U.S. patent application number 11/109463 was filed with the patent office on 2005-10-13 for compositions and methods for promoting nerve regeneration.
This patent application is currently assigned to Oregon Health and Science University. Invention is credited to Gold, Bruce G..
Application Number | 20050226871 11/109463 |
Document ID | / |
Family ID | 25498557 |
Filed Date | 2005-10-13 |
United States Patent
Application |
20050226871 |
Kind Code |
A1 |
Gold, Bruce G. |
October 13, 2005 |
Compositions and methods for promoting nerve regeneration
Abstract
FK506 and geldanamycin promote nerve regeneration by a common
mechanism that involves the binding of these compounds to
polypeptide components of steroid receptor complexes other than the
steroid hormone binding portion of the complex (FKBP52 and hsp90,
respectively). These and other agents cause hsp90 dissociation from
steroid receptor complexes or block association of hsp90 with
steroid receptor complexes.
Inventors: |
Gold, Bruce G.; (West Linn,
OR) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 SW SALMON STREET
SUITE 1600
PORTLAND
OR
97204
US
|
Assignee: |
Oregon Health and Science
University
|
Family ID: |
25498557 |
Appl. No.: |
11/109463 |
Filed: |
April 18, 2005 |
Related U.S. Patent Documents
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Application
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Filing Date |
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11109463 |
Apr 18, 2005 |
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10656701 |
Sep 4, 2003 |
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6881409 |
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10656701 |
Sep 4, 2003 |
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09825243 |
Apr 2, 2001 |
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6641810 |
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09825243 |
Apr 2, 2001 |
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09326728 |
Jun 7, 1999 |
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09326728 |
Jun 7, 1999 |
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08956691 |
Oct 24, 1997 |
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5968921 |
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Current U.S.
Class: |
424/143.1 ;
514/10.2; 514/17.7; 514/183; 514/291; 514/8.2; 514/8.3; 514/8.4;
514/8.6; 514/9.1 |
Current CPC
Class: |
A61P 25/00 20180101;
A61K 38/1883 20130101; A61K 31/395 20130101; A61K 45/06 20130101;
Y10T 436/14 20150115; A61P 43/00 20180101; G01N 33/743 20130101;
A61K 31/4745 20130101; G01N 2500/02 20130101; G01N 33/5058
20130101; A61K 31/395 20130101; A61K 2300/00 20130101; A61K 31/4745
20130101; A61K 2300/00 20130101; A61K 38/1883 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/143.1 ;
514/183; 514/012; 514/291 |
International
Class: |
A61K 039/395; A61K
038/18; A61K 031/4745 |
Claims
1. A pharmaceutical composition comprising a nerve growth
stimulating amount of a non-FKBP12-binding agent that binds to a
polypeptide component of a steroid receptor complex other than a
steroid hormone binding portion of the complex and a
pharmaceutically acceptable excipient.
2. The composition of claim 1 wherein binding of the agent to the
polypeptide component causes hsp90 dissociation from the complex or
prevents hsp90 association with the complex.
3. The composition of claim 1 wherein the agent is selected from
the group consisting of a non-FKBP12-binding FK506 analog, a
benzoquinone ansamycin, a peptide comprising a sequence of a
selected polypeptide component of the complex at a site of
interaction between the selected component and another polypeptide
component of the complex, an antibody that binds to a polypeptide
component of the complex, and combinations thereof.
4. The composition of claim 3 wherein the benzoquinone ansamycin is
geldanamycin or a derivative thereof.
5. The composition of claim 1 wherein the agent binds to hsp90 or
FKBP52.
6. The composition of claim 1 wherein the composition further
comprises a neurotrophic factor other than the agent.
7. The composition of claim 6 wherein the neurotrophic factor is
selected from the group consisting of NGF, IGF-1, aFGF, bFGF, PDGF,
BDNF, CNTF, GDNF, NT-3, NT 4/5, and mixtures thereof.
8. The composition of claim 1 wherein the composition further
comprises a steroid hormone that is a ligand of the steroid
receptor complex.
9-13. (canceled)
14. A method of stimulating nerve cell growth in a mammal
comprising administering to a mammal the pharmaceutical composition
according to claim 1.
15. A method of stimulating nerve cell growth in a mammal
comprising administering to a mammal the pharmaceutical composition
of claim 9.
16. A method of stimulating nerve cell growth in a mammal
comprising administering to a mammal the pharmaceutical composition
of claim 12.
17. A method of stimulating nerve cell growth in a mammal
comprising administering to a mammal the pharmaceutical composition
of claim 13.
18. A method for stimulating growth of a nerve cell comprising
contacting the nerve cell with a non-FKBP 12-binding agent that
binds to a polypeptide component of a steroid receptor complex
other than a steroid hormone binding portion of the complex and
causes hsp90 dissociation from the complex or blocks association of
hsp90 with the complex.
19. A method of treating a mammal having a transected peripheral
nerve or injured spinal cord, the method comprising: administering
to the mammal a pharmaceutical composition comprising a nerve
growth stimulating amount of a non-FKBP 12-binding agent that binds
to a polypeptide component of a Page 4 of 6 steroid receptor
complex other than a steroid hormone binding portion of the complex
and a pharmaceutically acceptable excipient; and grafting to the
peripheral nerve or spinal cord an allograft or an artificial nerve
graft.
20. The method of claim 19 wherein grafting the allograft or
artificial nerve graft to the peripheral nerve or spinal cord of
the mammal produces a gap between transected ends of the peripheral
nerve or spinal cord, wherein the non-FKBP 12-binding agent is
administered by filling the gap with a gap-filling composition
comprising the non-FKBP-12-binding agent.
21. A method of identifying a compound that stimulates nerve cell
growth comprising: assaying a plurality of test compounds for
binding to a polypeptide component of a steroid receptor complex
other than a steroid hormone binding portion of the complex,
thereby identifying binding compounds; assaying the binding
compounds for stimulation of nerve cell growth.
22. The method of claim 19 further comprising assaying the binding
compounds for dissociation of hsp90 from the complex or blocking
association of hsp90 with the complex.
Description
BACKGROUND OF THE INVENTION
[0001] Following traumatic or mechanically induced axonal
degeneration in the peripheral nervous system, axonal regeneration
ensues, resulting in functional recovery. However, the rate of
axonal elongation (3-4 mm/day) is slow. Consequently, recovery is
measured in weeks or months, depending upon the distance between
the site of injury and the target tissue. Therapies that speed
regeneration over long distances would be highly beneficial to
patients and would significantly reduce health care costs.
[0002] The immunosuppressant drug FK506 (USAN tacrolimus;
Prograf.RTM.) speeds functional recovery and axonal regeneration in
the rat in a dose-dependent manner following a sciatic nerve crush
lesion (Gold et al., J. Neurosci. 15:7505-7516, 1995; Gold ec al.,
Restor. Neurol. Neurosci. 6:287-296, 1994). FK506 was shown to
stimulate neuritic outgrowth in a rat pheochromocytoma cell line in
a concentration-dependent manner (Lyons et al., Proc. Natl. Acad.
Sci. USA 91:3191-3195, 1994).
[0003] Systemic administration of two synthetic FK506 analogs that
bind FKBP-12 but that do not inhibit calcineurin activity (and
which are not immunosuppressants) increases the size of myelinated
fibers (Steiner et al., Nature Medicine 3:1-8, 1997; Steiner et
al., Proc. Natl. Acad. Sci. USA 94:2019-2024, 1997). U.S. Pat. No.
5,654,332 (Armistead et al.) discusses immunosuppressive FK506
analogs that bind FKBP12 and that are said to stimulate neurite
outgrowth in the presence of NGF. It was stated that the
neurotrophic activity of these FKBP12 binding compounds "is
directly related to their affinity for FKBP12 and their ability to
inhibit FKBP12 rotomase activity" (id. at col. 7, lines 47-50).
[0004] It has been reported that androgens and estrogens stimulate
facial nerve regeneration in hamsters (Jones, "Androgenic
enhancement of motor neuron regeneration," In: Luine and Harding,
eds., Hormonal Restructuring of the Adult Brain, Ann. N.Y. Acad.
Sci. 85:141-164, 1994; Tanzer and Jones, Exp. Neurol. 146:258-264,
1997).
SUMMARY OF THE INVENTION
[0005] I have discovered that geldanamycin and FK506 stimulate
nerve regeneration via a common mechanism. Both compounds bind to
polypeptide components of steroid receptor complexes, hsp90 and
FKBP52, respectively. These and other compounds that cause hsp90
dissociation from steroid receptor complexes or that block
association of hsp90 with steroid receptor complexes stimulate
nerve cell growth and promote nerve regeneration. Such coupounds
can act directly by binding to hsp90 (as in the case of
geldanamycin) or indirectly by binding to another polypeptide in
the steroid receptor complex (as in the case of FK506 binding of
FKBP52).
[0006] According to one aspect of the invention, pharmaceutical
compositions are provided that include a nerve growth stimulating
amount of a non-FKBP12-binding agent that binds to a polypeptide
component of a steroid receptor complex other than the ligand
(i.e., steroid hormone) binding portion thereof (such polypeptide
components including, but not limited to, hsp90 or FKBP52) and a
pharmaceutically acceptable excipient. Without limitation to any
particular mechanism of action, binding of such agents to the
polypeptide component likely causes hsp90 dissociation from the
complex or prevents hsp90 association with the complex. Nerve
growth promoting agents according to the invention include, but are
not limited to non-FKBP12-binding FK506 analogs, benzoauinone
ansamycins (e.g., geldanamycin and derivatives thereof), peptides
that comprise a sequence of a selected polypeptide component of the
complex at a site of interaction between the selected component and
another component of the complex, antibodies that bind a
polypeptide component of the steroid receptor complex, and
combinations thereof.
[0007] According to another aspect of the invention, such
pharmaceutical compositions include other active ingredients,
including, but not limited to, neurotrophic factor other than the
nerve growth promoting agent (e.g., NGF, IGF-1, aFGF, bFGF, PDGF,
BDNF, CNTF, GDNF, NT-3, NT 4/5, or mixtures thereof), and a steroid
ligand of the steroid receptor complex (e.g., estrogen and
dexamethasone, as in the Examples below).
[0008] According to another aspect of the invention, a transection
(severing of the nerve) of a periphal nerve or a spinal cord injury
of a mammal is treated by methods that include administering a
nerve growth stimulating amount of a non-FKBP12-binding nerve
growth promoting agent to the mammal and grafting to the peripheral
nerve or spinal cord an allograft or an artificial nerve graft. In
the case of a transected peripheral nerve or spinal cord, the space
between the transected ends of the peripheral nerve or spinal cord
is preferably filled with a material such as collagen, methyl
cellulose, etc., or a cell suspension that promotes nerve cell
growth, such as Schwann cells and olfactory and sheathing cells.
The nerve growth promoting agent can be included together with with
such filling materials.
[0009] According to another aspect of the invention, pharmaceutical
compositions are provided that include a nerve growth stimulating
amount of a non-FKBP12-binding FK506 analog that binds to FKBP52
and a pharmaceutically suitable excipient.
[0010] According to another aspect of the invention, pharmaceutical
compositions are provided that include a nerve growth stimulating
amount of an agent that binds to hsp90 and causes hsp90
dissociation from a steroid receptor complex or prevents hsp90
association with the complex and a pharmaceutically suitable
excipient.
[0011] According to another aspect of the invention, methods of
stimulating nerve cell growth in a mammal are provided that include
administering a pharmaceutical composition as described herein.
[0012] According to another aspect of the invention, methods are
provided for stimulating growth of a nerve cell that include
contacting the nerve cell with a non-FKBP12-binding agent that
binds to a polypeptide component of a steroid receptor complex
other than the steroid hormone binding portion thereof and causes
hsp90 dissociation from the complex or blocks association of hsp90
with the complex.
[0013] According to another aspect of the invention, methods of
identifying compounds that stimulate nerve cell growth are provided
that include the steps of assaying test compounds for binding to a
component of a steroid receptor complex other than the steroid
hormone binding portion thereof, and assaying the binding compounds
for stimulation of nerve cell growth. In addition, the binding
compounds can be assayed for activity in dissociation of hsp90 from
the complex or blocking association of hsp90 with the complex.
[0014] The foregoing and various features and advantages of the
invention will become more apparent from the following detailed
description and accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows structures of FK506 (left) and a representative
FK506 analog, V-10,367 (right). The bracketed portion of FK506
represents the calcineurin-binding domain, which is absent in
V-10,367.
[0016] FIGS. 2-8 are histograms showing the stimulation of growth
of SH-SY5Y cells by geldanamycin and FK506 in the presence of NGF
(10 ng/mL) 168 hours after treatment. FIG. 2: control cells
(untreated). FIG. 3: NGF only (10 ng/mL). FIG. 4: geldanamycin (1
nM)+NGF (10 ng/mL). FIG. 5: geldanamycin (10 nM)+NGF (10 ng/mL).
FIG. 6: FK506 (10 nM)+NGF (10 ng/mL). FIG. 7: geldanamycin (1
nM)+FK506 (10 nM)+NGF (10 ng/mL). FIG. 8: geldanamycin (10
nM)+FK506 (10 nM)+NGF (10 ng/mL).
[0017] FIGS. 9-15 are histograms showing the stimulation of growth
of SH-SY5Y cells by geldanamycin and FK506 in the presence of NGF
(10 ng/mL) 168 hours after treatment. FIG. 9: control cells
(untreated). FIG. 10: NGF only (10 ng/mL). FIG. 11: FK506 (1
nM)+NGF (10 ng/mL). FIG. 12: FK506 (10 nM)+NGF (10 ng/mL). FIG. 13:
geldanamycin (0.1 nM)+NGF (10 ng/mL). FIG. 14: geldanamycin (0.1
nM)+FK506 (1 nM)+NGF (10 ng/mL). FIG. 15: geldanamycin (0.1
nM)+FK506 (10 nM)+NGF (10 ng/mL).
DETAILED DESCRIPTION
[0018] Members of the steroid/thyroid receptor family, including
steroid receptors such as the glucocorticoid and progesterone
receptors, act as ligand-inducible enhancers of specific gene
expression.
[0019] Upon translation, steroid receptors are assembled into
multiprotein complexes. Steroid receptors exist in two states that
are in dynamic equilibrium in the cell. An initial hormone receptor
complex includes the steroid receptor, hsp90, hsp70, and at least
two co-chaperones, p60 and Hip (p48). This initial complex is
localized in the cytosol and does not bind DNA or enhance specific
gene transcription. The initial complex is in equilibrium with a
metastable, nearly mature complex that lacks hsp90, p60, and Hip
but includes p23 and one of the three large immunophilins, FKBP52
(also called hsp56), FKBP54, or CyP40, and under some
circumstances, hsp 70 (Smith et al., Mol. Cell. Biol. 15:6804-6812,
1995; Dittmar et al., J. Biol. Chem. 271:12833-12839, 1996). The
nearly mature complex is competent to bind hormone; upon hormone
binding, the receptor is released as an active transcription factor
(Smith et al., Mol. Cell. Biol. 15:6804-6812, 1995; Dittmar et al.,
J. Biol. Chem. 271:12833-12839, 1996). Steroid receptor complexes
are constantly dissociating and reforming under physiological
conditions.
[0020] Hsp90-mediated conformational maturation is required for
nuclear hormone receptors to acquire or maintain a state competent
to bind hormone (Smith et al., Mol. Cell. Biol. 15:6804-6812, 1995;
Dittmar et al., J. Biol. Chem. 271:12833-12839, 1996).
Geldanamycin, a benzoquinone ansamycin antibiotic, binds in a
pharmacologically specific manner to hsp90 (Whitesell et al., Proc.
Natl. Acad. Sci. USA 91:8324-8328, 1994) and prevents association
of the p23 component of the heterocomplex assembly system with
hsp90 (Johnson and Toft, Mol. Endocrinol. 9:670-678, 1995).
Geldanamycin permits dissociation of a steroid receptor complex,
permitting receptors to be transformed (i.e., dissociated from
hsp90), but blocks reassembly of the hormone-responsive form of the
complex, thereby preventing hormone activation and ultimately
resulting in the degradation of the hormone receptor. Geldanamycin
blocks assembly of the progesterone receptor (PR) complex (Smith et
al., Mol. Cell. Biol. 15:6804-6812, 1995) and of the glucocorticoid
receptor (GR) complex (Czar et al., Biochem. 36:7776-7785, 1997) at
an intermediate stage of assembly where the hormone binding domain
is not properly folded and therefore cannot bind steroid.
Geldanamycin also is known to act on estrogen and androgen hormone
receptors (Smith et al., Mol. Cell. Biol. 15:6804--6812, 1995; Nair
et al., Cell Stress and Chaperones 1:237-250, 1996). Transformation
of GR and PR as measured either by 9S to 4S conversion or by
acquisition of DNA-binding activity is correlated with dissociation
of steroid receptors from hsp90 (see, e.g., Meshinchi et al., J.
Biol. Chem. 265:4863-4870, 1990; Kost et al., Mol. Cell. Biol.
9:3829-3838, 1989).
[0021] In addition to steroid receptors, other "substrate proteins"
for hsp90 include v-erbA, dioxin receptor, sim, myoD1, E12, heat
shock factor, tumor promoter-specific binding protein, hepatitis B
reverse transcriptase, p53 tumor suppressor mutant, various protein
kinases (e.g., the tyrosine kinases v-src, c-src, v-fps, v-yes,
v-fes, v-frg, c-frg, lck, Weel kinase, and sevenless PTK),
heme-regulated eIF-2.alpha., eEF-2 kinase, casein kinase II, v-raf,
c-raf, Gag-Mil, MEK, PI-4 kinase, actin, tubulin, centrin,
proteasome, and G.sub..beta..gamma. complex (reviewed in Pratt and
Toft, Endocrine Rev. 18:306-360, 1997).
[0022] FKBP52 is a member of the FK506-binding class of
immunophilins. Binding of FK506 to GR-associated FKBP52 caused
increased nuclear translocation of GR in response to dexamethasone
and potentiation of GR-mediated gene expression (Sanchez and Ning,
METHODS: A Companion to Meth. Enzymol. 9:188-200, 1996).
Dexamethasone-induced GR-specific gene expression is also
potentiated by cyclosporin A (CsA) (Renoir et al., Proc. Natl.
Acad. Sci. USA 92:4977-4981, 1995), rapamycin (Ning and Sanchez, J.
Biol. Chem. 268:6073-6076, 1993), and nonimmunosuppressive analogs
of FK506 (e.g., 15-.alpha.-desmethyl FK520) or of CsA (e.g., CsH
and SDZ220384) (Sanchez and Ning, METHODS: A Companion to Meth.
Enzymol. 9:188-200, 1996).
[0023] CyP40, rather than FKBP52, is the target for binding of CsA
and its analogs (Sanchez and Ning, METHODS: A Companion to Meth.
Enzymol. 9:188-200, 1996). FKBP52 and CyP40 bind directly to hsp90,
and CyP40-competes for FKBP52 binding to hsp90 and vice versa. The
immunophilins bind hsp90 in a mutually exclusive fashion, leading
to the formation of separate CyP40-hsp90 and FKBP52-hsp90 complexes
(Ratajczak and Carrello, J. Biol. Chem. 271:2961-2965, 1996).
Immunophilins such as FKBP52 and CyP40 and non-immunophilin
proteins such as PP5, p60, and Mas70p, have one or more
tetratricopeptide repeat (TPR) domains (Ratajczak et al., J. Biol.
Chem. 268:13187-13192, 1993) that bind to the TPR-binding domain of
hsp90. The number of TPR domains in a protein appears to correlate
with its hsp90-binding affinity. Regions bordering the TPR domain
also participate in binding, e.g., the acidic domain at the
N-terminal end of bovine CyP40 (residues 185-225) and FKBP52
(residues 232-271) and the calmodulin binding region at the
C-terminus of bovine CyP40 (Ratajczak and Carrello, J. Biol. Chem.
271:2961-2965, 1996). Binding of both FKBP52 and CyD40 to hsp90 is
competed by a purified fragment of human CyP40 comprising its three
TPR domains and by a fragment of rat PPS comprising its four TPR
domains (reviewed in Pratt and Toft, Endocrine Rev. 18:306-360,
1997).
[0024] In addition to multiple TPR binding domains, FKBP52 contains
a sequence (EDLTDDED in rabbit) that is retained with conservative
replacements in human and mouse. This negatively charged sequence
is electrostatically complementary to the receptor nuclear
localization signals (e.g., the NL1 sequence RKTKKKIK of rat GR).
An antibody raised against the conserved negatively charged
sequence impeded the dexamethasone-mediated shift of the GR into
the nucleus (reviewed in Pratt and Toft, Endocrine Rev. 18:306-360,
1997). It has also been reported that antibodies directed against a
conserved negatively-charged sequence of FKBP52 impede
dexamethasone-mediated cytophasmic-nuclear translocation of GR
(Czar et al., Mol. Endocrinol. 9:1549-1560, 1995).
[0025] The effects of FK506 and geldanamycin on nerve regeneration
likely result from the binding of these compounds to components of
steroid receptor complexes, causing the dissociation of hsp90 from
the steroid receptor complex either directly (by binding to hsp90
or interfering with the binding of hsp90 to the steroid receptor)
or indirectly (by binding to a polypeptide such as FKBP52 that
itself binds to hsp90), or, alternatively, by preventing
association of hsp90 with the steroid receptor complex. However,
interference with the ability of hsp90 to complex with and perform
its chaperone function for other hsp90 substrate proteins may also
be responsible for or contribute to the observed stimulation of
nerve regeneration by FK506 and/or geldanamycin.
[0026] Definitions and Methods
[0027] "Nerve growth promoting agent" (NGPA). A "nerve growth
promoting agent" or NGPA is defined as a substance that binds to a
polypeptide component of a steroid receptor complex other than the
steroid hormone binding portion thereof, such components including
but not limited to hsp90 and FKBP52, and promotes nerve
regeneration, without limitation to a particular mechanism of
action. Preferably, the NGPA does not bind FKBP12 and is
non-immunosuppressive. NGPA include, but are not limited to,
non-FKBP12-binding ("non-binding") analogs of FK506; benzoquinone
ansamycins, including geldanamycin, naturally occurring analogs of
geldanamycin, including, but not limited to, herbimycin A and
macbecin (DeBoer et al., J. Antibiot. (Tokyo) 23:442-447, 1970;
Omura et al., J. Antibiot. (Tokyo) 32:255-261, 1979; Ono et al.,
Gann. 73:938-944, 1992), and derivatives thereof; peptides
including an amino acid sequence of a particular polypeptide
component of a steroid receptor complex at a site of interaction
between that component and another component of the complex (such
as the TPR domain), and antibodies that bind specifically to
polypeptide components of steroid receptor complexes, e.g.,
anti-hsp90, anti-FKBP52, etc.) and interfere with the interaction
of the bound polypeptide with another polypeptide in the steroid
receptor complex.
[0028] "Steroid receptor comolex" or "steroid hormone receotor" and
"component" thereof: "transformation"; "activation". The term
"steroid receptor complex" is intended to encompass a multiprotein
complex associated with any steroid receptor, including, but not
limited to, the progesterone receptor, glucocorticoid receptor,
estrogen receptor, androgen receptor, and mineralocorticoid
receptor. A polypeptide "component" is a polypeptide other than the
steroid hormone binding portion of the steroid receptor complex,
and preferably other than steroid receptor (particularly the
steroid hormone binding portion thereof), such as hsp90, FKBP52,
etc., that is part of a steroid receptor complex.
[0029] The term "transformation" refers to the conversion of the 9S
non-DNA-binding form of a steroid receptor complex to the 4S
DNA-binding form. The term "activation" refers to the conversion of
a steroid receptor from a form that does not bind steroid to a
steroid-binding form.
[0030] Assays for Identifying NGPAs. There are a number of
well-known methods for assaying compounds that bind to hsp90,
FKBP52, and other polypeptide components of a steroid receptor
complex that can be used as an initial screen for candidate
compounds that stimulate nerve regeneration. Compounds can
subsequently be tested in vitro or in vivo for activity in
stimulating nerve regeneration.
[0031] For example, one may assay for the binding of a test
compound to a polypeptide that is a component of a steroid receptor
complex. An assay for binding to hsp90 is described, for example,
by Whitesell et al. (Proc. Natl. Acad. Sci. USA 91:8324-8328,
1994). Commercial hsp90 (StressGen Biotechnologies, Victoria, BC)
dissolved in 20 .mu.g/mL of TNESV buffer (50 mM Tris-HCl, pH 7.4/1%
Nonidet P-40/2 mM EDTA/100 mM NaCl/1 mM orthovanadate/1 mM
phenylmethylsulfonyl fluoride/20 .mu.g leupeptin per mL/20 .mu.g of
aprotinin per ml) and the test compound are incubated for 45 min at
4.degree. C. with geldanamycin immobilized on a conventional solid
support, e.g., geldanamycin-coupled agarose beads (Whitesell et
al., Proc. Natl. Acad. Sci. USA 91:8324-8328, 1994. The beads are
then washed with TNESV buffer and bound hsp90 is eluted by heating
in reducing loading buffer and can be analyzed by SDS/PAGE and
silver staining (Bio-Rad), for example. Alternatively, if the hsp90
is labeled, one can assay for bound label versus free label. Test
compounds that compete with geldanamycin for binding to hsp90
inhibit the binding of solubilized hsp90 to the beads.
[0032] Similar assays can be performed to identify compounds that
bind other steroid receptor complex polypeptide components. Binding
to FKBP52 can be assayed using recombinant FKBP52 (Peattie et al.,
Proc. Natl. Acad. Sci. USA 89:10974-10978, 1992) instead of hsp90
and immobilized FK506 or FK506 analogs or hsp90. Binding to p23 can
be assayed using recombinant human p23 (Johnson et al., Mol. Cell.
Biol. 14:1956-1963, 1994) and immobilized hsp90. Purified hsp70 and
recombinant p60 (Dittmar et al., J. Biol. Chem. 271:12833-12839,
1996) are also available for use in such binding assays.
[0033] Immunoassays can also be performed using conventional
immunoassay methodologies and antibodies that are specific for
steroid receptor complex components, e.g., antibodies against
FKBP52 (Tai et al., Biochem. 25:5269-5275, 1986), hsp90 (Sanchez et
al., J. Biol. Chem. 260:12398-12401, 1985; Catelli et al., EMBO J.
4:3131-3135, 1985; Schuh et al., J. Biol. Chem. 260:14292-14296,
1985), hsp70 (a serum that also recognizes hsp90 (Erhart et al.,
Oncogene 3:595-603, 1988), p23 (Johnson et al., Mol. Cell. Biol.
14:1956-1963, 1994), etc.
[0034] A well-accepted qualitative assay for receptor
transformation, which involves dissociation of hsp90 from the
receptor complex, is conversion of a receptor complex to a state
that binds polyanions such as phosphocellulose (Kalimi et al., J.
Biol. Chem. 250:1080-1086, 1975; Atger and Milgrom, Biochem.
15:4298-4304, 1976), ATP-Sepharose (Toft et al., J. Steroid
Biochem. 7:1053-1059, 1976; Miller and Toft, Biochem. 17:173-177,
1978), and carboxymethol-Sephadex (Milgrom et al., Biochem.
12:5198-5205, 1973; Parchman and Litwack, Arch. Biochem. Biophys.
183:374-382, 1977).
[0035] An in vitro assay for nerve cell growth (neurite outgrowth)
is provided in Example 1 below. In vivo assays for nerve
regeneration are discussed in, for example, Gold et al., Restor.
Neurol. Neurosci. 6:287-296, 1994; Cold et al., J. Neurosci.
15:7505-7516, 1995; Wang et al., J. Pharmacol. Exp. Therapeutics
282:1084-1093, 1997; Gold et al., Exp. Neurol. 147:269-278, 1997;
Gold et al., Soc. Neurosci. Abst. 23:1131, 1997, which examine the
effects of systematic administration of a test compound on nerve
regeneration and functional recovery following a crush injury to
the rat sciatic nerve. Briefly stated, the right sciatic nerve of
anaesthetized rats is exposed, and the nerve crushed twice using
forceps at the level of the hip. Following the sciatic nerve crush,
the test compound is administered to the rats, e.g., by
subcutaneous injection or oral administration. Functional recovery
is assessed by determining the number of days following nerve crush
until the animal demonstrates onset of an ability to right its foot
and move its toes, and the number of days until the animal
demonstrates an ability to walk on its hind feet and toes. Nerve
regeneration is also assessed by sampling tissues from the sciatic
nerve at known (0.5 cm) distances from the crush site and examining
the number of myelinated fibers and the size of axons by light
microscopy. The axons are also examined by electron microscopy.
Axonal areas of both myelinated and unmyelinated fibers are
determined by tracing the axolemma using a digitizing tablet
connected to a computer with appropriate software. Cumulative
histograms are constructed from these data and mean values and
standard errors are calculated to assess the effect of
administration of the test compound on axonal areas.
[0036] "Geldanamycin derivatives". "Geldanamycin derivatives"
include well-known synthetic derivatives (Schnur et al., J. Med.
Chem. 38:3813-3820, 1995; Schnur et al., J. Med. Chem.
38:3806-3812, 1995). Geldanamycin derivatives preferably have the
carbamate group and ansa ring of geldanamycin, which are necessary
for activity (Schur et al., J. Med. Chem. 38:3806-3812, 1995),
including modifications at the C23 methoxy and C22 methyl groups
(Stebbins et al., Cell 89:239-250, 1997). Geldanamycin derivatives
are also discussed in U.S. Pat. Nos. 5,3877,584, 4,261,989, and
3,987,035, and in Japanese Patent Applications 88041885, 5610-0766,
and 89002593, for example.
[0037] "FK506 analogs". As used herein, the term "FK506 analogs"
refers to compounds that are functionally analogous to FK506 in
their ability to stimulate neuritic outgrowth. Such FK506 analogs,
such as V-10,367, retain the FKBP12 binding domain but lack the
structural components of the effector domain (FIG. 1) and may
either bind FKBP12 or be non-binding. V-10,367, for example, binds
FKBP12 with high affinity (<1 nM) (Armistead et al., Acca
Cryscallogr. 51:522-528, 1995).
[0038] There has been an intense effort to design compounds that
are structurally related to FK506 and that share the ability of
FK506 to inhibit FKBP12 and thereby cause immunosuppression. See,
for example: Bierer et al., Science 250:556-559, 1990; Van Duyne et
al., Science 252:839-842, 1991; Van Duyne et al., J. Mol. Biol.
229:105-124, 1993; Hauske et al., J. Med. Chem. 35:4284-4296, 1992;
Holt et al., J. Am. Chem. Soc. 115:9925-9938, 1993; Holt et al.,
Bioorg. Med. Chem. Lett. 3:1977-1980, 1993; Teague and Stocks,
Bioorg. Med. Chem. Lett. 3:1947-1950, 1993; Wang et al., Bioorg.
Med. Chem. Lett. 4:1161-1166, 1994; Yamashita et al., Bioorg. Med.
Chem. Lett. 4:325-328, 1994; Stocks et al., Bioorg. Med. Chem.
Lett. 4:1457-1460, 1994; Goulet et al., Perspect. Drug Disc. Design
2:145-162, 1994; Wilson et al., Acta Cryst. D51:511-521, 1995;
Armistead et al., Acta Cryst. D51:522-528, 1995; U.S. Pat. Nos.
5,192,773, 5,330,993, 5,516,797, 5,612,350, 5,614,547, 5,622,970,
5,654,332; and published international patent applications WO
92/00278, WO 92/04370, WO 92/19593, WO 92/21313, WO 94/07858, and
WO 96/40633.
[0039] FK506 analogs include, but are not limited to:
[0040] (1) Compounds represented by the formula I (see U.S. Pat.
Nos. 5,622,970, 5,516,797, 5,330,993, 5,192,773, and WO 92/00278
regarding synthesis of these compounds, the disclosures of which
are incorporated herein by reference): 1
[0041] wherein A is O, NH, or N--(C1-C4 alkyl);
[0042] wherein B is hydrogen, CHL-Ar, (C1-C6)-straight or branched
alkyl, (C2-C6)-straight or branched alkenyl, (C5-C7)-cycloalkyl,
(C5-C7)-cycloalkenyl or Ar substituted (C1-C6)-alkyl or
(C2-C6)-alkenyl, or 2
[0043] wherein L and Q are independently hydrogen, (C1-C6)-straight
or branched alkyl or (C2-C6)-straight or branched alkenyl;
[0044] wherein T is Ar or substituted cyclohexyl with substituents
at positions 3 and 4 that are independently selected from the group
consisting of hydrogen, hydroxyl, O--(C1-C4)-alkyl or
O--(C2-C4)-alkenyl and carbonyl;
[0045] wherein Ar is selected from the group consisting of
1-naphthyl, 2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, 2-pyridyl,
3-pyridyl, 4-pyridyl and phenyl having one to three substituents
that are independently selected from the group consisting of
hydrogen, halo, hydroxyl, nitro, CF.sub.3, (C1-C6)-straight or
branched alkyl or (C2-C6)-straight or branched alkenyl,
O--(C1-C4)-straight or branched alkyl or O--(C2-C4)-straight or
branched alkenyl, O-benzyl, O-phenyl, amino and phenyl;
[0046] wherein D is U; E is either oxygen or CH--U, provided that
if D is hydrogen, then E is CH--U or if E is oxygen, then D is not
hydrogen;
[0047] wherein each U is independently selected from hydrogen,
O--(C1-C4)-straight or branched alkyl or 0-(C2-C4)-straight or
branched alkenyl, (C1-C6)-straight or branched alkyl or
(C2-C6)-straight or branched alkenyl, (C5-C7)-cycloalkyl or
(C5-C7)-cycloalkenyl substituted with (C1-C4)-straight or branched
alkyl or (C2-C4)-straight or branched alkenyl, 2-indolyl,
3-indolyl, [(C1-C4)-alkyl or (C2-C4)-alkenyl]-Ar or Ar;
[0048] wherein J is hydrogen or C1 or C2 alkyl or benzyl; K is
(C1-C4)-straight or branched alkyl, benzyl or cyclohexylmethyl; or
wherein J and K may be taken together to form a 5-7 membered
heterocyclic ring that may contain an oxygen (O), sulfur (S), SO or
SO.sub.2 substituent therein; and
[0049] the stereochemistry at position 1 is R or S.
[0050] (2) Compounds represented by the formula II (see U.S. Pat.
No. 5,654,332, WO 94/07858, and WO 92/19593 for synthesis of these
compounds, the disclosures of which are incorporated herein by
reference): 3
[0051] wherein A' is CH.sub.2, oxygen, NH, or N--(C1-C4 alkyl);
[0052] wherein B' and W are independently hydrogen, Ar',
(C1-C10)-straight or branched alkyl, (C2-C10)-straight or branched
alkenyl or alkynyl, (C5-C7)-cycloalkyl substituted (C1-C6)-straight
or branched alkyl, (C2-C6)-straight or branched alkenyl or alkynyl,
(C5-C7)-cycloalkenyl substituted (C1-C6)-straight or branched
alkyl, (C2-C6)-straight or branched alkenyl or alkynyl, or Ar'
substituted (C1-C6)-straight or branched alkyl, (C2-C6)-straight or
branched alkenyl or alkynyl wherein in each case, any one of the
CH.sub.2 groups of the alkyl, alkenyl, or alkynyl chains may be
optionally replaced by a heteroatom selected from the group
consisting of O, S, SO, SO.sub.2, N, and NR, wherein R is selected
from the group consisting of hydrogen, (C1-C4)-straight or branched
alkyl, (C2-C4)-straight or branched alkenyl or alkynyl, and (C1-C4)
bridging alkyl wherein a bridge is formed between the nitrogen and
a carbon atom of the heteroatom-containing chain to form a ring,
and wherein the ring is optionally fused to an Ar' group, or 4
[0053] wherein Q' is hydrogen, (C1-C6)-straight or branched alkyl
or (C2-C6)-straight or branched alkenyl or alkynyl;
[0054] wherein T' is Ar or substituted 5-7 membered cycloalkyl with
substituents at positions 3 and 4 that are independently selected
from the group consisting of oxo, hydrogen, hydroxyl,
O--(C1-C4)-alkyl, and O--(C2-C4)-alkenyl;
[0055] wherein Ar' is a carboxcyclic aromatic group selected from
the group consisting of phenyl, 1-naphthyl, 2-naphthyl, indenyl,
azulenyl, fluorenyl, and anthracenyl; or a heterocyclic aromatic
group selected from the group consisting of 2-furyl, 3-furyl,
2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrrolyl,
oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl,
isothiazolyl, 1,2,3-oxadiazolyl, 1,2,3-triazolyl,
1,3,4-thiadiazolyl, pyridazinyl, pyrimidinyl, pyrazinyl,
1,3,5-triazinyl, 1,3,5-trithianyl, indolizinyl, indolyl,
isoindolyl, 3H-indolyl, indolinyl, benzo[b]furanyl,
benzo[b]thiophenyl, 1H-indazolyl, benzimidazolyl, benzthiazolyl,
purinyl, 4H-quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl,
phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl,
pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, and
phenoxazinyl;
[0056] wherein Ar' may contain one to three substituents that are
independently selected from the group consisting of hydrogen,
halogen, hydroxyl, hydroxymethyl, nitro, trifluoromethyl,
trifluoromethoxy, (C1-C6)-straight or branched alkyl,
(C2-C6)-straight or branched alkenyl, O--[(C1-C4)-straight or
branched alkyl], O--[(C2-C4)-straight or branched alkenyl],
O-benzyl, O-phenyl, 1,2-methylenedioxy, amino, carboxyl,
N--[(C1-C5)-straight or branched alkyl or (C2-C5)-straight or
branched alkenyl)carboxamides, N,N-di((C1-C5)-straight or branched
alkyl or (C2-C5)-straight or branched alkenyl]carboxamides,
N-morpholinocarboxamide, N-benzylcarboxamide,
N-thiomorpholinocarboxamide- , N-picolinoylcarboxamide, O--X,
CH.sub.2--(CH.sub.2).sub.q--X, O--(CH.sub.2).sub.q--X,
(CH.sub.2).sub.q--O--X, and CH.dbd.CH--X;
[0057] wherein X is 4-methoxyphenyl, 2-pyridyl, 3-pyridyl,
4-pyridyl, pyrazyl, quinolyl, 3,5-dimethylisoxazoyl, isoxazoyl,
2-methylthiazoyl, thiazoyl, 2-thienyl, 3-thienyl, or pyrimidyl; and
a is 0-2;
[0058] wherein G is U';
[0059] wherein M is either oxygen or CH--U'; provided that if G is
hydrogen, then M is CH--U' or if M is oxygen, then G is U';
[0060] wherein U' is hydrogen, O--[(C1-C4)-straight or branched
alkyl] or O--[(C2-C4)-straight or branched alkenyl],
(C1-C6)-straight or branched alkyl or (C2-C6)-straight or branched
alkenyl, (C5-C7)-cycloalkyl or (C5--C7)-cycloalkenyl substituted
with (C1-C4)-straight or branched alkyl or (C2-C4)-straight or
branched alkenyl, [(C1-C4)-alkyl or (C2-C4)-alkenyl]-Y or Y;
[0061] wherein Y is selected from the group consisting of phenyl,
1-naphthyl, 2-naphthyl, indenyl, azulenyl, fluorenyl, anthracenyl,
2-pyrrolinyl, 3-pyrrolinyl, pyrolidinyl, 1,3-dioxolyl,
2-imidazolinyl, imidazolidinyl, 2H-pyranyl, 4H-pyranyl, piperidyl,
1,4-dioxanyl, morpholinyl, 1,4-dithianyl, thiomorpholinyl,
piperazinyl, quinuclidinyl, and heterocyclic aromatic groups as
defined for Ar' above;
[0062] wherein Y may contain one to three substituents that are
independently selected from the group consisting of hydrogen,
halogen, hydroxyl, hydroxymethyl, nitro, trifluoromethyl,
trifluoromethoxy, (C1-C6)-straight or branched alkyl,
(C2-C6)-straight or branched alkenyl, 0-[(C1-C4)-straight or
branched alkyl], O--[(C2-C4)-straight or branched alkenyl],
O-benzyl, O-phenyl, 1,2-methylenedioxy, amino, and carboxyl;
[0063] wherein J' is hydrogen, (C1-C2) alkyl or benzyl; wherein K
is (C1-C4)-straight or branched alkyl, benzyl or cyclohexylmethyl,
or wherein J' and K may be taken together to form a 5-7 membered
heterocyclic ring that may contain a heteroatom selected from the
group consisting of O, S, SO and SO.sub.2;
[0064] wherein m is 0-3; and
[0065] wherein the stereochemistry at position 1 is R or S and the
stereochemistry at position 2 is R or S.
[0066] (3) Compounds represented by the formula III (see Armistead
et al., Acta Crysc. D51:522-528, 1995, including a discussion of
selection of R and of the synthesis of these compounds, the
disclosure of which is incorporated herein by reference) 5
[0067] (4) Compounds represented by the formula IV (see WO
92/21313, including a discussion of the synthesis of these
compounds, the disclosure of which is incorporated herein by
reference) 6
[0068] wherein A is CH.sub.2, oxygen, NH or N-- (C1-C4 alkyl);
[0069] wherein B and D are independently Ar, hydrogen,
(C1-C6)-straight or branched alkyl, (C1-C6)-straight or branched
alkenyl, (C1-C6)-straight or branched alkyl or alkenyl that is
substituted with a (C5-C7)-cycloalkyl, (C1-C6)-straight or branched
alkyl or alkenyl that is substituted with a (C5-C7)-cycloalkenyl,
or Ar substituted (C1-C6)-straight or branched alkyl or alkenyl,
wherein, in each case, one or two of the CH.sub.2 groups of the
alkyl or alkenyl chains may contain 1-2 heteroatoms selected from
the group consisting of oxygen, sulfur, SO and SO.sub.2 in
chemically reasonable substitution patterns, or 7
[0070] provided that both B and D are not hydrogen;
[0071] wherein Q is hydrogen, (C1-C6)-straight or branched alkyl or
(C1-C6)-straight or branched alkenyl;
[0072] wherein T is Ar or substituted 5-7 membered cycloalkyl with
substituents at positions 3 and 4 that are independently selected
from the group consisting of hydrogen, hydroxyl, O--(C1-C4)-alkyl,
O--(C1-C4)-alkenyl and carbonyl;
[0073] wherein Ar is selected from the group consisting of phenyl,
1-naphthyl, 2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl,
2-pyridyl, 3-pyridyl, 4-pyridyl, monocyclic and bicyclic
heterocyclic ring systems with individual ring sizes being 5 or 6
that may contain in either or both rings a total of 1-4 heteroatoms
independently selected from O, N and S; wherein Ar may contain one
to three substituents that are independently selected from the
group consisting of hydrogen, halo, hydroxyl, nitro,
trifluoromethyl, trifluoromethoxy, (C1-C6)-straight or branched
alkyl, (C2-C6)-straight or branched alkenyl, O--(C1-C4)-straight or
branched alkyl, O--(C2-C4)-straight or branched alkenyl, O-benzyl,
O-phenyl, 1,2-methylenedioxy, amino, carboxyl and phenyl;
[0074] wherein E is (C1-C6)-straight or branched alkyl,
(C1-C6)-straight or branched alkenyl, (C5-C7)-cycloalkyl,
(C5-C7)-cycloalkenyl substituted with (C1-C4)-straight or branched
alkyl or (C1-C4)-straight or branched alkenyl, [(C2-C4)alkyl or
(C2-C4)-alkenyl)]-Ar or Ar (Ar as described above);
[0075] wherein J is hydrogen or C1 or C2 alkyl or benzyl; K is
(C1-C4)-straight or branched alkyl, benzyl or cyclohexylmethyl; or
wherein J and K may be taken together to form a 5-7 membered
heterocyclic ring that may contain an oxygen, sulfur, SO or
SO.sub.2 substituent therein; and;
[0076] wherein n is 0-3; and
[0077] wherein the stereochemistry at position 1 is R or S and the
stereochemistry at position 2 being R or S.
[0078] (5) Compounds represented by the formula V (see WO 92/04370,
including a discussion of the synthesis of these compounds, the
disclosure of which is incorporated herein by reference): 8
[0079] wherein A is NH, O, S, or CH;
[0080] wherein if A is NH, O, or S, B is PCO-- or POCO--, where P
is a C1-C6 straight or branched alkyl or alkenyl group, a C5-C6
cycloalkyl or cycloalkenyl, or a methyl substituted with a C5--C6
cycloalkyl, C5-C6 cycloalkenyl, phenyl, 1-naphthyl, 2-naphthyl,
9-fluorenyl, or 1-adamantyl;
[0081] wherein if A is CH, then B is connected via a trans double
bond and is a C2-C4 straight or branched alkyl or alkenyl group, or
is a methyl or ethyl substituted with either a C5-C6 cyclic alkyl
group or Ar, where Ar is selected from the group consisting of
1-naphthyl, 2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, phenyl and
phenyl having one to three substituents that are independently
selected from the group consisting of: hydroxyl, halo, nitro,
CF.sub.3, C1-C4 straight or branched alkyl or alkenyl, O--(C1-C4)
straight or branched alkyl or alkenyl, and Ar, where Ar is selected
from the group consisting of 1-naphthyl, 2-naphthyl, 2-furyl,
3-furyl, 2-thienyl, phenyl and phenyl having one to three
substituents that are independently selected from the group
consisting of: hydroxyl, halo, nitro, CF.sub.3, C1-C4 straight or
branched alkyl or alkenyl, O--(C1-C4) straight or branched alkyl or
alkenyl; wherein no more than two Ar groups may be linked
together;
[0082] wherein D is hydrogen, C1-C4 straight or branched alkyl or
alkenyl, hydroxy, tert-butyloxy, benzyloxy, 4-benzyloxyphenyl,
cyclohexyl, --(CH.sub.2).sub.n--CO.sub.2-Q, where n=0 or 1 and Q is
methyl, ethyl, i-propyl, t-butyl, benzyl, 1-naphthyl, 2-naphthyl,
or cyclohexyl; or Ar, where Ar is selected from the group
consisting of 1-naphthyl, 2-naphthyl, 2-furyl, 3-furyl, 2-thienyl,
phenyl and phenyl having one to three substituents that are
independently selected from the group consisting of: hydroxyl,
halo, nitro, CF.sub.3, C1-C4 straight or branched alkyl or alkenyl,
O--(C1-C4) straight or branched alkyl or alkenyl, and Ar, where Ar
is selected from the group consisting of 1-naphthyl, 2-naphthyl,
2-furyl, 3-furyl, 2-thienyl, phenyl and phenyl having one to three
substituents that are independently selected from the group
consisting of: hydroxyl, halo, nitro, CF.sub.3, C1-C4 straight or
branched alkyl or alkenyl, O--(C1-C4) straight or branched alkyl or
alkenyl; wherein no more than two Ar groups may be linked
together;
[0083] wherein E and K are independently hydrogen or methyl;
[0084] wherein G is either methyl or ethyl; J is hydrogen, C1-C6
straight or branched alkyl or alkenyl, C6-C6 cycloalkyl or
cycloalkenyl, sulfhydryl, hydroxy, phenyl, 3-indolyl, or benzyl;
wherein G and J may be connected by a bond to form a cycle of 5 or
6 members;
[0085] wherein L is O or an .alpha.-amino acid residue attached via
the .alpha.-nitrogen, and selected from the group consisting of:
alanine, 2-aminobutyric acid, valine, norvaline, leucine,
norleucine, isoleucine, phenylalanine, cyclohexylalanine,
tryptophan, 1-naphthylalanine, 2-naphthylalanine, threonine (side
chain benzyl or tert-butyl ether), methionine, or serine (side
chain benzyl or tert-butyl ether);
[0086] wherein if L is O, then M is C1-C6 straight or branched
alkyl or alkenyl, or --(CH.sub.2).sub.n--Ar, where n=1-6 and Ar is
selected from the group consisting of: 1l-naphthyl, 2-naphthyl,
2-furyl, 3-furyl, 2-thienyl, phenyl and phenyl having one to three
substituents that are independently selected from the group
consisting of: hydroxyl, halo, nitro, CF.sub.3, C1-C4 straight or
branched alkyl or alkenyl, O--(C1-C4) straight or branched alkyl or
alkenyl, and Ar, wherein Ar is selected from the group consisting
of: 1-naphthyl, 2-naphthyl, 2-furyl, 3-furyl, 2-thienyl, phenyl and
phenyl having one to three substituents that are independently
selected from the group consisting of: hydroxyl, halo, nitro,
CF.sub.3, C1-C4 straight or branched alkyl or alkenyl, O--(C1-C4)
straight or branched alkyl or alkenyl; wherein no more than two Ar
groups may be linked together;
[0087] wherein if L is an amino acid, then M is O--(C1-C4) straight
or branched alkyl, O-benzyl, NH-Phenyl, or NH-4-nitrophenyl and is
attached to the amino acid carbonyl;
[0088] the stereochemistry at all positions being R or S, and
preferably the stereochemistry is S at L if L is an .alpha.-amino
acid, and at those positions marked with asterisks; however, when J
is sulfhydryl, the preferred stereochemistry of the asterisked
position immediately adjacent to the nitrogen is R.
[0089] (6) Compounds represented by the formula VI (see WO
96/40633, including a discussion of the synthesis of these
compounds, the disclosure of which is incorporated herein by
reference): 9
[0090] wherein, R1 is selected from the group consisting of a C1-C9
straight or branched chain alkyl or alkenyl group optionally
substituted with C3-C8 cycloalkyl, C3 or C5 cycloalkyl, C5-C7
cycloalkenyl, or Ar1, where said alkyl, alkenyl, cycloalkyl or
cycloalkenyl groups may be optionally substituted with C1-C4 alkyl,
C1-C4 alkenyl, or hydroxy, where Ar1 is selected from the group
consisting of 1-naphthyl, 2-naphthyl, 2-indolyl, 3-indolyl,
2-furyl, 3-furyl, 2-thiazolyl, 2-thienyl, 3-thienyl, 2-, 3-,
4-pyridyl, and phenyl, having one to three substituents that are
independently selected from the group consisting of hydrogen, halo,
hydroxyl, nitro, trifluoromethyl, C1-C6 straight or branched alkyl
or alkenyl, C1-C4 alkoxy or C1-C4 alkenyloxy, phenoxy, benzyloxy,
and amino;
[0091] wherein X is selected from the group consisting of oxygen,
sulfur, methylene (CH.sub.2), or H.sub.2;
[0092] wherein Y is selected from the group consisting of oxygen or
NR.sub.2, where R.sub.2 is hydrogen or C1-C6 alkyl; and
[0093] wherein Z is selected from the group consisting of C2-C6
straight or branched chain alkyl or alkenyl, wherein the alkyl
chain is substituted in one or more positions with Ar.sub.1 as
defined above, C3-C8 cycloalkyl, cycloalkyl connected by a C1-C6
straight or unbranched alkyl or alkenyl chain and Ar.sub.1, where
Ar.sub.2 is selected from the group consisting of 2-indolyl,
3-indolyl, 2-furyl, 3-furyl, 2-thiazolyl, 2-thienyl, 3-thienyl, 2-,
3-, or 4-pyridyl, and phenyl, having one to three substituents that
are independently selected from the group consisting of hydrogen,
halo, hydroxyl, nitro, trifluoromethyl, C1-C6 straight or branched
alkyl or alkenyl, C1-C4 alkoxy or C1-C4 alkenyloxy, phenoxy,
benzyloxy, and amino;
[0094] wherein Z may also be the fragment: 10
[0095] where
[0096] R.sub.3 is selected from the group consisting of straight or
branched alkyl C1-C8 optionally substituted with C3-C8 cycloalkyl,
or Ar.sub.1 as defined above, and unsubstituted Ar.sub.1;
[0097] X is O or NR.sub.5, where R.sub.5 is selected from the group
consisting of hydrogen, CL-C6 straight or branched alkyl and
alkenyl;
[0098] R.sub.4 is selected from the group consisting of henyl,
benzyl, C1-C5 straight or branched alkyl or alkenyl, and C1-C5
straight or branched alkyl or alkenyl substituted with phenyl;
[0099] wherein the stereochemistry at position 1 is R or S.
[0100] Also encompassed are pharmaceutically acceptable derivatives
of the FK506 analogs, including, but not limited to, any
pharmaceutically acceptable salt, ester, salt of an ester, or any
other derivative which, upon administration to a patient, is
capable of providing directly or indirectly a non-binding FK506
analog or a metabolite or residue thereof that has the desired
neurotrophic activity. Included within the scope of the invention
are enantiomers, the tacemic form, and diastereoisomeric mixtures.
Enantiomers and diastereoisomers can be separated by conventional
methods.
[0101] Formulae I-VI above represent compounds that have a wide
range of binding affinities for FKBP12. The mechanism for
neurotrophic activity of FK506 presented herein indicates that the
effectiveness of FK506 and FK506 analogs in stimulating nerve cell
growth is unrelated to their ability to bind FKBP12. Instead, their
effectiveness in stimulating nerve cell growth relates to ability
of such compounds to bind FKBP52 and subsequently interfere with
the interaction of FKBP52 and hsp90 in a steroid receptor complex,
e.g., by competing for FKBP52 binding to hsp90, altering the
conformation of FKBP52, etc.
[0102] A "non-binding FK506 analog" is defined as an FK506 analog
that does not bind to FKBP12. Preferably, such FK506 analogs bind
FKBP12 with an apparent K.sub.d of greater than 10 .mu.M as
measured using well-known assays, and preferably greater than 30
.mu.M, and more preferably greater than 100 .mu.M. Values for the
apparent K.sub.d can be determined, for example, by a competitive
LH-20 binding assay performed as described, for example, in Harding
et al., Nature 341:758-760, 1989 (using 32-[1-.sup.14C]-benzoyl
FK506 as a reporting ligand; Siekierka et al., Nature 341:755-757,
1989, using [.sup.3H]dihydro-FK506 as a reporting ligand); and U.S.
Pat. No. 5,654,332.
[0103] Alternatively, a "non-binding FK506 analog" is defined as an
FK506 analog that does not significantly inhibit FKBP12 rotomase
activity when administered to a patient at dosage levels of about
0.01 to about 100 mg/kg body weight/day. Assays for inhibition of
FKBP12 rotamase activity are described in Harding et al. (Nature
341:758-760, 1989), Siekierka et al., Nature 341:755-757, 1989, and
U.S. Pat. No. 5,654,332, for example. The assays of Harding et al.
and Siekierka et al. employ a reaction mixture that includes the
cis form of N-succinyl-Ala-Ala-Pro-Phe-p-nitroa- nilide, FKBP12, a
test compound, and chymotrypsin, and spectrophotometrically measure
the release of p-nitroanilide as a result of isomerization of the
substrate.
[0104] Non-binding FK506 analogs are non-immunosuppressive, as can
be demonstrated by well-known assays, e.g., as discussed in U.S.
Pat. No. 5,516,797, WO 92/21313, WO 92/19593, and WO 92/04370.
[0105] Non-binding FK506 analogs can be used in the form of salts
preferably derived from inorganic or organic acids and bases,
including, but not limited to: acetate, adipate, alginate,
aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,
citrate, camphorate, camphorsulfonate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, fumarate,
glucoheptanoate, glycerophosphate, hemisulfate, heptanoate,
hexanoate, hydrochloride, hydrobromide, hydroiodide,
2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate,
persulfate, 3-phenylpropionate, picrate, pivalate, propionate,
succinate, tartrate, thiocyanate, tosylate, and undecanoate. Base
salts include, but are not limited to, ammonium salts, alkali metal
salts (such as sodium and potassium salts), alkaline earth metal
salts (such as calcium and magnesium salts), salts with organic
bases (such as dicyclohexylamine salts), N-methyl-D-glucamine, and
salts with amino acids (such as arginine, lysine, etc.). Basic
nitrogen-containing groups can be quaternized, e.g., with such
agents as lower alkyl halides (such as methyl, ethyl, propyl, and
butyl chlorides, bromides, and iodides), dialkyl sulfates (such as
dimethyl, diethyl, dibutyl, an diamyl sulfates), long-chain halides
(such as decyl, lauryl, myristyl, and stearyl chlorides, bromides,
and iodides), aralkyl halides (such as benzyl and phenethyl
bromides), etc. Water or oil-soluble or dispersible products are
produced thereby.
[0106] Non-binding FK506 analogs can be modified by appending
appropriate functionalities by well-known methods to enhance
selected biological properties, including increasing penetration of
the analogs into a given cellular compartment (e.g., blood,
lymphatic system, central nervous system, etc.), increase oral
availability, increase solubility to permit administration by
injection, alter metabolism, and alter rate of excretion, for
example.
[0107] Preferably, the non-binding FK506 analogs have a molecular
weight below about 750 atomic mass units (a.m.u.) (as the parent
compound, although the salts of such compounds can have higher
molecular weights).
[0108] "Effective amount" or "nerve growth stimulating amount." An
"effective amount" or a "nerve growth stimulating amount" of a
composition according to the invention is an amount sufficient to
achieve a statistically significant promotion of nerve cell growth
or regeneration compared to a control. Nerve cell growth or nerve
regeneration can be readily assessed using an in vitro assay, e.g.,
the assay described in the Examples below. Alternatively, nerve
cell growth or regeneration can be determined in an in vivo assay
or by direct or indirect signs of nerve cell growth and
regeneration in a patient. Preferably, the increase in nerve cell
growth or regeneration is at least 10%, preferably at least 30%,
and most preferably 50% or more compared to a control. Preferred
dosage levels are between about 0.1 to about 400 mg/kg per day of
the FK506 analog for subcutaneous delivery. For oral
administration, preferred dosage levels are between about 0.01 to
about 40 mg/kg/day.
[0109] Therapeutic and Prophylactic Uses
[0110] Pharmaceutical compositions according to the invention can
be periodically administered to a mammalian patient (e.g., a human
patient), in need of such treatment, to promote neuronal
regeneration and functional recovery and to stimulate neurite
outgrowth and thereby to treat various neuropathological states,
including damage to peripheral nerves and the central nervous
system caused by physical injury (e.g., spinal cord injury and
trauma, sciatic or facial nerve lesion or injury), disease (e.g.,
diabetic neuropathy), cancer chemotherapy (e.g., by vinca alkaloids
and doxorubicin), brain damage associated with stroke and ischemia
associated with stroke, and neurological disorders including, but
not limited to, various peripheral neuropathic and neurological
disorders related to neurodegeneration including, but not limited
to: trigeminal neuralgia, glossopharyngeal neuralgia, Bell's palsy,
myasthenia gravis, muscular dystrophy, amyotrophic lateral
sclerosis, progressive muscular atrophy, progressive bulbar
inherited muscular atrophy, herniated, ruptured or prolapsed
vertebral disk syndromes, cervical spondylosis, plexus disorders,
thoracic outlet destruction syndromes, peripheral neuropathies such
as those caused by lead, acrylamides, gamma-diketones
(glue-sniffer's neuropathy), carbon disulfide, dapsone, ticks,
porphyria, Gullain-Barre syndrome, Alzheimer's disease, Parkinson's
disease, and Huntington's chorea.
[0111] In addition, pharmaceutical compositions according to the
present invention display a wide range of other therapeutic or
prophylactic properties, including, treatment of stroke (see, e.g.,
Sharkey and Butcher, Nature 371:336-339, 1994, Vagita et. al., Life
Sciences 59:1643-1650, 1996; Tokime et al., Neurosci. Lett.
206:81-84, 1996; Drake et al., Acca. Physiol. Scand. 158:155-159,
1996; and Kuroda et al., Neurosci. Res. Comm. 19:83-90, 1996), AIDS
dementia (see, e.g., Dawson and Dawson, Adv. Neuroimmunol.
4:167-173, 1994; and Sekigawa et al., J. Clin. Immunol. 15:312-317,
1995); hair growth (Yamamoto et al., J. Investig. Dermatol.
102:160-164, 1994; Jiang et al., J. Investig. Dermatol.
104:523-525, 1995); and connective tissue disorders (see e.g.,
Steinmann et al., J. Biol. Chem. 266:1299-1303, 1991), and as a
male contraceptive (see e.g., Hisatomi et al., Toxicology
109:75-83, 1996).
[0112] A transection of a periphal nerve or a spinal cord injury
can be treated by administering a nerve growth stimulating amount
of a non-FKBP12-binding nerve growth promoting agent to the mammal
and grafting to the peripheral nerve or spinal cord an allograft
(Osawa et al., J. Neurocytol. 19:833-849, 1990; Buttemeyer et al.,
Ann. Plastic Surgery 35:396-401, 1995) or an artificial nerve graft
(Madison and Archibald, Exp. Neurol. 128:266-275, 1994; Wells et
al., Exp. Neurol. 146:395-402, 1997). The space between the
transected ends of the peripheral nerve or spinal cord is
preferably filled with a non-cellular gap-filling material such as
collagen, methyl cellulose, etc., or cell suspensions that promote
nerve cell growth, such as Schwann cells (Xu et al., J. Neurocytol.
26:1-16, 1997), olfactory cells, and sheathing cells (Li et al.
Science 277:2000-2002, 1997). The nerve growth promoting agent can
be included together with with such cellular or non-cellular
gap-filling materials.
[0113] Pharmaceutical Formulations
[0114] Pharmaceutical formulations according to the present
invention encompass formulations comprising (1) an amount (for
example, a unit dosage) of an NGPA together with (2) one or more
well-known non-toxic pharmaceutically acceptable excipients,
including carriers, diluents, and/or adjuvants, and optionally (3)
one or more biologically active ingredients. Standard
pharmaceutical formulation techniques are used, such as those
disclosed in Remington's Pharmaceutical Sciences, Mack Publishing
Co., Easton, Pa. (latest edition).
[0115] A pharmaceutical formulation according to the invention
includes one or more NGPAs and can also include, for example, one
or more other biologically active ingredients, including, but not
limited to FK506 or an FKBP12-binding FK506 analogs or one or more
other neurotrophic agents, including, for example, NGF, IGF-1,
aFGF, bFGF, PDGF, BDNF, CNTF, GDNF, NT-3, and NT 4/5; and so
on.
[0116] It is preferred that the pharmaceutical formulation includes
an amount of a neurotrophic agent (s), preferably NGF, such that
the patient receives a dosage of between about 0.01 to 100 .mu.g/kg
body weight/day of the neurotrophic agent, or that the neurotrophic
agent be administered separately, e.g., in separate single or
multiple dosage forms, preferably concurrently, consecutively, or
within less than about five hours of each other.
[0117] The compositions can be in the form of tablets, capsules,
powders, granules, lozenges, liquid or gel preparations, such as
oral, topical, or sterile parenteral solutions or suspensions
(e.g., eye or ear drops, throat or nasal sprays, etc.), transdermal
patches, and other forms known in the art.
[0118] Such pharmaceutical compositions can be administered
systemically or locally in any manner appropriate to the treatment
of a given condition, including orally, parenterally, rectally,
nasally, buccally, vaginally, topically, optically, by inhalation
spray, or via an implanted reservoir. The term "parenterally" as
used herein includes, but is not limited to subcutaneous,
intravenous, intramuscular, intrasternal, intrasynovial,
intrathecal, intrahepatic, intralesional, and intracranial
administration, for example, by injection or infusion. For
treatment of the central nervous system, the pharmaceutical
compositions preferably readily penetrate the blood-brain barrier
when peripherally administered or are administered
intraventricularly.
[0119] Pharmaceutically acceptable carriers include, but are not
limited to, ion exchangers, alumina, aluminum stearate, lecithin,
serum proteins (such as human serum albumin), buffers (such as
phosphates), glycine, sorbic acid, potassium sorbate, partial
glyceride mixtures of saturated vegetable fatty acids, water, salts
or electrolytes such as protamine sulfate, disodium hydrogen
phosphate, potassium hydrogen phosphate, sodium chloride, zinc
salts, colloidal silica, magnesium trisilicate, polyvinyl
pyrrolidone, cellulose-based substances, polyethylene glycol,
sodium carboxymethylcellulose, polyacrylates, waxes,
polyethylene-polyoxypropylene-block polymers, polyethylene glycol,
and wool fat, for example.
[0120] Tablets and capsules for oral administration can be in a
form suitable for unit dose presentation and can contain
conventional pharmaceutically acceptable excipients. Examples of
these include binding agents such as syrup, acacia, gelatin,
sorbitol, tragacanth, and polyvinylpyrrolidone; fillers such as
lactose, sugar, corn starch, calcium phosphate, sorbitol, or
glycine; tableting lubricants, such as magnesium stearate, talc,
polyethylene glycol, or silica; disintegrants, such as potato
starch; and dispersing or wetting agents, such as sodium lauryl
sulfate. The tablets can be coated according to methods well known
in normal pharmaceutical practice. Oral liquid preparations can be
in the form of, for example, aqueous or oily suspensions,
solutions, emulsions, syrups or elixirs, or can be presented as a
dry product for reconstitution with water or other suitable vehicle
before use. Such liquid preparations can contain conventional
additives such as suspending agents, e.g., sorbitol, syrup, methyl
cellulose, glucose syrup, gelatin, hydrogenated edible fats,
emulsifying agents, e.g., lecithin, sorbitan monooleate, or acacia;
non-aqueous vehicles (including edible oils), e.g., almond oil,
fractionated coconut oil, oily esters such as glycerine, propylene
glycol, or ethyl alcohol; preservatives such as methyl or propyl
p-hydroxybenzoate or sorbic acid, and, if desired, conventional
flavoring or coloring agents.
[0121] Pharmaceutical compositions according to the present
invention can also be administered parenterally in a sterile
aqueous or oleaginous medium. The composition can be dissolved or
suspended in a non-toxic parenterally-acceptable diluent or
solvent, e.g., as a solution in 1,3-butanediol. Adjuvants such as
local anesthetics, preservatives, and buffering agents can also be
dissolved in the vehicle. Commonly used vehicles and solvents
include water, physiological saline, Hank's solution, Ringer's
solution, and sterile, fixed oils, including synthetic mono- or
di-glycerides, etc. Fatty acids, such as oleic acid and its
glyceride derivatives are useful in the preparation of injectables,
as are natural pharmaceutically acceptable oils, such as olive oil
or castor oil, especially in their polyoxyethylated versions. These
oil solutions or suspensions may also contain a long-chain alcohol
diluent or dispersant, such as Ph. Helv or a similar alcohol.
[0122] For topical application, the drug may be made up into a
solution, suspension, cream, lotion, ointment in a suitable aqueous
or non-aqueous vehicle. Additives may also be included, e.g.,
buffers such as sodium metabisulphite or disodium edeate;
preservatives such as bactericidal and fungicidal agents, including
phenyl mercuric acetate or nitrate, benzalkonium chloride or
chlorhexidine, and thickening agents, such as hypromellose.
[0123] The dosage unit involved depends, for example, on the
condition treated, nature of the formulation, nature of the
condition, embodiment of the claimed pharmaceutical compositions,
mode of administration, and condition and weight of the patient.
Dosage levels on the order of about 0.1 to about 400 mg/kg per day
of the active ingredient are useful in the treatment of the
conditions listed above.
[0124] The invention will be better understood by reference to the
following examples, which are intended to merely illustrate the
best mode now known for practicing the invention. The scope of the
invention is not to be considered limited thereto, however.
EXAMPLES
[0125] FK506 and Geldanamycin Promote Nerve Regeneration by a
Common Mechanism
[0126] Materials and Methods
[0127] Cell cultures. SH-SY5Y human neuroblastoma cells were
maintained in DMEM medium (GIBCO) supplemented with 10% fetal calf
serum (SIGMA), 50 IU/mL penicillin, and 50 mg/mL streptomycin
(GIBCO) at 37.degree. C. in 7% CO.sup.2. Cells were plated in
six-well plates at 1.times.10.sup.6 cells/well and treated with 0.4
mM aphidicolin (SIGMA). At five days, cells were washed, treated
with nerve growth factor (NGF) (Boehringer Mannheim, Indianapolis,
Ind.) at 10 ng/mL (to induce process outgrowth) in the presence or
absence of FK506 (1 and 10 nM) (Calbiochem-Novabiochem Int'l., La
Jolla,. CA) and/or geldanamycin (0.1, 1, and 10 nM)
(Calbiochem-Novabiochem, La Jolla, Calif.). Media was changed at 96
hours and replaced with fresh media with the compounds (NGF plus
FK506 and/or geldanamycin) for an additional 72 hours (total time,
168 hours). The top 50% of axonal lengths were selected for
statistical analysis. All experiments were run in duplicate wells
and repeated at least twice for reproducibility.
[0128] Light morphometry of neurite lengths. For analysis of
process length, cells 20 fields per well) were randomly
photographed at 72 and 168 hours. Neurite lengths were measured on
photographic prints using a Houston Instrument HI-PAD digitizing
tablet connected to an IBM XT computer with appropriate software
(Bioquant IV, R&M Biometrics, Nashville, Tenn.); only those
processes greater than two times the cell body length were
measured. Data from identically treated wells were not different
and were therefore combined. Mean values and histograms were
constructed from these data. Histograms were compared using a
Mann-Whitney U test, which makes no assumptions about the shape of
the distribution.
[0129] Preparation of FK506 and geldanamycin. FK506 (mol. wt. 822)
and geldanamycin (mol. wt. 561) were dissolved in DMEM medium.
[0130] Results
[0131] In a first set of experiments, SH-SY5Y neuroblastoma cells
were plated in 6-well plates with DMEM plus 15% FCS and
differentiated with NGF (10 ng/ml) The effects of various
concentrations of geldanamycin and FK506 on neurite growth, alone
and in combination, were tested. The mean lengths of neuritic
processes of untreated and treated cells are shown in Table 1.
[0132] The cells developed long axonal-like processes upon exposure
to NGF (10 ng/mL) as measured at 168 hours after treatment. NGF
more than doubled the mean length of the processes compared to
untreated cells (compare FIGS. 1 and 2). An even greater increase
in the length of the processes was observed when the cells were
exposed to geldanamycin at 1 nM in the presence of NGF (FIG. 3).
However, geldanamycin at 10 .mu.M had no effect (FIG. 4). FK506 (10
nM) stimulated neurite outgrowth in the presence of NGF (FIG. 5) to
a greater degree than geldanamycin at either 1 nM or 10 nM. In
combination with FK506 (10 nM), geldanamycin (FIG. 6, 1 nM; FIG. 7,
10 nM) inhibited the effect of FK506 in a concentration-dependent
fashion.
[0133] In a second set of experiments, lower concentrations of
geldanamycin and FK506 were tested, alone and in combination. The
mean lengths of neuritic processes of untreated and treated cells
are shown in Table 2. NGF-treated cells (FIG. 9) had mean neuritic
lengths more than double the mean length of untreated control cells
(FIG. 8). Nerve growth stimulation was observed with FK506 (1 nM,
FIG. 10; 10 nM, FIG. 11) in the presence of NGF and geldanamycin
(0.1 nM, FIG. 12) in the presence of NGF. The lower concentration
of FK506 (1 nM) was more effective in stimulating neurite outgrowth
than the higher concentration (10 nM), and geldanamycin at 0.1 nM
was even more effective in stimulating neurite outgrowth than FK506
at either 1 nM or 10 nM. Combined treatment with geldanamycin (0.1
nM) and FK506 (1 nM, FIG. 13; 10 nM, FIG. 14) in the presence of
NGF showed that the effects of geldanamycin and FK506 were
additive, particularly at the lower FK506 concentration (FIG.
13).
[0134] Geldanamycyin and FK506 each stimulate neurite outgrowth in
a concentration-dependent fashion. Taken together, the similar
effects of geldanamycin and FK506 in stimulating neurite outgrowth,
their additive effects at low concentrations, and their inhibitory
effects at high concentrations (like high concentrations of either
compound alone), demonstrate that the two compounds act on nerve
cells via a common mechanism. That mechanism likely involves an
interaction of both compounds with components of steroid receptor
complexes. FKBP12 does not appear to play a role in the stimulation
of neurite outgrowth by either geldanamycin or FK506.
[0135] In further experiments, we have found that estrogen (10 nM)
and dexamethasone (10 nM) increased neurite outgrowth (without NGF)
of SH-SY5Y cells and produced an additive effect on neurite
outgrowth (neurotrophic action) with FK506 (10 nM).
[0136] This invention has been detailed both by example and by
direct description. It should be apparent that one having ordinary
skill in the relevant art would be able to surmise equivalents to
the invention as described in the claims which follow but which
would be within the spirit of the foregoing description. Those
equivalents are to be included within the scope of this
invention.
1TABLE 1 Mean Length of Top 50% of Neuritic Processes of SH-SY5Y
168 Hours After Treatment with Geldanamycin (1 nm or 10 nM) and/or
FK506 (10 nM) in the Presence of NGF Treatment Mean Length (.mu.M)
S.E.M. Untreated 41.61 1.25 NGF (10 ng/mL) 53.99 2.26 Geldanamycin
(1 nM) + 64.00 2.36 NGF (10 ng/mL) Geldanamycin (10 nM) + 54.81
2.10 NGF (10 ng/mL) FK506 (10 nM) + 77.82 2.70 NGF (10 ng/mL)
Geldanamycin (1 nM) + 72.63 2.01 FK506 (10 nM) + NGF (10 ng/mL)
Geldanamycin (10 nM) + 67.41 1.67 FK506 (10 nM) + NGF (10
ng/mL)
[0137]
2TABLE 2 Mean Length of Top 50% of Neuritic Processes of SH-SY5Y
168 Hours After Treatment with Geldanamycin (0.1 nM) and/or FK506
(1 nM or 10 nM) in the Presence of NGF Treatment Mean Length
(.mu.M) S.E.M. Untreated 31.86 1.56 NGF (10 ng/mL) 70.38 6.61
Geldanamycin (0.1 nM) + 98.07 5.72 NGF (10 ng/mL) FK506 (1 nM) +
89.92 6.40 NGF (10 ng/mL) FK506 (10 nM) + 82.68 5.22 NGF (10 ng/mL)
Geldanamycin (0.1 nM) + 110.51 6.13 FK506 (1 nM) + NGF (10 ng/mL)
Geldanamycin (0.1 nM) + 92.50 6.40 FK506 (10 nM) + NGF (10
ng/mL)
* * * * *