hr. at 37°C. Uncoated HSP90 was removed and the wells washed
twice in 1 x
PBS (phosphate-buffered saline) buffer. Compound 5 (biotinylated geldanamycin)
was then added to the wells, and the reaction was further incubated for lhr at
37°C.
The wells were washed twice with 1 x PBS, before the addition of 20ug/ml
streptavidin-phycoerythrin, and incubated for lhr at 37°C. The wells
were again
washed twice with 1 x PBS. The fluorescence was then measured in a Gemini
Spectrofluorometer (Molecular Devices Corp, Sunnyvale, CA, USA) using.an
excitation of 485nm and emission of 580 nm.
Figures 1-3 show assay embodiments employing different HSP90 ligands.
Details of the assays are as follows:
CA 02549463 2006-06-12
WO 2004/054624 PCT/US2003/018776
Figure 1. Competition of biotinylated-GM (compound S) binding by free
geldanamycin (GM). Native HSP90 that was coated onto 96-well plates was pre-
incubated with increasing concentrations of 0 nM (B3), 100 nM(C3), 300 nM
(D3),
1000 nM (E3), 3000 nM (F3), 10,000 nM (G3), and 100,000nM (closed diamonds) of
geldanamycin and then S was added. Binding of S was detected by measuring the
fluorescence of streptavidin-phycoerythrin (excitation: 48S run; emission: 510-
6S0
run). The background fluorescence without any HSP90 present (A3) was minimal.
Increasing concentrations of GM inhibits the peak fluorescence at 580 run.
Figure 2. Competition of biotinylated-GM (compound 5) binding by 17 -allyl
amino geldanamycin (17-AAG). Native Hsp90 that was coated onto 96-well plates
was pre-incubated with increasing concentrations of 0 nM (B4), 100 nM (C4),
300
nM (D4), 1000 nM (E4), 3000 nM (F4), 10,000 nM (G4), and 100,000 nM (closed
diamonds) of 17-AAG and then 5 was added. Binding of 5 was detected by
measuring the fluorescence of streptavidin-phycoerythrin (excitation: 48Snm;
1 S emission: 510-650 nm). The background fluorescence without any HSP90
present
(A4) was minimal. Increasing concentrations of 17-AAG inhibits-the peak
fluorescence at 580 nm
Figure 3. Competition ofbiotinylated-GM (compound 5) binding by
geldanamyein (GM) and 17-allyl amino geldanamycin (17-AAG). Native Hsp90 that
was coated onto 96-well plates was pre-intubated, with increasing
concentrations of 0-
100,000 nM of either GM or 17 -AAG and then S was added Binding of S was
detected by measuring the fluorescence of streptavidin-phycoerythrin
(excitation: 485
nm; emission: 580 nm). The background fluorescence without any HSP90 present
was
minimal. Increasing concentrations of GM or 17 -AAG inhibits the peak
fluorescence
2S at 580 nm.
Example 5: HSP90s taken from tumor cell lines more avidly bind HSP90 ligands
Purified native HSP90 protein (Stressgen) or cell lysates prepared in lysis
buffer (20mM Hepes, pH 7.3, 1 mM EDTA, SmM MgCl2, lOOmM I~Cl) wexe
incubated in the absence or presence of CF7 (17-AAG) or test compound for 1
Smin at
4°C. Biotin- geldanarnycin (biotin-GM) was then added to the mixture as
discussed
previously, and the reaction was further incubated by rotating for lhr at
4°C. '
BioMagTM streptavidin magnetic beads were then added to the mixture, and the
31
CA 02549463 2006-06-12
WO 2004/054624 PCT/US2003/018776
reaction was incubated by rotating for another lhr at 4°C. Tubes were
placed on a
magnetic rack, and the unbound supernatant removed. The magnetic beads were
washed three times in lysis buffer, and the washes discarded. SDS-PAGE sample
buffer was added to the beads and boiled for 5 min at 95°C. Samples
were analyzed
on 10% SDS protein gels (Novex), and then Western blots using anti-HSP90
monoclonal antibody (Stressgen SPA-830). The bands in the Western Blots were
quantitated using the Bio-rad Fluor-S Imager, and the % inhibition of binding
of CF7
or test compound calculated. The IC50 reported is the concentration of the
compound
needed to cause half maximal inhibition of binding. For experiments that
utilized
shocked HSP90, the purified HSP90 native protein was incubated for 15 min at
90°C.
For experiments that utilized bis-ANS treated HSP90, the purified HSP90
protein was
incubated with bis-ANS (Molecular Probes) for 30 min at 37°C. The
results are
shown in Figures 4-6.
Example 6: Pharmacokinetic demonstration of selective accumulation and
persistence of CF237 in human tumors relative to normal human cells and
normal mouse tissues
Figures 7 and 8 respectively show the pharmacokinetic profile of CF237 in
human tumor xenografts relative to liver/intestine and serum of the host
animals
Figure 9 shows the in vitro cellular drug uptake kinetics of CF237 in a human
breast
tumor cell line and in normal human epithelial and endothelial cells.
The preparation of CF237 is described in commonly owned application
PCT/US03/0483, as is the preparation of many other ansamycins, including many
geldanamycin derivatives that can be used in connection with various aspects
and
embodiments of the present invention.
Tumor tissue was a human tumor cell line, MDA435, obtained from the
American Type Culture Collection (ATCC), Rockville, MD, IJSA. MDA-MB-435 is
a human breast tumor line isolated in 1976 by R. Cailleau, et al. from the
pleural
effusion of a 31 year old female with metastatic, ductal adenocarcinoma of the
breast.
Cailleau R , et al. Long-term human breast carcinoma cell lines of metastatic
origin:
preliminary c~aaracterization, In Vitro 14: 911-915, 1978; PubMed: 730202.
Normal
human tissues were purchased from Clonetics, the BT474 tumor cells from ATCC.
32
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Briefly, drug was administered in a single dose of 30mg/kg body weight via
the intraperitoneal route in a formulation comprising 6 parts PEG400, 1 part
ethanol
and 3 parts Tween 80 ("PET formulation"). Test animals (3/group) were
sacrificed at
the indicated times after dosing (as indicated in Figures 7 and 8). Tumors
were
excised, minced into lmrn3 fragments and frozen at -80°C in cryovials
until ready for
use, at which time they were thawed and homogenized in acetonitrile, and the
solvent
then evaporated off under nitrogen. Extractions were then performed on the
homogenate using ethyl acetatelwater. Blood samples were taken at the times
indicated in Figure 8 by serial bleed from the retro-orbital sinus or the tail
vein. Blood
was collected into tubes, allowed to clot at room temperature for one hour and
serum
was collected as the supernatant after centrifugation at 300 X g for 5
minutes. The
usual volume of serum available for analysis from the pharmacokinetics studies
is
15~.L, although smaller volumes can be used. 485p.L acetonitrile and 15~.L
serum
sample (from each time point) are added to a l.SmL siliconized microcentrifuge
tube
and the mixture vortexed briefly at high speed. The tubes are then centrifuged
at
21,OOOxg for 10 minutes, after which the organic layer is transferred to a
glass test
tube. The tubes containing the organic phase are then placed in a Turbovap set
at
40°C for 5-10 min. and the acetonitrile blown off using 25PSI nitrogen.
Once the
tubes are totally dry, the sample is reconstituted with 150p.L of Mobile Phase
(30%
Mobile Phase B Acetonitrile w! 1%Acetic Acid and 0.5% TEA/70% Mobile Phase A
H20 w/ 1% Acetic Acid and 0.5% TEA for ansamycins , 5% Mobile Phase B
Acetonitrile with 0.05% TFA/95% Mobile Phase B H20 with 0.1% TFA). The tubes
ar well mixed and the contents then transferred to HPLC vials or 96-well
microplates
with a micro-insert. HPLC is then performed on the samples at ~160Bar using an
Agilent 1100 series device with a Zorbax 300SB-CI$ column (5uM particle size;
150 x
4.6mm), UV photodiode array detector, and the following phases of solvent (30%
Mobile Phase B Acetanitrile w/ 1 %Acetic Acid and 0.5% TEA/70% Mobile Phase A
H20 w/ 1 % Acetic Acid and 0.5% TEA for ansamycins, 5% Mobile Phase B
Acetoniixile with 0.05% TFA/95% Mobile Phase B H20 with 0.1% TFA). The
analysis takes approximately 15-25 minutes per sample, based on method. As
controls, spiked serum standards using pooled Balb/C or Nu/Nu mouse serum
containing 10, 25, 50, 500 and 5000 ng/mL of the compounds of interest were
run for
each analytical run per the following table.
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Standard ConcentrationMouse Serum1 mg/mL Dilution
(n$~~') (~,I,) Standard Scheme
(Compound dissolved
in
Mobile Phase)
5000 497.5 2.5 NA
500 1001tL Mouse
Serum +
l O 1tL 5000
ng/mL
Standard
50 200 ~L Mouse
Serum +
201tL 500
nglmL
Standard
25 SO ~I Mouse
Serum +
SO ltl 50
nglmL Standard
100 pl Mouse
Serum
20 ttl 50
ng/mL Standard
Resulting data is expressed as ng/ml or nM for serum pharmacokinetics (PK) and
as
ng drug/g tissue for tumor PK.
Figure 9. 18 x T75 of BT474 (Breast carcinoma) cells were compared to 9 x
5 T75 of RPTEC and HUVEC (normal cells). CF237 was added to flasks of these
cells
(80-90°!o confluent) at indicated times to a final concentration of luM
in 20m1
volumes. Incubations were at 37°C. Cells were harvested using Sml
trypsin/flask at
the times indicated in Figure 9 (i.e. 0.5, 2, 4, 8, 16 and 24 hours)
neutralized with Sml
medium, spun down, resuspended in 1mI PBS buffer, spun down again in an
10 Eppendorf centrifuge for lmin @ 13000 rpm, the supernatant aspirated and
the
product stored at -20°C for later analysis. Analysis was made by
suspending the
pellets in 1 mL of lysing buffer on ice for 30 min, after which centrifugation
was
performed @ 14000 Rl'M for 15 min @ 4°C, and the product suspended in 3
mL of
ethylacetate. Extracts were evaporated @ 20 psi f~r 15 min @a 30°C,
reconstituted
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WO 2004/054624 PCT/US2003/018776
in MPA (25 mM Tris-HCl pH 7.0 40% ACN) and assayed by HPLC as described
above. The results are displayed in the following table and in Figure 9:
CA 02549463 2006-06-12
WO 2004/054624 PCT/US2003/018776
CF-237 CF-237 CF-237 CF-237 CF-237 BT474-
Time Hour BT 474- RPTEC HUVEC BT BT SD
mean 474-O1 474-02
0.5 8570 203 2603 8838 8302 379.07
2 26500 894 7470 26759 26242 365.37
4 41647 _ 905 9100 42530 40764 1248.61
8 61970 1520 15483 57883 66056 5779.42
16 _1_01_90_21450 16706 77287 126517 34810.46
24 f 97677 1485 11026 94726 100628 4173.41
Example 7: Prophetic Examples of methods fox preparing radionuclide-
functionalized or cytotoxin-functionalized geldanamycin derivatives
Methods for synthesizing nuclides and intermediates are described in Boehm
et al., J. Org. Chem., 1986, 51, 5447-5450; Eng et al., Bull. Chim. Soc.
Belges, 1986,
95, 895-914; Prestwich et al., Tetrahedron 1984, 40, 529-537; Hanson et al.,
Int. J.
Appli. Radiat. Isot., 1984, 35, 810-812; Corey et al., J. Am. Chem. Soc.,
1974, 96,
5581-5583; and Bottaro et al., J. Org. Chern., 1967, 174, 424-427, Synthesis
and
Applications of Isotopically Labelled Compounds, Volume 7 Ulrich Pleiss
(Editor),
Rolf Voges (Editor), ISBN: 0-471-49501-8, January 2001; Jalilian et al. J
Pharm
Pharmaceut Sci. 2000, 3, 114-124. Two additional citations for preparing
radioactive
iodine compounds include John et al., An improved synthesis -of [125I]N-
(diethylaminoethyl)-4-iodobenzarnide: a potential ligand for imaging malignant
melanoma, Nuclear Medicine and Biology (1993), 20(1), 75-9. CODEN: NMBIEO
ISSN:0883-2897. CAN 118:212605 AN 1993:212605 CAPLUS, and Chumpradit,
S.; Kung, M. P.; Billings, J.; Mach, R.; Kung, H. F. Fluorinated and iodinated
dopamine agents: D2 imaging agents for PET and SPELT. Journal of Medicinal
Chemistry (1993), 36(2), 221-8. CODEN: JMCMAR ISSN:0022-2623. CAN
118:254670 AN 1993:254670 CAPLUS. Based on the foregoing, the following
prophetic synthetic schemes and products are hypothesized to be produced and
have
useful effect according to various aspects and embodiments of the invention.
Scheme I
36
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WO 2004/054624 PCT/US2003/018776
O OH
H2N
I I O H2N ~ I 0
'H I _Na2S204
HO ~2NOC0 I EtOAc HQ OHZ OCO
I
i0 _ i0 i0 _ i0
Bu3Sn ~ I H OH Bu3Sn ~ H '
O
Bu Sn ~ N
3 w I CI O W I O W N I I O
O
---° Y H I A~~ H I
THF, MS 4A H~ ~z OCO I MeON, SiOz HO ~2NOC0_
\ : I
i0 _ i0 i0 _ i0
1251
0
N
I I 0
NatzSi, i-izOz O N
H I
CN3COzH,~ HO ~2NOCQ
buffer, 12 h I
i0 ' ,O
37
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Scheme II
H v
~cHz)~ Nhi2 ~cH2)~~N
Bu3Sn~
Bu3Sn' v
'N
H
HO OH2NOC
TNF
- ~ 0
Na~2s~ ~z5~
-----
Scheme III
NH ~ZSI
2
~(CH2)L
125~~
THF
38
,v
CA 02549463 2006-06-12
WO 2004/054624 PCT/US2003/018776
Scheme IV
O
Ba(OH)2
THFlH20, 65°C
VIVIC
VIVIe
Tf2O ~g(OH)2
(i-Pr NEt CH CI p H
)a ~ a 2 Pd2dba3, CsBr off HzNOCO I
CsF, dioxane
Me0
vme _
OMe
Scheme V
H ~ H O
HzN~N I I O HN~N O
H I ~~CHa~ I~CHa I I N
ii
O H l
HO_ ~ZNOCO HO HZNOCO
I NaH, THF
i0 _ i0 r0 _ s0
Scheme VI
N N O ttCHs~ ~~CH3 H O
I I O -----~. /~N~N O
NaH, THF I I
'H I H I
HO ~zNOCO I HO ~~NOCO I
s0 _ i0 i0 _ i0
39
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Scheme VII
H H O
NON (Kl2.2.2JtaF- H H O
I I o ~ NON I I p
NaN, THF
o H I H I
HO HZNOCO O
I HO HZNOCO_ I
O ' \ -
TosO ~O ~O
yaF i0
([K/2.2.2J ~ BF - is an aminopolyether potassium complex [Kryotofix 2.2.2] ~
8F -
Scheme VIII
H v
,~, taF~
R~sN
Kl2.2.2Jt aF-
HO HZNOCO
NaN, TNF
i0 _. i0
/v
R = -OTs or CF3S03-
Scheme IX
H a
HZN~N I I O ~aF~N~
IaF~OTs H
HO H2NOC0
Na OEt, DMF
_ ~ ~O
40
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Scheme X
(CHa)zN a H O CF3SOa -(CHa)aN+
W I N O ~/ I H O
O I I II N O
CHzCIz, CF~S03CH3 O I I
HO H NOCO I ~ p H
HO HZNOCO
,O
i0 - ,O
'BF
H O
I N O
Kryptofixzzz. DMSO, O I I N
SepPak ~ p H
HO H2NOC0
,O
Scheme XI
H v H v
I W N I I O I ~ N I I O
(CHa)zN / O H I CFaSOa -(CHa)aN+ i O
HO HzNOCO I CN2CIz, HO H2NOC l0
O _ O CF3S~ O ~
i i s - i0
H v
N
I I I O
Krypfofixzza DMSO, ~BF
SepPak O H
HO HzNOCO
,O _ ,O
Scheme XII
41
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CF3SO3 (CH3)3N+
~aF
\ O
O H H O \ I
O N ~ N ~ N Kryptofix222, DMSO O H O H O
O
I H I 1 I I N SepPak ° I I N~N~N I I\ °
OCONH~ OH O H I I \H II H I
I , / HO HpNOCO I I OCONH~ OH HO ~aNOCO
\ I
Ow _ Ow i0 _ i0 _ / \
O~ _ O~ i0 _ i0
Scheme XIII
iCHs)zN
CF3S03
/~N~O H O
O ~ _N J1
CHzCIz, CF3SO3CH3
---
HO HZNOCO I HO OHzNOCO
0 - i0 \ _
~O
Kryptofixzz2, DMSO,
SepPak
42
i' s~
CA 02549463 2006-06-12
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Scheme XIV
~zsl
/ \
H O
~N O
O
Naizsi yz02 AcOH
> p
pN 9 buffer HO H2NOC0
_ I o
i
Scheme XV
HzN ~ n HzN
O H ~ 2 z a r H
HO H NOCO > OH
z ~ EtOAc HO HzNOCO
i0 _ i0 i0 _ i0
CI C~N CI
H OH CI ~CI
N a ~N o H O
O
CI \ I CI O \ ~ W I N
O
~H I Air ox. O N
HO OHNOCO
TNF, MS 4A z I MeOH, SiOz O H
HO HZNOCO
,O - ,O
i0 - ,Q
43
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Scheme XVI
cl
0
ie0 CIA / H O
I I O THF \ ~ I~ N
N ----~ I I p
H I ~'~
HO pHZNOCO I H2N ~ ~ N.~CI O H l
HO HzNOCO
,O _ ~p CI
,O _ i
Scheme XVII
H a
I I O TNF CI~N~N
O H I I I N O
HO HZNOCO I HZN~N-SCI CI O H
HO HZNOCO
CI
3 _ i0
,O - ,O
* * *
The foregoing examples are not limiting and are merely illustrative of various
aspects and embodiments of the present invention. All documents cited are
indicative
of the levels of skill in the art to which the invention pertains. The
reagents used,
other than those novel reagents of the invention, are commercially available
and/or
readily synthesized or acquired by one of ordinary skill in the art without
undue
effort. The disclosure of each document is incorporated by reference herein to
the
same extent as if each had been incorporated by reference in its entirety
individually,
although none of the documents is admitted to be prior art.
One skilled in the art will readily appreciate that the present invention is
well
adapted to carry out the objects and obtain the ends and advantages mentioned,
as
44
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WO 2004/054624 PCT/US2003/018776
well as those inherent therein. The methods and compositions described
illustrate
preferred embodiments, are exemplary, and are not intended as limitations on
the
scope of the invention. Certain modifications and other uses will occur to
those skilled
in the art, and are encompassed within the spirit of the invention, as defined
by the
scope of the claims.
It will be readily apparent to one skilled in the art that varying
substitutions
and modifications may be made to the invention without departing from the
scope and
spirit of the invention. Thus, such additional embodiments are within the
scope of the
invention and the following claims.
The invention illustratively described herein suitably may be practiced in the
absence of any element or elements, limitation or limitations which is not
specifically
disclosed herein. Thus, for example, while the terms "comprising", "consisting
essentially of ' and "consisting of," each carries a different meaning as a
transition
phrase, each such phrase may be used in lieu of the others to demonstrate a
different
aspect or embodiment of the invention. The terms and expressions which have
been
employed are used as terms of description and not of limitation, and there is
no
intention in the use of such terms and expressions of excluding any
equivalents of the
features shown and described, or portions thereof. It is recognized that
various
modifications are possible within the scope of the invention claimed. Thus, it
should
be understood that although the present invention has been specifically
disclosed by
preferred embodiments, optional features, modifications and variations of the
concepts herein disclosed may be resorted to by those skilled in the art, and
that such
modifications and variations are considered to be within the scope of this
invention as
defined by the description and the appended claims.
In addition, where features or aspects of the invention are described in terms
of Markush groups or other grouping of alternatives, those skilled in the art
will
recognize that the invention is also thereby described in terms of any
individual
member or subgroup of members of the Markush group or other group, and
exclusions of individual members as appropriate.
Other embodiments are within the following claims.
We claim:
