U.S. patent application number 14/935985 was filed with the patent office on 2016-03-03 for treatments for neuropathy.
The applicant listed for this patent is Children's Medical Center Corporation. Invention is credited to Gabriel Corfas, Joshua C. Murtie.
Application Number | 20160058749 14/935985 |
Document ID | / |
Family ID | 40939434 |
Filed Date | 2016-03-03 |
United States Patent
Application |
20160058749 |
Kind Code |
A1 |
Corfas; Gabriel ; et
al. |
March 3, 2016 |
TREATMENTS FOR NEUROPATHY
Abstract
Small fiber neuropathy is treated or prevented by topically
administering to a subject in need thereof topically active
quinoline compounds or pharmaceutically acceptable salts thereof
under conditions effective to treat or prevent neuropathy in the
subject. Glial cell-derived neurotrophic factor (GDNF) receptors
are modulated with the subject active quinoline compounds, which
may be formulated in topical lotions.
Inventors: |
Corfas; Gabriel; (Brookline,
MA) ; Murtie; Joshua C.; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Children's Medical Center Corporation |
Boston |
MA |
US |
|
|
Family ID: |
40939434 |
Appl. No.: |
14/935985 |
Filed: |
November 9, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12970891 |
Dec 16, 2010 |
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14935985 |
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12350751 |
Jan 8, 2009 |
7863295 |
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12970891 |
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61028024 |
Feb 12, 2008 |
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Current U.S.
Class: |
514/313 |
Current CPC
Class: |
A61K 9/20 20130101; A61K
9/0019 20130101; A61P 43/00 20180101; A61P 25/02 20180101; A61K
31/47 20130101; A61K 9/007 20130101; A61P 25/00 20180101; A61K
31/4706 20130101; A61K 9/0053 20130101 |
International
Class: |
A61K 31/4706 20060101
A61K031/4706; A61K 9/20 20060101 A61K009/20; A61K 9/00 20060101
A61K009/00 |
Goverment Interests
[0002] The subject matter of this application was made with support
from the National Institute for Health (NIH) Grant No. NS035884.
The U.S. Government has certain rights.
Claims
1-20. (canceled)
21. A method of treating or preventing a small fiber peripheral
neuropathy in a subject determined to be in need thereof
comprising: administering to the subject
N.sup.4-{7-chloro-2-[(E)-2-(2-chloro-phenyl)-vinyl]-quinolin-4-yl}-N.sup.-
1,N.sup.1-diethyl-pentane-1,4-diamine (XIB4035) or a
pharmaceutically acceptable salt thereof under conditions effective
to treat or prevent the peripheral neuropathy in the subject.
22. The method of claim 21, wherein the XIB4035 is administered
orally.
23. The method of claim 21, wherein the XIB4035 is administered
parenterally.
24. The method of claim 21, wherein the XIB4035 is administered by
injection.
25. The method of claim 21, wherein the XIB4035 is administered by
a route selected from the group consisting of subcutaneously,
intravenously, intramuscularly, intraperitoneally, by intranasal
instillation, by inhalation and by application to mucous
membranes
26. The method of claim 21, wherein the XIB4035 is applied to a
mucous membrane selected from the group consisting of that of the
nose, throat and bronchial tube.
27. The method of claim 21, wherein the XIB4035 delivers
systemically.
28. The method of claim 21, wherein between 1 and 250 mg of XIB4035
is administered to the subject in a single dose.
29. The method of claim 21, wherein the subject is human.
30. The method of claim 21, further comprising the antecedent step
of determining that the subject is in need of said method by
detecting the small fiber neuropathy in the subject.
31. The method of claim 21, further comprising the subsequent step
of detecting a resultant diminution of the small fiber
neuropathy.
32. A formulation comprising
N.sup.4-{7-chloro-2-[(E)-2-(2-chloro-phenyl)-vinyl]-quinolin-4-yl}-N.sup.-
1,N.sup.1-diethyl-pentane-1,4-diamine (XIB4035) or a
pharmaceutically acceptable salt thereof in an amount effective to
treat or prevent a peripheral neuropathy in a subject, and a
pharmaceutically acceptable carrier formulated for
administration.
33. The formulation of claim 32, wherein the formulation contains
between about 1 to 250 mg of XIB4035.
34. The formulation of claim 32, wherein the formulation comprises
a dosage of XIB4035 effective for treatment of peripheral
neuropathy in the subject.
35. The formulation of claim 32, formulated for parenteral
administration.
36. The formulation of claim 32, formulated for injection.
37. The formulation of claim 32, formulated for oral
administration.
38. The formulation of claim 32, formulated as a tablet.
39. The formulation of claim 32, formulated for inhalation.
40. The formulation of claim 32, wherein XIB4035 is present at
about 2% to about 60% of the weight of the formulation.
Description
[0001] This application claims priority to U.S. Ser. No.
61/028,024, filed Feb. 12, 2008 by the same inventors: Gabriel
Corfas and Joshua C. Murtie, both of Boston, Mass.
FIELD OF THE INVENTION
[0003] This invention relates to methods of treating or preventing
small fiber neuropathy.
BACKGROUND OF THE INVENTION
[0004] Small fiber neuropathy (SFN) is a disorder characterized by
degeneration or dysfunction of small diameter unmyelinated nerve
fibers in the peripheral nervous system (1,2). Patients with SFN
have sensory defects with a variety of symptoms, including loss of
sensation or chronic pain. Despite its prevalence, the etiology of
SFN is poorly understood and there are currently no effective
treatments. Since the initial stages of SFN commonly involve
degeneration of nerve terminals without loss of neuronal cell
bodies (3), we wondered if delivery of therapeutic agents at the
target of innervation, i.e. the skin, would be an effective
non-invasive approach that could minimize the side effects commonly
observed with systemic drug delivery methods.
[0005] When considering molecules that could be used in this
fashion, we focused on neurotrophic factors, which have been viewed
as potentially useful therapeutic agents in the treatment of
peripheral neuropathies because they regulate the survival and
function of peripheral nerves during development (4, 5). In
addition, decreases in expression of certain trophic factors have
been observed in multiple models of peripheral neuropathy (6, 7)
suggesting that low levels of these factors may be involved in
disease etiology. One of these trophic factors, GDNF, is necessary
for proper development and survival of small diameter unmyelinated
nerve fibers (5, 8). In early postnatal life, a large proportion of
developing unmyelinated nerve fibers switch from dependence on
nerve growth factor (NGF) to dependence on GDNF (5). This
transition coincides with a gradual decrease in expression of the
NGF receptor (TrkA) with a corresponding increase in the expression
of GDNF family receptors by dorsal root ganglion sensory neurons
(4). Based on the known roles of the GDNF pathway in peripheral
nerve development and function and the pattern of expression of
GDNF family ligands and receptors, we decided to test if
application of GDNF receptor ligands to the skin could be used to
treat SFN. To address this question, we used two mouse models of
SFN arising from different pathogenic processes. In one model,
progressive SFN results from disruption of non-myelinating Schwann
cell (NMSC) function (6). In the other, rapid onset SFN is caused
by treatment with a toxin (resiniferatoxin, RTX) that activates
TRPV1 channels in c-fiber nerve terminals, inducing loss of
unmyelinated fiber nerve terminals in the skin and loss of thermal
nociception (9). Here we show that topical delivery of GDNF
receptor ligands to affected skin areas is sufficient to prevent
degeneration and maintain sensory function in both types of SFN. In
addition, we demonstrate that the non-peptidyl GFR.alpha.1 agonist
XIB4035 and related quinolines are capable of providing trophic
support to peripheral nerves in vivo and thus are useful
therapeutic agents in the treatment of SFN.
[0006] Use of
N4-{7-chloro-2-[(E)-2-(2-chloro-phenyl)-vinyl]-quinolin-4-yl}-N1,N1-dieth-
yl-pentane-1,4-diamine (XIB4035), also known as
7-chloro-2-(o-chlorostyryl)-4-[4diethylamino-1-methylbutyl]aminoquiinolin-
e phosphate), and
2-(2-Chlorostyryl)-4-(delta-diethylamino-alpha-methylbutylamino)-7-chloro-
quinazoline (CAS RN 57942-32-2; CAS 10023-54-8) has been described,
e.g. Tokugawa et al., Neurochem Intnl 2003, 42, 81-86; WO01003649;
and JP 2008-230974.
SUMMARY OF THE INVENTION
[0007] The invention provides methods and compositions for treating
or preventing small fiber peripheral neuropathy in a subject
determined to be in need thereof, and generally comprising: (a)
topically administering to the subject an anti-peripheral
neuropathic quinoline compound of the formula:
##STR00001##
wherein R.sub.1-R.sub.7 are each independently H, hydroxy, halogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted aryl alkyl, substituted or un substituted amine,
substituted or un substituted alkylamine, substituted or
unsubstituted dialkylamine, substituted or unsubstituted alkoxy,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted alkoxy, substituted or
unsubstituted haloalkyl, or a pharmaceutically acceptable salt
thereof, under conditions effective to treat or prevent the
peripheral neuropathy in the subject.
[0008] In particular embodiments the method further comprises the
antecedent step of determining that the subject is in need thereof,
e.g. by detecting or diagnosing the small fiber neuropathy in the
subject or patient, or that the subject is at particular risk of
developing the neuropathy.
[0009] In particular embodiments, the method further comprises the
subsequent step of detecting a resultant effect on the subject,
such as a diminution in the severity of the neuropathy, or delayed
onset of the neuropathy.
[0010] The invention encompasses all alternative combinations of
particular embodiments:
R.sub.2 is halogen, particularly Cl; R.sub.5 is a substituted
amine, particularly optionally-substituted alkyl substituted
secondary amine, particularly wherein the alkyl is substituted with
a dialkylamine such as 1-methyl-3-diethylaminobutyl,
1-methyl-4-dimethylaminobutyl, 1-ethyl-4-dimethylaminobutyl,
1-ethyl-4-diethylaminobutyl, or 1-methyl-4-diethylaminobutyl:
##STR00002##
R.sub.7 is a substituted alkenyl, particularly
optionally-substituted phenyl substituted ethenyl, particularly
such as
##STR00003##
and/or wherein R.sub.8 is hydrogen or halogen, such as Cl,
particularly ortho-chloro: such as wherein the compound has
formula:
##STR00004##
[0011] In another aspect, the invention provides methods and
compositions for treating or preventing a small fiber peripheral
neuropathy in a subject determined to be in need thereof, and
generally comprising: topically administering to the subject an
agonist of glial cell-derived neurotrophic factor (GDNF) receptor
GFR.alpha.1 or GFR.alpha.2, under conditions effective to treat or
prevent the peripheral neuropathy in the subject, particularly,
wherein the agonist is XIB4035.
[0012] In another aspect, the invention provides a topical lotion
comprising (a) a subject anti-peripheral neuropathic quinoline
compound, or a pharmaceutically acceptable salt thereof; and (b) a
pharmaceutically acceptable carrier formulated for topical
application.
DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION
[0013] In one embodiment, the invention provides methods of
treating or preventing small fiber neuropathy in a subject,
particularly a human, determined to be in need thereof, the method
comprising the step of (a) topically administering to the subject a
compound of the formula:
##STR00005##
wherein R.sub.1-R.sub.7 are each independently H, hydroxy, halogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted arylalkyl, substituted or unsubstituted amine,
substituted or unsubstituted alkylamine, substituted or
unsubstituted dialkylamine, substituted or unsubstituted alkoxy,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted alkoxy, substituted or
unsubstituted haloalkyl, or a pharmaceutically acceptable salts
thereof under conditions effective to treat or prevent the small
fiber peripheral neuropathy in the subject.
[0014] "Alkyl" as used herein refers to a saturated hydrocarbon
radical which may be straight-chain or branched-chain (for example,
ethyl, isopropyl, t-amyl, or 2,5-dimethylhexyl) or cyclic (for
example cyclobutyl, cyclopropyl or cyclopentyl) and contains from 1
to 24 carbon atoms. This definition applies both when the term is
used alone and when it is used as part of a compound term, such as
"haloalkyl" and similar terms. In some embodiments, preferred alkyl
groups are those containing 1 to 4 carbon atoms, which are also
referred to as "lower alkyl." In some embodiments preferred alkyl
groups are those containing 5 or 6 to 24 carbon atoms, which may
also be referred to as "higher alkyl".
[0015] "Alkenyl," as used herein, refers to a straight or branched
chain hydrocarbon containing from 2 to 24 carbons and containing at
least one carbon-carbon double bond formed by the removal of two
hydrogens. Representative examples of "alkenyl" include, but are
not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl,
3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl,
3-decenyl and the like. "Lower alkenyl" as used herein, is a subset
of alkenyl and refers to a straight or branched chain hydrocarbon
group containing from 1 to 4 carbon atoms.
[0016] "Alkynyl," as used herein, refers to a straight or branched
chain hydrocarbon group containing from 2 to 24 carbon atoms and
containing at least one carbon-carbon triple bond. Representative
examples of alkynyl include, but are not limited, to acetylenyl,
1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl, 1-butynyl and the
like. "Lower alkynyl" as used herein, is a subset of alkyl and
refers to a straight or branched chain hydrocarbon group containing
from 1 to 4 carbon atoms.
[0017] "Alkoxy" refers to an alkyl radical as described above which
also bears an oxygen substituent which is capable of covalent
attachment to another hydrocarbon radical (such as, for example,
methoxy, ethoxy and t-butoxy).
[0018] "Alkylthio" as used herein refers to an alkyl group, as
defined herein, appended to the parent molecular moiety through a
thio moiety, as defined herein. Representative examples of
alkylthio include, but are not limited, methylthio, ethylthio,
tert-butylthio, hexylthio, and the like.
[0019] "Aryl" or "aromatic ring moiety" refers to an aromatic
substituent which may be a single ring or multiple rings which are
fused together, linked covalently or linked to a common group such
as an ethylene or methylene moiety. The aromatic rings may each
contain heteroatoms and hence "aryl" encompasses "heteroaryl" as
used herein. Representative examples of aryl include, azulenyl,
indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, biphenyl,
diphenylmethyl, 2,2-diphenyl-1-ethyl, thienyl, pyridyl and
quinoxalyl. "Aryl" means substituted or unsubstituted aryl unless
otherwise indicated and hence the aryl moieties may be optionally
substituted with halogen atoms, or other groups such as nitro,
carboxyl, alkoxy, phenoxy and the like. Additionally, the aryl
radicals may be attached to other moieties at any position on the
aryl radical which would otherwise be occupied by a hydrogen atom
(such as, for example, 2-pyridyl, 3-pyridyl and 4-pyridyl).
[0020] "Heteroaryl" means a cyclic, aromatic hydrocarbon in which
one or more carbon atoms have been replaced with heteroatoms. If
the heteroaryl group contains more than one heteroatom, the
heteroatoms may be the same or different. Examples of heteroaryl
groups include pyridyl, pyrimidinyl, imidazolyl, thienyl, furyl,
pyrazinyl, pyrrolyl, pyranyl, isobenzofuranyl, chromenyl,
xanthenyl, indolyl, isoindolyl, indolizinyl, triazolyl,
pyridazinyl, indazolyl, purinyl, quinolizinyl, isoquinolyl,
quinolyl, phthalazinyl, naphthyridinyl, quinoxalinyl, is
othiazolyl, and benzo[b]thienyl. Preferred heteroaryl groups are
five and six membered rings and contain from one to three
heteroatoms independently selected from 0, N, and S. The heteroaryl
group, including each heteroatom, can be unsubstituted or
substituted with from 1 to 4 substituents, as chemically
feasible.
[0021] "Halo" or "halogen," as used herein, refers to --Cl, --Br,
--I or --F.
[0022] "Haloalkyl," as used herein, refers to at least one halogen,
as defined herein, appended to the parent molecular moiety through
an alkyl group, as defined herein. Representative examples of
haloalkyl include, but are not limited to, chloromethyl,
2-fluoroethyl, trifluoromethyl, pentafluoroethyl,
2-chloro-3-fluoropentyl, and the like.
[0023] "Hydroxy," as used herein, refers to an --OH group.
[0024] "Amine" or "amino" as used herein, refers to a nitrogen atom
attached by single bonds to hydrogen atoms, alkyl groups, aryl
groups, or a combination of these three. An organic compound that
contains an amino group is called an amine. Amines are derivatives
of the inorganic compound ammonia, NH.sub.3. When one, two, or all
three of the hydrogens in ammonia are replaced by an alkyl or aryl
group, the resulting compound is known as a primary, secondary, or
tertiary amine, respectively.
[0025] In certain embodiments, R.sub.2 may be halogen, R.sub.5 may
be a substituted amine, and/or R.sub.7 may be a substituted alkenyl
such as
##STR00006##
wherein R.sub.8 may be H or halogen, for example, Cl.
[0026] In preferred embodiments, R.sub.2 is Cl, R.sub.5 is
##STR00007##
[0027] The subject topically-active, anti-peripheral neuropathic
quinoline compounds are commercially available and/or readily
produced using convention organic synthesis. Relevant
derivitization schemes are known in the art, such as described in
"Synthesis of substituted
4-(.delta.-diethylamino-.alpha.-methylbutylamino)-2-styrylquinolines",
Berenfel'd, V. M.; Yakhontov, L. N.; Yanbukhtin, N. A.;
Krasnokutskaya, D. M.; Vatsenko, S. V.; Rubtsov, M. V. Zhurnal
Obshchei Khimii (1962), 32 2169-77. CODEN: ZOKHA4 ISSN: 0044-460X;
"Syntheses in the isoquinoline series. Hofmann degradation of
1-phenyl-substituted 1,2,3,4-tetrahydroisoquinolines," Rheiner, A.,
Jr.; Brossi, A. F. Hoffmann-La Roche & Co., A.-G., Basel,
Switz. Helvetica Chimica Acta (1962), 45 2590-600. CODEN: HCACAV
ISSN: 0018-019X; "Synthesis and antileishmaniasis activity of
2-(2'-chlorostyryl)-4-(.delta.-diethylamino-.alpha.-methylbutylamino)-7-c-
hloroquinazoline diphosphate," Yakhontov, L. N.; Zhikhareva, G. P.;
Mastafanova, L. I.; Evstratova, M. I.; Pershin, G. N.; Moskalenko,
N. Yu.; Pushkina, T. V.; Kutchak, S. N.; Fadeeva, N. I.; et al.
VNIFI, Moscow, USSR. Khimiko-Farmatsevticheskii Zhurnal (1987),
21(1), 38-49. CODEN: KHFZAN ISSN: 0023-1134; and "Reaction products
of 4-[[4-(diethylamino)-1-methylbutyl]amino]-7-chloroquinaldine
with o-chlorobenzaldehyde," Uritskaya, M. Ya.; Anisimova, O. S.;
Tubina, I. S.; Vinokurova, T. Yu.; Pershin, G. N.; Moskalenko, N.
Yu.; Gus'kova, T. A.; Kutchak, S. N.; Stebaeva, L. F. Vses.
Nauchno-Issled. Khim.-Farm. Inst., Moscow, USSR.
Khimiko-Farmatsevticheskii Zhurnal (1983), 17(11), 1334-40. CODEN:
KHFZAN ISSN: 0023-1134.
[0028] Anti-peripheral neuropathic activity is readily confirmed in
topical formulations and the convenient animal models, as
demonstrated below. The subject compounds are topically-active,
antineuropathic quinolines, particularly aminoquinolines,
particularly 4- and 8-aminoquinolines, particularly chloroquines
(chloroquine and derivatives thereof), and include compounds of
Tables 1-3:
TABLE-US-00001 TABLE 1 Exemplary anti-peripheral neuropathic
compounds of the invention are shown in Tables 1 and 2 below:
##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012##
##STR00013## ##STR00014## ##STR00015## ##STR00016## ##STR00017##
##STR00018## ##STR00019## ##STR00020## ##STR00021## ##STR00022##
##STR00023## ##STR00024## ##STR00025## ##STR00026## ##STR00027##
##STR00028## ##STR00029## ##STR00030## ##STR00031## ##STR00032##
##STR00033## ##STR00034## ##STR00035## ##STR00036## ##STR00037##
##STR00038## ##STR00039## ##STR00040## ##STR00041## ##STR00042##
##STR00043##
TABLE-US-00002 TABLE 2 ##STR00044##
1,4-Pentanediamine,N4-[7-chloro-2-[(1E)-2-(2-
chlorophenyl)ethenyl]-4-quinolinyl]-N1,N1- diethyl- ##STR00045##
1,4-Pentanediamine,N4-[7-chloro-2-[2-(2-
chlorophenyl)ethenyl]-4-quinolinyl]-N1,N1- diethyl- ##STR00046##
1,4-Pentanediamine,N4-[7-chloro-2-[2-(2,6-
dichlorophenyl)ethenyl]-4-quinolinyl]-N1,N1- diethyl- ##STR00047##
##STR00048## ##STR00049## ##STR00050## ##STR00051## ##STR00052##
##STR00053## ##STR00054## ##STR00055## ##STR00056##
TABLE-US-00003 TABLE 3
7-chloro-4-(4-diethylamino-1-methylbutylamino)quinoline
(chloroquine);
7-hydroxy-4-(4-diethylamino-1-methylbutylamino)quinoline;
chloroquine phosphate;
7-chloro-4-(4-diethylamino-1-butylamino)quinoline
(desmethylchloroquine);
7-hydroxy-4-(4-diethylamino-1-butylamino)quinoline;
7-chloro-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;
7-hydroxy-4-(1-carboxy-4-diethylamino-1-butylamino)quinoline;
7-chloro-4-(1-carboxy-4-diethylamino-1-methylbutylamino)quinoline;
7-hydroxy-4-(1-carboxy-4-diethylamino-1-methylbutylamino)
quinoline;
7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutylamino)
quinoline (hydroxychloroquine);
7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-methylbutyl amino)
quinoline; hydroxychloroquine phosphate;
7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)quinoline
(desmethylhydroxychloroquine);
7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)
quinoline;
7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-butylamino)
quinoline; 7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-
butylamino)quinoline;
7-chloro-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-
methylbutylamino)quinoline;
7-hydroxy-4-(1-carboxy-4-ethyl-(2-hydroxyethyl)-amino-1-
methylbutylamino)quinoline;
8-[(4-aminopentyl)amino]-6-methoxydihydrochloride quinoline;
1-acetyl-1,2,3,4-tetrahydroquinoline; 8-[4-aminopentyl)amino]-
6-methoxyquinoline dihydrochloride;
1-butyryl-1,2,3,4-tetrahydroquinoline;
7-chloro-2-(o-chlorostyryl)-4-[4-diethylamino-1-methylbutyl]
aminoquiinoline phosphate;
3-chloro-4-(4-hydroxy-.alpha.,.alpha.'-bis(2-methyl-1-pyrrolidinyl)-2,5-
xylidinoquinoline, 4-
[(4-diethylamino)-1-methylbutyl)amino]-6-methoxyquinoline;
3,4-dihydro-1 (2H)-quinolinecarboxyaldehyde;
1,1'-pentamethylenediquinoleinium diiodide; 8-quinolinol sulfate;
Chloroquine 4-acetaminosalicylate; Chlorquinaldol;
3-Methylchloroquine; 3-Carboxy-4-hydroxy-7-chloroquinoline;
4,7-Dichloroquinoline; 7-Chloro-4-hydroxyquinoline;
6-Chloroquinaldine; N,2,6-Trichloro-4-benzoquinone imine;
Hydroxychloroquine; Chloranil; Clioquinol; Cloxyquin; Chloroquine
sulfate; 8-Chloroquinoline; 4-Chloroquinoline; 3-Chloroquinoline;
6-Chloroquinoline; 2-Chloroquinoline; 2-Chloro-1,4-hydroxyquinone;
5-Chloroquinoline; 2-Chloro-1,4-benzoquinone;
2,6-Dichlorobenzoquinone; Hydroxychloroquine sulfate; Chloroxine;
7-Chloroquinolin-8-ol; Chloroquinine phosphate;
2-Chloroquinoxaline; Desethylchloroquine;
2,3-Dichloroquinoxaline-6-carbonylchloride;
2,3-Dichloroquinoxaline; 2-Chloroquinoline-4-carbonyl chloride;
4,11-Dichloroquinacridonequinone;
2,9-Dichloroquino(2,3-b)acridine-6,7,13,14(5H,12H)-tetrone;
2,3,6-Trichloroquinoxaline; Chlorquinox; Chloroquine hydrochloride;
Glafenine; Chloroquine mustard; N,N-Dideethylchloroquine;
Cletoquine; Chloroquine-ethyl phenyl mustard; 4-Chloroquinazoline;
4-(3',5'-Bis(pyrrolidinomethyl)-4-hydroxyanilino)-7-chloroquinoline;
6-Chloroquinoxaline; 6-Chloro-8-aminoquinoline;
2-Chloromethyl-4-phenyl-6-chloroquinazoline-3-oxide;
2-Chloroquinazoline; 4-(2-Methyl-1-pyrrolidyl)-7-chloroquinoline;
6,7-Dichloroquinoline-5,8-dione; 6,7-Dichloroquinoxaline-2,3-dione;
Cloquinate; 8-Quinolinol, 7-bromo-5-chloro-; Collagenan;
Dichlorquinazine; 4,7-Dichloroquinolinium tribromide;
Chloroquinoline; Chloroquine diorotate;
2,4,6-Triamino-5-chloroquinazoline;
Methyl-8-(5,7-dichloroquinolyl)carbonic acid ester;
6-Amino-7-chloro-5,8-dioxoquinoline; 4,8-Dichloroquinoline;
5-Chloroquinolin-8-ol hydrochloride;
3-Phenyl-4-hydroxy-7-chloroquinolin-2(1H)-one;
N-Methyl-6-chloroquinolinium iodide; 3-Chloroquinuclidine
hydrochloride; Halacrinate; 1-Phenacyloxime-4,5-dichloroquinolinium
chloride hydrate; Chloroquine diascorbate;
2-(7-Chloroquinolin-4-yl)anthranilic acid hydrochloride;
Tripiperaquine;
2-(2-Chlorostyryl)-4-(delta-diethylamino-alpha-methylbutylamino)-7-
chloroquinazoline; (+)-Chloroquine; (-)-Chloroquine;
7-Chloro-4-(3-octylaminopropyl)aminoquinoline 1-oxide; Ethyl
chloroquine mustard; L-Chloroquine;
2,6-Dianilino-6-chloroquinoxaline;
2-(2-(5-Nitrofuryl)vinyl)-4-(delta-diethylamino-alpha-
methylbutylamino)-7-chloroquinazoline; D-Chloroquine;
2,3-Bis(allylamino)-6-chloroquinoxaline; 7-Chloroquinolin-4-ol
hydrochloride; 2-Amino-3,4-dichloroquinoline; Quizalofop; Presocyl;
Tris(5,7-dichloroquinolin-8-olato-N1,O8)aluminium; Contramibial;
Quinclorac;
N-(4((7-Chloroquinolin-4-yl)amino)pentyl)-N-ethylacetamide;
7-Bromo-5-chloroquinolin-ol; Chlorsulfaquinoxaline;
1-Dimethylaminopropyl-3-methyl-6-chloroquinoxaline-2(1H)-one;
Propaquizafop; 3-Chloroquinoline-8-carboxylic acid;
5,10,15,20-Tetraphenyl-1-3-(4-(4-aminobutyl)-7-
chloroquinoline)propioamidoporphine;
4-((Carboxymethyl)amino)-5,7-dichloroquinoline-2-carboxylic acid;
4-((Carboxymethyl)oxy)-5,7-dichloroquinoline-2-carboxylic acid;
5,7-Dichlorokynurenic acid;
N1,N2-Bis(7-chloroquinolin-4-yl)cyclohexane-1,2-diamine;
Meclinertant;
5-(2-(1-(3-(2-(7-Chloroquinolin-2-yl)ethenyl)benzyl)indol-7-yl)ethyl)-
1H-tetrazole;
(N1-(7-Chloroquinolin-4-yl)-3-(N3,N3-diethylamino)propylamine)
dihydrochloride trihydrate; and enantiomers thereof, and mixtures
thereof, and suitable pharmaceutical salts thereof.
[0029] Another aspect of the invention is directed toward methods
of treating or preventing neuropathy in a subject including
administering to the subject a modulator of glial cell-derived
neurotrophic factor (GDNF) receptor.
[0030] In certain embodiments, the modulator may be an agonist of
the GDNF receptor GFR.alpha.1 and/or GFR.alpha.2. The agonist may
be a non-peptidyl agonist, particularly is X1134035.
[0031] A further aspect of the present invention is directed toward
topical lotions including a subject anti-peripheral neuropathic
quinoline compound, or a pharmaceutically acceptable salt thereof;
and a pharmaceutically acceptable carrier formulated for topical
application.
[0032] In another aspect, the present invention provides
"pharmaceutically acceptable" compositions, that include a
therapeutically effective amount of one or more of the compounds
described herein, formulated together with one or more
pharmaceutically acceptable carriers (additives) and/or diluents.
The pharmaceutical compositions of the present invention may be
specially formulated for administration in solid or liquid form,
including those adapted for topical application, for example, as a
cream, ointment, drops, gels, or a controlled-release patch or
spray or sustained-release formulation applied to the skin, for
example, as a cream or foam.
[0033] Certain aspects of the invention include topical lotion
formulations. A topical lotion comprises a therapeutically
effective amount of one or more of the compounds described herein
and a topical carrier. Topical carriers include but are not limited
to creams, ointments, drops, gels, or a controlled-release patch or
spray or sustained-release formulation applied to the skin.
Suitable carrier components include, but are not limited to,
mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters
wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, liquid
petroleum, white petroleum, propylene glycol, polyoxyethylene
polyoxypropylene compound, emulsifying wax and water.
[0034] The phrase "pharmaceutically acceptable" is employed herein
to refer to those materials, compositions, and/or dosage forms
which are, within the scope of sound medical judgment, suitable for
use in contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk
ratio.
[0035] The phrase "pharmaceutically-acceptable carrier" as used
herein means a pharmaceutically-acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient, or
solvent encapsulating material, involved in carrying or
transporting the subject extract from one organ, or portion of the
body, to another organ, or portion of the body. Each carrier must
be "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not injurious to the patient.
Some examples of materials which can serve as
pharmaceutically-acceptable carriers include: sugars, such as
lactose, glucose and sucrose; starches, such as corn starch and
potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; alginic acid; sterile distilled
water; pyrogen-free water; isotonic saline; Ringer's solution;
ethyl alcohol; pH buffered solutions; polyesters, polycarbonates
and/or polyanhydrides; and other non-toxic compatible substances
employed in pharmaceutical formulations.
[0036] Wetting agents, emulsifiers and lubricants, such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
release agents, coating agents, and perfuming agents, preservatives
and antioxidants can also be present in the compositions.
[0037] The compounds of the present invention can be administered
orally, parenterally, for example, subcutaneously, intravenously,
intramuscularly, intraperitoneally, by intranasal instillation, by
inhalation, or by application to mucous membranes, such as, that of
the nose, throat, and bronchial tubes. They may be administered
alone or with suitable pharmaceutical carriers, and can be in solid
or liquid form such as, tablets, capsules, powders, solutions,
suspensions, or emulsions.
[0038] The active compounds of the present invention may be orally
administered, for example, with an inert diluent, or with an
assimilable edible carrier, or they may be enclosed in hard or soft
shell capsules, or they may be compressed into tablets, or they may
be incorporated directly with the food of the diet. For oral
therapeutic administration, these active compounds may be
incorporated with excipients and used in the form of tablets,
capsules, elixirs, suspensions, syrups, and the like. Such
compositions and preparations should contain at least 0.1% of
active compound. The percentage of the compound in these
compositions may, of course, be varied and may conveniently be
between about 2% to about 60% of the weight of the unit. The amount
of active compound in such therapeutically useful compositions is
such that a suitable dosage will be obtained. Preferred
compositions according to the present invention are prepared so
that an oral dosage unit contains between about 1 and 250 mg of
active compound.
[0039] The tablets, capsules, and the like may also contain a
binder such as gum tragacanth, acacia, corn starch, or gelatin;
excipients such as dicalcium phosphate; a disintegrating agent such
as corn starch, potato starch, alginic acid; a lubricant such as
magnesium stearate; and a sweetening agent such as sucrose,
lactose, or saccharin. When the dosage unit form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier, such as a fatty oil.
[0040] Various other materials may be present as coatings or to
modify the physical form of the dosage unit. For instance, tablets
may be coated with shellac, sugar, or both. A syrup may contain, in
addition to active ingredient, sucrose as a sweetening agent,
methyl and propylparabens as preservatives, a dye, and flavoring
such as cherry or orange flavor.
[0041] These active compounds may also be administered
parenterally. Solutions or suspensions of these active compounds
can be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof in
oils. Illustrative oils are those of petroleum, animal, vegetable,
or synthetic origin, for example, peanut oil, soybean oil, or
mineral oil. In general, water, saline, aqueous dextrose and
related sugar solution, and glycols such as, propylene glycol or
polyethylene glycol, are preferred liquid carriers, particularly
for injectable solutions. Under ordinary conditions of storage and
use, these preparations contain a preservative to prevent the
growth of microorganisms.
[0042] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol, and liquid polyethylene glycol),
suitable mixtures thereof, and vegetable oils.
[0043] The compounds of the present invention may also be
administered directly to the airways in the form of an aerosol. For
use as aerosols, the compounds of the present invention in solution
or suspension may be packaged in a pressurized aerosol container
together with suitable propellants, for example, hydrocarbon
propellants like propane, butane, or isobutane with conventional
adjuvants. The materials of the present invention also may be
administered in a non-pressurized form such as in a nebulizer or
atomizer.
[0044] The compounds of the present invention may also be
administered directly to the airways in the form of a dry powder.
For use as a dry powder, the compounds of the present invention may
be administered by use of an inhaler. Exemplary inhalers include
metered dose inhalers and dry powdered inhalers. A metered dose
inhaler or "MDI" is a pressure resistant canister or container
filled with a product such as a pharmaceutical composition
dissolved in a liquefied propellant or micronized particles
suspended in a liquefied propellant. The correct dosage of the
composition is delivered to the patient. A dry powder inhaler is a
system operable with a source of pressurized air to produce dry
powder particles of a pharmaceutical composition that is compacted
into a very small volume. For inhalation, the system has a
plurality of chambers or blisters each containing a single dose of
the pharmaceutical composition and a select element for releasing a
single dose.
[0045] Suitable powder compositions include, by way of
illustration, powdered preparations of the active ingredients
thoroughly intermixed with lactose or other inert powders
acceptable for intrabronchial administration. The powder
compositions can be administered via an aerosol dispenser or
encased in a breakable capsule which may be inserted by the patient
into a device that punctures the capsule and blows the powder out
in a steady stream suitable for inhalation. The compositions can
include propellants, surfactants, and co-solvents and may be filled
into conventional aerosol containers that are closed by a suitable
metering valve.
EXAMPLES
[0046] First we used a transgenic mouse line (GFAP-DN-erbB4) in
which erbB receptor function in non-myelinating cells (NMSCs) is
eliminated by expression of a dominant negative erbB receptor (6).
Around 21 days of age, these mice begin to show symptoms of SFN,
including loss of thermal nociception, breakdown of Remak bundles
(unmyelinated axons surrounded by a NMSC), degeneration of c-fibers
and death of NMSCs. This degenerative process coincides with
dramatic reduction in the levels of GDNF expression in peripheral
nerves, suggesting that loss of GDNF could be implicated in the
pathogenesis of SFN (6) in this model.
[0047] As a first test of the effects of target-derived trophic
support via GDNF receptor activation in this model we crossed
GFAP-DN-erbB4 mice with a previously characterized mouse line that
over-expresses GDNF in the skin under the control of the keratin 14
promoter (K14-GDNF) (8). In these mice GDNF expression in the skin
is .about.6-fold higher than that found in wild type skin and leads
to increased numbers of sensory terminals (8), indicating that the
transgenic GDNF is active and has an impact on sensory neurons.
Importantly, despite this hyper-innervation, K14-GDNF mice have
normal sensory behavior and are otherwise normal (8), indicating
that GDNF over-expression in the skin has no deleterious
effects.
[0048] GFAP-DN-erbB4 and K14-GDNF double and single transgenic mice
were tested for sensory function using a hot plate test. As shown
previously, adult (6-week-old) K14-GDNF mice responded normally in
this assay (8) while GFAP-DN-erbB4 mice had a dramatic loss in
thermal-nociception and needed to be removed from the hot plate
after 30 seconds (6). In contrast, withdrawal latencies in
GFAP-DN-erbB4::K14-GDNF double transgenic mice were
indistinguishable from those of wild types, indicating that the
sensory deficit in GFAP-DN-erbB4 mice was rescued by
over-expression of GDNF in the skin.
[0049] In our previous characterization of GFAP-DN-erbB4 mice we
showed that loss of thermal nociception correlates with dramatic
alterations in peripheral nerve structure, including disruption of
Remak bundles (6). Therefore, we used electron microscopy to
examine Remak bundle structure in mice of the four genotypes.
Remarkably, GFAP-DN-erbB4::K14-GDNF double transgenic mice
contained many normal-appearing Remak bundles. Since the ability of
mice to respond to noxious thermal stimuli depends on c-fiber
sensory terminals in the footpads and patients with symptomatic SFN
have reduced epidermal nerve fiber density (10), we tested whether
these terminals were disrupted in GFAP-DN-erbB4 mice, and if so,
can they be rescued by GDNF over-expression. Quantification of free
nerve endings identified by expression of the neuronal marker
protein gene product 9.5 (PGP9.5) at P30 confirmed previous reports
of hyper-innervation in the skin of K14-GDNF mice compared to wild
type skin (8), while GFAP-DN-erbB4 mice showed reduced nerve
terminals in the skin at this age. Consistent with the rescue of
thermal nociception in double transgenic mice, we observed
preservation of nerve terminals in the skin of
GFAP-DN-erbB4::K14-GDNF mice compared to GFAP-DN-erbB4 mice.
Over-expression of GDNF not only improved the axonal phenotyes of
GFAP-DN-erbB4 mice but also reduced the extent of Schwann cell
death, indicating that trophic support delivered at the target of
innervation preserves the health of glial cells along the nerve.
Importantly, levels of DN-erbB4 expression in sciatic nerves of
double transgenic mice were similar to those in GFAP-DN-erbB4 mice,
indicating that reversion of the SFN in double transgenic mice was
not due to an effect of GDNF on DN-erbB4 expression. These results
show that GDNF over-expression in the skin also rescues the
anatomical phenotypes along the nerve produced by DN-erbB4
expression in NMSCs.
[0050] The results described above suggest that GDNF
over-expression in the skin could be used to treat progressive SFNs
like the one found in GFAP-DN-erbB4 mice. However, since K14-GDNF
mice overexpress this factor from embryonic development, the
possibility that alterations in development could have contributed
to the phenotypic rescue could not be overlooked. Furthermore, when
considering treatment of humans, GDNF over-expression would require
some type of gene therapy, which has its own drawbacks and
complications. Thus, we felt it was necessary to test alternative
approaches to activate GDNF signaling in the skin as a treatment
for SFN. Since proteins such as GDNF do not readily diffuse through
the skin, we tested if XIB4035, a non-peptidyl small molecule
agonist for the GDNF receptor GFR.alpha.1 (11) would be a useful
alternative. XIB4035 had previously been shown to activate the Ret
co-receptor by binding to the GFR 1 GDNF receptor in both human and
murine cells (11).
[0051] We generated a cream containing XIB4035 (1.2 mM) and applied
it directly to the hind paws of GFAP-DN-erbB4 and wild type mice
twice daily for a period of 4 weeks starting at the time of weaning
(P21). Importantly, by this age GFAP-DN-erbB4 mice already exhibit
neuropathic symptoms (6). Thus, these experiments would test
whether activation of Ret signaling has therapeutic effects after
disease onset. To control for potential effects of the control
cream, mice of both genotypes were treated in the same way with the
base cream without the drug. Mice were tested for responses to
noxious thermal stimulus prior to the initiation of treatment and
every 7 days throughout the treatment period. Over the 4 week
treatment period, the behavior of wild type mice remained normal,
independent of the presence of XIB4035 in the cream while
GFAP-DN-erbB4 mice treated with control cream progressively lost
thermal nociception as we found in untreated animals (6). In
contrast, when treated with cream containing XIB4035, GFAP-DN-erbB4
mice showed dramatic preservation of thermal nociception, their
behavior being similar to that of wild type mice. As expected from
our previous characterization of GFAP-DN-erbB4 mice (6), response
thresholds to punctate mechanical stimuli remained normal in all
groups after the 4 weeks of treatment, indicating that the drug had
no effect on mechanoreception. Together, these findings indicate
that topical treatment with XIB4035 preserves the ability of mice
with SFN to respond to noxious heat stimulus without inducing
hypersensitivity. They also show that treatment of unaffected mice
with XIB4035 had no deleterious effects on their ability to respond
to heat or mechanical stimuli.
[0052] From the anatomical point of view, similarly to what we
found in the double transgenic mice, 4-week treatment with XIB4035
prevented the degeneration of Remak bundles and c-fiber axons.
Morphometric analysis showed that XIB4035 treatment preserved both
the size of c-fiber axons and the number of c-fibers per Remak
bundle in GFAP-DN-erbB4 mice. Furthermore, NMSC apoptosis was also
significantly reduced in GFAP-DN-erbB4 mice treated with XIB4035.
Interestingly, like GDNF over-expression, drug treatment not only
prevented degeneration of sensory nerve terminals in the footpad
skin of GFAP-DN-erbB4 mice, but it increased the density of nerve
terminals in wild type skin.
[0053] As it is unlikely that SFN is always caused by glial
dysfunction, we considered it necessary to test the effectiveness
of XIB4035 treatment on SFN resulting from a different pathogenic
mechanism. Therefore we tested the efficacy of our therapeutic
approach in resiniferatoxin (RTX)-induced SFN (9). RTX-induced SFN
involves intraperitoneal injection of an ultrapotent capsaicin
analog that targets c-fiber terminals by binding to and activating
TRPV1 channels resulting in calcium influx, which induces loss of
skin innervation and loss of thermal nociception (9). After RTX
injection, mice were either treated immediately with XIB4035 or
treatment was delayed by 12 hours. Both treatment paradigms yielded
similar results.
[0054] Similar to the effect seen in GFAP-DN-erbB4 mice, treatment
with XIB4035 greatly improved thermal nociception in the RTX model.
Seven days after RTX treatment paw withdrawal latencies were
greatly increased in animals treated with control cream. In
contrast, RTX-injected animals that were treated with XIB4035 had
remarkable preservation of their ability to respond to noxious
heat. Furthermore, innervation of the skin was preserved as a
result of XIB4035 treatment in RTX-injected mice. As expected based
on the preservation of thermal nociception and skin innervation,
Remak bundle structure was also significantly improved in
RTX-injected animals treated with XIB4035. Both c-fiber area and
the number of fibers per Remak bundle were improved by treatment
with XIB4035 in RTX-induced SFN.
[0055] Together, these results indicate that SFNs may be treated by
supplying ligands for neurotrophic factor receptors at the target
of innervation, providing an alternative to invasive or systemic
routes of delivery. Using either "gene therapy" or a "topical"
pharmacological approach we show that application of GDNF receptor
ligands to the skin results in preservation of nerve structure and
function in two mouse models of SFN. Since SFN occurs frequently as
a late-onset complication of a number of diseases, e.g. diabetes,
topical application of GDNF receptor ligands can be considered as a
preventive therapy after the first disease symptoms, e.g.
hyperglycemia, appear. Nevertheless, since topical application of
the drug to mice was effective after the onset of sensory defects,
our studies indicate that this type of treatment could help even
after patients develop SFN symptoms.
[0056] The results presented herein together with our previous
analysis of GFAP-DN-erbB4 mice (6) also provide insights into the
roles of trophic factors in adult nerves. The emerging picture is
that GDNF produced by Schwann cells along the nerve is necessary
for the maintenance of c-fiber structure and function, and that
endogenous GDNF can be replaced by application of GDNF or synthetic
ligands to the sensory nerve endings. If similar mechanisms are at
work in other nerves, this type of therapeutic intervention could
be used to treat other neuropathies, including those involving
central nerves, i.e. optic nerve. Furthermore, the use of this type
of drug to treat degenerative processes involving other neurons
that express receptors for the GDNF family, e.g. spinal cord
motorneurons, using intramuscular injections, should be considered.
Furthermore, these results demonstrate that tyrosine kinase
signaling in cells or cell segments within the skin can be modified
by the topical application of a non-peptidyl molecule. Therefore,
in addition to the results presented herein, this strategy might
also be useful to locally treat other disorders such as skin cancer
(12, 13).
[0057] Previous animal tests of the effectiveness of systemic or
intrathecal injection of neurotrophic factors as therapeutic agents
for peripheral neuropathy have provided encouraging results
(14-16). However, human trials using injections of trophic factors
to treat peripheral neuropathies have either resulted in severe
side effects or have been ineffective altogether. For example,
trials examining the efficacy of NGF treatment in diabetic patients
with peripheral neuropathy or patients with HIV neuropathy have
shown some improvement in the patient's perception of symptom
severity, but side effects including myalgia, peripheral edema, and
hyperalgesia were also observed (17-19). Test of GDNF injections
for other neurological disorders have been marred by serious side
effects. For example, intracerebroventricular administration of
GDNF results in weight loss, anorexia, and nausea while providing
little benefit to Parkinson's disease patients (20). Our results
indicate that topical treatment with small molecule agonists for
neurotrophic factor receptors, e.g. GDNF receptor ligands, can
provide an effective treatment for peripheral neuropathies without
the side effects associated with generalized delivery.
[0058] Animals and Induction of Neuropathy Using RTX.
[0059] Transgenic mouse lines used were as previously described (6,
8). Animals were kept in the animal facility with free access to
food and water. Behavioral experiments were performed in a quiet
environment at the same time of day. The hot plate test was
performed using a "controlled hot-plate analgesia meter" (Columbus
Instruments) heated to 55.degree. C. Paw withdrawal latency was
measured as the time required for the mouse to visibly respond to
the thermal stimulus, e.g. licking paws, lifting paws, or jumping
off of the plate. Mechanical sensitivity was tested by simulation
of the plantar surface of the hind paw with a series of von Frey
filaments while the animal was placed on an elevated wire grid. The
threshold was determined as the lowest force that evoked a visible
withdrawal response. 8-week-old ICR mice weighing at least 30 g
were injected intraperitoneally with RTX (50 mg/kg) or vehicle (10%
Tween-80, 10% ethanol in isotonic saline) (9).
[0060] Preparation and Use of XIB4035.
[0061] The cream containing XIB4035 (1.2 mM, Matrix Scientific,
Columbia, S.C.) consisted of N-methyl-pyrrolidone (5%), isopropyl
myristate (5%) and petroleum jelly (90%). Control cream had the
same ingredients without XIB4035. Cream was applied twice daily to
the hind paws of isoflurane anesthetized mice starting at P21 for a
period of 9 days (for analysis of cell death in sciatic nerves) or
4 weeks (all other studies using GFAP-DN-erbB4). Treatment of
RTX-induced neuropathy began at the time of RTX injection or 12
hours after RTX injection and proceeded twice daily for a period of
one week.
[0062] Plastic Embedding and Electron Microscopy.
[0063] Tissue was prepared as in (6). Briefly, mice were perfused
intracardially with 2% paraformaldehyde, 2.5% gluteraldehyde and
0.03% picric acid in 0.1 M cacodylate buffer (pH 7.2). Tissue was
post-fixed overnight at 4.degree. C. and embedded in Epon.
Ultrathin sections were cut, collected on cellodin-coated grids and
stained using uranyl acetate and lead citrate. Photographs were
taken using the Tecnai G.sup.2 Spirit BioTWIN transmission electron
microscope.
[0064] Immunohistochemistry.
[0065] Mice were anesthetized with 2.5% Avertin and footpad skin
was removed and immersion fixed in 4% paraformaldehyde, 14% picric
acid in 0.1 M phosphate buffer (pH 7.3) overnight at 4.degree. C.
and cryoprotected in 20% sucrose overnight at 4.degree. C. Tissues
were embedded in OCT and sectioned at 30 m and stained as floating
sections by washing 3 times for 5 minutes in PBS+0.1% Triton-X 100,
blocked for 30 minutes in PBS+0.1% Triton-X 100+10% normal goat
serum and then incubated, PGP9.5 rabbit polyclonal antibody
(Ultraclone, 1:1000), overnight at 4.degree. C. Sections were
washed 3 times for 10 minutes in PBS+0.1% Triton-X 100 then
incubated in donkey anti-rabbit Alexa-488 (Invitrogen) 1:300 for 2
hours at room temperature. Nuclei were stained with DAPI and
sections were mounted with Gel-Mount.
[0066] Cell Death Detection.
[0067] Sciatic nerves were dissected from mice perfused with 4%
paraformaldehyde in PBS and fixed overnight at 4.degree. C. Nerves
were embedded in OCT and 16 m thick transverse sections were
processed for TUNEL as follows. Sections were washed 3 times for 5
minutes with PBS followed by proteinase K digestion (0.02 U/mL) in
10 mM TRIS/HCl pH 7.5 for 30 minutes at 37.degree. C. Digested
sections were fixed with 4% paraformaldehyde at room temperature
for 20 minutes followed by 3 washes in PBS for 5 minutes. Apoptotic
nuclei were identified using the In Situ Cell Death Detection Kit
(Roche). Nuclei were stained with DAPI and sections were mounted
with Gel-Mount.
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[0088] Although preferred embodiments have been depicted and
described in detail herein, it will be apparent to those skilled in
the relevant art that various modifications, additions,
substitutions, and the like can be made without departing from the
spirit of the invention and these are therefore considered to be
within the scope of the invention as defined in the claims which
follow. All references cited herein, and references cited therein
are incorporated by reference as if each was specifically and
individually indicated to be incorporated by reference.
* * * * *