U.S. patent application number 10/805139 was filed with the patent office on 2004-11-18 for compositions and methods for promoting nerve regeneration.
This patent application is currently assigned to Oregon Health & Science University. Invention is credited to Gold, Bruce G..
Application Number | 20040229800 10/805139 |
Document ID | / |
Family ID | 32232867 |
Filed Date | 2004-11-18 |
United States Patent
Application |
20040229800 |
Kind Code |
A1 |
Gold, Bruce G. |
November 18, 2004 |
Compositions and methods for promoting nerve regeneration
Abstract
Neurite outgrowth and nerve regeneration are promoted by
disruption of the steroid receptor complex and stimulation of MAP
kinase/kinase activity. This disruption can take the form of
disruption of the physical assembly or function of the steroid
receptor complex, such as the mature complex or a precursor of the
mature complex that is required for assembly of the mature complex.
Geldanamycin and its analogs, bastadin and members of the bastadin
family, and radicicol and its analogs, as well as FKBP-52 antibody,
are shown to disrupt the complex and promote nerve growth. Assays
for finding neurotrophic compounds, as well as compounds found by
these assays, pharmaceutical compositions into which they are
incorporated, and methods of treating subjects having neuronal
dysfunction caused by injury or desease are disclosed. Any of these
compounds can be used in combination with a therapeutically
effective amount of heat, such as heat applied locally to an area
where nerve growth is desired, or systemically in an organism in
which neurite growth is desired. Alternatively, these compounds can
he used in association with a template, such as a tubular member
that defines an anatomic pathway along which nerve regeneration is
desired (particularly around transected or partially transected
nerve).
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 & Science
University
|
Family ID: |
32232867 |
Appl. No.: |
10/805139 |
Filed: |
March 19, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10805139 |
Mar 19, 2004 |
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10030904 |
Apr 29, 2002 |
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6734211 |
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10030904 |
Apr 29, 2002 |
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PCT/US00/18539 |
Jul 7, 2000 |
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60143180 |
Jul 9, 1999 |
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Current U.S.
Class: |
514/513 ;
514/8.2; 514/8.4; 514/8.6; 514/9.1 |
Current CPC
Class: |
A61K 31/436 20130101;
A61K 31/165 20130101; A61K 31/365 20130101; G01N 2500/00 20130101;
C12Q 1/48 20130101; A61K 31/21 20130101; A61F 7/00 20130101; A61K
31/00 20130101; A61K 38/185 20130101; A61K 31/395 20130101; A61K
31/444 20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/17 |
Claims
1-30. (Canceled)
31. A method for stimulating nerve cell growth in a subject
comprising administering to the subject a therapeutically effective
amount of a bastadin subunit or an analog thereof that stimulates
nerve cell growth.
32. The method of claim 1 wherein the bastadin subunit is a
bromotyrosine or an analog thereof or a bromotyrosine dimer or an
analog thereof.
33. The method of claim 32, wherein the bastadin subunit is a
bromotyrosine dimer or an analog thereof.
34. The method of claim 31, wherein the bromotryosine dimer is a
hemibastadin or analog thereof.
35. The method of claim 34, wherein the hemibastadin is a
hemibastadin having the structure: 28wherein each R is
independently selected from the group consisting of H, C1-8 alkyl,
or sulfato, W is selected from the group consisting of H, OH, or
C1-8 alkoxy, X and Y are selected independently from the group
consisting of hydrogen, halogen, hydroxyl, or C1-8 alkoxy, and A
and B are carbon atoms that are joined by a single or a double
bond.
36. The method of claim 35, wherein the hemibastadin is
hemibastadin 1, 2 or 3, or an analog thereof.
37. The method of claim 33, wherein the bromotyrosine dimer or
analog thereof is a hemibastadinol or analog thererof.
38. The method of claim 37, wherein the hemibastadin is a
hemibastadinol having the structure: 29wherein each R is
independently selected from the group consisting of H, C1-8 alkyl,
or sulfato, W is selected from the group consisting of H, OH, or
C1-8 alkoxy, X and Y are selected independently from the group
consisting of hydrogen, halogen, hydroxyl, or C1-8 alkoxy, and A
and B are carbon atoms that are joined by a single or a double
bond.
39. The method of claim 38, wherein the hemibastadin is
hemibastadinol 1, 2 or 3, or an analog thereof.
40. The method of claim 32, wherein the bastadin subunit is a
bromotryosine or analog thereof.
41. The method of claim 40, wherein the bromotryosine or analog
thereof has the structure: 30wherein each R is independently
selected from the group consisting of H, C1-8 alkyl, or sulfato, W
is selected from the group consisting of H, OH, or C1-8 alkoxy, X
is selected from the group consisting of hydrogen, halogen,
hydroxyl, or C1-8 alkoxy, and A and B are carbon atoms that are
joined by a single or a double bond.
42. The method of claim 41, wherein the bastadin subunit is the
3-bromotyramine amide of oxalic acid amide.
43. The method of claim 31 further comprising applying a
therapeutically effective amount of heat to an area where nerve
cell growth is desired, wherein the therapeutically effective
amount of heat enhances nerve growth.
44. The method of claim 31, further comprising providing a template
in an area where nerve growth is desired, wherein the template
provides a pathway along which nerve growth is desired.
45. The method of claim 44, wherein the template is a tubular
member that defines an anatomical pathway along which nerve growth
is desired.
46. The method of claim 44, wherein the template is placed between
opposing ends of a transected or partially transected nerve.
47. The method of claim 41 further comprising applying to the
template a therapeutically effective amount of heat, wherein the
therapeutically effective amount of heat enhances nerve growth.
48. The method of claim 31 further comprising administering a
second neurotrophic agent other than the bastadin subunit or analog
thereof.
49. The method of claim 48, wherein the second neurotrophic agent
is NGF, IGF-1, .alpha.-FGF, .beta.-FGF, PDGF, BDNF, CNTF, GDNF,
NT-3, NT4/5, or a mixture thereof.
50. The method of claim 48 further comprising applying a
therapeutically effective amount of heat to an area where nerve
cell growth is desired, wherein the therapeutically effective
amount of heat enhances nerve growth.
51. The method of claim 50 further comprising providing a template
in an area where nerve growth is desired, wherein the template
provides a pathway along which nerve growth is desired.
52. The method of claim 48 further comprising providing a template
in an area where nerve growth is desired, wherein the template
provides a pathway along which nerve growth is desired.
53. A pharmaceutical composition, comprising: a bastadin or an
analog thereof; and a pharmaceutically acceptable carrier.
54. The pharmaceutical composition of claim 53, wherein the
bastadin or analog thereof is a bastadin.
55. The pharmaceutical composition of claim 53, wherein the
bastadin or analog thereof is a bastadin or its analog having the
structure: 31wherein each R is independently selected from the
group consisting of H, C1-8 alkyl, or sulfato, W is selected from
the group consisting of H, OH, or C1-8 alkoxy, X, Y, and Z are
selected independently from the group consisting of hydrogen,
halogen, hydroxyl, or C1-8 alkoxy, and A and B are carbon atoms
that are joined by a single or a double bond.
56. The pharmaceutical composition of claim 53 wherein the bastadin
or analog therof is a bastadin or its analog having the structure:
32wherein each R is independently selected from the group
consisting of H, C1-8 alkyl, or sulfato, W is selected from the
group consisting of H, OH, or C1-8 alkoxy, X, Y, and Z are selected
independently from the group consisting of hydrogen, halogen,
hydroxyl, or C1-8 alkoxy, and A and B are carbon atoms that are
joined by a single or a double bond.
57. The pharmaceutical composition of claim 53 wherein the bastadin
or analog thereof is a bastadin or its analog having the structure:
33wherein each R is independently selected from the group
consisting of H, C1-8 alkyl, or sulfato, W is selected from the
group consisting of H, OH, or C1-8 alkoxy, X, Y, and Z are selected
independently from the group consisting of hydrogen, halogen,
hydroxyl, or C1-8 alkoxy, and A and B are carbon atoms that are
joined by a single or a double bond.
58. The pharmaceutical composition of claim 53, wherein the
bastadin or analog thereof is a bastadin or its analog having the
structure: 34wherein each R is independently selected from the
group consisting of H, C1-8 alkyl, or sulfato, W is selected from
the group consisting of H, OH, or C1-8 alkoxy, X and Y are selected
independently from the group consisting of hydrogen, halogen,
hydroxyl, or C1-8 alkoxy, and A and B are carbon atoms that are
joined by a single or a double bond.
59. The pharmaceutical composition of claim 53, wherein the
bastadin or analog thereof is a bastadin subunit or an analog
thereof.
60. The pharmaceutical composition of claim 59, wherein the
bastadin subunit or analog thereof is a bromotyrosine or an analog
thereof or a bromotyrosine dimer or an analog thereof.
61. The pharmaceutical composition of claim 60, wherein the
bastadin subunit is a bromotryosine dimer or an analog thereof.
62. The pharmaceutical composition of claim 61, wherein the
bromotyrosine dimer or analog thereof is a hemibastadin or an
analog thereof.
63. The pharmaceutical composition of claim 62, wherein the
hemibastadin or analog thereof is a hemibastadin or its analog
having the structure: 35wherein each R is independently selected
from the group consisting of H, C1-8 alkyl, or sulfato, W is
selected from the group consisting of H, OH, or C1-8 alkoxy, X and
Y are selected independently from the group consisting of hydrogen,
halogen, hydroxyl, or C1-8 alkoxy, and A and B are carbon atoms
that are joined by a single or a double bond.
64. The pharmaceutical composition of claim 61, wherein the
bromotyrosine dimer or analog thereof is a hemibastadinol or an
analog thereof.
65. The pharmaceutical composition of claim 64, wherein the
hemibastadinol or analog thereof is a hemibastidinaol or its analog
having the structure: 36wherein each R is independently selected
from the group consisting of H, C1-8 alkyl, or sulfato, W is
selected from the group consisting of H, OH, or C1-8 alkoxy, X and
Y are selected independently from the group consisting of hydrogen,
halogen, hydroxyl, or C1-8 alkoxy, and A and B are carbon atoms
that are joined by a single or a double bond.
66. The pharmaceutical composition of claim 60, wherein the
bastadin subunit or an analog thereof is a bromotryosine or an
analog thereof.
67. The pharmaceutical composition of claim 66, wherein the
bromotryorsine or analog thereof is a bromotyrosine or its analog
having the structure: 37wherein each R is independently selected
from the group consisting of H, C1-8 alkyl, or sulfato, W is
selected from the group consisting of H, OH, or C1-8 alkoxy, X is
selected from the group consisting of hydrogen, halogen, hydroxyl,
or C1-8 alkoxy, and A and B are carbon atoms that are joined by a
single or a double bond.
68. The pharmaceutical composition of claim 53 further comprising a
second neutrotrophic agent other than the bastadin or an analog
thereof.
69. The pharmaceutical composition of claim 68, wherein the second
neurotrophoic agent is NGF, IGF-1, .alpha.-FGF, .beta.-FGF, PDGF,
BDNF, CNTF, GDNF, NT-3, NT4/5, or a mixture thereof.
70. A template that provides a pathway along which nerve growth is
desired that is impregnated with the pharmaceutical composition of
claim 53.
Description
FIELD OF THE INVENTION
[0001] This invention concerns neurotrophic compounds and agents
useful in the treatment of neurological injury and disease.
BACKGROUND OF THE INVENTION
[0002] Following traumatic or mechanically induced axonal
degeneration in the peripheral nervous system, axonal regeneration
often ensues, resulting in functional recovery. However, the rate
of axonal elongation (3-4 mm/day) is slow, and sometimes does not
result in recovery of full neurological function. If neurological
function is restored, recovery usually occurs 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.
[0003] Other neurological conditions result from dysfunction of
neurons in the peripheral or central nervous systems that is caused
by chronic disease or injury. Chronic disease processes can
permanently and progressively damage the nervous system, and
(particularly in the central nervous system) usually results in
permanent loss of function. Such loss of neurological function is a
major cause of physical incapacitation and death throughout the
world.
[0004] The immunosuppressant drug FK506 (USAN tacrolimus;
Prograf.RTM.) induces immunosuppression by binding the immunophilin
FKBP-12. This binding prevents calcineurin from dephosphorylating
the transcription factor NF/AT (nuclear factor of activated
T-cells), which blocks translocation of calcineurin into the
nucleus, and prevents a receptor-mediated increase in the synthesis
and secretion of cytokines, such as interleukin-2 (IL-2), which are
required for T-cell proliferation. FK506 has also been found to
speed functional recovery and axonal regeneration in the rat in a
dose-dependent manner following a sciatic nerve crush lesion.
[0005] U.S. Pat. No. 5,654,332 (Armistead et al.) discusses
immunosuppressive FK506 analogs that bind FKBP-12, and are said to
stimulate neurite outgrowth in the presence of NGF. The
neurotrophic activity of these FKBP-12 binding compounds was said
to be "directly related to their affinity for FKBP-12 and their
ability to inhibit FKBP-12 rotamase activity" (id. at col. 7, lines
47-50). Rotamase activity measures peptidylisomerase cis-trans
isomerization, and inhibition of this activity has been accepted as
an indication of the immunosuppresant and neurotrophic activity of
therapeutic agents. See U.S. Pat. No. 5,614,547 (Hamilton et
al.).
[0006] Systemic administration of two synthetic PK506 analogs that
bind FKBP-12 but that do not inhibit calcineurin activity (and
which are not immunosuppressants) have been reported to increase
the size of myelinated fibers (Gold et al., Exp. Neurol.
147:269-278, 1997: Steiner et al., Nature Medicine 3:1-8, 1997;
Steiner et al., Proc. Natal. Acad. Sci. USA 94:2019-2024, 1997). It
has also been reported that androgens and estrogens stimulate
facial nerve regeneration in hamsters (e.g. Tanzer and Jones, Exp.
Neurol. 146:258-264, 1997).
[0007] Many of the compounds previously shown to stimulate nerve
regeneration have undesired side-effects, such as immunosuppression
(FK506 and analogs that retain immunosuppressant activity) or
androgenic or estrogenic stimulation. There is therefore a need to
provide a class of nerve growth stimulating compounds that are well
tolerated by subjects who take them.
SUMMARY OF THE INVENTION
[0008] The mechanism by which FK506 and other analogs induces nerve
growth stimulation has previously been misunderstood, which has
been an obstacle to the development of new drugs for this
purpose.
[0009] The present invention takes advantage of the surprising
discovery that nerve growth stimulation is promoted by disruption
of the mature steroid receptor complex, and not by interaction with
FKBP-12, as was previously thought. Disruption of the complex can
include inhibition of physical assembly, promotion of disassembly,
or functional interference with the steroid receptor complex, for
example the mature steroid receptor complex, or a less mature form
of the complex that is a predecessor to the mature complex. The
participation of MAP kinase/kinase (MEK) in stimulating nerve
growth, and the role heat plays in increasing neurite outgrowth
when combined with nerve growth stimulating compounds, are
additional parameters that can be exploited as part of this
invention.
[0010] In view of the discovery of the biochemical mechanism by
which neurite outgrowth is promoted, assays have been developed for
selecting new compounds that may have activity in promoting nerve
growth. Such assays may include determining if a test compound,
other than a steroid ligand such as an androgen or an estrogen,
disrupts assembly of the steroid receptor complex, and selecting a
compound that disrupts assembly of the steroid receptor complex.
Alternatively, the assay may include determining the ability of
test compounds to stimulate MEK activity, and selecting compounds
on this basis. Examples of specific classes of compounds that can
be screened include geldanamycin and its structural analogs,
rapamycin and its structural analogs, and FK506 and its structural
analogs, radicicol and its analogs and bastadins and their analogs.
Compounds selected by this assay for further investigation may be
tested in additional assays to measure actual neurite outgrowth
induced by the compound. In this way neurotrophic compounds have
been identified by the assay for disruption of the steroid receptor
complex or stimulation of MEK.
[0011] Methods have been designed for stimulating nerve cell growth
in a subject by administering to the subject a compound (including
a compound discovered by the assay) that disrupts assembly or
function of the steroid receptor complex, for example of the mature
steroid receptor complex, (for example by inhibiting association or
promoting dissociation), or stimulates MEK activity, wherein the
compound is other than a ligand for the steroid hormone binding
portion of the steroid receptor complex (such as an androgen or an
estrogen), and in some specific embodiments does not bind with high
affinity to FKBP-12. A therapeutic amount of heat may be
administered in combination with nerve growth stimulating
compounds. In particular embodiments, the compound is administered
to disrupt association of a p23 component of the steroid receptor
complex with an hsp-90 component or disrupt association of FKBP-52
with hsp-90. In other embodiments, the compound is administered to
competitively bind with ATP at an amino terminal ATP binding site
of hsp-90, for example at a geldanamycin binding site of the
steroid receptor complex. In yet other embodiments, the compound is
administered to stimulate MEK activity. These methods include
administration of a benzoquinone ansamycin, such as geldanamycin or
a structural analog or mimetic thereof, an anti-FKBP-52 antibody,
radicicol or a structural analog or mimetic thereof, or a bastadin
or a structural analog or mimetic thereof. The method can also
include administering a second neurotrophic factor, other than the
compound that disrupts association of the steroid receptor
complex.
[0012] The method is useful in the treatment of animals (including
mammals such as humans) having a neurological condition associated
with neuronal dysfunction caused by disease or injury to neurons in
either the central or peripheral nervous systems. Compounds or
compositions are administered, with or without heat, to the animal
in a therapeutically effective neurotrophic amount to bind to the
mature steroid receptor complex (for example at a geldanamycin
binding site of hsp-90) to disrupt association of the mature
steroid receptor complex or stimulate MEK activity, and promote
neurite outgrowth from neurons. The method can also be used in
association with procedures such as a surgical nerve graft, or
other implantation of neurological tissue, to promote healing of
the graft or implant, and promote incorporation of the graft or
implant into adjacent tissue.
[0013] Certain pharmaceutical compounds that are not a ligand for
the steroid hormone binding portion of the steroid receptor complex
can disrupt assembly of a steroid receptor complex. These compounds
can be geldanamycin and its structural analogs, rapamycin and its
structural analogs, FK506 and its structural analogs, radicicol or
a structural analog or mimetic thereof, or a bastadin or a
structural analog or mimetic thereof, but more particular
embodiments of the compound may have low rotamase inhibition
activity, may be other than an FK506 or rapamycin analog, may not
bind with high affinity to FKBP-12, or are not immunosuppressive.
In particular embodiments, the compound specifically disrupts
formation of the steroid receptor complex (for example the mature
steroid receptor complex) either by inhibiting association or
promoting dissociation of the steroid receptor complex, for example
by disrupting association of a p23 component of the steroid
receptor complex with an hsp-90 component, or disrupting
association of FKBP-52 with hsp-90, or inhibiting interaction of
p23, FKBP-52 or hsp-90 with the complex. Certain embodiments of the
compound competitively bind with ATP at an amino terminal ATP
binding site of hsp-90, which is also the binding site for
geldanamycin binding to the steroid receptor complex. In particular
embodiments the compound is radicicol or a radicicol analog that
binds to a geldanamycin binding site of hsp-90. In other
embodiments, the compound is an anti-FKBP-52 antibody, or another
agent that specifically causes FKBP-52 to dissociate from hsp-90 of
the steroid receptor complex.
[0014] The compound can be incorporated into a pharmaceutical
composition, which can also include another neurotrophic factor,
such as NGF, IGF-1, .alpha.FGF, .beta.FGF, PDGF, BDNF, CNTF, GDNF,
NT-3, NT 4/5, and mixtures thereof, or a steroid hormone that is a
ligand of the steroid receptor complex (such as an estrogen, an
androgen or a corticosteroid such as dexamethasone).
[0015] In a more specific aspect, the compound is a nerve growth
stimulating amount of an agent that binds to a polypeptide of a
steroid receptor complex other than a steroid hormone binding
portion of the complex, the agent being selected from the group
consisting of an FK506 analog having low binding affinity for
FKBP-12 and low rotamase activity, for example a benzoquinone
ansamycin and structural analogs thereof, 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, and combinations
thereof, wherein the agent disrupts assembly or interferes with
function of the steroid receptor complex by causing p23 or FKBP-52
dissociation from the complex, or inhibiting p23 or FKBP-52
association with the complex, or inhibiting interaction of p23,
FKBP-52 or hsp-90 with the complex.
[0016] Compounds of the present invention need not have significant
calcineurin inhibition or rotamase inhibition. The compounds may
have an IC.sub.50 for rotamase inhibtion of greater than 1 nM, for
example greater than 10 nM, 25 nM, or even 50-100 nM.
[0017] Nerve cell growth can be stimulated in a subject by
administering to the subject a compound that stimulates nerve cell
growth, wherein the compound is one or more of radicicol or its
analogs; a bastadin or its analogs; or an agent that stimulates MAP
kinase/kinase activity. In particular embodiments, the compound is
a radicicol analog, or a bastadin such as bastadin 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20,
particularly bastadin 10, or an analog of a bastadin.
[0018] The method also includes administering radicicol or its
analogs, or the bastadin or its analogs, in combination with a
neurotrophic factor other than the compound that disrupts
association of the mature steroid receptor complex or stimulates
MAP kinase/kinase activity. The neurotrophic factor may be, for
example, NGF, IGF-1, .alpha.-FGF, .beta.-FGF, PDGF, BDNF, CNTF,
GDNF, NT-3, NT 4/5, and mixtures thereof.
[0019] Another aspect is screening for agents that stimulate nerve
cell growth, by detecting agents that stimulate MAP kinase/kinase
activity, such as radicicol analogs, or platelet derived growth
factor BB (PDGFBB) or analogs thereof.
[0020] The method also includes applying a sufficient amount of
heat to an area where nerve cell growth is desired, for example
along a normal anatomic pathway, or in an anatomic region, of a
transected, partially transected or otherwise damaged nerve.
Alternatively, the body temperature of a subject can be
systemically elevated, for example by inducing a fever or placing
the body in a heated environment. The invention can also include
providing a template in an area where nerve growth is desired, for
example a tubular member that defines an anatomical pathway along
which nerve growth is desired. If desired, a therapeutically
effective amount of the neurotrophic compound may be provided in
association with the template to promote nerve growth. The template
may be placed between opposing ends of a transected or partially
transected nerve. Heat can be applied to the template in a
therapeutically sufficient amount, effective to enhance nerve
growth. Alternatively, the template along the desired anatomical
path can be impregnated with the neurotrophic compound, or the
impregnated template can be heated.
[0021] In some embodiments, the radicicol compound or its analogs
are of the formula: 1
[0022] where X, Y, Z, R1, R2 and R3 are as defined in Example 14,
and in particular embodiments is not radicicol.
[0023] In some embodiments, the neurotrophic compound is a complete
bastadin or bastadin analog, such as, 2
[0024] where each R is independently selected from the group
consisting of H, C1-8 alkyl, or sulfato, W is selected from the
group consisting of H, OH, or C1-8 alkoxy, X, Y, and Z are
independently selected from the group consisting of hydrogen,
halogen, hydroxyl, or C1-8 alkoxy, and A and B are carbon atoms
that are joined by a single or a double bond. Specific complete
bastadin structures (which also show the naturally occurring
bastadin macrocylic ring structures) are found in Example 15.
[0025] Also included are hemibastadins and their analogs, of the
formula 3
[0026] where A, B, R, W, X, and Y are defined as above for the
complete bastadins. Specific hemibastadin structures are found in
Example 15
[0027] Also included are hemibastadinols and their analogs of the
formula 4
[0028] where A, B, R, W, X, and Y are defined as above for the
complete bastadins. Specific hemibastadinol structures are found in
Example 15
[0029] In other embodiments, the compound is a bastadin subunit
such as 5
[0030] where A, B, R, W, and X are defined as above for the
complete bastadins. A specifice example of a bastadin subunit is
shown in Example 15.
[0031] 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 FIGURES
[0032] FIG. 1 is a schematic diagram illustrating receptor-hsp-90
heterocomplex assembly of the steroid receptor complex.
[0033] FIG. 2 is a schematic diagram illustrating the mature
steroid receptor complex, and the binding sites of some of the
agents of the present invention that promote nerve growth.
[0034] FIG. 3 is a cumulative histogram showing neurite outgrowth
lengths after 72 hours for untreated hippocamapal cells and for
hippocampal cells treated with either NGF or radicicol at the
concentrations indicated.
[0035] FIG. 4 is a cumulative histogram showing neurite outgrowth
lengths after 72 hours for untreated hippocampal cells and for
hippocampal cells treated with heat, NGF, or a combination of heat
and NGF.
[0036] FIG. 5 is a cumulative histogram showing neurite outgrowth
lengths after 72 hours for untreated hippocampal cells and for
hippocampal cells treated with NGF, a combination of heat and NGF,
a combination of heat and Hsp-90 antibodies (Ab), and a combination
of heat, NGF, and Hsp-90 antibodies.
[0037] FIG. 6 is a histogram showing the mean neurite length after
72 hours for untreated hippocampal cells and for hippocampal cells
treated with NGF, FK506, combinations of NGF and the MAP
kinase/kinase inhibitor PD 098059 (2 concentrations), and
combinations of FK506 and PD 098059 (2 concentrations).
[0038] FIG. 7 is a histogram showing the mean neurite length after
72 hours for untreated hippocampal cells and for hippocampal cells
treated with NGF and various combinations of radicicol (2
concentrations) and PD 098059 (2 concentrations).
[0039] FIG. 8 is a cumulative histogram showing neurite outgrowth
lengths after 72 hours for hippocampal cells treated with NGF,
radicicol and two combinations of radicicol and the MAP
Kinase/Kinase inhibitor PD 098059.
[0040] FIGS. 9A, 9B, and 9C are light micrographs showing
hippocampal cells after 72 hours for untreated cells, cells treated
with radicicol, and cells treated with a bastadin 10 analog,
respectively.
[0041] FIG. 10 is a cumulative histogram of neurite outgrowth
lengths for hippocamapal cells after 72 hours when untreated and
treated with either radicicol or a bastadin 10 analog.
DETAILED DESCRIPTION OF PARTICULAR EMBODIMENTS
[0042] Steroid receptors are part of a superfamily of molecules
that regulate gene expression by direct interaction with the
upstream region of specific structural genes. It is essential to
hormone action that a receptor must be able to assume both an
active and an inactive state. This regulation is accomplished by
association of the receptor (the steroid ligand binding component)
with a multimeric complex of chaperone proteins, such as heat shock
proteins (hsp-90), p23 and FKBP-52, which form the steroid receptor
complex (SRC). When the steroid receptor binds its ligand, the
receptor is activated, the chaperone proteins of the SRC are
dissociated, and a DNA binding domain of the receptor is exposed
for interaction with gene regulatory sequences. Members of the
steroid receptor family that are regulated in this fashion include
mineralocorticoids (such as aldosterone), glucocorticoids (such as
dexamethasone), progestins (such as progesterone), androgens (such
as testosterone), and estrogens (including estrogen, .beta.-estriol
and .beta.-estradiol).
[0043] A model of steroid receptor complex assembly is shown in
FIG. 1. ATP-dependent association of the steroid receptor SR with
the hsp-90/p-60/hsp-70 folding complex (foldosome) yields an
intermediate (SR/hsp-90/p-60/hsp-70) complex that is unstable in
the absence of p23 or molybdate. Once p23 has associated with the
complex, and hsp-90 binds tetratricopeptide repeat (TPR) domain
proteins, such as the immunophilins (e.g. FKBP-52 and CyP40), the
nearly mature, metastable SRC is formed (SR/hsp-90/p23/FKBP-52). In
FIG. 1, the TPR domain is indicated by the solid black crescent to
which FKBP-52 is bound. Assembly of the mature steroid receptor
complex is the last step shown in FIG. 1, in which the complex of
chaperone proteins hsp-90/p23/FKBP-52 is assembled with the steroid
receptor SR.
[0044] The immunophilins are a highly conserved family of chaperone
proteins that are known to be mediators of immunosuppressant drug
activity. The best characterized immunophilin is FKBP-12, which
interacts with the immunosuppressant drug FK-506 in T lymphocytes,
to prevent calcineurin from dephosphorylating the nuclear factor of
activated T-cells (NF/AT), thereby blocking synthesis and secretion
of cytokines required for immune function. Immunophilins have
peptidylisomerase (PPIase) activity, and inhibitors of this
activity can be detected with a rotamase assay which measures
inhibition of cis-trans isomerization of the peptidylprolyl.
However, FKBP-12 immunosupression is not mediated by an ability to
inhibit rotamase activity. Rotamase activity has nonetheless been
accepted as an indication of immunosuppressant activity of
immunophilins, even though it does not measure the
dephosphorylation of calcineurin activity by which
immunosuppression is actually mediated. Previous researchers had
taken advantage of the rotamase assay to look for FKBP-12 binding
drugs that would promote nerve regeneration (as FK506 had been
found to do).
[0045] The present invention takes advantage of the surprising
finding that disrupting assembly of the SRC, and not binding to
FKBP-12, is what promotes nerve regeneration. Hence previous
efforts to find FKBP-12 analogs that promote nerve regeneration by
measuring rotamase or immunosuppressive activity was misdirected,
and was likely to find drugs that had unwanted side effects (such
as immunosuppression and cardiomyopathy). The present invention has
taken advantage of the discovery of the actual biological mechanism
by which nerve regeneration is promoted to provide a superior assay
for finding new neurotrophic drugs that are superior to those in
the prior art.
[0046] One such new compound is geldanamycin, a benzoquinone
ansamycin antibiotic, which binds in a pharmacologically specific
manner to hsp-90 (Whitesell et al., Proc. Natal. Acad. Sci. USA
91:8324-8328, 1994) and inhibits association of the p23 component
of the heterocomplex assembly system with hsp-90 (Johnson and Toft,
Mol. Endocrinol. 9:670-678, 1995). Geldanamycin and radicicol
thereby promote dissociation of a steroid receptor complex, and
block reassembly of the hormone-responsive form of the complex,
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 with high
affinity (for example, does not bind steroid ligand that is present
in concentrations of less than about 10 nM). 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 hsp-90 (see, e.g., Meshinchi et al., J. Biol. Chem.
265:4863-4870, 1990; Kost et al., Mol. Cell. Biol. 9:3829-3838,
1989).
[0047] Another class of neurotrophic agents that have been found in
accordance with the invention are those that act at the FKBP-52
component of the SRC. It has surprisingly been found that the
action of the neurotrophic immunosuppressant FK506 is via an
interaction with FKBP-52, which induces a conformational change in
hsp-90, enabling dissociation of p23 from hsp-90, thereby
interfering with assembly of the mature SRC. The present invention
also includes bastadins that may disrupt the SRC by binding to
FKBP-52. Mack et al. have shown that bastadin 5 stimulates
[.sup.3H]ryanodine binding to ryanodine receptors, but that such
stimulation is antagonized by FK506. (Mack et al., J. Biol. Chem.,
269(37) pp. 23236-49, 1994). The model in FIG. 2 of the present
application shows how FK506 binds to FKBP-52. By competing with
bastadin 5 for this binding site or by inducing changes in the
conformation of FKBP-52, FK506 may block the action of bastadin
5.
[0048] As shown in FIG. 10, bastadin 10 stimulates neurite
outgrowth.
[0049] An anti-FKBP-52 antibody also inhibits assembly of the
mature SRC. Binding of FK506 to GR-associated FKBP-52 causes
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).]
[0050] FKBP-52 and CyP40 bind directly to hsp-90, and CyP40
competes for FKBP-52 binding to hsp-90 and vice versa. CyP40 is an
example of a protein targeted by cyclosporin A (CsA) and its
analogs. These immunophilins bind hsp-90 in a mutually exclusive
fashion, leading to the formation of separate CyP40-hsp-90 and
FKBP-52-hsp-90 complexes (Ratajczak and Carrello, J. Biol. Chem.
271:2961-2965, 1996). Immunophilins such as FKBP-52, CyP40 and PP5
and non-immunophilin proteins such as 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 hsp-90. An increased number of TPR domains in a protein
appears to correlate with increased hsp-90-binding affinity. Hence
peptides having one or more TPR domains would be expected to have
increased hsp-90 binding affinity, and would interfere with FKBP-52
association with hsp-90, which is required for assembly of the
mature steroid receptor complex.
[0051] For example, binding of both FKBP-52 and CyP40 to hsp-90 is
competitively inhibited by a purified fragment of human CyP40
comprising its three TPR domains, and by a fragment of rat PP5
comprising its four TPR domains (reviewed in Pratt and Toft,
Endocrine Rev. 18:306-360, 1997). Such purified fragments, or other
peptides such as PP5, p60 and Mas70, containing one or more TPR
domains (particularly two, three or more TPR domains) are therefore
suitable for interfering with assembly or function of the steroid
receptor complex, and are included within the scope of this
invention.
[0052] The effects of radicicol, radicicol analogs, bastadins,
bastadin analogs, geldanamycin and the other neurotrophic agents
that do not bind directly to the steroid binding domain of the
steroid receptor complex are believed to result from binding of
these compounds to components of steroid receptor complexes,
causing the dissociation of hsp-90 from the steroid receptor
complex either directly (by binding to hsp-90 or interfering with
the binding of hsp-90 to the steroid receptor) or indirectly (by
binding to a polypeptide such as FKBP-52 that itself binds to
hsp-90, or a polypeptide that binds to p23), or alternatively by
preventing association of hsp-90 or p23 with the steroid receptor
complex. Interference with the ability of hsp-90 to complex with
and perform its chaperone function for other hsp-90 substrate
proteins is believed to also be responsible for or contribute to
the observed stimulation of nerve regeneration by FK506 and/or
geldanamycin. Any agent that interferes with the function of the
mature steroid receptor complex (including interference with a
precursor of the mature complex, such as the nearly mature complex
or foldosome) is also included in the scope of this invention. Also
included in the scope of this invention are compounds that
stimulate MAP kinase/kinase.
[0053] Specific embodiments of the neurotrophic compounds of this
invention can be substantially free of calcineurin inhibition, and
can have low rotamase inhibition, for example an IC.sub.50 of
greater than about 1 nM, or even 5 or 10 nM.
[0054] Identification of the role that MAP kinase/kinase (MEK)
plays in stimulating neurite outgrowth also provides a basis for
screening compounds that may stimulate nerve growth. Compounds
which stimulate MEK activity are included in the scope of the
invention.
Abbreviations and Definitions
[0055] AR: Androgen Receptor
[0056] ER: Estrogen receptor
[0057] GR: Glucocorticoid receptor
[0058] PR: Progesterone receptor
[0059] SRC: Steroid receptor complex. 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.
[0060] TPR: Tetratricopeptide repeats are Domain III of FKBP-52;
TPRs were first identified by Sikorski et al., Cell 60:307-317,
1990, as degenerate consensus sequences of 34 amino acids.
[0061] Mimetic: A biological compound (such as a peptide) that
mimics the effect of a pharmaceutical, for example a peptide that
mimics the effect of a benzoquinone ansamycin by binding to a
geldanamycin binding site on hsp-90.
Ligand for the Steroid Hormone Binding Portion of the Steroid
Receptor Complex
[0062] An already recognized ligand for the receptor subtype. For
example, dexamethasone for the GR; estrogen for the ER,
testosterone for the AR, progesterone for the PR.
[0063] Immunophilins: A highly conserved family of chaperone
proteins that have PPIase activity, producing cis-trans
isomerization. The immunophilins are divided into low molecular
weight (less than 40 kD) and high molecular weight (40-65 kD)
immunophilins. The high molecular weight immunophilins (e.g.,
FKBP-52), in contrast to FKBP-12, contain three or more
tetratricopeptide repeats (TPRs) which mediate binding to hsp-90.
The immunophilins may be subdivided into two classes on the basis
of their abilty to bind either cyclosporin A (cyclophilins) or
FK506 and rapamycin (the FK binding proteins, FKBPs). Members of
the FKBP family of immunophilins include FKBP-12, FKBP-13, FKBP-25,
FKBP-52 (also referred to as FKBP-59), and FKBP-65. PP5 is also
considered a member of this family, because it binds FK506 weakly,
as reported by Silverstein et al., J. Biol. Chem. 272:16224-16230,
1997.
[0064] NGPA: Nerve growth promoting agent. A "nerve growth
promoting agent" or NGPA is defined as a substance that binds to a
polypeptide component of a steroid receptor complex, such
components including but not limited to hsp-90 and FKBP-52, and
promotes nerve regeneration, without limitation to a particular
mechanism of action. In particular embodiments, the NGPA does not
bind FKBP-12 (or binds it with low affinity, with a Kd of greater
than 1 .mu.M), has low rotamase inhibitory activity (an apparent Ki
of more than 2500 nM), binds with low affinity to calcineurin
(requires concentrations greater than 30 .mu.M to bind), or is
non-immunosuppressive, as measured by the substantial absence of a
drop in total blood lymphocyte counts in subjects to whom the agent
is administered. NGPAs include, but are not limited to, non-FKBP
12-binding ("non-binding") or low affinity FKBP-12 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
structural analogs and derivatives thereof, such as geldampicin,
tunicamycin, and dihydrogeldanamycin, as well as the compounds
listed in Example 12; 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), which interferes or
competitvely binds with the component of the SRC; and antibodies
that bind specifically to polypeptide components of steroid
receptor complexes, e.g., anti-hsp-90, anti-FKBP-52, etc.) that
interfere with the interaction of the bound polypeptide with
another polypeptide in the steroid receptor complex. The
neurotrophic agents include compounds that either physically
disrupt association of the mature SRC (either by inhibiting
association or promoting dissociation of the SRC), or inhibit
interaction of components (such as p23, FKBP-52 or hsp-90) of the
SRC. Further, the neurotrophic agents include compounds which
stimulate MAP kinase/kinase (MEK) activity. Heat applied for
physiologically tolerable periods to boost a mammal's body
temperature to a physiologically tolerable temperature in
combination with administering compounds which stimulate nerve
growth is yet another neurothophic agent.
[0065] Neurotrophic: Promoting Nerve Growth.
[0066] Transformation: 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 ligand to a steroid-ligand-binding
form.
[0067] Rotamase Activity: Rotamase (PPIase) activity can be
determined, for example, as in WO 92/04370, and can be expressed as
a K.sub.i. The cis-trans isomerization of the alanine-proline
peptide bond in a model substrate,
N-succinyl-Ala-Ala-Pro-Phe-4-nitroanalide, may be monitored
spectrophotemetrically in a coupled assay with chymotrypsin, which
releases 4-nitroanalide from the trans form of the substrate. The
inhibitory effect upon the addition of different concentrations of
inhibitor on the extent of the reaction is determined, and analysis
of the change in the first order rate constant as a function of
inhibitor concentration yields an estimate of the apparent K.sub.i.
In the assay of the present invention, rotamase activity has been
found to be unrelated to the neurotrophic activity of the
compounds, and need not be determined.
[0068] "Known" or "recognized" compounds (such as FKBP-12 binding
compounds or FK506 analogs) are those that have previously been
reported in patents or publications, or that otherwise qualify as
prior art.
[0069] Analogs are structurally similar compounds that retain
biological activity of the parent compound, such as stimulation of
neurite outgrowth.
EXAMPLE 1
Assays for Identifying Nerve Growth Promoting Agents
[0070] There are a number of well-known methods for assaying
compounds that bind to hsp-90, FKBP-52, 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.
[0071] Examples include assays for the binding of a test compound
to a polypeptide that is a component of a steroid receptor complex.
An assay for detecting binding to hsp-90 is described, for example,
by Whitesell et al. (Proc. Natal. Acad. Sci. USA 91:8324-8328,
1994). Commercial hsp-90 (StressGen Biotechnologies, Victoria, BC)
dissolved in 20 .mu.g/mL of TNESV buffer (50 mM Tris-HCl, pH 7.4/1%
Nonidet P40/2 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. Natal. Acad. Sci. USA 91:8324-8328, 1994). The beads are
then washed with TNESV buffer and bound hsp-90 is eluted by heating
in reducing loading buffer, and can be analyzed by SDS/PAGE and
silver staining (Bio-Rad). Alternatively, if the hsp-90 is labeled,
the assay can be performed for the bound label instead of the free
label. Test compounds that compete with geldanamycin for binding to
hsp-90 inhibit the binding of solubilized hsp-90 to the beads.
[0072] Similar assays can be performed to identify compounds that
bind other steroid receptor complex polypeptide components. Binding
to FKBP-52 can be assayed using recombinant FKBP-52 (Peattie et
al., Proc. Natal. Acad. Sci. USA 89:10974-10978, 1992). Binding to
p23 can be assayed using recombinant human p23 (Johnson et al.,
Mol. Cell. Biol. 14:1956-1963, 1994) and immobilized hsp-90.
Purified hsp70 and recombinant p60 (Dittmar et al., J. Biol. Chem.
271:12833-12839, 1996) are also available for use in such binding
assays.
[0073] Immunoassays can also be performed using conventional
immunoassay methodologies and antibodies that are specific for
steroid receptor complex components, e.g., antibodies against
FKBP-52 (Tai et al., Biochem. 25:5269-5275, 1986), hsp-90 (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 hsp-90)(Erhart et al.,
Oncogene 3:595-603, 1988), and p23 (Johnson et al., Mol. Cell.
Biol. 14:1956-1963, 1994).
[0074] A well-accepted qualitative assay for receptor
transformation, which involves dissociation of hsp-90 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.
15:42984304, 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).
[0075] An in vitro assay for nerve cell growth (neurite outgrowth)
is provided in Example 2, and in Gold et al., Exp. Neurol.
147:269-278, 1997. In vivo assays for nerve regeneration are
discussed in, for example, Gold et al., Restor. Neurol. Neurosci.
6:287-296, 1994; Gold 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 and Gold et al., Soc.
Neurosci. Abst. 23:1131, 1997, which examine the effects of
systemic administration of a test compound on nerve regeneration
and functional recovery following a crush injury to the rat sciatic
nerve. The sciatic nerves of anaesthetized rats are exposed, and
the nerves crushed 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.
[0076] Nerve regeneration is also assessed by sampling tissues from
the sciatic nerve at known (0.5 cm) distances from the crush site
and counting the number of myelinated fibers by light microscopy.
The size of axons is calculated 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.
EXAMPLE 2
FK506 and Geldanamycin Promote Nerve Regeneration by a Common
Mechanism
[0077] This Example illustrates that FK506 and geldanamycin promote
nerve regeneration by a common mechanism. 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.sub.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, Calif.)
and/or geldanamycin (0.1, 1, and 10 nM) (Calbiochem-Novabiochem, La
Jolla, Calif.) dissolved in the DMEM medium. Media was changed at
96 hours and replaced with fresh media containing 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.
[0078] For analysis of neurite process length of 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.
[0079] The mean lengths of neuritic processes of untreated and
treated cells are shown in Table 1:
1TABLE 1 Mean Length of Top 50% of Neuritic Processes 168 Hours
After Treatment with Geldanamycin in the Presence of NGF Treatment
Mean Length (.mu.M) S.E.M. Untreated 83.22 2.50 NGF (10 ng/mL)
107.98 4.52 Geldanamycin (1 nM) + NGF 128.00 4.72 (10 ng/mL)
Geldanamycin (10 nM) + NGF 109.62 4.20 (10 ng/mL) FK506 (10 nM) +
NGF 155.64 5.40 (10 ng/mL) Geldanamycin (1 nM) + FK506 145.26 4.02
(10 nM) + NGF (10 ng/mL) Geldanamycin (10 nM) + FK506 134.82 3.34
(10 nM) + NGF (10 ng/mL)
[0080] Geldanamycin and FK506 each stimulate neurite outgrowth in a
concentration dependent manner. The similar neurotrophic effects of
geldanamycin and FK506, their additive effects at very low
concentrations (e.g. 0.1 nM; data not shown), and their inhibitory
effects at high concentrations (like high concentrations of either
compound alone) demonstrate that the two compounds act on nerve
cells to promote nerve outgrowth by a common mechanism. As the
following examples will illustrate, that mechanism has now been
found to involve an interaction of both compounds with components
of the steroid receptor complex. FKBP12 does not appear to have a
role in the stimulation of neurite outgrowth by either geldanamycin
or FK506.
[0081] Cell lines other than the SH-SY5Y human neuroblastoma cells
can be used in the nerve growth assays. Examples of suitable other
cell lines include PC-12 (rat pheochromocytoma), LA-N-5 cells
(human neuroblastoma cells less differentiated than SY5Y cells),
and Neuro-2a and NS20Y cells (mouse neuroblastoma).
EXAMPLE 3
FKBP-12 Knockout Mice Demonstrate FKBP-12 Not Involved in
Neurotrophic Activity
[0082] FKBP-12 knockout mice (Shou, et al., Nature 391:489, 1998)
were used to test whether FKBP-12 is necessary for FK506's ability
to increase nerve elongation. Such mice usually die from severe
cardiomyopathy between embryonic day 14.5 (E14.5) and birth,
consistent with the known association between FKBP-12 and calcium
release channels. No gross pathology has been noted in brains of
these mice. Primary neuronal hippocampal cultures were prepared
from E18.5 homozygote FKBP-12 knockout and wild-type mice. No
difference was found in FK506's regenerative-promoting response of
neurons in FKBP-12 knockout and wild-type mice. Mean axonal lengths
of hippocampal neurons were not significantly different between
FKBP-12 knockout and wild-type mice in drug-free cell cultures
(203.+-.9.5 and 219.+-.8.0, respectively; mean.+-.S.E.M.) [two-way
ANOVA and Scheffe's test of least significant differences; p=0.68,
df=230)] or FK506-treated cultures (264.+-.18.2 and 276.+-.11.1,
respectively) [two-way ANOVA and Scheffe's test of least
significant differences; p=0.94, df=112)].
[0083] FK506 elicited a similarly significant increase over
non-treated values in cells from FKBP-12 knockout [two-way ANOVA
and Scheffe's test of least significant differences; p<0.006,
df=144] and wild-type mice [two-way ANOVA and Scheffe's test of
least significant differences; p<0.002, df=198]; i.e., 30% and
26%, respectively. Thus, neuronal cells from FKBP-12 knockout mice
retain their responsiveness to the neurite outgrowth-promoting
property of FK506. FKBP-12 is therefore not required for FK506 to
promote neurite outgrowth in vitro.
EXAMPLE 4
FKBP-52 Blocks FK506 Neurotrophic Activity
[0084] Neuroblastoma SH-SY5Y cells were used to examine human
neurite outgrowth in vitro and to explore which neuroimmunophilin
mediates the effect. SH-SY5Y cells do not extend processes in the
absence of exogenous nerve growth factor (NGF), with optimal
neurotrophic activity at 10 ng/ml NGF. Initial studies showed that
FK506 increases neurite outgrowth in SH-SY5Y cells in a
concentration-dependent manner. Cumulative histograms of neurite
lengths show that 10 pM-10 nM FK506 significantly [Mann-Whitney U
test (.alpha.=0.05)] increases neurite outgrowth. However, 100 nM
was less effective and, at 1000 nM or greater concentrations,
neurite outgrowth was inhibited.
[0085] The involvement of FKBP-52 was demonstrated using a mouse
monoclonal antibody, FKBP-52 Ab (StressGen Biotechnologies Corp.,
British Columbia, Canada) that does not interact with FKBP-12. To
get the antibody into the cells, SH-SY5Y cells were permeabilized
with saponin (30 .mu.g/.mu.l) for 10 min in the presence of the
antibody; preliminary experiments showed that saponin treatment did
not alter the response of the cells to NGF alone. The FKBP-52
antibody significantly [Mann-Whitney U test (.alpha.=0.05)] blocked
the ability of FK506 (1 and 10 nM) to promote neurite outgrowth
from SH-SY5Y cells in a concentration-dependent manner between 50
and 100 nM. Cumulative histograms of neurite lengths show that 100
nM FKBP-52 antibody completely blocks the action of FK506 at these
concentrations. Surprisingly, the antibody blocked not only the
effect of FK506 but also NGF's effect, suggesting a convergence of
their signal transduction pathways perhaps involving the MAP kinase
pathway (ERK2). Regardless of the underlying mechanism involved,
FK506's neurite outgrowth-promoting property is totally dependent
on its interaction with the immunophilin FKBP-52.
EXAMPLE 5
FKBP-52 Antibody Promotes Neurite Outgrowth
[0086] The FKBP-52 antibody (FKBP-52 Ab) itself possesses agonistic
properties on neurite outgrowth. Cumulative histograms of neurite
lengths show that FKBP-52 Ab significantly [Mann-Whitney U test
(.alpha.=0.05)] shifted the distribution of neurite lengths to the
right in a concentration-dependent manner, indicating longer
processes. In fact, the FKBP-52 Ab elicited even longer neurites
per unit time than those maximally observed with FK506 (10 nM),
producing some of the fastest growing neurites found to date
(maximal length, 880 .mu.m). These findings demonstrate that it is
possible to develop compounds which can distinguish between FXBP-52
and FKBP-12 (i.e., do not substantially bind to both immunophilins)
while maintaining the ability to increase neurite outgrowth. This
finding enables the development of a new class of neuroimmunophilin
ligands: neuroimmunophilin compounds, having low or absent FKBP-12
binding affinity, which specifically bind to components of the SRC,
and increase nerve regeneration by interacting selectively with
FKBP-52 (or other components of the SRC, such as p23 or hsp-90)
[0087] The synthetic glucocorticoid dexamethasone and O-estradiol
both significantly increased neurite outgrowth in SH-SY5Y cells in
a concentration-dependent manner. .beta.-Estradiol (50 nM) produced
a significantly [Mann-Whitney U test (.alpha.=0.05)] greater
positive effect on neurite outgrowth than dexamethasone (50 nM),
suggesting a greater involvement of the estrogen receptor complex
in SH-SY5Y cells. This is supported by the data also showing that
the combination of .beta.-estradiol and FK506 did not produce a
further significant [Mann-Whitney U test (.alpha.=0.05)] increase
in neurite outgrowth, indicating that these compounds act at the
same steroid receptor sub-type. In contrast, the combination of
dexamethasone and FK506 produced neurites (maximal length, 960
.mu.m) that grew at least as, if not more, rapidly than those under
FKBP-52 antibody modulation, indicating that dexamethasone and
FK506 act at different steroid receptor sub-types. Taken together,
the finding that maximal neurite outgrowth elicited by FK506 and ,
.beta.-estradiol is not additive suggests the estrogen receptor
complex plays a greater role than the glucocorticoid receptor
complex in human SH-SY5Y neurite outgrowth promotion by FK506.
EXAMPLE 6
Geldanamycin Promotes Neurite Outgrowth by Disruption of Steroid
Receptor Complex
[0088] SH-SY5Y cells were treated with geldanamycin, a benzoquinone
antibiotic that blocks the reassociation of the mature steroid
complex (containing FKBP-52 and p23), thereby preventing nuclear
translocation and activation of steroid response elements.
Geldanamycin (0.1-10 nM) alone significantly [Mann-Whitney U test
(.alpha.=0.05)] increased neurite outgrowth in a
concentration-dependent fashion. Thus, disruption of the steroid
receptor complex is sufficient to increase neurite outgrowth. Since
geldanamycin and steroid hormones have opposite effects on the
translocation of the steroid receptor ligand component to the
nucleus, these results demonstrate that the promotional effect of
these compounds on neurite outgrowth is mediated by a mechanism
other than nuclear translocation of the steroid receptor
ligand-binding component and activation of steroid response
elements. Using this information, it is now possible to exploit the
structure of geldanamycin to develop a new class of hsp-90-binding
compounds for use in nerve regeneration.
[0089] Assays for determining disruption of the steroid receptor
complex are set forth in Example 10.
EXAMPLE 7
Neurotrophic Effect of Geldanamycin Combined with Other
Compounds
[0090] To explore the interaction of other neurotrophic substances
with geldanamycin, SH-SY5Y cells were co-treated with 1 nM (not
shown) or 10 nM geldanamycin and various other compounds. On the
one hand, geldanamycin (10 nM) significantly [Mann-Whitney U test
(.alpha.=0.05)] inhibited neurite outgrowth promotion by FK506,
dexamethasone or .beta.-estradiol; at 0.1 nM, geldanamycin was less
effective in inhibiting the neurite outgrowth-promoting effect of
all these compounds. On the other hand, geldanamycin (10 nM)
significantly [Mann-Whitney U test (.alpha.=0.05)] enhanced the
neurite outgrowth-promoting effect of the FKBP-52 antibody. The
combined effect of FKBP-52 antibody and geldanamycin is consistent
with their different binding sites on hsp-90; geldanamycin binds to
the N-terminus and FKBP-52 to the C-terminus portions of hsp-90
(Scheigel et al., J. Bio. Chem. 272:8007, 1997). This finding shows
an interaction at the steroid level complex for all tested
compounds, yet reveals that the antibody acts somewhat
differently.
[0091] These findings can be explained by the model shown in FIG. 2
in which geldanamycin produces a conformational change (not
dissociation) in hsp-90 which, via its adenosine triphosphatase
activity, leads to an activation (adenosine diphosphate) state in
which p23 dissociates from the complex. In contrast, this
conformational change may be blocked, thereby preventing release of
p23, when FKBP-52 is bound to FK506, because FK506 does not
dissociate FKBP-52 from the complex; a similar interaction may
occur in the presence of steroid hormones to prevent the
conformational change in hsp-90. This model indicates that the
FKBP-52 antibody dissociates FKBP-52 from the complex, perhaps by
altering its degree of phosphorylation and thereby reducing its
binding to hsp-90, and leads to a conformational change in hsp-90
that results in release of p23. Thus, the combination of
geldanamycin and the FKBP-52 antibody are additive (not inhibitory)
because dissociation of FKBP-52 from hsp-90 would not prevent the
geldanamycin-induced conformational change that releases p23.
[0092] In addition, the association of FKBP-52 with microtubules
(Czar et al., Mol. Endocrinol 8:1731, 1994) and perhaps
microfilaments such as actin (Tai et al., Biochem. 32:8842, 1993)
following its dissociation from hsp-90, would promote the greater
neurite outgrowth seen with FKBP-52 antibody than with FK506. The
microtubules are involved with neurite outgrowth and axonal
elongation, hence association of FKBP-52 with those elements (after
dissociation from the SRC) is a particularly effective mechanism of
promoting neurite growth.
EXAMPLE 8
Stabilization of Steroid Receptor Complex Inhibits Neurite
Outgrowth
[0093] This Example shows that prevention of the dissociation of
the steroid receptor complex inhibits neurite outgrowth, as
predicted by the model shown in FIG. 2. SH-SY5Y cells were treated
with sodium molybdate, a transition metal oxyanion, that at a
concentration of 20 mM prevents dissociation of the complex in
intact cells. Surprisingly, molybdate (20 mM) itself exhibited a
modest but significant [Mann-Whitney U test (.alpha.=0.05)] agonist
effect on neurite outgrowth. As predicted, molybdate (20 mM)
reduced the neurite outgrowth promotion elicited by FK506, the
distribution of neurite lengths produced by FK506 in the presence
of molybdate being not significantly [Mann-Whitney U test
(.alpha.=0.05)] different from that with molybdate alone.
Furthermore, molybdate (20 mM) significantly [Mann-Whitney U test
(.alpha.=0.05)] inhibited the neurite outgrowth-promoting effects
of FKBP-52 antibody.
[0094] The neurite outgrowth-promoting effect of molybdate (20 mM)
in the presence of dexamethasone was significantly [Mann-Whitney U
test (.alpha.=0.05)] reduced compared to molybdate alone.
Furthermore, molybdate (20 mM) completely [Mann-Whitney U test
(.alpha.=0.05)] inhibited the neurite outgrowth-promoting effect of
.beta.-estradiol and geldanamycin; the larger degree of interaction
between molybdate and .beta.-estradiol compared to molybdate and
dexamethasone is consistent with a greater involvement of the
estrogen receptor complex in human SH-SY5Y neurite outgrowth.
Molybdate produced similar but less marked effects at a lower (2
mM) concentration (not shown).
[0095] While it is unclear how molybdate alone increases outgrowth,
the data (showing that molybdate inhibits the activity of all
agents, including FKBP-52 antibody) indicate that dissociation of
the receptor complex is an essential step for activation of the
neurite development pathway. Prevention of this dissociation by
molybdate inhibits the neurotrophic activity of geldanamycin and
other neurotrophic agents that act by disrupting association of the
complex. Hence inhibition of neurotrophic activity by adding
molybdate to an assay can constitute a test for determining whether
a neurotrophic agent is structurally or functionally disrupting the
SRC.
EXAMPLE 9
Determination of Rotamase Inhibition Activity
[0096] Some embodiments of the present invention have low or absent
inhibition of peptidyl-prolyl isomerase (rotamase) activity.
Inhibition of this activity can be evaluated by techniques known in
the art, such as that described in U.S. Pat. No. 5,614,547.
Inhibition is expressed as an apparent Ki for cis-trans
isomerization of an alanine-proline bond in a model substrate,
N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, which is monitored
spectrophometrically in a chymotrypsin-coupled assay, which
releases para-nitroanilide from the trans form of the'substrate.
The inhibition of this reaction caused by the addition of different
concentrations of inhibitor is determined, and the data is analyzed
as a change in the first-order rate constant as a function of
inhibitor concentration to yield the apparent Ki values.
[0097] In a plastic cuvette are added 950 ml of ice cold assay
buffer (25 mM HEPES, pH 7.8, 100 nM NaCl), 10 ml of FKBP (2.5 mM in
10 mM Tris-Cl pH j7.5, 100 mM NaCl, 1 mM dithiothreitol), 25 ml of
chymotrypsin (50 mg/ml in 1 mM HCl) and 10 ml of test compound at
various concentrations in dimethyl sulfoxide. The reaction is
initiated by the addition of 5 ml of substrate
(succinyl-Ala-Phe-Pro-Phe-para-nitroanilide, 5 mg/ml in 2.35 mM
LiCl in trifluroethanol).
[0098] Absorbance at 390 nm versus time is monitored for 90 seconds
using a spectrophotometer and the rate constants are determined
from the absorbance versus time data files. Inhibitors that have an
apparent Ki of 2500 or greater, for example greater than 5000 or
even 10,000, are considered to have "low" rotamase inhibition.
EXAMPLE 10
Assays for Determining if Agent Binds to and Disrupts SRC
Function
[0099] Assays for detecting binding to components of the SRC are
given in Example 1. Detection of disruption of the SRC can be
assessed by additional assays.
[0100] Disruption of the SRC by release of p23 can be assessed by
the techniques disclosed in Whitesell and Cook, 1996, where
benzoquinone ansamycin binding to hsp-90 was shown to result in
complete loss of p23 protein from glucocorticoid receptor
immunoprecipitates, which was associated with a rapid,
noncompetitive loss of dexamethasone binding activity, and a slower
(2-8 hours) marked decline in the cellular level of glucocorticoid
receptor protein. Using this approach, drug treatment did not
disrupt coprecipitation of hsp-90 with glucocorticoid receptor, and
a complete loss of detectable p23 from glucocorticoid receptor
precipitates.
[0101] Whitesell and Cook performed affinity precipitation by
lysing cells in TNESV buffer (50 mM Tris-HCl, pH 7.4%/1% Nonidet
P-40/2 mM EDTA/100 mM NaCl/1 mM orthovanadate/1 mM
phenylmethysulfonyl fluoride/20 .mu.g/ml leupeptin/20 .mu.g/ml
aprotinin) and lysates (0.75 mg of total protein per precipitation)
incubated with geldanamycin-coupled beads. Bound proteins are
eluted by heating in reduced loading buffer and analyzed by
SDS-PAGE followed by Coomassie blue staining. Immnoprecipitation
from cell lysates is performed using a specific monoclonal antibody
BuGR-2, and protein G Sepharose beads (Pharmacia). For experiments
involving coprecipitation of GR with heteroprotein complex
components, cells are lysed in detergent-free hypotonic buffer with
10 mM sodium molbydate. Immunoblot detection of proteins in total
cells lysates, geldanamycin affinity precipitates, and
glucocorticoid receptor immunoprecipitates are performed after
SDS-PAGE and electrophoretic transfer of proteins to
nitrocellulose. BuGR-2 hybridoma supernatant (1:40) is used for
detection of rodent derived GR while a peptide-derived rabbit
polyclonal antibody (1:250; PA1-512, Affinity Bioreagents; Golden,
Colo.) is used for the human GR. Hsp-90 and hsp-70 are detected
with antibodies AC88 and N27F3-4 respectively (1:5000; StressGen;
Victoria, BC, Canada). Ascites containing antibody JJ3 (1:1000) is
used to blot for p23. Polyclonal rabbit anti-ubiquitin antiserum
(1:500; Sigma Chemcial Colo.) is used to detect ubiquitinated
proteins after blots are autoclaved for 20 minutes to fully
denature ubiquitinated proteins and enhance their detection.
Detection is achieved using appropriate peroxidase-conjugated
secondary antibodies (1:20,000) and chemiluminescent substrate
(Kierkegaard and Perry Laboratories, Gaithersburg, Md.).
[0102] Loss of dexamethasone binding activity can be determined
with a binding assay in which HeLa cells (2.times.10.sup.5/well,
24-well plate) are treated with various concentrations of
geldanamycin for varying periods of time in complete medium at 37
degrees C. At the end of the treatment interval, medium is
aspirated and monolayers washed twice with ice-cold PBS containing
1% BSA and 0.1% sodium azide (binding buffer). Monolayers are then
incubated for 60 minutes on ice with 1.0 .mu.Ci/well (48 nM)
[.sup.3H]dexamethasone (Amersham, 82 Ci/mmol) in binding buffer
with or without 5 mM non-radioactive dexamethasone. After this
binding interval, wells are washed four times with cold binding
buffer and then extracted with 0.5 ml ethanol for 30 minutes. The
ethanol solution is transferred to scintillation vials and
evaporated to dryness before standard liquid scintillation
counting. Specific binding is calculated as the counts per minute
bound in the absence of excess nonradioactive dexamethasone less
the counts per minute bound in its presence. Similar measurements
can be made with estrogen and other steroid receptors, to detect
disruption in ligand binding, by substituting those steroids for
dexamethasone.
[0103] A loss of detectable p23 (disappearance of the p23 band)
from receptor immunoprecipitates can indicate loss of p23 in
association with disruption of the SRC.
EXAMPLE 11
Preparation of Antibodies
[0104] The present invention also contemplates the preparation of
antibodies against components of the SRC. The components of the SRC
can be purified by techniques known in the art, such as
immunoprecipitation. Monoclonal or polyclonal antibodies may be
produced to either the SRC component proteins, peptide fragments,
or mutant forms of these proteins. Optimally, antibodies raised
against the protein will specifically detect the protein. That is,
antibodies raised against the protein would recognize and bind the
protein and would not substantially recognize or bind to other
proteins found in human cells. The determination that an antibody
specifically detects a protein is made by any one of a number of
standard immunoassay methods; for instance, the Western blotting
technique (Sambrook et al., 1989).
[0105] To determine that a given antibody preparation specifically
detects the protein by Western blotting, total cellular protein is
extracted from human cells (for example, lymphocytes) and
electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel. The
proteins are then transferred to a membrane (for example,
nitrocellulose) by Western blotting, and the antibody preparation
is incubated with the membrane. After washing the membrane to
remove non-specifically bound antibodies, the presence of
specifically bound antibodies is detected by the use of an
anti-mouse antibody conjugated to an enzyme such as alkaline
phosphatase; application of the substrate 5-bromo4-chloro-3-indolyl
phosphate/nitro blue tetrazolium results in the production of a
dense blue compound by immuno-localized alkaline phosphatase.
[0106] Antibodies which specifically detect the protein will, by
this technique, be shown to bind to the protein band (which will be
localized at a given position on the gel determined by its
molecular weight). Non-specific binding of the antibody to other
proteins may occur and may be detectable as a weak signal on the
Western blot. The non-specific nature of this binding will be
recognized by one skilled in the art by the weak signal obtained on
the Western blot relative to the strong primary signal arising from
the specific protein binding.
[0107] Antibodies that specifically bind to a protein component of
the SRC belong to a class of molecules that are referred to herein
as "specific binding agents." Specific binding agents that are
capable of specifically binding to the SRC protein may include
polyclonal antibodies, monoclonal antibodies (including humanized
monoclonal antibodies) and fragments of monoclonal antibodies such
as Fab, F(ab')2 and Fv fragments, as well as any other agent
capable of specifically binding to a protein component of the
SRC.
[0108] Monoclonal antibody to epitopes of the SRC protein
components identified and isolated as described can be prepared
from murine hybridomas according to the classical method of Kohler
and Milstein (1975) or derivative methods thereof. Briefly, a mouse
is repetitively inoculated with a few micrograms of the selected
protein over a period of a few weeks. The mouse is then sacrificed,
and the antibody-producing cells of the spleen isolated. The spleen
cells are fused by means of polyethylene glycol with mouse myeloma
cells, and the excess unfused cells destroyed by growth of the
system on selective media comprising aminopterin (HAT media). The
successfully fused cells are diluted and aliquots of the dilution
placed in wells of a microtiter plate where growth of the culture
is continued. Antibody-producing clones are identified by detection
of antibody in the supernatant fluid of the wells by immunoassay
procedures, such as ELISA, as originally described by Engvall
(1980), and derivative methods thereof. Selected positive clones
can be expanded and their monoclonal antibody product harvested for
use. Detailed procedures for monoclonal antibody production are
described in Harlow and Lane (1988). In addition, protocols for
producing humanized forms of monoclonal antibodies (for therapeutic
applications) and fragments of monoclonal antibodies are known in
the art.
[0109] Polyclonal antiserum containing antibodies to heterogeneous
epitopes of a single protein can be prepared by immunizing suitable
animals with the expressed protein, which can be unmodified or
modified to enhance immunogenicity. Effective polyclonal antibody
production is affected by many factors related both to the antigen
and the host species. For example, small molecules tend to be less
immunogenic than others and may require the use of carriers and
adjuvant. Also, host animals vary in response to site of
inoculations and dose, with both inadequate or excessive doses of
antigen resulting in low titer antisera. Small doses (ng level) of
antigen administered at multiple intradermal sites appears to be
most reliable. An effective immunization protocol for rabbits can
be found in Vaitukaitis et al. (1971).
[0110] Booster injections can be given at regular intervals, and
antiserum harvested when antibody titer thereof, as determined
semi-quantitatively, for example, by double immunodiffusion in agar
against known concentrations of the antigen, begins to fall. See,
for example, Ouchterlony et al. (1973). Plateau concentration of
antibody is usually in the range of 0.1 to 0.2 mg/ml of serum
(about 12 .mu.M). Affinity of the antisera for the antigen is
determined by preparing competitive binding curves, as described,
for example, by Fisher (1980).
[0111] A third approach to raising antibodies against the SRC
proteins is to use synthetic peptides synthesized on a commercially
available peptide synthesizer based upon the predicted amino acid
sequence of the protein components of the SRC.
EXAMPLE 12
Geldanamycin Analogs and Derivatives
[0112] The term "geldanamycin derivative" refers to compounds that
are structurally analogous to geldanamycin in their ability to
stimulate neurite outgrowth. Geldanamycin consists of a closed ansa
ring with a planar benzoquinone embedded in it. 6
[0113] The ansa ring is sterically hindered because its backbone
includes a planar amide and three carbon-carbon double bonds (two
of them arranged in a 1,3-diene), and nine of its sixteen backbone
atoms carry nonhydrogen substitutents such as a carbonyl, a
carbamate (--OC(O)NH.sub.2), a hydroxyl, two methoxy and four
methyl groups. The crystal structure of geldanamycin has been set
forth, and the structure/activity relationships of the benzoquinone
ansamycins have been described in Stebbins et al., Cell 89:239-250,
1997; Schnur et al., J. Med. Chem. 38:3813-3820, 1995; and Schnur
et al., J. Med. Chem. 38:3806-3812, 1995. Geldanamycin derivatives
may have the carbamate group and ansa ring of geldanamycin (Schnur
et al., J. Med. Chem. 38:3806-3812, 1995), and/or have
modifications at functional groups such as 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, 56100766, and 89002593, for example.
[0114] The structures of some of the benzoquinone ansamycins that
are structural analogs of geldanamycin are set forth in Table
2.
2TABLE 2 Some Structural Analogs of Geldanamycin 7 R.sub.1 R.sub.2
R.sub.3 R.sub.4 GA --OH --H --OCH.sub.3 --H HA --OCH.sub.3
--OCH.sub.3 --H --H HD --OH --H --NHC.sub.6H.sub.12NH.sub.2 --H GM
--OH --H --OCH.sub.3 --CHN--N N--CH.sub.3
[0115] Additional benzoquinone ansamycin analogs of geldanamycin
are shown in Tables 3 and 4. Table 3 illustrates several synthesis
schemes for geldanamycin derivatives, while Table 4 sets forth
substitutions for the derivatives, as described more fully in
Schnur, et al., J. Med. Chem. 38:3813-3820, 1995.
3TABLE 3 Synthesis Scheme for Geldanamycin Derivatives With
Ansa-Ring Modifications 8 9 10 11 12
[0116]
4TABLE 4 Substitutions for Compounds Shown in Table 3 compd.sup.a R
R' HBM A GDM OCH.sub.3 DHGDM OCH.sub.3 1a --N(CH.sub.2).sub.3-- 1b
NH.sub.2 1c NHCH.sub.2CH.dbd.CH.sub.2 1d NHCH(CH.sub.3).sub.2 1e
NHCH.sub.2 1f* --N(CH.sub.2).sub.3-- 1g* NH.sub.2 1h*
NHCH.sub.2CH.dbd.CH.sub.2 2a phenacyl NH.sub.2 2b
3',4'-dichlorophenacyl NH.sub.2 2c 3'-iodo-4'-azidophenacyl
NH.sub.2 2d 2'-methoxyphenacyl NH.sub.2 2e* 2'-methoxyphenacyl
NH.sub.2 2f 4'-methoxyphenacyl NH.sub.2 2g 4'-nitrophenacyl
NH.sub.2 2h 1'-napthacyl NH.sub.2 2i 2'-napthacyl NH.sub.2 2j
4'-azidophenacyl NH.sub.2 2k 4'-azidophenacyl --N(CH.sub.2).sub.3--
2l 2'-oxopropyl NH.sub.2 2m 2'-pyridylmethyl NH.sub.2 3a COCH.sub.8
--N(CH.sub.2).sub.3-- 3b CONHSO.sub.2NHCH(CH.sub.3).sub.2
--N(CH.sub.2).sub.3-- 3c CONHSO.sub.2N[(CH.sub.2).sub.2].sub.2NC-
H.sub.3 --N(CH.sub.2).sub.3-- 3d CONH.sub.2 --N(CH.sub.2).sub.3--
3e CONHSO.sub.2N(CH.sub.2).sub.3 NHCH.sub.2CH.dbd.CH.sub.2 3f
CONHSO.sub.2NHCH(CH.sub.3).sub.2 NHCH.sub.2CH.dbd.CH.sub.2 3g
CONHSO.sub.2N(CH.sub.2).sub.2NCH.sub.3 NHCH.sub.2CH.dbd.CH.sub.2 4a
--N(CH.sub.2).sub.3-- 4b NH.sub.2 5a --N(CH.sub.2).sub.3-- 6a
NHCH(CH.sub.2).sub.3 7a COCO.sub.2H --N(CH.sub.2).sub.3-- 7b
CONHCH.sub.2CH.sub.3 NHCH.sub.2CH.dbd.CH.sub.2 7c
COCH.sub.2NH.sub.2 NHCH.sub.2CH.dbd.CH.sub.2 8a
--N(CH.sub.2).sub.3-- 8b* --N(CH.sub.2).sub.3-- 8c
NHCH.sub.2CH.dbd.CH.sub.2 8d* NHCH.sub.2CH.dbd.CH.sub.2 8e NH.sub.2
8f NHCH(CH.sub.2).sub.2 9a NHCH.sub.2CH.dbd.CH.sub.2 9b
--N(CH.sub.2).sub.3-- 9c NHCH.sub.3 10a S --N(CH.sub.2).sub.3-- 10b
O --N(CH.sub.2).sub.3-- 11a --N(CH.sub.2).sub.3-- 11b
NHCH(CH.sub.3).sub.2 12a CH.sub.2CH.dbd.CH.sub.2 NHCH.sub.3 12b
CH.sub.2C.sub.6H.sub.5 NHCH.sub.3
[0117]
5TABLE 5 Several Synthesis Schemes for Aminogeldanamycin
Derivatives (Scheme 1), and Geldanamycin with Quinone Modifications
(Scheme 2) 13 14 15 16 17
[0118]
6TABLE 6 Some Substitutions for Compounds Shown in Table 5
compd.sup.a R R' HBM A GDM OCH.sub.3 DHGDM OCH.sub.3 1a
--N(CH.sub.2).sub.3-- 1b NH.sub.2 1c NHCH.sub.2CH.dbd.CH.sub.2 1d
NHCH(CH.sub.3).sub.2 1e NHCH.sub.2 1f* --N(CH.sub.2).sub.3-- 1g*
NH.sub.2 1h* NHCH.sub.2CH.dbd.CH.sub.2 2a phenacyl NH.sub.2 2b
3',4'-dichlorophenacyl NH.sub.2 2c 3'-iodo-4'-azidophenacyl
NH.sub.2 2d 2'-methoxyphenacyl NH.sub.2 2e* 2'-methoxyphenacyl
NH.sub.2 2f 4'-methoxyphenacyl NH.sub.2 2g 4'-nitrophenacyl
NH.sub.2 2h 1'-napthacyl NH.sub.2 2i 2'-napthacyl NH.sub.2 2j
4'-azidophenacyl NH.sub.2 2k 4'-azidophenacyl --N(CH.sub.2).sub.3--
2l 2'-oxopropyl NH.sub.2 2m 2'-pyridylmethyl NH.sub.2 3a COCH.sub.8
--N(CH.sub.2).sub.3-- 3b CONHSO.sub.2NHCH(CH.sub.3).sub.2
--N(CH.sub.2).sub.3-- 3c CONHSO.sub.2N[(CH.sub.2).sub.2].sub.2NC-
H.sub.3 --N(CH.sub.2).sub.3-- 3d CONH.sub.2 --N(CH.sub.2).sub.3--
3e CONHSO.sub.2N(CH.sub.2).sub.3 NHCH.sub.2CH.dbd.CH.sub.2 3f
CONHSO.sub.2NHCH(CH.sub.3).sub.2 NHCH.sub.2CH.dbd.CH.sub.2 3g
CONHSO.sub.2N(CH.sub.2).sub.2NCH.sub.3 NHCH.sub.2CH.dbd.CH.sub.2 4a
--N(CH.sub.2).sub.3-- 4b NH.sub.2 5a --N(CH.sub.2).sub.3-- 6a
NHCH(CH.sub.2).sub.3 7a COCO.sub.2H --N(CH.sub.2).sub.3-- 7b
CONHCH.sub.2CH.sub.3 NHCH.sub.2CH.dbd.CH.sub.2 7c
COCH.sub.2NH.sub.2 NHCH.sub.2CH.dbd.CH.sub.2 8a
--N(CH.sub.2).sub.3-- 8b* --N(CH.sub.2).sub.3-- 8c
NHCH.sub.2CH.dbd.CH.sub.2 8d* NHCH.sub.2CH.dbd.CH.sub.2 8e NH.sub.2
8f NHCH(CH.sub.2).sub.2 9a NHCH.sub.2CH.dbd.CH.sub.2 9b
--N(CH.sub.2).sub.3-- 9c NHCH.sub.3 10a S --N(CH.sub.2).sub.3-- 10b
O --N(CH.sub.2).sub.3-- 11a --N(CH.sub.2).sub.3-- 11b
NHCH(CH.sub.3).sub.2 12a CH.sub.2CH.dbd.CH.sub.2 NHCH.sub.3 12b
CH.sub.2C.sub.6H.sub.5 NHCH.sub.3
[0119] Analogs can also 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.
[0120] In some particular embodiments, 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).
EXAMPLE 13
FK506 and Rapamycin Analogs
[0121] The term "FK506 analogs" refers to compounds that are
structurally analogous to FK506 in their ability to stimulate
neuritic outgrowth. Some FK506 analogs, such as V-10,367, retain
the FKBP12 binding domain but lack the structural components of the
effector domain that interacts with calcineurin. The FK506 analogs
may bind FKBP12 with low or high affinity. V-10,367, for example,
binds FKBP12 with high affinity (K.sub.d<1 nM) (Armistead et
al., Acta Crystallogr. 51:522-528, 1995).
[0122] 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. 35:42844296, 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. These references set forth the structure of FK506 and
some of its known analogs, such as V-10,367, which lacks the
effector domain (shown in brackets) that inhibits calcineurin. A
"known" or "recognized" compound is a compound (such as an FK506
analog) that has previously been reported in patents or
publications that qualify as prior art. 18
[0123] FK506 analogs have a wide range of binding affinities for
FKBP-12. The 5 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 FKBP-12. Instead, their effectiveness in
stimulating nerve cell growth relates to ability of such compounds
to physically or functionally disrupt the steroid receptor complex,
for example by interfering with the interaction of FKBP-52 and
hsp-90 in a steroid receptor complex, or by promoting dissociation
of p23 from the complex.
[0124] A "non-binding FK506 analog" is defined as an FK506 analog
that does not substantially bind to FKBP-12. An FK506 analog with
low affinity for binding FKBP-12 refers to an analog that binds
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.
[0125] Alternatively or additionally, the analog may be one that
does not significantly inhibit FKBP-12 rotamase 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 Example 9.
[0126] 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.
[0127] "Rapamycin analogs" include compounds structurally similar
to rapamycin, for example WAY-124,466 shown in Ocain et al.,
Biochem. Biophys. Res. Commun. 192-1340-1346, 1993. This analog is
identical to rapamycin, except that it has been modified in the
triene region, and has a Ki for PPIase activity of 12.5 nM, as
determined by the methods shown in that reference. It is also
non-immunosuppressive, as determined in that reference by an
inability to inhibit the proliferation of murine thymocytes. Other
rapamycin analogs include other macrocyclic trienes, mono- and
diacylated derivatives esterified at the 31 and 42 positions (U.S.
Pat. No. 4,313,885) and water soluble prodrugs of rapamycin (U.S.
Pat. No. 4,650,803); hydrogenated derivatives in which one, two, or
three of the double bonds at the 1-, 3-, or 5-positions have been
reduced to the corresponding alkane, or a pharmaceutically
acceptable salt thereof (U.S. Pat. No. 5,023,262); oxidized
derivatives wherein a hydroxyl group (such as the group at the
42-position) has been oxidized to the corresponding ketone (for
example 42-oxorapamycin or a pharmaceutically acceptable salt, as
described in U.S. Pat. No. 5,023,263); 7,29-bisdesmethyl rapamycin
(U.S. Pat. No. 5,093,338); and rapamycin cleaved between the C-31
and C-32 carbons due to base degradation by a reverse aldol
reaction (U.S. Pat. No. 5,138,051).
EXAMPLE 14
Radicicol and Its Analogs
[0128] "Radicicol analogs" refers to compounds structurally similar
to radicicol, for example those shown in U.S. Pat. No. 5,731,343,
U.S. Pat. No. 5,650,430, U.S. Patent No. 4,228,079, and published
international patent applications W096/33989 and W098/18780 which
are all incorporated by reference. Particular examples of such
compounds are those shown in the following structural formula:
19
[0129] where R.sub.1, R.sub.2, and R.sub.3 are independently
selected from the group consisting of H, C1-C8 alkyl or COR.sub.5.
R.sub.5 is chosen from the group consisting of hydrogen,
substituted alkyl, alkoxy, alkenyl, substituted alkenyl,
alkenyloxy, alkynyl, substituted alkynyl, aryl with 6 to 14 ring
atoms, arlyoxy with 6 to 14 ring atoms, heterocyclic groups with 5
or 6 ring atoms, heterocylic groups with 5 or 6 ring atoms fused to
an aryl group, cycloalkyl, cycloalkenyl, and cycloalkyl fused to an
aryl group. Specific examples of COR.sub.5 are given in Table 7;
any of the groups appearing in Table 7 can be used for R.sub.1,
R.sub.2, and R.sub.3. X is halogen. Y is chosen from the group
consisting of O, and N--O--R.sub.4, wherein R.sub.4 is chosen from
the group consisting of hydrogen and C1-8 alkyl. Z is halogen.
Alternatively, Z can be combined with R.sub.3 to form an epoxide
ring.
7TABLE 7 Cpd. No. R.sup.1 R.sup.2 1 CH.sub.3CH.sub.2CO--
CH.sub.3CH.sub.2CO-- 2 CH.sub.3CH.sub.2CO-- H 3 H
CH.sub.3CH.sub.2CO-- 4 CH.sub.3CH.sub.2CH.sub.2CO--
CH.sub.3CH.sub.2CH.sub.2CO-- 5 CH.sub.3(CH.sub.2).sub.3CO--
CH.sub.3(CH.sub.2).sub.3CO-- 6 CH.sub.3(CH.sub.2).sub.4CO--
CH.sub.3(CH.sub.2).sub.4CO-- 7 CH.sub.3(CH.sub.2).sub.5CO--
CH.sub.3(CH.sub.2).sub.5CO-- 8 H CH.sub.3(CH.sub.2).sub.5CO-- 9
CH.sub.3(CH.sub.2).sub.6CO-- CH.sub.3(CH.sub.2).sub.6CO-- 10 H
CH.sub.3(CH.sub.2).sub.6CO-- 11 CH.sub.3(CH.sub.2).sub.7CO--
CH.sub.3(CH.sub.2).sub.7CO-- 12 H CH.sub.3(CH.sub.2).sub.7CO-- 13
CH.sub.3(CH.sub.2).sub.8CO-- CH.sub.3(CH.sub.2).sub.8CO-- 14 H
CH.sub.3(CH.sub.2).sub.8CO-- 15 CH.sub.3(CH.sub.2).sub.9CO--
CH.sub.3(CH.sub.2).sub.9CO-- 16 H CH.sub.3(CH.sub.2).sub.9CO-- 17
CH.sub.3(CH.sub.2).sub.10CO-- CH.sub.3(CH.sub.2).sub.10CO-- 18 H
CH.sub.3(CH.sub.2).sub.10CO-- 19 CH.sub.3(CH.sub.2).sub.11CO--
CH.sub.3(CH.sub.2).sub.11CO-- 20 H CH.sub.3(CH.sub.2).sub.11CO-- 21
CH.sub.3(CH.sub.2).sub.12CO- -- CH.sub.3(CH.sub.2).sub.12CO-- 22 H
CH.sub.3(CH.sub.2).sub.12CO-- 23 CH.sub.3(CH.sub.2).sub.13CO--
CH.sub.3(CH.sub.2).sub.13CO-- 24 H CH.sub.3(CH.sub.2).sub.13CO-- 25
CH.sub.3(CH.sub.2).sub.14- CO-- CH.sub.3(CH.sub.2).sub.14CO-- 26
CH.sub.3(CH.sub.2).sub.14CO-- H 27 H CH.sub.3(CH.sub.2).sub.14CO--
28 CH.sub.3(CH.sub.2).sub.15CO-- CH.sub.3(CH.sub.2).sub.15CO-- 29
CH.sub.3(CH.sub.2).sub.15CO-- H 30 H CH.sub.3(CH.sub.2).sub.15CO--
31 CH.sub.3(CH.sub.2).sub.16CO-- CH.sub.3(CH.sub.2).sub.16CO-- 32
CH.sub.3(CH.sub.2).sub.16CO-- H 33 H CH.sub.3(CH.sub.2).sub.-
16CO-- 34 CH.sub.3(CH.sub.2).sub.17CO--
CH.sub.3(CH.sub.2).sub.17CO- -- 35 H CH.sub.3(CH.sub.2).sub.17CO--
36 CH.sub.3(CH.sub.2).sub.18CO-- CH.sub.3(CH.sub.2).sub.18CO-- 37 H
CH.sub.3(CH.sub.2).sub.18CO-- 38 CH.sub.3(CH.sub.2).sub.19CO--
CH.sub.3(CH.sub.2).sub.19CO-- 39 H CH.sub.3(CH.sub.2).sub.19CO-- 40
CH.sub.3(CH.sub.2).sub.20CO-- CH.sub.3(CH.sub.2).sub.20CO-- 41 H
CH.sub.3(CH.sub.2).sub.20CO-- 42 (CH.sub.3).sub.2CHCH.sub.2C- O--
(CH.sub.3).sub.2CHCH.sub.2CO-- 43 H (CH.sub.3).sub.2CHCH.sub.2C-
O-- 44 (CH.sub.3).sub.3CCO-- (CH.sub.3).sub.3CCO-- 45 H
(CH.sub.3).sub.3CCO-- 46 CH.sub.2.dbd.CHCH.sub.2CO--
CH.sub.2.dbd.CHCH.sub.2CO-- 47 H CH.sub.2.dbd.CHCH.sub.2CO-- 48
CH.sub.3CH.dbd.CHCO-- CH.sub.3CH.dbd.CHCO-- 49 H
CH.sub.3CH.dbd.CHCO-- 50 (CH.sub.3).sub.2.dbd.CHCO--
(CH.sub.3).sub.2.dbd.CHCO-- 51 H (CH.sub.3).sub.2.dbd.CHCO-- 52
EtCH.dbd.CHCO-- EtCH.dbd.CHCO-- 53 H EtCH.dbd.CHCO-- 54
H.sub.2C.dbd.CHCH.sub.2CH.sub.2CO--
H.sub.2C.dbd.CHCH.sub.2CH.sub.2CO-- 55 H
H.sub.2C.dbd.CHCH.sub.2CH.sub.2CO-- 56 PrCH.dbd.CHCO--
PrCH.dbd.CHCO-- 57 H PrCH.dbd.CHCO-- 58 EtCH.dbd.CHCH.sub.2CO--
EtCH.dbd.CHCH.sub.2CO-- 59 H EtCH.dbd.CHCH.sub.2CO-- 60
PnCH.dbd.CHCO-- PnCH.dbd.CHCO-- 61 H PnCH.dbd.CHCO-- 62
HxCH.dbd.CHCO-- HxCH.dbd.CHCO-- 63 H HxCH.dbd.CHCO-- 64
H.sub.2C.dbd.CH(CH.sub.2).sub.7CO--
H.sub.2C.dbd.CH(CH.sub.2).sub.7CO-- 65 H H.sub.2C.dbd.CH(CH.sub.2)-
.sub.7CO-- 66 H.sub.2C.dbd.CH(CH.sub.2).sub.8CO--
H.sub.2C.dbd.CH(CH.sub.2).sub.8CO-- 67 H H.sub.2C.dbd.CH(CH.sub.2)-
.sub.8CO-- 68 BuCH.dbd.CH(CH.sub.2).sub.7CO--
BuCH.dbd.CH(CH.sub.2).sub.7CO-- 69 H BuCH.dbd.CH(CH.sub.2).sub.7CO-
-- 70 HxCH.dbd.CH(CH.sub.2).sub.7CO--
HxCH.dbd.CH(CH.sub.2).sub.7CO- -- 71 H
HxCH.dbd.CH(CH.sub.2).sub.7CO-- 72 OcCH.dbd.CH(CH.sub.2).sub.7CO--
OcCH.dbd.CH(CH.sub.2).sub.7CO-- 73 H
OcCH.dbd.CH(CH.sub.2).sub.7CO-- 74 Bu(CH.sub.2CH.dbd.CH).sub.2(C-
H.sub.2).sub.7CO-- Bu(CH.sub.2CH.dbd.CH).sub.2(CH.sub.2).sub.7CO--
75 H Bu(CH.sub.2CH.dbd.CH).sub.2(CH.sub.2).sub.7CO-- 76
Me(CH.sub.2CH.dbd.CH).sub.3(CH.sub.2).sub.7CO--
Me(CH.sub.2CH.dbd.CH).sub- .3(CH.sub.2).sub.7CO-- 77 H
Me(CH.sub.2CH.dbd.CH).sub.3(CH.sub.2).s- ub.7CO-- 78
Bu(CH.sub.2CH.dbd.CH).sub.3(CH.sub.2).sub.4CO--
Bu(CH.sub.2CH.dbd.CH).sub.3(CH.sub.2).sub.4CO-- 79
Pn(CH.dbd.CHCH.sub.2).sub.4(CH.sub.2).sub.2CO--
Pn(CH.dbd.CHCH.sub.2).sub- .4(CH.sub.2).sub.2CO-- 80
Me(CH.sub.2CH.dbd.CH).sub.6(CH.sub.2).sub- .2CO--
Me(CH.sub.2CH.dbd.CH).sub.6(CH.sub.2).sub.2CO-- 81 H
Pn(CH.dbd.CHCH.sub.2).sub.4(CH.sub.2).sub.2CO-- 82
HxC(OH)H--CH.sub.2CH.dbd.CHCO-- HxC(OH)H--CH.sub.2CH.dbd.CHCO-- 83
H HxC(OH)H--CH.sub.2CH.dbd.CHCO-- 84 HOCH.sub.2(CH.sub.2).sub.14CO-
-- HOCH.sub.2(CH.sub.2).sub.14CO-- 85 H
HOCH.sub.2(CH.sub.2).sub.14- CO-- 86 (4-MeOPh).sub.2PhC--
(4-MeOPh).sub.2PhC--OCH.sub.2(CH.sub.2- ).sub.14CO--
OCH.sub.2(CH.sub.2).sub.14CO-- 87 H
(4-MeOPh).sub.2PhC--OCH.sub.2(CH.sub.2).sub.14CO-- 88 HC C.CO HC
C.CO 89 H HC C.CO 90 MeC C.CO MeC C.CO 91 H MeC C.CO 92 PnC C.CO
PnC C.CO 93 H PnC C.CO 94 MeOCH.sub.2 CO MeOCH.sub.2 CO 95
MeOCH.sub.2 CO H 96 H MeOCH.sub.2 CO 97 DdcOCH.sub.2CH.sub.2 CO
DdcOCH.sub.2CH.sub.2 CO 98 H DdcOCH.sub.2CH.sub.2 CO 99
MeO(CH.sub.2).sub.9 CO MeO(CH.sub.2).sub.9 CO 100
MeO(CH.sub.2).sub.11 CO MeO(CH.sub.2).sub.11 CO 101
MeO(CH.sub.2).sub.13 CO MeO(CH.sub.2).sub.13 CO 102
MeO(CH.sub.2).sub.15 CO MeO(CH.sub.2).sub.15 CO 103 H
MeO(CH.sub.2).sub.13 CO 104 PhOCH.sub.2CO PhOCH.sub.2CO 105 H
PhOCH.sub.2CO 106 Bz.CO Bz.CO 107 H Bz.CO 108 2-PhPrn 2-PhPrn 109 H
2-PhPrn 110 3-PhPrn 3-PhPrn 111 H 3-PhPrn 112 6-PhHxo 6-PhHxo 113
PhCH.dbd.CH.CO PhCH.dbd.CH.CO 114 H PhCH.dbd.CH.CO 115 -Np.CH.sub.2
CO -Np.CH.sub.2 CO 116 H -Np.CH.sub.2 CO 117 Boz Boz 118 H Boz 119
2-Fur.CO 2-Fur.CO 120 H 2-Fur.CO 121 2-Fur.CH.dbd.CH.CO
2-Fur.CH.dbd.CH.CO 122 3-Thi.CO 3-Thi.CO 123 H 3-Thi.CO 124
2-Thi.CH.dbd.CH.CO 2-Thi.CH.dbd.CH.CO 125 3-Thi.CO H 126 2-Thi.CO
2-Thi.CO 127 H 2-Thi.CO 128 2-ThiCH.sub.2CO 2-ThiCH.sub.2 CO 129 H
2-ThiCH.sub.2 CO 130 2-NH.sub.2-4-Thiz.CH.sub.2 CO
2-NH.sub.2-4-Thiz.CH.sub.2 CO 131 H 2-NH.sub.2-4-Thiz.CH.sub.2 CO
132 5-oxo-2-Pyrd.CO 5-oxo-2-Pyrd.CO 133 H 5-oxo-2-Pyrd.CO 134
3-Isox.CO 3-Isox.CO 135 H 3-Isox.CO 136 4-Isox.CO 4-Isox.CO 137 H
4-Isox.CO 138 6-oxo-2-Pip.CO 6-oxo-2-Pip.CO 139 H 6-oxo-2-Pip.CO
140 3NH.sub.2Prn 3NH.sub.2Prn 141 H 3NH.sub.2Prn 142 6-NH.sub.2Hxo
6-NH.sub.2Hxo 143 H 6-NH.sub.2Hxo 144 H.sub.2N(CH.sub.2).sub.11 CO
H.sub.2N(CH.sub.2).sub.11CO 145 H.sub.2N(CH.sub.2).sub.11 CO H 146
H.sub.2N(CH.sub.2).sub.15 CO H.sub.2N(CH.sub.2).sub.15CO 147
H.sub.2N(CH.sub.2).sub.15 CO H 148 TrocNH(CH.sub.2).sub.2 CO
TrocNH(CH.sub.2).sub.2 CO 149 TrocNH(CH.sub.2).sub.2 CO H 150
TrocNH(CH.sub.2).sub.5 CO TrocNH(CH.sub.2).sub.5 CO 151
TrocNH(CH.sub.2).sub.5 CO H 152 TrocNH(CH.sub.2).sub.11 CO
TrocNH(CH.sub.2).sub.11 CO 153 H TrocNH(CH.sub.2).sub.11CO 154
TrocNH(CH.sub.2).sub.15 CO TrocNH(CH.sub.2).sub.15 CO 155 H
TrocNH(CH.sub.2).sub.15 CO 156 AocNH(CH.sub.2).sub.2 CO
AocNH(CH.sub.2).sub.2 CO 157 H AocNH(CH.sub.2).sub.2 CO 158
AocNH(CH.sub.2).sub.5 CO AocNH(CH.sub.2).sub.5 CO 159
AocNH(CH.sub.2).sub.5 CO H 160 AocNH(CH.sub.2).sub.11 CO
AocNH(CH.sub.2).sub.11 CO 161 H AocNH(CH.sub.2).sub.11 CO 162
AocNH(CH.sub.2).sub.15 CO AocNH(CH.sub.2).sub.15 CO 163 H
AocNH(CH.sub.2).sub.15 CO 164 FmocNH(CH.sub.2).sub.11 CO
FmocNH(CH.sub.2).sub.11 CO 165 Ph.sub.3C.S.NH(CH.sub.2).sub.11 CO
Ph.sub.3C.S.NH(CH.sub.2).sub.11 CO 166 CICH.sub.2CO CICH.sub.2CO
167 H CICH.sub.2CO 168 FCH.sub.2CO FCH.sub.2CO 169 H FCH.sub.2CO
170 BrCH.sub.2CO BrCH.sub.2CO 171 H BrCH.sub.2CO 172 BrCH.sub.2CO H
173 ICH.sub.2CO ICH.sub.2CO 174 ICH.sub.2CO H 175 H ICH.sub.2CO 176
MeSCH.sub.2CO MeSCH.sub.2CO 177 H MeSCH.sub.2CO 178
MeS(CH.sub.2).sub.2CO MeS(CH.sub.2).sub.2CO 179
MeS(CH.sub.2).sub.9CO MeS(CH.sub.2).sub.9CO 180
MeS(CH.sub.2).sub.11CO MeS(CH.sub.2).sub.11CO 181
MeS(CH.sub.2).sub.15CO MeS(CH.sub.2).sub.15CO 182
MeSO.sub.2CH.sub.2CO MeSO.sub.2CH.sub.2CO 183 H
MeSO.sub.2CH.sub.2CO 184 MeSO.sub.2(CH.sub.2).sub.9CO
MeSO.sub.2(CH.sub.2).sub.9CO 185 MeSO.sub.2(CH.sub.2).sub.12CO
MeSO.sub.2(CH.sub.2).sub.12CO 186 MeSO.sub.2(CH.sub.2).sub.15CO
MeSO.sub.2(CH.sub.2).sub.15CO 187 MeSO.CH.sub.2CO MeSO.CH.sub.2CO
188 H MeSO.CH.sub.2CO 189 MeSO(CH.sub.2).sub.9CO
MeSO(CH.sub.2).sub.9CO 190 MeSO(CH.sub.2).sub.11CO
MeSO(CH.sub.2).sub.11CO 191 MeSO.sub.2(CH.sub.2).sub.15CO
MeSO.sub.2(CH.sub.2).sub.15CO 192 HXS.CH.sub.2CO HXS.CH.sub.2CO 193
H HXS.CH.sub.2CO 194 DdcS.CH.sub.2CO DdcS.CH.sub.2CO 195 H
DdcS.CH.sub.2CO 196 PhS.CH.sub.2CO PhS.CH.sub.2CO 197 H
PhS.CH.sub.2CO 198 PhS(CH.sub.2).sub.9CO PhS(CH.sub.2).sub.9CO 199
PhS(CH.sub.2).sub.11CO PhS(CH.sub.2).sub.11CO 200
PhS(CH.sub.2).sub.15CO PhS(CH.sub.2).sub.15CO 201
2-PhEt.S.CH.sub.2CO 2-PhEt.S.CH.sub.2CO 202 H 2-PhEt.S.CH.sub.2CO
203 Bz.SS.CH.sub.2CO Bz.SS.CH.sub.2CO 204 H Bz.SS.CH.sub.2CO 205
Bz.SS.(CH.sub.2).sub.9CO Bz.SS.(CH.sub.2).sub.9CO 206
Bz.SS.(CH.sub.2).sub.11CO Bz.SS.(CH.sub.2).sub.11CO 207
Bz.SS.(CH.sub.2).sub.15CO Bz.SS.(CH.sub.2).sub.15CO 208
Et.sub.2NCH.sub.2CO Et.sub.2NCH.sub.2CO 209 Et.sub.2NCH.sub.2CO H
210 Et.sub.2N(CH.sub.2).sub.9CO Et.sub.2N(CH.sub.2).sub.9CO 211
Et.sub.2N(CH.sub.2).sub.11CO Et.sub.2N(CH.sub.2).sub.11CO 212
Et.sub.2N(CH.sub.2).sub.15CO Et.sub.2N(CH.sub.2).sub.15CO 213
1-Me-4-Piz.CH.sub.2CO 1-Me-4-Piz.CH.sub.2CO 214 H
1-Me-4-Piz.CH.sub.2CO 215 1-Me-4-Piz.(CH.sub.2).sub.15CO
1-Me-4-Piz.(CH.sub.2).sub.15CO 216 Me.sub.2N(CH.sub.2).sub.9CO
Me.sub.2N(CH.sub.2).sub.9CO 217 Me.sub.2N(CH.sub.2).sub.11CO
Me.sub.2N(CH.sub.2).sub.11CO 218 Me.sub.2N(CH.sub.2).sub.15CO
Me.sub.2N(CH.sub.2).sub.15CO 219 4-Mor.CH.sub.2CO 4-Mor.CH.sub.2CO
220 4-Mor.CH.sub.2CO H 221 4-Mor.(CH.sub.2).sub.15CO
4-Mor.(CH.sub.2).sub.15CO 222 4-Mor.(CH.sub.2).sub.15CO H 223
1-Pyrd.CH.sub.2CO 1-Pyrd.CH.sub.2CO 224 H 1-Pyrd.CH.sub.2CO 225
1-Pyrd.(CH.sub.2).sub.15CO 1-Pyrd.(CH.sub.2).sub.15CO 226 H
1-Pyrd.(CH.sub.2).sub.15CO 227 Etc(CH.sub.2).sub.12CO
Etc(CH.sub.2).sub.12CO 228 H Etc(CH.sub.2).sub.12CO 229
Mec(CH.sub.2).sub.10CO Mec(CH.sub.2).sub.10CO 230
Car(CH.sub.2).sub.12CO Car(CH.sub.2).sub.12CO 231 H
Car(CH.sub.2).sub.12CO 232 Car(CH.sub.2).sub.10CO
Car(CH.sub.2).sub.10CO 233 Car(CH.sub.2).sub.14CO
Car(CH.sub.2).sub.14CO 234 HOOC(CH.sub.2).sub.12CO
HOOC(CH.sub.2).sub.12CO 235 HOOC(CH.sub.2).sub.12CO H 236
NC(CH.sub.2).sub.10CO NC(CH.sub.2).sub.10CO 237
NC(CH.sub.2).sub.15CO NC(CH.sub.2).sub.15CO 238
HO(CH.sub.2).sub.2CO HO(CH.sub.2).sub.2CO 239 H
HO(CH.sub.2).sub.2CO 240 HO(CH.sub.2).sub.5CO HO(CH.sub.2).sub.5CO
241 H HO(CH.sub.2).sub.5CO 242 HO(CH.sub.2).sub.9CO
HO(CH.sub.2).sub.9CO 243 H HO(CH.sub.2).sub.9CO 244
HO(CH.sub.2).sub.11CO HO(CH.sub.2).sub.11CO 245 H
HO(CH.sub.2).sub.11CO 246 HO(CH.sub.2).sub.15CO
HO(CH.sub.2).sub.15CO 247 H HO(CH.sub.2).sub.15CO 248
MemO(CH.sub.2).sub.9CO MemO(CH.sub.2).sub.9CO 249
MemO(CH.sub.2).sub.11CO MemO(CH.sub.2).sub.11CO 250
MemO(CH.sub.2).sub.15CO MemO(CH.sub.2).sub.15CO 251
MomO(CH.sub.2).sub.9CO MomO(CH.sub.2).sub.9CO 252
MomO(CH.sub.2).sub.11CO MomO(CH.sub.2).sub.11CO 253
MomO(CH.sub.2).sub.15CO MomO(CH.sub.2).sub.15CO 254
HS(CH.sub.2).sub.2CO HS(CH.sub.2).sub.2CO 255 HS(CH.sub.2).sub.2CO
H 256 HS(CH.sub.2).sub.5CO HS(CH.sub.2).sub.5CO 257
HS(CH.sub.2).sub.5CO H 258 HS(CH.sub.2).sub.11CO
HS(CH.sub.2).sub.11CO 259 HS(CH.sub.2).sub.11CO H 260
HS(CH.sub.2).sub.15CO HS(CH.sub.2).sub.15CO 261
HS(CH.sub.2).sub.15CO H 262 Ac.S(CH.sub.2).sub.9CO
Ac.S(CH.sub.2).sub.9CO 263 Ac.S(CH.sub.2).sub.11CO
Ac.S(CH.sub.2).sub.11CO 264 Ac.S(CH.sub.2).sub.15CO
Ac.S(CH.sub.2).sub.15CO 265 4-PrBoz 4-PrBoz 266 H 4-PrBoz 267
4-PhByr 4-PhByr 268 4-PhByr H 269 6-PhHxo 6-PhHxo 270 6-PhHxo H 271
MecCH(NHAc).(CH.sub.2).sub.10CO MecCH(NHAc).(CH.sub.2).sub.10CO 272
MecCH(NHAc).(CH.sub.2).sub.12C- O MecCH(NHAc).(CH.sub.2).sub.12CO
273 MecCH(NHAc).(CH.sub.2).sub.16- CO
MecCH(NHAc).(CH.sub.2).sub.16CO 274 1-Ind.CO 1-Ind.CO 275 1-Ind.CO
H 276 1-(4H-cOc)CO 1-(4H-cOc)CO 277 1-(4H-cOc)CO H 278 2-PhOPrn
2-PhOPrn 279 2-PhOPrn H 280 3-Pyr.CH.dbd.CHCO 3-Pyr.CH.dbd.CHCO 281
3-Pyr.CH.dbd.CHCO H 282 2-Pyr.CH.sub.2CO 2-Pyr.CH.sub.2CO 283
2-Pyr.CH.sub.2CO H 284 2-HXDco 2-HXDco 285 H 2-HXDco 286 2-PnHpo
2-PnHpo 287 H 2-PnHpo 288 Me(CH.sub.2).sub.14CO
Me(CH.sub.2).sub.16CO 289 Me(CH.sub.2).sub.16CO
Me(CH.sub.2).sub.14CO 290 Me(CH.sub.2).sub.16CO
OcCH.dbd.CH(CH.sub.2).sub.7CO 291 Me(CH.sub.2).sub.16CO
Me(CH.sub.2).sub.12CO 292 Me(CH.sub.2).sub.16CO
CH.sub.2.dbd.CH(CH.sub.2).sub.8CO 293 cPrCO cPrCO 294 H cPrCO 295
cBuCO cBuCO 296 H cBuCO 297 cPnCO cPnCO 298 H cPnCO 299 cPnCO H 300
cHxCO cHxCO 301 H cHxCO 302 cHxCO H 303 cHpCO cHpCO 304 H cHpCO 305
cOcCO H 306 cOcCO cOcCO 307 H cOcCO 308 1-cPenCO 1-cPenCO 309 H
1-cPenCO 310 3-cHexCO 3-cHexCO 311 H 3-cHexCO 312 3-cHexCO H 313
4-cHexCO 4-cHexCO 314 1-cHexCO 1-cHexCO 315 2-HOcPnCO 2-HOcPnCO 316
2-HOcPnCO H 317 4-NH.sub.2cHxCO 4-NH.sub.2cHxCO 318 4-NH.sub.2cHxCO
H 319 2-ClcHxCO 2-ClcHxCO 320 Retio Retio 321 H Retio 322 Retio H
323 Me(CH.sub.2).sub.14CO OcCH.dbd.CH(CH.sub.2).sub.7CO 324
Me(CH.sub.2).sub.14CO Me(CH.sub.2).sub.12CO 325
Me(CH.sub.2).sub.14CO CH.sub.2.dbd.CH(CH.sub.2).sub.8CO 326
Me.sub.3SiCH.sub.2CH.sub.2OC- H.sub.2O(CH.sub.2).sub.15
Me.sub.3SiCH.sub.2CH.sub.2OCH.sub.2O(CH.sub.2).s- ub.15CO CO 327
MeSCH.sub.2O(CH.sub.2).sub.9CO MeSCH.sub.2O(CH.sub.2).sub.9CO 328
MeSCH.sub.2O(CH.sub.2).sub.11CO MeSCH.sub.2O(CH.sub.2).sub.11CO 329
MeSCH.sub.2O(CH.sub.2).sub.15C- O
MeSCH.sub.2O(CH.sub.2).sub.15CO
EXAMPLE 15
Bastadins and Their Analogs
[0130] "Bastadins" refer to any bastadin presently known, or
discovered in the future. A compound from the bastadin family
refers to subunits of the bastadins, such as halogenated tyrosines,
including bromotyrosines and the 3-bromotyramine amide of oxalic
acid amide. A bastadin also includes the hemibastadins, which
represent bromotyrosine dimers. The hemibastadins include
hemibastadins 1, 2, and 3 as well as hemibastadinols 1,2, and
3.
[0131] The methods of the present invention, for improving neurite
outgrowth, include the use of any bastadin or any member of the
bastadin family, or their analogs in any aspect of the invention
(including their use in assays to search for other neurotrophic
compounds).
[0132] Particular examples of bastadins and other members of the
bastadin family include those shown in Pettit et al., J. Nat.
Prod., 59(10): 927-34, 1996; Franklin et al., J. Nat. Prod.,
59(12): 1121-7, 1996; Pettit et al., J. Nat. Prod., 58(5): 680-8,
1995; Mack et al., J. Biol. Chem., 269(37): 23236-49, 1994;
Gulavita et al., J. Nat. Prod., 56(9): 1613-7, 1993; Carney and
Scheuer, J. Natal. Prod., 56(1): 153-7, 1993; and Miao and
Andersen, J. Nat. Prod., 53(6), 1990, which are incorporated by
reference.
[0133] Particular examples of bastadins and their analogs include
bastadins of the formula where the identity and substitution
patterns of some particular bastadins and bastadin analogs are
given in Table 8 below.
8TABLE 8 20 Substitution Patterns for Some Bastadins and Bastadin
Analogs compound R.sub.1 R.sub.2 W X Y Z C5,6 Basdadin 4 H H H Br
Br H .DELTA. Bastadin 4 tetra-O-Me Me Me H Br Br H .DELTA. Bastadin
5 H H H Br Br H -- Bastadin 5 di-O-Me Me H H Br Br H -- Bastadin 5
tetra-O-Me Me Me H Br Br H -- Bastadin 6 H H H Br Br Br -- Bastadin
6 tetra-O-Me Me Me H Br Br Br -- Bastadin 7 H H H H Br H .DELTA.
15,34-disulfatobastadin 7 H SO.sub.3Na H H Br H .DELTA. Bastadin 8
H H OH Br Br H -- Bastadin 8 tetra-O-Me Me Me OH Br Br H --
Bastadin 9 H H OH H Br Br -- Bastadin 9 tetra-O-Me Me Me OH H Br Br
-- Bastadin 10 H H OH H Br H -- Bastadin 11 H H H Br H H .DELTA.
Bastadin 12 H H OH H Br Br -- Bastadin 14 H H H H Br Br .DELTA.
Bastadin 14 tetra-O-Me Me Me H H Br Br .DELTA. Bastadin 15 H H H H
Br Br -- Bastadin 15 di-O-Me Me H H H Br Br -- Bastadin 15
tetra-O-Me Me Me H H Br Br -- Bastadin 18 H H H H Br Br -
[0134] Other particular examples of bastadins include compounds of
the structure, where the identity and substitution patterns of some
particular bastadins and bastadin analogs are given in Table 9
below.
9TABLE 9 21 Substitution Patterns for Some Bastadins and Bastadin
Analogs Compound R X Y Z Iso-bastadin 6 H Br Br Br Iso-bastadin 6
teter-O-Me Me Br Br Br Bastadin 19 H Br H Br Bastadin 19 tetra-O-Me
Me Br H Br Bastadin 20 H H H Br Bastadin 20 tetra-O-Me Me H H
Br
[0135] Examples of bastadin compounds include bastadin 13 and
34-sulfatobastadin 13 which have the following structure, where R=H
and R=SO.sub.3Na, respectively. 22
[0136] Particular examples of bastadins that are of an open-ring
structure exist and include bastadins 1 and 2 which have the
following structure, where X=H and X=Br respectively. 23
[0137] Bastadin 3 and 10-sulfatobastadin 3 also possess an
open-ring structure. They have the following formulae, where R=H
for bastadin 3 and R=SO.sub.3Na for 10-sulfatobastadin 3. 24
[0138] Another example of a compound in the bastadin family is the
3-bromotyramine amide of oxalic acid amid which has the following
structure, where R can for example be H or COCH.sub.3. 25
[0139] The bastadins also include the hemibastadins, some of which
can be described by the following structure, where the identities
and substitution patterns are defined in Table 10.
10TABLE 10 26 Substitution Patterns for Some Hemibastadins and
Analogs Thereof Compound X Y R.sub.1 R.sub.2 Hemibastadin 1 H H H H
Hemibastadin 2 H Br H H Hemibastadin 3 Br H H H Analog 1 H Br Me Me
Analog 2 H H Me Me Analog 3 Br H Me Me Analog 4 H H Me H Analog 5 H
Br Me H
[0140] The bastadins also include the hemibastadinols, some of
which have the following structure. The identity and substitution
patterns of several examples are given below in Table 11.
11TABLE 11 27 Substitution Patterns for Some Hemibastadinols and
Their Analogs compound X Y R.sub.1 R.sub.2 Hemibastadinaol 1 H H H
H Analog 1 H H Me H Hemibastadinol 2 H Br H H Hemibastadlnol 3 Br H
H H Analog 2 H Br Me H Analog 3 Br H Me H
EXAMPLE 16
Effect of Radicicol on Neurite Outgrowth
[0141] Utilizing the in vitro assay described in Example 2, the
effect of radicicol on neurite outgrowth was investigated.
Hippocampal cells were treated with radicicol at a concentration of
0.1 ng/mL and at a concentration of 10 ng/mL. The neurite outgrowth
for radicicol treated cells was compared to the outgrowth for
untreated cells and for cells treated with 10 ng/mL of NGF. The
results are shown in FIG. 3. A shift of the histogram to the right
indicates increased stimulation of neurite outgrowth and it can be
seen that radicicol alone (at both concentrations) is more
effective than NGF alone in stimulating outgrowth.
EXAMPLE 17
Effect of Heat on Neurite Outgrowth
[0142] Utilizing the in vitro assay described in Example 2, the
effect of heat alone or in combination with various compounds on
neurite outgrowth was investigated. FIG. 4 shows that heat in
combination with NGF treatment is more effective than either heat
or NGF treatment alone in stimulating neurite outgrowth for the
hippocampal cells tested. FIG. 5 shows that heat treatment is more
effective when administered in combination with NGF than when
administered in combination with an Hsp-90 Antibodies. FIG. 5 also
illustrates that a combination of heat treatment and treatment with
both NGF, and Hsp-90 antibodies is not as effective as the
combination of heat and NGF treatment. Involvment of Hsp-90 as a
mediator of the heat shock effect is demonstrated by the ability of
the Hsp-90 antibody to inhibit the increase in neurite
outgrowth.
EXAMPLE 18
Role of MAP Kinase/Kinase
[0143] The role of MAP Kinase/Kinase was investigated utilizing the
in vitro assay described in Example 2. Hippocampal cells were
treated with the MAP kinase/kinase inhibitor PD 098059 in
combination with NGF, FK506, and radicicol to determine if there is
down-stream involvement of the MAP kinase pathway in the action of
each of these compounds. In FIG. 6 it can be seen that the
selective MAP kinase/kinase (MEK) inhibitor blocks neurite
outgrowth by NGF and FK-506 in a concentration dependent fashion.
FIG. 7 illustrates that PD 098059 also blocks the action of
radicicol in a concentration dependent fashion and the highest
concentration almost completely blocks activity.
EXAMPLE 19
Effect of Bastadin on Neurite Outgrowth
[0144] Utilizing the in vitro assay described in Example 2, the
effect of treating hippocampal cells with a bastadin 10 analog was
compared to the effect of treating hippocampal cells with
radicicol. FIG. 9A is a light micrograph of untreated hippocampal
cells after 72 hours. FIG. 9B, another light micrograph,
illustrates the effect radicicol has on neurite outgrowth and 9C
demonstrates the same for a bastadin 10 analog (Davis B-10,
bastadin). Quantitative measurements of the lengths of neurite
outgrowths for untreated cells, cells treated with radicicol, and
cells treated with the bastadin 10 analog are presented in FIG. 10.
Taken together, FIGS. 9 and 10 illustrate the ability of bastadin
to stimulate neurite outgrowth.
EXAMPLE 20
Methods of Use
[0145] The neurotrophic compounds of the invention are administered
in an effective amount sufficient to stimulate nerve growth or
regeneration compared to a control. Suitable local concentrations
for nerve cell growth or nerve regeneration can be readily assessed
using an in vitro assay, e.g., the assay described in Example 1.
Alternatively, nerve cell growth or regeneration can be determined
by an in vivo assay, or by direct or indirect signs of nerve cell
growth and regeneration in a subject (for example a restoration of
motor and/or sensory function in the hand in the area of
innervation of a previously transected median nerve). Preferably,
the increase in nerve cell growth or regeneration rate 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, dosage level examples are
between about 0.01 to about 40 mg/kg/day. Alternatively, the dose
can be sufficient to achieve tissue concentrations that have been
shown to be neurotrophic in vitro.
[0146] Pharmaceutical compositions according to the invention can
be periodically administered to a mammalian subject (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, limb
transplantation following amputation), disease (e.g., diabetic
neuropathy), cancer chemotherapy (e.g., neuropathy induced by
acrylamide, taxol, vinca alkaloids and doxorubicin), brain damage
associated with stroke and ischemia, 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-Barr syndrome, Alzheimer's disease, Parkinson's
disease, and Huntington's chorea.
[0147] 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., Acta. 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. Immnunol.
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).
[0148] A transection of a peripheral nerve or a spinal cord injury
can be treated by administering a nerve growth stimulating amount
of the agent to the mammal and grafting to the peripheral nerve or
spinal cord a nerve graft such as an allograft (Osawa et al., J.
Neurocytol. 19:833-849, 1990; Buttemeyer et al., Ann. Plastic
Surgery 35:396401, 1995) or an artificial nerve graft (Madison and
Archibald, Exp. Neurol. 128:266275, 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 such cellular or non-cellular gap-filling
materials, or administered systemically before, during or after the
nerve graft procedure.
EXAMPLE 21
Pharmaceutical Formulations
[0149] Pharmaceutical formulations according to the present
invention encompass formulations that include an amount (for
example, a unit dosage) of the neurotrophic agent together with one
or more non-toxic pharmaceutically acceptable excipients, including
carriers, diluents, and/or adjuvants, and optionally other
biologically active ingredients. Standard pharmaceutical
formulation techniques are used, such as those disclosed in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa. (19th Edition).
[0150] A pharmaceutical formulation according to the invention
includes one or more of the neurotrophic agents of the present
invention, and can also include, for example, one or more other
biologically active ingredients, such as FK506 or an FKBP12-binding
FK506 analog, NGF, IGF-1, .alpha.-FGF, .beta.-FGF, PDGF, BDNF,
CNTF, GDNF, NT-3, and NT 4/5. When the SRC disrupting agents of the
present invention are combined with a second neurotrophic agent,
the two agents are ideally selected such that they structurally or
functionally disrupt the SRC by acting at different SRC components.
For example, the first agent may be geldanamycin (which promotes
dissociation of p23) and the second agent may be FKBP52-Ab (which
interferes with association of FKBP-52 to hsp-90). In particular
embodiments, the composition includes NGF, or another agent that
stimulates nerve growth in combination with the SRC disrupting
complex, or the neurotrophic action of which is augmented by
administration in combination with the SRC disrupting agent.
[0151] The dosage of the combined biologically active agents is
sufficient to achieve tissue concentrations at the site of action
that are similar to those that are shown to achieve in vivo nerve
regeneration. Pharmaceutical formulations may include, for example,
an amount of a NGF, such that the subject receives a dosage of
between about 0.01 to 100 .mu.g/kg body weight/day. The NGF (or
other adjuvant) can be administered separately, concurrently,
consecutively, or within less than about five hours of each
other.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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. 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.
[0156] The pharmaceutical compositions 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. 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. For topical application, the drug may be made up into a
solution, suspension, cream, lotion, or 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.
[0157] 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 are typically sufficient to achieve a tissue
concentration at the site of action that is at least the same as a
concentrations that has been shown to be neurotrophic in vitro. For
example, a dosage of about 0.1 to about 400 mg/kg per day of the
active ingredient may be useful in the treatment of the conditions
listed above.
[0158] The compounds 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 C1-8 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.
[0159] Pharmaceutical compositions can be included in a kit
accompanied by instructions for intended use, for example
instructions required by a pharmaceutical regulatory agency, such
as the Food and Drug Administration in the United States.
SUMMARY
[0160] The foregoing examples illustrate that neurotrophic
properties of neuroimmunophilin ligands (FK506) and steroid
hormones are mediated by physical or functional disruption of
steroid receptor complexes. Some of the components of the complex
that can act as targets for disruption include FKBP-52, hsp-90 and
p23, which are all present together in mature steroid receptor
complexes, which can be disrupted by geldanamycin. Since FKBP-52
can associate with microtubules and dynein, and via its TPR motifs
also associate with kinesin, it can also have a direct role in the
movement (axonal transport) of cytoskeletal elements and,
consequently, axonal elongation.
[0161] In view of the many possible embodiments to which the
principles of our invention may be applied, it should be recognized
that the illustrated embodiments are only examples of the invention
and should not be taken as a limitation on the scope of the
invention. Rather, the scope of the invention is defined by the
following claims, and equivalents thereof. We therefore claim as
our invention all that comes within the scope and spirit of these
claims, including equivalents thereof.
* * * * *