U.S. patent application number 15/137801 was filed with the patent office on 2016-09-15 for compositions for treating cmt and related disorders.
The applicant listed for this patent is Pharnext. Invention is credited to Ilya Chumakov, Daniel Cohen, Serguei Nabirochkin.
Application Number | 20160263104 15/137801 |
Document ID | / |
Family ID | 41168603 |
Filed Date | 2016-09-15 |
United States Patent
Application |
20160263104 |
Kind Code |
A1 |
Cohen; Daniel ; et
al. |
September 15, 2016 |
COMPOSITIONS FOR TREATING CMT AND RELATED DISORDERS
Abstract
The present invention relates to compositions and methods for
the treatment of the Charcot-Marie-Tooth disease and related
disorders.
Inventors: |
Cohen; Daniel; (Le Vesinet,
FR) ; Nabirochkin; Serguei; (Chatenay Malabry,
FR) ; Chumakov; Ilya; (Vaux Le Penil, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pharnext |
Issy Les Moulineaux |
|
FR |
|
|
Family ID: |
41168603 |
Appl. No.: |
15/137801 |
Filed: |
April 25, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13375288 |
Nov 30, 2011 |
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PCT/EP2010/057438 |
May 28, 2010 |
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15137801 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/047 20130101;
A61K 31/575 20130101; A61P 25/28 20180101; A61K 45/06 20130101;
A61K 31/567 20130101; A61P 25/00 20180101; A61K 31/436 20130101;
A61K 31/439 20130101; A61K 9/0053 20130101; A61K 31/7004 20130101;
A61K 31/192 20130101; A61K 31/555 20130101; A61K 31/195 20130101;
A61K 31/4164 20130101; A61K 31/4178 20130101; A61K 31/197 20130101;
A61K 31/485 20130101; A61K 31/192 20130101; A61K 2300/00 20130101;
A61K 31/197 20130101; A61K 2300/00 20130101; A61K 31/4164 20130101;
A61K 2300/00 20130101; A61K 31/4178 20130101; A61K 2300/00
20130101; A61K 31/436 20130101; A61K 2300/00 20130101; A61K 31/485
20130101; A61K 2300/00 20130101; A61K 31/575 20130101; A61K 2300/00
20130101; A61K 31/7004 20130101; A61K 2300/00 20130101; A61K 31/567
20130101; A61K 2300/00 20130101; A61K 31/047 20130101; A61K 2300/00
20130101; A61K 31/555 20130101; A61K 2300/00 20130101; A61K 31/195
20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 31/485 20060101
A61K031/485; A61K 31/047 20060101 A61K031/047; A61K 9/00 20060101
A61K009/00; A61K 31/197 20060101 A61K031/197 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 2, 2009 |
EP |
09305506.9 |
Claims
1-13. (canceled)
14. A pharmaceutical composition comprising, as active ingredients:
(i) baclofen or a salt thereof, (ii) sorbitol or a salt thereof,
and (iii) naltrexone or a salt thereof; in a relative weight ratio
of the baclofen, sorbitol, and naltrexone of 8.6:300:1; and a
pharmaceutically suitable excipient or carrier.
15. The composition according to claim 14, wherein the sorbitol is
D-sorbitol.
16. The composition according to claim 14, wherein said composition
is formulated for an oral administration.
17. The composition according to claim 14, wherein said composition
comprises a dose of baclofen in an amount from 1 to 300 .mu.g/kg of
a human subject.
18. The composition according to claim 14, wherein said composition
comprises a dose of naltrexone in an amount from 1 to 100 .mu.g/kg
of a human subject.
19. The composition according to claim 18, wherein said composition
comprises naltrexone in an amount from 1 to 50 .mu.g/kg of a human
subject.
20. The composition according to claim 14, wherein said composition
comprises baclofen in an amount from 1 to 300 .mu.g/kg of a human
subject and naltrexone in an amount from 1 to 100 .mu.g/kg of a
human subject.
21. The composition according to claim 20, wherein said composition
comprises naltrexone in an amount from 1 to 50 .mu.g/kg of a human
subject.
22. A method for treating CMT in a human subject in need thereof,
said method comprising the step of administering to said human
subject the composition according to claim 14.
23. The method according to claim 22, wherein the CMT is CMT1A.
Description
[0001] The present invention relates to compositions and methods
for the treatment of the Charcot-Marie-Tooth disease and related
disorders.
[0002] Charcot-Marie-Tooth disease ("CMT") is an orphan genetic
peripheral poly neuropathy. Affecting approximately 1 in 2,500
individuals, this disease is the most common inherited disorder of
the peripheral nervous system. Its onset typically occurs during
the first or second decade of life, although it may be detected in
infancy. Course of disease is chronic with gradual neuromuscular
degeneration. The disease is invalidating with cases of
accompanying neurological pain and extreme muscular disability. CMT
is one of the best studied genetic pathologies with approximately
30,000 cases in France. While a majority of CMT patients harbour a
duplication of a chromosome 17 fragment containing a myelin gene:
PMP22 (form CMT1A), two dozens of genes have been implicated in
different forms of CMT. Accordingly, although monogenic in origin,
this pathology manifests clinical heterogeneity due to possible
modulator genes. The genes mutated in CMT patients are clustering
around tightly connected molecular pathways affecting
differentiation of Schwann cells or neurons or changing interplay
of these cells in peripheral nerves.
[0003] Mining of publicly available data, describing molecular
mechanisms and pathological manifestations of the CMT1A disease,
allowed us to prioritize a few functional cellular modules
transcriptional regulation of PMP22 gene, PMP22 protein
folding/degradation, Schwann cell proliferation and apoptosis,
death of neurons, extra-cellular matrix deposition and remodelling,
immune response--as potential legitimate targets for CMT-relevant
therapeutic interventions. The combined impact of these deregulated
functional modules on onset and progression of pathological
manifestations of Charcot-Marie-Tooth justifies a potential
efficacy of combinatorial CMT treatment.
[0004] International patent application n.degree. PCT/EP2008/066457
describes a method of identifying drug candidates for the treatment
of the Charcot-Marie-Tooth disease by building a dynamic model of
the pathology and targeting functional cellular pathways which are
relevant in the regulation of CMT disease.
[0005] International patent application n.degree. PCT/EP2008/066468
describes compositions for the treatment of the Charcot-Marie-Tooth
disease which comprise at least two compounds selected from the
group of multiple drug candidates.
SUMMARY OF INVENTION
[0006] The purpose of the present invention is to provide new
therapeutic combinations for treating CMT and related disorders.
The invention thus relates to compositions and methods for treating
CMT and related disorders, in particular toxic neuropathy and
amyotrophic lateral sclerosis, using particular drug
combinations.
[0007] An object of this invention more specifically relates to a
composition comprising Baclofen, Sorbitol and a compound selected
from Pilocarpine, Methimazole, Mifepristone, Naltrexone,
Rapamycine, Flurbiprofen and Ketoprofen, salts or prodrugs thereof,
for simultaneous, separate or sequential administration to a
mammalian subject.
[0008] A particular object of the present invention relates to a
composition comprising Baclofen, Sorbitol and Naltrexone, for
simultaneous, separate or sequential administration to a mammalian
subject.
[0009] Another object of the invention relates to a composition
comprising (a) rapamycin, (b) mifepristone or naltrexone, and (c) a
PMP22 modulator, for simultaneous, separate or sequential
administration to a mammalian subject.
[0010] In a particular embodiment, the PMP22 modulator is selected
from Acetazolamide, Albuterol, Amiloride, Aminoglutethimide,
Amiodarone, Aztreonam, Baclofen, Balsalazide, Betaine, Bethanechol,
Bicalutamide, Bromocriptine, Bumetanide, Buspirone, Carbachol,
Carbamazepine, Carbimazole, Cevimeline, Ciprofloxacin, Clonidine,
Curcumin, Cyclosporine A, Diazepam, Diclofenac, Dinoprostone,
Disulfiram, D-Sorbitol, Dutasteride, Estradiol, Exemestane,
Felbamate, Fenofibrate, Finasteride, Flumazenil, Flunitrazepam,
Flurbiprofen, Furosemide, Gabapentin, Galantamine, Haloperidol,
Ibuprofen, Isoproterenol, Ketoconazole, Ketoprofen, L-carnitine,
Liothyronine (T3), Lithium, Losartan, Loxapine, Meloxicam,
Metaproterenol, Metaraminol, Metformin, Methacholine, Methimazole,
Methylergonovine, Metoprolol, Metyrapone, Miconazole, Mifepristone,
Nadolol, Naloxone, Naltrexone; Norfloxacin, Pentazocine,
Phenoxybenzamine, Phenylbutyrate, Pilocarpine, Pioglitazone,
Prazosin, Propylthiouracil, Raloxifene, Rapamycin, Rifampin,
Simvastatin, Spironolactone, Tacrolimus, Tamoxifen, Trehalose,
Trilostane, Valproic acid, salts or prodrugs thereof.
[0011] Another object of this invention is a composition comprising
Rapamycin and mifepristone, for simultaneous, separate or
sequential administration to a mammalian subject.
[0012] A further object of this invention is a composition as
disclosed above further comprising one or several pharmaceutically
acceptable excipients or carriers (i.e., a pharmaceutical
composition).
[0013] Another object of the present invention relates to a
composition as disclosed above for treating CMT or a related
disorder.
[0014] A further object of this invention relates to the use of a
combination of compounds as disclosed above for the manufacture of
a medicament for the treatment of CMT or a related disorder.
[0015] A further object of this invention is a method for treating
CMT or a related disorder, the method comprising administering to a
subject in need thereof an effective amount of a composition as
defined above.
[0016] A further object of this invention is a method of preparing
a pharmaceutical composition, the method comprising mixing the
above compounds in an appropriate excipient or carrier.
[0017] A more specific object of this invention is a method of
treating CMT1a in a subject, the method comprising administering to
the subject in need thereof an effective amount of a compound or
combination of compounds as disclosed above.
[0018] A further specific object of this invention is a method of
treating toxic neuropathy in a subject, the method comprising
administering to the subject in need thereof an effective amount of
a compound or combination of compounds as disclosed above.
[0019] A further specific object of this invention is a method of
treating ALS in a subject, the method comprising administering to
the subject in need thereof an effective amount of a compound or
combination of compounds as disclosed above.
[0020] Any of the various uses or methods of treatment disclosed
herein can also include an optional step of diagnosing a patient as
having CMT or a related disorder, particularly CMT1A, or
identifying an individual as at risk of developing CMT or a related
disorder, particularly CMT1A.
[0021] In this regard, a further object of this invention is a
method of treating CMT, particularly CMT1a, the method comprising
(1) assessing whether a subject has CMT, particularly CMT1a and (2)
treating the subject having CMT, particularly CMT1a with an
effective amount of a combination of compounds as described above.
Determining whether a subject has CMT, particularly CMT1a, can be
done by various tests known per se in the art, such as DNA
assays.
[0022] The invention may be used for treating CMT or a related
disorder in any mammalian subject, particularly human subjects,
more preferably CMT1a.
LEGEND TO THE FIGURES
[0023] FIG. 1. Synergistic effect of drug combination, dose 1:
effect of A) Mix7 (dose 1, day 10), B) d-Sorbitol (SRB, 500 .mu.M,
day 10), C) (R/S)-Baclofen (BCL, 5 .mu.M, day 10) and D) Naltrexone
(NTX, 5 .mu.M, day 10) on MBP expression, *:p<0.05:
significantly different from control (=ascorbic acid) (One-Way
ANOVA followed by Fisher Post-hoc test); ns: not statistically
different
[0024] FIG. 2. Synergistic effect of drug combination, dose 6 A)
Mix7 (dose 6, day 10), B) SRB (160 nM, day 10), C) BCL (1.6 nM, day
10) and D) NTX (1.6 nM, day 10) on MBP expression, *:p<0.05:
significantly different from control (=ascorbic acid) (One-Way
ANOVA followed by Fisher Post-hoc test); ns: not statistically
different
[0025] FIG. 3. Positive effect of Mix7 (7 doses) A) on day 10 and
B) on day 11 in co-incubation with ascorbic acid in PMP22 TG
co-cultures on MBP expression in percentage of control (=ascorbic
acid). One-Way Anova followed by Fisher post-hoc test.
[0026] FIG. 4. Positive effect on male rats of the 3 and 6 weeks
treatment with Mix1 measured using bar test. Latencies were
measured as the mean of two first assays of the tests (white bars
represent control rats treated with placebo; black bars represent
transgenic rats treated with placebo; grey bars represent
transgenic rats treated with Mix1. p<0.01. Statistics are
realised with the Student bilateral test).
[0027] FIG. 5. Positive effect on gait of male rats of the 3 and
6-week (respectively left and right graph treatment with Mix1
composition (white bars represent fluid gait; grey bars represent
not fluid gait; black bars represent rats with a severe incapacity
to walk. Statistics are realised with the Student bilateral
test).
[0028] FIG. 6. Positive effect on male rats of the Mix1 composition
in rats using inclined plane test (25.degree.). Rats were examined
after 3, 6 9 and 12 weeks of treatment (white bars represent
control rats treated with placebo; black bars represent transgenic
rats treated with placebo; grey bars represent transgenic rats
treated with Mix1. *p<0.05. Statistics are realised with the
Student bilateral test).
[0029] FIG. 7. Positive effect on female rats of the 3 weeks
treatment with the Mix2 composition in rats, using an inclined
plane test (white bars represent control rats treated with placebo;
black bars represent transgenic rats treated with placebo; grey
bars represent transgenic rats treated with Mix2. ** p<0.01.
Statistics are realised with the Student bilateral test).
[0030] FIG. 8. Protective effect on male rats of Mix1 on
oxaliplatin-induced neuropathy (white bars represent wild type rats
treated with placebo; black bars represent wild type rats treated
with reference product gabapentin; grey bars represent wild type
rats treated with Mix1. * p<0.05; ** p<0.01. Statistics are
realised with the Student bilateral test).
[0031] FIG. 9. Significant decrease of pmp22 RNA expression in
treated transgenic animals compared to PMP22 transgenic rats,
observed after 9 weeks of treatment with the Mix7-dose 3 (MPZ as
reference gene, Sereda et al, 1996) (p=0.0015). The transgene
integration and the overexpression of pmp22 gene have also been
confirmed; pmp22 RNA in transgenic PMP22 rats was 1.8 fold
overexpressed compared to their wild type littermates controls
(p<1.10-4). Extraction of pmp22 RNA was performed on sciatic
nerves of 16 weeks old male rats (n=18 for the Wild Type, n=20 for
the transgenic rats and n=18 for TG treated with Mix7-dose3).
Statistical analysis was performed by using the Welch t-test.
[0032] FIG. 10. A clustering analysis was performed on the inclined
plane test score at 35.degree. (to distribute in the poor,
intermediate and good performance classes at all time points of
evaluation (3, 6 and 9 weeks of treatment analyzed together). A
significant difference was observed between WT and TG placebo: 68%
of WT belonged to the good performances group and only 5% of TG
placebo belonged to this group (p=0.0003). Mix7-dose 2 and dose 3
improved the performances of TG rats. Statistical analysis were
performed by applying a trend-test at the 5% significance level
(n=18 for WT placebo rats, n=20 for TG placebo rats, n=17 for TG
treated with Mix7-dose 2, n=18 for TG treated with Mix7-dose3).
[0033] FIG. 11. The fall latencies of TG rats in the bar test after
9 weeks of treatment with Mix7-dose3 were analyzed using a Cox
model with a sandwich variance estimator, and compared to the
reference TG placebo by applying a log rank-test at the 5%
significance level. Mix7-dose 3 significantly increased the fall
latency of TG rats after 9 weeks of treatment.
[0034] FIG. 12. The grip strength of groups of wild type,
transgenic placebo and transgenic animals treated with Mix7-dose 3
daily for 9 weeks was modelized using a Cox model with a sandwich
variance estimator over all the times after treatment (3, 6 and 9
weeks) and compared to the reference TG placebo by applying a log
rank-test at the 5% significance level. The corresponding p-values
were presented on Kaplan-Meier curves A significant decrease of the
fore paws grip strength of transgenic placebo rats was observed
(black plain line, n=21) compared to WT rats (grey plain line,
p=1.45,10-5, n=19). The treatment with Mix7-dose 3 significantly
increased the strength of the fore paws (black dashed line; p=0.03,
n=18).
[0035] FIG. 13. A Pearson correlation test showed a significant
correlation between the fall latency time in the bar test (after 9
weeks of treatment) and the pmp22 RNA expression level:
(p=1.6,10.sup.-4 (WT, TG placebo and TG treated with the Mix7-dose
3 analysed together); p=0.07 (TG placebo and TG treated with the
Mix7-dose 3 analysed together). [0036] The lower the pmp22 RNA
expression was, the better the bar test performances were. Male
rats were 16 weeks old (n=18 for WT it white circles; n=20 for the
TG placebo, black circles and n=18 for TG treated with the
Mix7-dose3, white triangles).
[0037] FIG. 14. A Pearson correlation test showed a significant
correlation between the fall latency time in the bar test (after 9
weeks of treatment) and the conduction velocity of the sensitive
nerve (NCV): p=1.34.10-6 (WT, TG placebo and TG treated with
Mix7-dose3 analysed together) and p=0.04 (TG placebo and TG treated
with Mix7-dose3 analysed together). The higher the conduction
velocity was, the better the performances in bar test were. Male
rats were 16 weeks old (n=18 for WT rats, white circles; n=20 for
the TG placebo, black circles and n=18 for TG treated with the
Mix7-dose3, white triangles).
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention provides new therapeutic approaches
for treating CMT or related disorders. The invention discloses
novel drug combinations which allow an effective correction of such
diseases and may be used in any mammalian subject.
[0039] Within the context of this invention, CMT includes CMT1A,
CMT1B, CMT1C, CMT1D, CMT1X, CMT2A, CMT2B, CMT2D, CMT2E, CMT2-P0,
CMT4A, CMT4B1, CMT4B2, CMT4D, CMT4F, CMT4, or AR-CMT2A, more
preferably CMT1a.
[0040] Within the context of the present invention, the term "CMT
related disorder" designates other diseases associated with
abnormal expression of PMP22 leading to abnormal myelination and
loss of neurons. The term "CMT related disorder" more particularly
includes Alzheimer's disease (AD), senile dementia of AD type
(SDAT), Parkinson's disease, Lewis body dementia, vascular
dementia, autism, mild cognitive impairment (MCI), age-associated
memory impairment (AAMI) and problem associated with ageing,
post-encephalitic Parkinsonism, schizophrenia, depression, bipolar
disease and other mood disorders, Huntington s disease, motor
neurone diseases including amyotrophic lateral sclerosis (ALS),
multiple sclerosis, idiopathic neuropathies, diabetic neuropathy,
toxic neuropathy including neuropathy induced by drug treatments,
neuropathies provoked by HIV, radiation, heavy metals and vitamin
deficiency states, prion-based neurodegeneration, including
Creutzfeld-Jakob disease (CJD), bovine spongiform encephalopathy
(BSE), GSS, FFI, Kuru and Alper's syndrome.
[0041] In a preferred embodiment, "CMT related disorder" designates
a toxic neuropathy, particularly drug-induced neuropathies, or
ALS.
[0042] As used herein, "treatment" of a disorder includes the
therapy, prevention, prophylaxis, retardation or reduction of pain
provoked by the disorder. The term treatment includes in particular
the control of disease progession and associated symptoms.
[0043] Also, the term "compound" designates the chemical compounds
as specifically named in the application, as well as any
pharmaceutically composition with acceptable salt, hydrate, ester,
ether, isomers, racemate, conjugates, pro-drugs thereof. The
compounds listed in this application may also be identified with
its corresponding CAS number.
[0044] Thus, the preferred compounds used in the invention are
Baclofen (CAS 134-47-0) and its possible salts enantiomers,
racemates, prodrugs and derivatives; Sorbitol (CAS 50-70-4) and its
possible salts, enantiomers, racemates, prodrugs and derivatives;
Naltrexone (CAS 16590-41-3) and its possible salts, enantiomers,
racemates, prodrugs and derivatives; Mifepristone (CAS 84371-65-3)
and its possible salts, enantiomers, racemates, prodrugs and
derivatives; Pilocarpine (CAS 54-71-7) and its possible salts,
enantiomers, racemates, prodrugs and derivatives; Methimazole (CAS
60-56-0) and its possible salts, enantiomers, racemates, prodrugs
and derivatives; Ketoprofen (CAS 22071-15-4) and its possible
salts, enantiomers, racemates, prodrugs and derivatives;
Flurbiprofen (5104-49-4) and its possible salts, enantiomers,
racemates, prodrugs and derivatives and Rapamycin (CAS 53123-88-9)
and its possible salts, enantiomers, racemates, prodrugs and
derivatives.
[0045] Further compounds used in the invention are Acetazolamide
(CAS 59-66-5) and its possible salts, enantiomers, prodrugs and
derivatives; Albuterol (CAS 18559-94-9) and its possible salts,
enantiomers, prodrugs and derivatives; Amiloride (CAS 2016-88-8)
and its possible salts, enantiomers, prodrugs and derivatives;
Aminoglutethimide (CAS 125-84-8) and its possible salts,
enantiomers, prodrugs and derivatives; Amiodarone (CAS 1951-25-3)
and its possible salts, enantiomers, prodrugs and derivatives;
Aztreonam (CAS 78110-38-0) and its possible salts, enantiomers,
prodrugs and derivatives; Baclofen (CAS 1134-47-0) and its possible
salts, enantiomers, prodrugs and derivatives; Balsalazide (CAS
80573-04-2) and its possible salts, enantiomers, prodrugs and
derivatives; Betaine (CAS 107-43-7) and its possible salts,
enantiomers, prodrugs and derivatives; Bethanechol (CAS 674-38-4)
and its possible salts, enantiomers, prodrugs and derivatives;
Bicalutamide (CAS 90357-06-5) and its possible salts, enantiomers,
prodrugs and derivatives; Bromocriptine (CAS 25614-03-3) and its
possible salts, enantiomers, prodrugs and derivatives; Bumetanide
(CAS 28395-03-1) and its possible salts, enantiomers, prodrugs and
derivatives; Buspirone (CAS 36505-84-7) and its possible salts,
enantiomers, prodrugs and derivatives; Carbachol (CAS 51-83-2) and
its possible salts, enantiomers, prodrugs and derivatives;
Carbamazepine (CAS 298-46-4) and its possible salts, enantiomers,
prodrugs and derivatives; Carbimazole (CAS 22232-54-8) and its
possible salts, enantiomers, prodrugs and derivatives: Cevimeline
(CAS 107233-08-9) and its possible salts, enantiomers, prodrugs and
derivatives; Ciprofloxacin (CAS 85721-13-1) and its possible salts,
enantiomers, prodrugs and derivatives; Clonidine (CAS 4205-90-7)
and its possible salts, enantiomers, prodrugs and derivatives;
Curcumin (CAS 458-37-7) and its possible salts, enantiomers,
prodrugs and derivatives; Cyclosporine A (CAS 59865-13-3) and its
possible salts, enantiomers, prodrugs and derivatives; Diazepam
(CAS 439-14-5) and its possible salts, enantiomers, prodrugs and
derivatives; Diclofenac (CAS 15307-86-5) and its possible salts,
enantiomers, prodrugs and derivatives; Dinoprostone (CAS 363-24-6)
and its possible salts, enantiomers, prodrugs and derivatives;
Disulfiram (CAS 97-77-8) and its possible salts, enantiomers,
prodrugs and derivatives; D-Sorbitol (CAS 50-70-4) and its possible
salts, enantiomers, prodrugs and derivatives; Dutasteride (CAS
164656-23-9) and its possible salts, enantiomers, prodrugs and
derivatives; Estradiol (CAS 50-28-2) and its possible salts,
enantiomers, prodrugs and derivatives; Exemestane (CAS 107868-30-4)
and its possible salts, enantiomers, prodrugs and derivatives;
Felbamate (CAS 25451-15-4) and its possible salts, enantiomers,
prodrugs and derivatives; Fenofibrate (CAS 49562-28-93 and its
possible salts, enantiomers, prodrugs and derivatives; Finasteride
(CAS 98319-26-7) and its possible salts, enantiomers, prodrugs and
derivatives; Flumazenil (CAS 78755-81-4) and its possible salts,
enantiomers, prodrugs and derivatives; Flunitrazepam ((CAS
1622-62-4) and its possible salts, enantiomers, prodrugs and
derivatives; Flurbiprofen (CAS 5104-49-4) and its possible salts,
enantiomers, prodrugs and derivatives; Furosemide (CAS 54-31-9) and
its possible salts, enantiomers, prodrugs and derivatives;
Gabapentin (CAS 60142-96-3) and its possible salts, enantiomers,
prodrugs and derivatives; Galantamine (CAS 357-70-0) and its
possible salts, enantiomers, prodrugs and derivatives; Haloperidol
(CAS 52-86-8) and its possible salts, enantiomers, prodrugs and
derivatives; Ibuprofen (CAS 15687-27-1) and its possible salts,
enantiomers, prodrugs and derivatives; Isoproterenol (CAS
7683-59-2) and its possible salts, enantiomers, prodrugs and
derivatives; Ketoconazole (CAS 65277-42-1) and its possible salts,
enantiomers, prodrugs and derivatives; Ketoprofen ((AS 22071-15-4)
and its possible salts, enantiomers, prodrugs and derivatives;
L-carnitine ((AS 541-15-1) and its possible salts, enantiomers,
prodrugs and derivatives; Liothyronine (T3) (CAS 6893-02-3) and its
possible salts, enantiomers, prodrugs and derivatives; Lithium (CAS
7439-93-2) and its possible salts, enantiomers, prodrugs and
derivatives; Losartan (CAS 114798-26-4) and its possible salts,
enantiomers, prodrugs and derivatives; Loxapine (CAS 1977-10-2) and
its possible salts, enantiomers, prodrugs and derivatives;
Meloxicam (CAS 71125-38-7) and its possible salts, enantiomers,
prodrugs and derivatives; Metaproterenol (CAS 586-06-1) and its
possible salts, enantiomers, prodrugs and derivatives; Metaraminol
(CAS 54-49-9) and its possible salts, enantiomers, prodrugs and
derivatives; Metformin (CAS 657-24-9) and its possible salts,
enantiomers, prodrugs and derivatives; Methacholine (CAS 55-92-5)
and its possible salts, enantiomers, prodrugs and derivatives;
Methimazole (CAS 60-56-0) and its possible salts, enantiomers,
prodrugs and derivatives; Methylergonovine (CAS 113-42-8) and its
possible salts, enantiomers, prodrugs and derivatives; Metoprolol
(CAS 37350-58-6) and its possible salts, enantiomers, prodrugs and
derivatives; Metyrapone (CAS 54-36-4) and its possible salts,
enantiomers, prodrugs and derivatives: Miconazole (CAS 22916-47-8)
and its possible salts, enantiomers, prodrugs and derivatives;
Mifepristone (CAS 84371-65-3) and its possible salts, enantiomers,
prodrugs and derivatives; Nadolol (CAS 42200-33-9) and its possible
salts, enantiomers, prodrugs and derivatives; Naloxone (CAS
465-65-6) and its possible salts, enantiomers, prodrugs and
derivatives; Naltrexone (CAS 16590-41-3) and its possible salts,
enantiomers, prodrugs and derivatives; Norfloxacin (CAS 70458-96-7)
and its possible salts, enantiomers, prodrugs and derivatives;
Pentazocine (CAS 359-83-1) and its possible salts, enantiomers,
prodrugs and derivatives; Phenoxybenzamine (CAS 59-96-1) and its
possible salts, enantiomers, prodrugs and derivatives;
Phenylbutyrate (CAS 1821-12-1) and its possible salts, enantiomers,
prodrugs and derivatives; Pilocarpine (CAS 54-71-7) and its
possible salts, enantiomers prodrugs and derivatives; Pioglitazone
(CAS 111025-46-8) and its possible salts, enantiomers, prodrugs and
derivatives; Prazosin (CAS 19216-56-9) and its possible salts,
enantiomers, prodrugs and derivatives; Propylthiouracil (CAS
51-52-5) and its possible salts, enantiomers, prodrugs and
derivatives; Raloxifene (CAS 84449-90-1) and its possible salts,
enantiomers, prodrugs and derivatives; Rapamycin (CAS 53123-88-9)
and its possible salts, enantiomers, prodrugs and derivatives;
Rifampin (CAS 13292-46-1) and its possible salts, enantiomers,
prodrugs and derivatives; Simvastatin (CAS 79902-'63-9) and its
possible salts, enantiomers, prodrugs and derivatives;
Spironolactone (CAS 52-01-7) and its possible salts, enantiomers,
prodrugs and derivatives; Tacrolimus (CAS 104987-11-3) and its
possible salts, enantiomers, prodrugs and derivatives; Tamoxifen
(CAS 10540-29-1) and its possible salts, enantiomers, prodrugs and
derivatives; Trehalose (CAS 99-20-7) and its possible salts,
enantiomers, prodrugs and derivatives; Trilostane (CAS 13647-35-3)
and its possible salts, enantiomers, prodrugs and derivatives;
Valproic acid (CAS 99 66-1) and its possible salts, enantiomers,
prodrugs and derivatives.
[0046] The term "combination" designates a treatment wherein
several drugs are co-administered to a subject to cause a
biological effect. In a combined therapy, the drugs may be
administered together or separately, at the same time or
sequentially. Also, the drugs may be administered through different
routes and protocols.
[0047] The invention now discloses the identification and
activities of particular drug combinations which provide an
efficient treatment for CMT. More specifically, the invention
discloses novel ternary combinations which provide a significant
effect in vitro and in vivo on CMT or related disorders.
[0048] In this regard, the invention relates to a composition
comprising Baclofen, Sorbitol and a compound selected from
Pilocarpine, Methimazole, Mifepristone, Naltrexone, Rapamycine,
Flurbiprofen and Ketoprofen, salts, enantiomers, racemates, or
prodrugs thereof.
[0049] More preferably, the invention relates to a composition
comprising Baclofen. Sorbitol and a compound selected from
Pilocarpine, Methimazole, Mifepristone, Naltrexone, and
Ketoprofen.
[0050] In the most preferred embodiment, the present invention
relates to a composition comprising Naltrexone, Baclofen and
Sorbitol, for simultaneous, separate or sequential administration
to a mammalian subject.
[0051] Preferably, in the above compositions, Sorbitol is
D-Sorbitol and Baclofen is RS-Baclofen or S-Baclofen, more
preferably RS-baclofen.
[0052] Another preferred object of the invention relates to a
composition comprising: [0053] (a) rapamycin, [0054] (b)
mifepristone or naltrexone, and [0055] (c) a PMP22 modulator,
[0056] for simultaneous, separate or sequential administration to a
mammalian subject.
[0057] Another preferred object of this invention is a composition
comprising: [0058] (a) rapamycin, [0059] (b) mifepristone, and
[0060] (c) a PMP22 modulator, [0061] for simultaneous, separate or
sequential administration to a mammalian subject.
[0062] The PMP22 modulator may be any compound that modulates PMP22
pathway in a cell and essentially causes or contributes to
normalization of myelin organization and/or inhibition of neuron
loss. The PMP22 modulator may be selected from Acetazolamide,
Albuterol, Amiloride, Aminoglutethimide, Amiodarone, Aztreonam,
Baclofen, Balsalazide, Betaine, Bethanechol, Bicalutamide,
Bromocriptine, Bumetanide, Buspirone, Carbachol, Carbamazepine,
Carbimazole, Cevimeline, Ciprofloxacin, Clonidine, Curcumin,
Cyclosporine A, Diazepam, Diclofenac, Dinoprostone, Disulfiram,
D-Sorbitol, Dutasteride, Estradiol, Exemestane, Felbamate,
Fenofibrate, Finasteride, Flumazenil, Flunitrazepam, Flurbiprofen,
Furosemide, Gabapentin, Galantamine, Haloperidol, Ibuprofen,
Isoproterenol, Ketoconazole, Ketoprofen, L-carnitine, Liothyronine
(T3), Lithium, Losartan, Loxapine, Meloxicam, Metaproterenol,
Metaraminol, Metformin, Methacholine, Methimazole,
Methylergonovine, Metoprolol, Metyrapone, Miconazole, Mifepristone,
Nadolol, Naloxone, Naltrexone; Norfloxacin, Pentazocine,
Phenoxybenzamine, Phenylbutyrate, Pilocarpine, Pioglitazone,
Prazosin, Propylthiouracil, Raloxifene, Rapamycin, Rifampin,
Simvastatin, Spironolactone, Tacrolimus, Tamoxifen, Trehalose,
Trilostane, Valproic acid salts or prodrugs thereof.
[0063] In a preferred embodiment, compound (c) is selected from
pilocarpin, methimazole and baclofen. In this regard, a most
preferred composition of this invention comprises: [0064] (a)
rapamycin, [0065] (b) mifepristone, and [0066] (c) a compound
selected from pilocarpin, methimazole and baclofen, for
simultaneous, separate or sequential administration to a mammalian
subject.
[0067] Specific examples of such compositions include compositions
comprising: [0068] Rapamycin; mifepristone and pilocarpin; [0069]
Rapamycin; mifepristone and Baclofen; [0070] Rapamycin;
mifepristone and methimazole; or [0071] Rapamycin; Naltrexone and
methimazole.
[0072] The experimental section shows these particular drug
combinations are able to efficiently correct PMP22 expression in
vitro, to restore normal myelination and neuron integrity, and thus
to ameliorate CMT in animals in vivo. The results also show these
combinations can protect animals from chemotherapy-induced
neuropathy. As a result, these compositions may be used to prevent
or reduce chemotherapy-induced neuropathy, thereby allowing
patients to receive chemotherapy for longer periods.
[0073] Another object of this invention is a composition comprising
Naltrexone, Baclofen and a further distinct PMP22 inhibitor as
defined above.
[0074] A further object of this invention is a composition as
disclosed above further comprising one or several pharmaceutically
acceptable excipients or carriers (i.e., a pharmaceutical
composition).
[0075] Another object of the present invention relates to a
composition as disclosed above for treating CMT or a related
disorder.
[0076] A further object of this invention relates to the use of a
combination of compounds as disclosed above for the manufacture of
a medicament for the treatment of CMT or a related disorder.
[0077] A further object of this invention is a method for treating
CMT or a related disorder, the method comprising administering to a
subject in need thereof an effective amount of a composition as
defined above.
[0078] A further object of this invention is a method of preparing
a pharmaceutical composition, the method comprising mixing the
above compounds in an appropriate excipient or carrier.
[0079] A more specific object of this invention is a method of
treating CMT1a in a subject, the method comprising administering to
the subject in need thereof an effective amount of a compound or
combination of compounds as disclosed above.
[0080] A further specific object of this invention is a method of
treating toxic neuropathy in a subject, the method comprising
administering to the subject in need thereof an effective amount of
a compound or combination of compounds as disclosed above.
[0081] A further specific object of this invention is a method of
treating ALS in a subject, the method comprising, administering to
the subject in need thereof an effective amount of a compound or
combination of compounds as disclosed above.
[0082] Therapy according to the invention may be performed as drug
combination and/or in conjunction with any other therapy. It and
may be provided at home, the doctor's office, a clinic, a
hospital's outpatient department, or a hospital, so that the doctor
can observe the therapy's effects closely and make any adjustments
that are needed.
[0083] The duration of the therapy depends on the stage of the
disease being treated, the age and condition of the patient, and
how the patient responds to the treatment.
[0084] Additionally, a person having a greater risk of developing
an additional neuropathic disorder (e.g., a person who is
genetically predisposed to or have, for example, diabetes, or is
being under treatment for an oncological condition, etc.) may
receive prophylactic treatment to alleviate or to delay eventual
neuropathic response.
[0085] The dosage, frequency and mode of administration of each
component of the combination can be controlled independently. For
example, one drug may be administered orally while the second drug
may be administered intramuscularly. Combination therapy may be
given in on-and-off cycles that include rest periods so that the
patient's body has a chance to recovery from any as yet unforeseen
side-effects. The drugs may also be formulated together such that
one administration delivers both drugs.
[0086] Formulation of Pharmaceutical Compositions
[0087] The administration of each drug of the combination may be by
any suitable means that results in a concentration of the drug
that, combined with the other component, is able to ameliorate the
patient condition (which may be determined e.g., in vitro by an
effect on elevated expression of PMP22 upon reaching the peripheral
nerves).
[0088] While it is possible for the active ingredients of the
combination to be administered as the pure chemical it is
preferable to present them as a pharmaceutical composition, also
referred to in this context as pharmaceutical formulation. Possible
compositions include those suitable for oral, rectal, topical
(including transdermal, buccal and sublingual), or parenteral
(including subcutaneous, intramuscular, intravenous and
intradermal) administration.
[0089] More commonly these pharmaceutical formulations are
prescribed to the patient in "patient packs" containing a number
dosing units or other means for administration of metered unit
doses for use during a distinct treatment period in a single
package, usually a blister pack. Patient packs have an advantage
over traditional prescriptions, where a pharmacist divides a
patient's supply of a pharmaceutical from a bulk supply, in that
the patient always has access to the package insert contained in
the patient pack, normally missing in traditional prescriptions.
The inclusion of a package insert has been shown to improve patient
compliance with the physician's instructions. Thus, the invention
further includes a pharmaceutical formulation, as herein before
described, in combination with packaging material suitable for said
formulations. In such a patient pack the intended use of a
formulation for the combination treatment can be inferred by
instructions, facilities, provisions, adaptations and/or other
means to help using the formulation most suitably for the
treatment. Such measures make a patient pack specifically suitable
for and adapted for use for treatment with the combination of the
present invention.
[0090] The drug may be contained in any appropriate amount in any
suitable carrier substance, and is may be present in an amount of
1-99% by weight of the total weight of the composition. The
composition may be provided in a dosage form that is suitable for
the oral, parenteral (e.g., intravenously, intramuscularly),
rectal, cutaneous, nasal, vaginal, inhalant, skin (patch), or
ocular administration route. Thus, the composition may be in the
form of, e.g., tablets, capsules, pills, powders, granulates,
suspensions, emulsions, solutions, gels including hydrogels,
pastes, ointments, creams, plasters, drenches, osmotic delivery
devices, suppositories, enemas, injectables, implants, sprays, or
aerosols.
[0091] The pharmaceutical compositions may be formulated according
to conventional pharmaceutical practice (see, e.g., Remington: The
Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro,
Lippincott Williams & Wilkins, 2000 and Encyclopedia of
Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan,
1988-1999, Marcel Dekker, New York).
[0092] Pharmaceutical compositions according to the invention may
be formulated to release the active drug substantially immediately
upon administration or at any predetermined time or time period
after administration.
[0093] The controlled release formulations include (i) formulations
that create a substantially constant concentration of the drug
within the body over an extended period of time (ii) formulations
that after a predetermined lag time create a substantially constant
concentration of the drug within the body over an extended period
of time (iii) formulations that sustain drug action during a
predetermined time period by maintaining a relatively, constant,
effective drug level in the body with concomitant minimization of
undesirable side effects associated with fluctuations in the plasma
level of the active drug substance; (iv) formulations that localize
drug action by e.g., spatial placement of a controlled release
composition adjacent to or in the diseased tissue or organ; and (v)
formulations that target drug action by using carriers or chemical
derivatives to deliver the drug to a particular target cell type.
[0094] Administration of drugs in the form of a controlled release
formulation is especially preferred in cases in which the drug in
combination, has (i) a narrow therapeutic index (i.e., the
difference between the plasma concentration leading to harmful side
effects or toxic reactions and the plasma concentration leading to
a therapeutic effect is small; in general, the therapeutic index,
TI, is defined as the ratio of median lethal dose (LD50) to median
effective dose (ED50)); (ii) a narrow absorption window in the
gastro-intestinal tract; or (iii) a very short biological half-life
so that frequent dosing during a day is required in order to
sustain the plasma level at a therapeutic level.
[0095] Any of a number of strategies can be pursued in order to
obtain controlled release in which the rate of release outweighs
the rate of metabolism of the drug in question. Controlled release
may be obtained by appropriate selection of various formulation
parameters and ingredients, including, e.g., various types of
controlled release compositions and coatings. Thus, the drug is
formulated with appropriate excipients into a pharmaceutical
composition that, upon administration, releases the drug in a
controlled manner (single or multiple unit tablet or capsule
compositions, oil solutions, suspensions, emulsions, microcapsules,
microspheres, nanoparticles, patches, and liposomes).
[0096] Solid Dosage Forms for Oral Use
[0097] Formulations for oral use include tablets containing the
active ingredient(s) in a mixture with non-toxic pharmaceutically
acceptable excipients. These excipients may be, for example, inert
diluents or fillers (e.g., sucrose, microcrystalline cellulose,
starches including potato starch, calcium carbonate, sodium
chloride, calcium phosphate, calcium sulfate, or sodium phosphate);
granulating and disintegrating agents (e.g., cellulose derivatives
including microcrystalline cellulose, starches including potato
starch, croscarmellose sodium, alginates, or alginic acid); binding
agents (e.g., acacia, alginic acid, sodium alginate, gelatin,
starch, pregelatinized starch, microcrystalline cellulose,
carboxymethylcellulose sodium, methylcellulose, hydroxypropyl
methylcellulose ethylcellulose, polyvinylpyrrolidone, or
polyethylene glycol); and lubricating agents, glidants, and
antiadhesives e.g., stearic acid, silicas, or talc). Other
pharmaceutically acceptable excipients can be colorants, flavoring
agents, plasticizers, humectants, buffering agents, and the like.
[0098] The tablets may be uncoated or they may be coated by known
techniques, optionally to delay disintegration and absorption in
the gastrointestinal tract and thereby providing a sustained action
over a longer period. The coating may be adapted to release the
active drug substance in a predetermined pattern (e.g., in order to
achieve a controlled release formulation) or it may by adapted not
to release the active drug substance until after passage of the
stomach (enteric coating). The coating may be a sugar coating, a
film coating (e.g., based on hydroxypropyl methylcellulose,
methylcellulose, methyl hydroxyethylcellulose,
hydroxypropylcellulose, carboxymethylcellulose, acrylate
copolymers, polyethylene glycols and/or polyvinylpyrrolidone), or
an enteric coating (e.g., based on methacrylic acid copolymer,
cellulose acetate phthalate, hydroxypropyl methylcellulose
phthalate, hydroxypropyl methylcellulose acetate succinate,
polyvinyl acetate phthalate, shellac, and/or ethylcellulose). A
time delay material such as e.g., glyceryl monostearate or glyceryl
distearate may be employed.
[0099] The solid tablet compositions may include a coating adapted
to protect the composition from unwanted chemical changes, (e.g.,
chemical degradation prior to the release of the active drug
substance). The coating may be applied on the solid dosage form in
a similar manner as that described in Encyclopedia of
Pharmaceutical Technology.
[0100] The drugs may be mixed together in the tablet, or may be
partitioned. For example, a first drug is contained on the inside
of the tablet, and a second drug is on the outside, such that a
substantial portion of the second drug is released prior to the
release of the first drug.
[0101] Formulations for oral use may also be presented as chewable
tablets, or as hard gelatin capsules wherein the active ingredient
is mixed with an inert solid diluent (e.g., potato starch,
microcrystalline cellulose, calcium carbonate, calcium phosphate or
kaolin), or as soft gelatin capsules wherein the active ingredient
is mixed with water or an oil medium, for example, liquid paraffin,
or olive oil. Powders and granulates may be prepared using the
ingredients mentioned above under tablets and capsules in a
conventional manner.
[0102] Controlled release compositions for oral use may, e.g., be
constructed to release the active drug by controlling the
dissolution and/or the diffusion of the active drug substance.
[0103] Dissolution or diffusion controlled release can be achieved
by appropriate coating of a tablet, capsule, pellet, or granulate
formulation of drugs, or by incorporating the drug into an
appropriate matrix. A controlled release coating may include one or
more of the coating substances mentioned above and/or, e.g.,
shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl
alcohol, glyceryl monostearate, glyceryl distearate, glycerol
palmitostearate, ethylcellulose, acrylic resins, dl-polylactic
acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl
acetate, vinyl pyrrolidone, polyethylene, polymethacrylate,
methylmethacryate, 2-hydroxymethacrylate, methacrylate hydrogels,
1,3 butylene glycol, ethylene glycol methacrylate, and/or
polyethylene glycols. In a controlled release matrix formulation,
the matrix material may also include, e.g., hydrated
metylcellulose, carnauba wax and stearyl alcohol, carbopol 934,
silicone, glyceryl tristearate, methyl acrylate-methyl
methacrylate, polyvinyl chloride, polyethylene, and/or halogenated
fluorocarbon.
[0104] A controlled release composition containing one or more of
the drugs of the claimed combinations may also be in the form of a
buoyant tablet or capsule (i.e., a tablet or capsule that, upon
oral administration, floats on top of the gastric content for a
certain period of time). A buoyant tablet formulation of the
drug(s) can be prepared by granulating a mixture of the drug(s)
with excipients and 20-75% w/w of hydrocolloids, such as
hydroxyethylcellulose, hydroxypropylcellulose, or
hydroxypropylmethylcellulose. The obtained granules can then be
compressed into tablets. On contact with the gastric juice, the
tablet forms a substantially water impermeable gel barrier around
its surface. This gel barrier takes part in maintaining a density
of less than one thereby allowing the tablet to remain buoyant in
the gastric juice.
[0105] Liquids for Oral Administration
[0106] Powders, dispersible powders, or granules suitable for
preparation of an aqueous suspension by addition of water are
convenient dosage forms for oral administration. Formulation as a
suspension provides the active ingredient in a mixture with a
dispersing or wetting agent, suspending agent, and one or more
preservatives. Suitable suspending agents are, for example, sodium
carboxymethylcellulose, methylcellulose, sodium alginate, and the
like.
[0107] Parenteral Compositions
[0108] The pharmaceutical composition may also be administered
parenterally by injection, infusion, or implantation (intravenous,
intramuscular, subcutaneous, or the like) in dosage forms,
formulations, or via suitable delivery devices or implants
containing conventional, non-toxic pharmaceutically acceptable
carriers and adjuvants. The formulation and preparation of such
compositions are well known to those skilled in the art of
pharmaceutical formulation.
[0109] Compositions for parenteral use may be provided in unit
dosage forms (e.g., in single-dose ampoules), or in vials
containing several doses and in which a suitable preservative may
be added (see below). The composition may be in form of a solution,
a suspension, an emulsion, an infusion device, or a delivery device
for implantation or it may be presented as a dry powder to be
reconstituted with water or another suitable vehicle before use.
Apart from the active drug(s), the composition may include suitable
parenterally acceptable carriers and/or excipients. The active
drug(s) may be incorporated, into microspheres, microcapsules,
nanoparticles, liposomes, or the like for controlled release. The
composition may include suspending, solubilizing, stabilizing,
pH-adjusting agents, and/or dispersing agents. [0110] The
pharmaceutical compositions according to the invention may be in
the form suitable for sterile injection. To prepare such a
composition, the suitable active drug(s) are dissolved or suspended
in a parenterally acceptable liquid vehicle. Among acceptable
vehicles and solvents that may be employed are water, water
adjusted to a suitable pH by addition of an appropriate amount of
hydrochloric acid, sodium hydroxide or a suitable buffer,
1,3-butanediol, Ringer's solution, and isotonic sodium chloride
solution. The aqueous formulation may also contain one or more
preservatives methyl, ethyl or n-propyl p-phdroxybenzaoate). In
cases where one of the drugs is only sparingly or slightly soluble
in water, a dissolution enhancing or solubilizing agent can be
added, or the solvent may include 10-60% w/w of propylene glycol or
the like. [0111] Controlled release parenteral compositions may be
in form of aqueous suspensions, microspheres, microcapsules,
magnetic microspheres, oil solutions, oil suspensions, or
emulsions. Alternatively, the active drug(s) may be incorporated in
biocompatible carriers, liposomes, nanoparticles, implants, or
infusion devices. Materials for use in the preparation of
microspheres and/or microcapsules are, e.g.,
biodegradable/bioerodible polymers such as polygalactin,
poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutamnine).
Biocompatible carriers that may be used when formulating a
controlled release parenteral formulation are carbohydrates (e.g.,
dextrans), proteins (e.g., albumin), lipoproteins, or antibodies.
Materials for use in implants can be non-biodegradable (e.g.,
polydimethyl siloxane) in biodegradable (e.g., poly(caprolactone),
poly(glycolic acid) or poly(ortho esters)).
[0112] Rectal Compositions
[0113] For rectal application, suitable dosage forms for a
composition include suppositories (emulsion or suspension type),
and metal gelatin capsules (solutions or suspensions). In a typical
suppository formulation, the active drug(s) are combined with an
appropriate pharmaceutically acceptable suppository base such as
cocoa butter, esterified fatty acids, glycerinated gelatin, and
various water-soluble or dispersible bases like polyethylene
glycols. Various additives, enhancers, or surfactants may be
incorporated.
[0114] Percutaneous and Topical Compositions [0115] The
pharmaceutical compositions may also be administered topically on
the skin for percutaneous absorption in dosage forms or
formulations containing conventionally non-toxic pharmaceutical
acceptable carriers and excipients including microspheres and
liposomes. The formulations include creams, ointments, lotions,
liniments, gels, hydrogels, solutions, suspensions, sticks, sprays,
pastes, plasters, and other kinds of transdermal drug delivery
systems. The pharmaceutically acceptable carriers or excipients may
include emulsifying agents, antioxidants, buffering agents,
preservatives, humectants, penetration enhancers, chelating agents,
gel-forming agents, ointment bases, perfumes, and skin protective
agents.
[0116] The Emulsifying Agents May Be Naturally Occurring Gums
(e.g., Gum Acacia or Gum Tragacanth)
[0117] The preservatives, humectants, penetration enhancers may be
parabens, such as methyl or propyl p-hydroxybenzoate, and
benzalkonium chloride, glycerin, propylene glycol, urea etc.
[0118] The pharmaceutical compositions described above for topical
administration on the skin may also be used in connection with
topical administration onto or close to the part of the body that
is to be treated. The compositions may be adapted for direct
application or for application by means of special drug delivery
devices such as dressings or alternatively plasters, pads, sponges,
strips, or other forms of suitable flexible material.
[0119] Dosages and Duration of the Treatment
[0120] It will be appreciated that the drugs of the combination may
be administered concomitantly, either in the same or different
pharmaceutical formulation or sequentially. If there is sequential
administration, the delay in administering one of the active
ingredients should not be such as to lose the benefit of the
efficacious effect of the combination of the active ingredients. A
minimum requirement for a combination according to this description
is that the combination should be intended for combined use with
the benefit of the efficacious effect of the combination of the
active ingredients. The intended use of a combination can be
inferred by facilities, provisions, adaptations and/or other means
to help using the combination according to the invention.
[0121] Therapeutically effective amounts of the drugs that are
subject of this invention can be used together for the preparation
of a medicament useful for reducing the effect of increased
expression of PMP22 gene; restoration of normal myelination and
nerve integrity, preventing or reducing the risk of developing CMT
disease, halting or slowing the progression of CMT disease once it
has become clinically manifest, and preventing or reducing the risk
of a first or subsequent occurrence of an neuropathic event.
[0122] Although the active drugs of the present invention may be
administered in divided doses, for example two or three times
daily, a single daily dose of each drug in the combination is
preferred, with a single daily dose of all drugs in a single
pharmaceutical composition (unit dosage form) being most
preferred.
[0123] Administration can be one to several time daily for several
days to several years, and may even be for the life of the patient.
Chronic or at least periodically repeated long-term administration
will be indicated in most cases. [0124] The term "unit dosage form"
refers to physically discrete units (such as capsules, tablets, or
loaded syringe cylinders) suitable as unitary dosages for human
subjects, each unit containing a predetermined quantity of active
material or materials calculated to produce the desired therapeutic
effect, in association with the required pharmaceutical
carrier.
[0125] The amount of each drug in the combination preferred for a
unit dosage will depend upon several factors including the
administration method, the body weight and the age of the patient,
the severity of the neuropathic damage caused by CMT disease or
risk of potential side effects considering the general health
status of the person to be treated.
[0126] Additionally, pharmacogenomic (the effect of genotype on the
pharmacokinetic, pharmacodynamic or efficacy profile of a
therapeutic) information about a particular patient may affect the
dosage used.
[0127] Except when responding to especially impairing CMT disease
cases when higher dosages may be required, or when treating
children when lower dosages should be chosen, the preferred dosage
of each drug in the combination will usually lie within the range
of doses not above the usually prescribed for long-term maintenance
treatment or proven to be safe in the large phase 3 clinical
studies.
[0128] For example, [0129] for Rapamycin, from about 1 to about 100
.mu.g/kg per day, typically from 1 to 50 .mu.g/kg, for instance
between 5 and 30 .mu.g/kg/day. [0130] for Mifepristone, from about
1 to about 300 .mu.g/kg per day, typically from 10 to 200 .mu.g/kg,
for instance between 10 and 80 .mu.g/kg/day. [0131] for Naltrexone,
from about 1 to about 100 .mu.g/kg per day, typically from 1 to 50
.mu.g/Kg, for instance between 1 and 20 .mu.g/kg/day. [0132] for
Pilocarpin, from about 1 to about 100 .mu.g/kg per day, typically
from 1 to 50 .mu.g/Kg, for instance between 1 and 20 .mu.g/kg/day.
[0133] for Baclofen, from about 1 to about 300 .mu.g/kg per day,
typically from 10 to 200 .mu.g/kg, for instance between 20 and 100
.mu.g/kg/day. [0134] for Methimazole, from about 1 to about 100
.mu.g/kg per day, typically from 1 to 50 .mu.q/kg, for instance
between 1 and 20 .mu.g/kg/day.
[0135] The most preferred dosage will correspond to amounts from 1%
up to 10% of those usually prescribed for long -term maintenance
treatment.
[0136] It will be understood that the amount of the drug actually
administered will be determined by a physician. in the light of the
relevant circumstances including the condition or conditions to be
treated, the exact composition to be administered, the age, weight,
and response of the individual patient, the severity of the
patient's symptoms, and the chosen route of administration.
Therefore, the above dosage ranges are intended to provide general
guidance and support for the teachings herein, but are not intended
to limit the scope of the invention.
[0137] The following examples are given for purposes of
illustration and not by way of limitation.
EXAMPLES
[0138] A. Preparation of Drug Combinations
[0139] The following drug combinations were prepared:
TABLE-US-00001 Molecule dose Mix1 Sorbitol 2.1 mg/kg/day S-Baclofen
(-) 60 .mu.g/kg/day Naltrexone 7 .mu.g/kg/day
TABLE-US-00002 Molecule dose Mix2 Rapamycin 15 .mu.g/kg/day
Mifepristone 40 .mu.g/kg/day
TABLE-US-00003 Molecule dose Mix3 Rapamycin 15 .mu.g/kg/day
Mifepristone 40 .mu.g/kg/day Pilocarpin 7 .mu.g/kg/day
TABLE-US-00004 Molecule dose Mix4 Rapamycin 15 .mu.g/kg/day
Mifepristone 40 .mu.g/kg/day Baclofen 60 .mu.g/kg/day
TABLE-US-00005 Molecule dose Mix5 Rapamycin 15 .mu.g/kg/day
Mifepristone 40 .mu.g/kg/day Methimazole 4.2 .mu.g/kg/day
TABLE-US-00006 Molecule dose Mix6 Rapamycin 15 .mu.g/kg/day
Naltrexone 7 .mu.g/kg/day Methimazole 4.2 .mu.g/kg/day
TABLE-US-00007 Molecule dose 1 dose 2 dose 3 Mix7 Sorbitol 10.5
mg/kg/day 2.1 mg/kg/day 1.05 mg/kg/day (RS) 0.3 mg/kg/day 60
.mu.g/kg/day 30 .mu.g/kg/day Baclofen Naltrex- 35 .mu.g/kg/day 7
.mu.g/kg/day 3.5 .mu.g/kg/day one
[0140] B. In Vitro Experiments
1. PMP22 Expression Assays on Schwann Cells Treated with Mix1-6
[0141] 1.1 Cell Culture
[0142] 1.1.1: Commercially Available Rat Primary Schwann Cells
[0143] Vials of rat Schwann cells (SC) primary culture (Sciencell #
R1700) are defrost and seeded at the density of 10 000 cells/cm2in
"Sciencell Schwann cell medium" (basal medium from Sciencell #
R1701) in poly-L-lysine pre-coated 75 cm.sup.2 flasks. The culture
medium is composed of basal medium, 5% Fetal Bovine Serum
(3H-Biomedical AB #1701-0025), 1% Schwann cell growth supplement
(3H Biomedical AB #1701-1752), 1% Gentamicin (Sigma #G1397) and 10
.mu.M of Forskolin (Sigma # F6886) to promote their proliferation.
[0144] After reaching confluency (4 to 10 days depending on cell
batch), Schwann cells are purified by gentle agitation or b thy1.1
immunopanning that allow SC isolation from adherent fibroblasts, to
produce cultures that are at least 95% pure. SC are then counted
(Tryptan blue method) and seeded in poly-L-lysine pre-coated 75
cm.sup.2 flask in the same SC medium. At confluency, cells are
rinsed, trypsinized (trypsin-EDTA 1.times. diluted from Invitrogen
#1540054), diluted in PBS without calcium and magnesium) counted
and platted in 12 well-dishes (140 000 cells/well) in Sciencell
Schwann cell medium with 5% of FBS, 1% of cell growth supplement
(CGS), 40 .mu.g/ml of gentamicin and 4 .mu.M Forskolin.
[0145] 1.1.2 Custom -Made Rat Primary Schwann Cells [0146] Primary
Schwann cell cultures (SC) are established from Sprague-Dawley
newborn rats (between P0 and P2) sciatic nerves. All newborn rats
are sacrificed and isolated in a Petri dish. Dissection is
performed under sterile conditions. [0147] The dorsal skin is
removed from the hind paw and the lower torso. The sciatic nerve is
isolated and transferred to a culture dish containing ice-cold
Leibovitz (L15, Invitogen #11415) supplemented with 1%
penicillin/streptomycin solution (50 UI/ml and 50 .mu.g/ml,
respectively; Invitrogen #15070) and 1% of bovine serum albumin
(BSA, Sigma A6003). Both nerves per rats are transferred in a 15 ml
tube containing ice-cold L15. The L15 medium is then removed and
replaced by 2.4 ml of DMEM (Invitrogen #21969035) with 10 mg/ml of
collagenase (Sigma #A6003). Nerves are incubated in this medium for
30 minutes at 37.degree. C. The medium is then removed and both
nerves are dissociated by trypsin (10% trypsin EDTA 10.times.,
Invitrogen #15400054) diluted in PBS without calcium and magnesium
(Invitrogen # 2007-03) for 20 min at 37.degree. C. The reaction is
stopped by addition of DMEM containing DNase I grade II (0.1 mg/ml
Roche diagnostic #104159) and foetal calf serum (FCS 10%,
Invitrogen #10270). The cell suspension was triturated with a 10 ml
pipette and passed through a filter in a 50 ml tube (Swinnex 13 mm
filter units. Millipore, with 20 .mu.m nylon-mesh filters, Fisher).
The cell suspension is centrifuged, at 350 g for 10 min at room
temperature (RT) and the pellets are suspended in DMEM with 1.0%
FCS and 1% penicillin/streptomycin. Cells are counted (Tryptan blue
method) and seeded in Falcon 100 mm Primaria tissue culture plates
at the density of 5.10.sup.5 to 10.sup.6 cells/dish. [0148] After
one day of culture, the medium is changed with DMEM, 10% FCS, 1%
penicillin/streptomcin and 10 .mu.M of cytosine
b-D-arabinofuranoside (Sigma #C1768). 48 hrs later, medium is
eliminated and cells are washed three times with DMEM. The SC
growth medium is then added, composed of DMEM, 10% FCS, 1%
penicillin/streptomycin, 2 .mu.M of Forskolin (Sigma #F6886), 10
.mu.g/ml of bovine pituitary extract (PEX, Invitrogen #3028). The
medium is replaced every 2-3 days. [0149] After 8 days of culture
(4 to 10 days depending on cell batches), Schwann cells reach
confluency and the culture, containing a large amount of
contaminating fibroblasts, is purified by the thy1.1 immunopanning
method. After this purification, cells are suspended in growth
medium at 10 000 cells/cm2 in poly-L-lysine pre-coated 75 cm.sup.2
flasks. Once they reach confluency, cells are rinsed, trypsinized
(trypsin-EDTA), counted and platted in 12 well-dishes (100 000
cells/well).
[0150] 1.1.3 Drug Incubation [0151] After cells being platted in
12well-dishes, the medium is replaced by a defined medium
consisting in a mix of DMEM -F12 (Invitrogen # 21331020)
complemented by 1% of N2 supplement (Invitrogen # 17502), I%
L-Glutamine (Invitrogen #25030024) 2.5% FBS (Sciencell #0025), 0.02
.mu.g/ml of corticosterone (Sigma # C2505), 4 .mu.M Forskolin and
50 .mu.g/ml of gentamycin. Growth factors are not added to this
medium, to promote SC differentiation [0152] 24 hours later, the
medium is replaced by a defined medium (DMEM-F12) complemented with
1% Insulin-Transferrin-Selenium--X (ITS, Invitrogen # 51300), 16
.mu.g/ml of Putrescine (Sigma # P5780), 0.02 .mu.g/ml of
corticosterone and 50 .mu.g/ml of gentamicin. At this step, neither
progesterone nor forskolin are present in the medium. [0153] One
day later, Schwann cells are stimulated by combinations of drugs
during 24 hrs (3 wells/condition). The preparation of each compound
is performed just prior to its addition to the cell culture medium.
[0154] Drugs are added to a defined medium composed of DMEM-F12,
with. 1% Insulin-Transferrin-Selenium--X (ITS, Invitrogen # 51300),
16 .mu.g/ml of Putrescine, 0.02 .mu.g/ml of corticosterone, 10 nM
Progesterone and 50 .mu.g/ml of gentamicin. The absence of
Forskolin during drug stimulation avoids adenylate cyclase
saturation.
[0155] 1.2. Schwann Cells Purification by Thy1.1 Immunopanning
[0156] To prevent fibroblast culture contamination, Schwann cells
are purified using the clone Thy1.1 (ATCC TIB-103.TM.)
immunopanning protocole. [0157] Antibody pre-coated 100 mm bacteria
Petri dishes are prepared as follows: these dishes are washed three
times with PBS and treated by 20 ml of Tris HCl solution 50 mM, pH
9.5, with 10 .mu.g/ml of goat Anti-Mouse IgM MU antibody (Jackson
ImmunoResearch #115-005-020) overnight at 4.degree. C.; then rinsed
3 times with PBS and treated by a solution of PBS with 0.02% of BSA
and supernatant obtained from T11D7e2 hybridoma culture (ATCC
#TIB-103) containing the Thy1.1 IgM antibody for 2 hours at room
temperature. Finally, the plates are washed three times with PBS
before the cell suspensions are added. [0158] SC are detached with
trypsin EDTA. As soon as the majority of cells are in suspension,
the trypsin is neutralized with DMEM-10% FBS and the cells are
centrifuged. The pellet of dissociated cells is resuspended in 15
ml of medium with 0.02% BSA at the density of 0.66.times.10.sup.6
cells per ml (maximum) and transferred to Petri dish (about 6.6
million of cells/10 ml/dish of 100 mm). [0159] The cell suspension
is incubated in the Thy 1.1 coated Petri dish during 45 min at
37.degree. C. with gentle agitation every 15 mm to prevent
non-specific binding. The majority of fibroblast cells expressing
Thy1.1. adhere on the dish. At the end of the incubation, the cell
suspension is recovered and centrifuged. This cell suspension
contains in theory only Schwann cells. Cells are centrifuged and
cell pellet is suspended in growth medium with 10 .mu.M of
Forskolin at 16 000 cells/cm.sup.2 in T75 cm.sup.2 flask
Poly-L-Lysine treated.
[0160] 1.3 Quantitative Reverse Transcriptase Polymerase Chain
Reaction (Q-RT-PCR) [0161] Quantitative RT-PCR is used to compare
the levels of PMP22 mRNA after drug stimulation, relative with
housekeeping Ribosomal L13A mRNA in rat Schwann cell primary
culture. [0162] After rinsing with cold sterilized PBS, total RNAs
from each cell sample are extracted and purified from SC using the
Qiagen RNeasy micro kit (Qiagen #74004). Nucleic acids are
quantified by Nanodrop spectrophotometer using 1 .mu.l of RNA
sample. The RNA integrity is determined through a BioAnalyzer
(Agilent) apparatus. [0163] RNAs are reverse-transcribed into cDNA
according to standard protocol. cDNA templates for PCR
amplification are synthesized from 200 ng of total RNA using
SuperScript II reverse-transcriptase (Invitrogen # 18064-014) for
60 min at 42.degree. C. in the presence of oligo(dT), in a final
volume of 20 .mu.l. [0164] cDNAs are subjected to PCR amplification
using the <<LightCycler.RTM.480>> system (Roche
Molecular Systems Inc.) Each cDNA are diluted five times before
being used for PCR amplification. 2.5 .mu.l of this cDNAs enters
the PCR reaction solution (final volume of 10 .mu.l). Preliminary
experiments ensured that quantitation was done in the exponential
phase of the amplification process for both sequences and that
expression of the reference gene was uniform in the different
culture conditions. [0165] PCR reaction is performed by
amplification of 500 nM of forward primer of rat PMP22
(NM_0170371), 5-GGAAACGCGAATGAGGC-3 (SEQ ID NO: 1), and 500 nM of
reverse primer 5-GTTCTGTTTGGTTTGGCTT-3 (SEQ ID NO: 2)
(amplification of 148-bp). A 152-bp fragment of the RPL13A
ribosomal (NM_173340) RNA is amplified in parallel in separate
reactions for normalization of the results by using a 500 nM of
forward primer 5-CTGCCCTCAAGGTTGTG-3 (SEQ ID NO 3), and a 500 nM of
reverse primer 5-CTTCTTCTTCCGGTAATGGAT-3 (SEQ ID NO: 4). [0166] We
used FRET chemistry to perform RT-Q-PCR analysis. FRET probes are
composed of 0.3 .mu.M of Pmp22-FL-5-GCTCTGAGCGTGCATAGGGTAC (SEQ ID
NO: 5) or Rp113A-FL-5-TCGGGTGGAAGTACCAGCC (SEQ ID NO: 6), labelled
at their 3' end with a donor fluorophore dye (Fluorescein). 0.15
.mu.M Red640 probes are defined as follows:
Pmp22-red-5'-AGGGAGGGAGGAAGGAAACCAGAAA (SEQ ID NO: 7) or
Rp113A-red-5'-TGACAGCTACTCTGGAGGAGAAACGGAA (SEQ ID NO: 8), labelled
at their 5' end with an acceptor fluorophore dye (Rhodamine Red
640). [0167] Each PCR reaction contained 2.5 .mu.l cDNA template in
a final volume of 10 .mu.l of master mix kit (Roche #04-887301001).
[0168] The following PCR conditions are used: 10 sec at 95.degree.
C., 10 sec at 63.degree..degree.C. and 12 sec at 72.degree. C. and
30 sec at 40.degree. C. (Forty amplification cycles). The relative
levels of PMP22 gene expression is measured by determining the
ratio between the products generated from the target gene PMP22 and
the endogenous internal standard RPL13A.
[0169] 1.4PMP22 Protein Expression Analysis by Flow Cytometry
(FACS) [0170] 8 hrs, 24 hrs and 48 hrs after drugs incubation,
supernatants are recovered, centrifuged and frozen. SC are detached
with trypsin-EDTA. As soon as the majority of cells are in
suspension, the trypsin is neutralised using DMEM with 10% FCS.
[0171] Supernatants with cells are recovered and centrifuged. The
pellets of cells are transferred in micro tubes, washed in PBS once
and fixed with a specific solution (AbCys #Reagent A BUF09B). 10
minutes later, cells are rinsed once with PBS and kept at 4.degree.
C. [0172] Five days after cell fixation, all cell preparations with
different incubation times are labelled using the following
protocol. [0173] Cells are centrifuged at 7000 rpm for 5 minutes
and the pellets are suspended in a solution of permeabilization
(AbCys #Reagent B BUF09B) and labelled with primary PMP22 antibody
(Abeam #ab61220, 1/50) for 1 hr room at temperature. Cells are then
centrifuged at 7000 rpm for 5 minutes and cell pellets are rinsed
once in PBS. A secondary antibody is added, coupled to Alexa Fluor
48 8(goat anti-rabbit IgG, Molecular Probes #A11008,, 1/100), for
one hour at room temperature. Cells are then centrifuged at 7000
rpm for 5 minutes and cell pellets are rinsed once in PBS. The
labelling is increased adding a tertiary antibody coupled to Alexa
Fluor 488 (chicken anti-goat IgG, Molecular Probes #A21467, 1/100)
for one hour incubation, at room temperature. Cells are then rinsed
once in PBS. Control without any antibody (unlabelled cells) is
performed to determine the level of autofluorescence and adapted
the sensitivity of the photomultiplicators. Control with both
secondary and tertiary antibodies but without primary antibody, is
performed to assess non specific binding of antibodies. [0174] Data
acquisition and analysis are performed with a FACS Array cytometer
and FACS Array software (Becton Dickinson) on 5000 cells. Forward
Scatter (FSC) correlated with cell volume (sire) and Side Scatter
(SSC) depending on inner complexity of cells (granularity) are
analysed. For expression of PMP22, analysis is performed within the
total cells and percent of positive cells is calculated. Positive
cells are cells with fluorescence intensity higher than the control
with secondary antibody. [0175] In order to quantify the number of
SC, cells in control medium are analysed using antibodies anti-S100
Protein. [0176] Cells are prepared according to the following
protocol: Schwann cells are stained with antibody anti-S100 Protein
(Dako #S0311, 1/100) for 1 hr room at temperature. This antibody is
labelled according to protocol described above for PMP22
immunostaining but without incubation with tertiary antibody.
[0177] 1.5. Drug Incubation and Activity
[0178] Drugs are incubated for 24 hrs or 48 hrs in the same defined
medium than described above (3 wells/condition) in absence of
Forskolin to avoid adenylate cyclase stimulation saturation, but in
presence of 10 nM of progesterone. After drug incubation,
supernatants are recovered and Schwann cells are frozen for
RT-Q-PCR analysis. [0179] These experiments are summarized in Table
1.
TABLE-US-00008 [0179] TABLE 1 Combination PMP22 expression Mix1
down regulation Mix2 down regulation Mix3 down regulation Mix4 down
regulation Mix5 down regulation Mix6 down regulation
2. Assessment of Synergistic Effect of Compounds in Mix7 in a
Co-Culture Model for CMT
[0180] A model of co-culture was used as an in vitro model of
CMT1A. This model of myelination consists in co-culturing sensory
neurons and Schwann cells from male PMP22 Transgenic (TG)
dissociated Dorsal Root Ganglia (DRG). [0181] The aim of this study
is to assess the effect of 3 test compounds (+/-Baclofen,
Naltrexone and Sorbitol) and Mix7 (a mixture of these 3 drugs) on
myelination process. The effect of the 3 test compounds, and their
mixture on myelination, are assessed by evaluating Myelin Basic
Protein (MBP) expression in presence of ascorbic acid.
[0182] 2.1 Materials and Methods [0183] 15 days gestation pregnant
female rats are killed by cervical dislocation. The embryos are
removed from the uterus and are at similar fetal stage of
development.
2.1.1 Genotyping
[0183] [0184] A piece of each embryo head (3 mm.sup.3 ) is placed
in a 2 ml tube DNase free. The DNA is extracted with the SYBR
Green. Extract-N-Amp tissue PCR kit (Sigma, ref XNATG-1KT). 120
.mu.l of extraction solution (Kit Sigma, ref XNATG-1KT) was put on
each piece of embryo head. The heads are incubated for 10 min at
room temperature. At the end of this incubation, the heads are
incubated for 5 min at 95.degree. C. in the extraction solution.
Immediately after this last incubation, 100 .mu.l of neutralizing
solution are added, each DNA extract is diluted at 1/140 with
sterile ultrapure water (Biosolve, ref: 91589) and stored at
+4.degree. C. until use. The genotyping of female (F) and male (M)
embryos is performed during the dissection of the DRG, with the kit
Fast SYBR Green Master Mix (Applied Biosystem, 4385612). The gender
of each embryo is determined using the male SRY gene. The SRY
primers are supplied by Pharnext (SRY-F (SEQ ID NO:9):
5'-GAGAGAGGCACAAGTTGGC-3'; SRY-R (SEQ ID NO: 10):
5'-GCCTCCTGGAAAAAGGGCC-3'), SRY primers are diluted at 3 .mu.M in
sterile ultrapure water (Biosolve, ref: 91589). A mix for PCR is
prepared with ultrapure water (4 .mu.l per sample), primer at 3
.mu.M (2 .mu.l per sample) and Master Mix (10 .mu.l per sample). In
a PCR 96 wells plate, 16 .mu.l of PCR mix is deposited in each
well, 4 .mu.l of each diluted DNA is added according to a plan
deposit. The PCR is run using the 7500 fast RT-PCR system (Applied
Biosystem), with the following program: [0185] Beginning:
95.degree. C.--20 sec [0186] 45 cycles: 95.degree. C.--10 sec,
65.degree. C.--10 sec, 72.degree. C.--30 sec (data acquisition).
[0187] Melt curve: 95.degree. C.--15 sec, 64.degree. C.--1 min,
90.degree. C.--30 sec (continuous data acquisition), 60.degree. C.
15 sec. The amplification plots and melt curves are analyzed with
the 7500 software (Applied Biosystems). [0188] The results for each
sample are compared to negative control (ultrapure water) and to
the positive control (TG/Male and WT/Female), to conclude on the
genotype of each embryo.
[0189] 2.1.2 Sensory Neurons and Schwann Cells Co-Cultures [0190]
Rat Dorsal root ganglions are cultured as previously described by
Cosgaya et at., 2002 and Rangaraju et al., 2008. [0191] Each embryo
is dispatched on numerating petri dish (35 mm of diameter). The
head of embryo is cut, placed on 1.5 ml tube DNAase free; the ADN
is extracted with the Extract-N-Amp Tissue Kit (Sigma Aldrich). The
genotyping (Male (M) and female (F), wild type and PMP22
transgenic) is performed with the kit Fast SYBR Green Master Mix
(Applied Biosystem). This genotyping is performed in parallel of
the dissection of dorsal root ganglia (DRG), so that at the end of
the dissection, only one type of culture (DRG from transgenic male)
is done. DRG of each embryo is collected, placed in ice-cold medium
of Leibovitz (L15 Invitrogen). At the end of the dissection, DRG of
TGM are pooled and dissociated by trypsinization (trypsin EDTA,
Invitrogen) for 20 min at 37.degree. C. The reaction is stopped by
addition of DMEM containing 10% of fetal bovine serum (FBS) in the
presence of DNAase I (Roche). The suspension is triturated with a
10 ml pipette. Cells are then centrifuged at 350 .times. g for 10
min at room temperature. The pellet of dissociated cells is
resuspended in neurobasal medium (Invitrogen) containing 2% of B27
(Invitrogen), 1% of penicillin-streptomycin (Invitrogen), 1% de L
glutamine and 50 ng/ml NGF (Sigma). This medium is the neuronal
medium. Viable cells are counted in a Neubauer cytometer using the
trypan blue exclusion test (Sigma) and seeded on the basis of 10
000 cells per well in 96 well-plates (Greiner) treated s n th
poly-L-lysine. The plates are maintained at 37.degree. C. in a
humidified incubator, in an atmosphere of air (95%)-CO2 (5%). Half
of the standard neuronal culture medium is changed every other day.
The cultures are maintained in standard neurobasal medium for 7
days to allow Schwann cells to populate the sensory neuron
neurites. On day 7, the cultures are fed with standard neuronal
medium supplemented or not with 50 .mu.g/ml ascorbic acid in order
to initiate basal lamina formation and myelination.
[0192] 2.13. Drug Incubation [0193] On day 7, the following test
compounds (alone or in combination) are added in the medium with 50
.mu.g/ml ascorbic acid: [0194] (RS)-Baclofen [0195] Natrexone
[0196] d-Sorbitol [0197] These compounds or compound combination
are tested at the following concentrations (Table 2):
TABLE-US-00009 [0197] TABLE 2 Concentration of individual drugs or
in combination used for in vitro studies of MBP expression in TG
DRG/SC co-cultures. Dose 1 Dose 2 Dose 3 Dose 4 Dose 5 Dose 6 Dose
7 Individual Naltrexone 5 .mu.M 1 .mu.M 200 nM 40 nM 8 nM 1.6 nM
320 pM drugs d-Sorbitol 500 .mu.M 100 .mu.M 20 .mu.M 4 .mu.M 800 nM
160 nM 32 nM (RS)- 5 .mu.M 1 .mu.M 200 nM 40 nM 8 nM 1.6 nM 320 pM
Baclofen Mix7 Naltrexone 5 .mu.M 1 .mu.M 200 nM 40 nM 8 nM 1.6 nM
320 pM d-Sorbitol 500 .mu.M 100 .mu.M 20 .mu.M 4 .mu.M 800 nM 160
nM 2 nM (RS)- 5 .mu.M 1 .mu.M 200 nM 40 nM 8 nM 1.6 nM 320 pM
Baclofen
[0198] The test compounds are incubated for 5 different times: 5,
9, 10, 11 and 13 days. Three separate and independent cultures of
DRG (from TG embryos male rats) are done. These conditions are
assessed in presence of ascorbic acid, 6 wells per condition. The
solution ready to use of all test compounds are extemporaneously
prepared from a stock solution, stored at -20.degree. C. This
solution is prepared once a week. Half of the standard neuronal
medium supplemented with test compounds and ascorbic acid (each at
the concentration 1.times.) are changed every other day.
[0199] 2.1.4 Staining Protocol [0200] After 5, 9, 10, 11 and 13
days of incubation, cells are fixed by a cold solution of ethanol
(95%) and acetic acid (5%) for 10 min. The cells are permeabilized
and blocked with PBS containing 0.1% saponin and 10% goat serum for
15 min. Then, the cells are incubated with a specific marker of
myelin: polyclonal antibody anti-myelin basic protein (MBP)
antibody (Sigma 1181(0431). [0201] This antibody is revealed with
Alexa Fluor 568 goat anti-rabbit IgG and Alexa Fluor 488 goat
anti-mouse IgG (Molecular probe 687621, 623962). Nuclei of neurons
are labeled by a fluorescent marker (Hoechst solution, SIGMA ref
131155).
[0202] 2.1.5. Data Processing [0203] Per well, 20 pictures are
taken using InCell Analyzer.TM. 1000 (GE Healthcare) with 20.times.
magnification. All images are taken in the same conditions.
Analysis of total length of myelinated axons was automatically done
(length and area around axons) using Developer software (GE
Healthcare). All values will be expressed as mean +/- s.e.mean.
Statistic analyses are done on different conditions (ANOVA followed
by Fisher's PLSD test when allowed).
[0204] 2.2. Results [0205] Synergistic effect of drugs in the
efficacy of Mix7 [0206] An important synergistic effect of drugs
composing the Mix7 combination is observed on MBP expression.
Indeed, on day 10 (=17 days of culture), combination of
(RS)-Baclofen, Naltrexone and d-Sorbitol significantly increases
MBP expression at doses 1 and 6 as shown in FIGS. 1A and 2A. By
contrast, the above drugs used individually have no substantial
effect compared to control (FIGS. 1B-D and 2B-D). A significant
effect on MBP expression is also recorded after 10 days of
incubation at doses 2, 3, 4, 5 and 7 of Mix7 (FIG. 3A). [0207] This
effect is still observed on day 11 with doses 2-7 (FIG. 3) in form
of a clear bell shape curve.
C. Experiments In Vivo in CMT Animal Model
[0207] [0208] We tested the compounds for therapeutic effect in a
rat model. [0209] The experimental groups are formed with young
rats of both genders separately. The rats are assigned to the
groups following randomization schedule based on the body weight.
In some experiments the randomization is based on the performances
of the rats in the bar test. Both genders are represented by
separate control groups that are numerically equal or bigger than
the treatment groups. [0210] The rats are treated chronically with
drugs--force fed or injected by Alzet osmotic subcutaneous pump
(DURECT Corporation Cupertino, Calif.), depending on each drug
bioavailability during 3 or to weeks. In all the in vivo
experiments performed, the Mix7 is administered by gavage. [0211]
The animals are weighted twice a week in order to adjust the doses
to growing body weight. If the osmotic pump is chosen for the
treatment administration, the doses of the drug are calculated on
the basis of the estimated mean body weight of the animals expected
for their age over the period of the pump duration (6 weeks). The
pumps are re-implanted if necessary, with the appropriated
anesthesia protocol.
Behavioural Tests
[0211] [0212] Each three or four weeks the animals are subjected to
a behavioural test. Each test is conducted by the same investigator
in the same room and at the same time of the day; this homogeneity
is maintained throughout entire experiment. All treatments and
genotype determination are blinded for the investigator. "Bar test"
and "Grip strength" has been mainly used to access the performance
throughout study. The schedule of the bar test may change as the
animal growth (in order to avoid the bias due to the learning, for
example). [0213] The assay of the grip strength allows detection of
subtle differences in the grip performance that seems to be
composed of the muscle force, sensitivity status (for instance,
painful tactile feelings may change measured values of the force),
behavioural component ("motivation"). The values differ between
fore and hind limbs and greatly depend on the age of the animals.
[0214] The grip strength test measures the strength with which an
animal holds on to a grip with its forepaws or its hindpaws
separately. A dynamometer is placed with a grip to measure the
strength (Force Gauge FG-5000A). The rat is held by the
experimenter in a way that it grasps the grip either with its
forepaws or with its hind paws and pulls gently the rat backwards
until it releases the grip. The force measured when the animal
releases the grip is recorded. [0215] Two successive trials
measuring the forepaws and two successive trials measuring the
hindpaws strength per animal are processed; only the maximum score
(one for forepaws and one for hindpaws) is noticed (in N).
The Bar Test
[0215] [0216] The bar test evaluates rats' ability to hold on a fix
rod. Pmp22 rats which display muscular weakness, exhibit a
performance deficit in this test (Sereda et al. 1996). The rat is
placed on its four paws on the middle of the rod (diameter; 2.5 cm;
length: 50 cm: 30 cm above the table). Trials are performed
consecutively; the number and the duration of trials in our
experiments have been depending on batches of the animals. This
variability in the testing has been introduced in order to
determine the schedule appropriated to the best detection of the
motor deficiency in the CMT rats in the course of the
experiments.
Performance Indices are Recorded on Each Session:
[0216] [0217] The number of trials needed to hold for 60 sec (or 30
sec for batch 1, session 1 and 2) on the rod. [0218] The time spent
on the bar (i.e. the fall latency) in each trial and the average on
the session. In the experimental procedures where the session ends
after the rat has staved for a cut-off time, i.e. 30 or 60 s, on
the bar, a performance of the cut-off time (30 s or 60 s) is
assigned to trials not completed (eg: for batch 8, for an animal
which stays on the bar less than 10 sec on trials 1, 2 and 3, then
for 60 see on trials 4 and 5, 60 s is assigned to trials 6 to 10).
[0219] The number of falls.
General Health Assessment
[0219] [0220] Body weights, overt signs (coat appearance, body
posture, gait, tremor etc.) of the animals are monitored throughout
the experiment. The rating scale is used for recording: 0=normal,
1=abnormal.
The Gait
[0220] [0221] Each rat is observed in a novel rat cage (dimensions
55.times.33.times.18 cm) without litter for five minutes. The gait
of rats is evaluated with 4 parameters: [0222] Score 0: normal gait
(fluid) [0223] Score 1: abnormal gait (not fluid or the rat has a
slight limp) [0224] Score 2: moderate incapacity (the rat drags
one's leg and is able to put it right and walk) [0225] Score 3:
serious incapacity (the rat drags its one's or both hindpaws but is
unable to put it/them right).
Inclined Plane Test
[0225] [0226] The sliding apparatus had a 30.times.50 cm Plexiglas
plane that could be inclined at an angle of 0.degree. (horizontal)
to 60.degree.. Each rat was initially placed on the
25.degree.-angled inclined plane in the up headed position (head-up
orientation); two trials separated by 1 min are performed. 30 min
later, the same experiment is realized on a 35.degree.-angled
inclined plane then on 40.degree.-angled inclined plane. During
this time the rat was returned to its cage. The plane is cleaned
after each trial. [0227] The performances of rats are evaluated by
4 different scores: [0228] Score 0: no slide [0229] Score 1: a
little slide (one or two paws) [0230] Score 2: a moderate slide (4
paws) but not until the end of the plane [0231] Score 3: the rat is
sliding until the very bottom of the plane.
Further Tests
[0231] [0232] When appropriate, the rats are subjected to
electrophysiological evaluation, histological measurement and the
pmp22 RNA expression level in the sciatic nerve is quantified.
Quantification of pmp22 RNA in Sciatic Nerve by Quantitative RT-PCR
[0233] Total RNA was isolated from left sciatic nerves using Qiazol
(ref N.degree.79306, Qiagen Gmbh, Germany) followed by the
single-step purification method with RNeasy Mini Kit (ref N.degree.
74106, Qiagen Gmbh, Germany) described by the manufacturer's
protocol (Qiagen-RNeasy Fibrous tissue Handbook). DNA contamination
was removed by digestion with RNase-free DNase I by use of the
DNA-free kit (Qiagen-Rnase-free dnase set 1500 Kunits, ref
N.degree. 1023460). [0234] RNA concentrations are estimated by
NanoDrop ND-1000 and a test of quality control was done by Agilent
RNA 6000 nano chips on Agilent 2100 Bioanalyzer. [0235] Reverse
transcription and real-time PCR: Quantitative RT-PCR (RT-Q-PCR) was
performed as follows: 80 ng of total RNA was reverse transcribed
using SuperScript.TM. II Reverse Transcriptase (Invitrogen,
Carlsbad, Calif.) with Oligo(dT)12-18 (Invitrogen, Carlsbad,
Calif.) in a 20-0 reaction volume. [0236] Real-time PCR was
performed with a rapid thermal cycler system (LightCycler.RTM. 480
II, 384 -Well, Roche, Switzerland). Amplifications are performed in
a 10 .mu.l total volume with primers concentration optimized
between 130 nM and 1 .mu.M. Primers and template are supplemented
with LightCycler.RTM. 480 SYBR Green I Master (2.times. conc.
Roche, Cat. Ref N.degree. 04 887 352 001). Nucleotides, MgCl.sub.2,
Tag DNA polymerase and buffer are included in the mix. An
amplification protocol incorporated an initial incubation at
95.degree. C. for 10 min for the activation of Tag DNA polymerase
followed by 45 cycles, with a 95.degree. C. denaturation for 10s,
60.degree. C. annealing for 40s and 72.degree. C. extension for 10s
(detection of the fluorescent product was performed at the end of
the 72.degree. C. extension period by a single acquisition mode)
and ended by a cycle of melting curse with 95.degree. C.
denaturation for 5s, 63.degree. C. annealing for 60s and 95.degree.
C. (from 63.degree. C. to 95.degree. C. the ramp rate is
0.11.degree. C./s and detection of the fluorescent product was
continuous). To confirm the amplification specificity, the PCR
product from each primer pair was subjected to a melting curve
analysis. Relative quantification was performed based on the
crossing point (Cp value) for each of the PCR samples. The point at
which the fluorescence of a sample rises above the background
fluorescence is called the "crossing point (Cp) of the sample.
Rattus norvegicus Myelin Protein Zero (MPZ) gene was used for
normalization (Sereda et al, 2006). The sequences of the primers
(synthesized by Eurofins MWG Operon, Germany) used for the RT-Q-PCR
analysis are:
TABLE-US-00010 [0236] PMP22-forward: (SEQ ID NO: 11)
5'-TGTACCACATCCGCCTTGG-3' and PMP22-reverse: (SEQ ID NO: 12)
5'-GAGCTGGCAGAAGAACAGGAAC-3'. MPZ-forward: (SEQ ID NO: 13)
5'-TGTTGCTGCTGTTGCTCTTC-3' and MPZ-reverse: (SEQ ID NO: 14)
5'-TTGTGAAATTTCCCCTTCTCC-3'.
Results
[0237] Mix1 composition improves bar test performances throughout
the treatment procedure (FIG. 4). [0238] Mix1 improves the gait
score of transgenic rats after 3 and 6 weeks of treatment as shown
in FIG. 5. [0239] Mix 1 increases the performances of transgenic
rats after 3, 6, 9 and 12 weeks of treatment in the inclined plane
test at 25.degree. described in the FIG. 6. [0240] FIG. 7
illustrates the positive effect of Mix2 on gait score of transgenic
rats at 25, 35 and 40.degree. in the inclined plane test. [0241]
Mix7 (dose 3) significantly decreases the pmp22 RNA gene expression
in the sciatic nerve of pmp22 transgenic rats (FIG. 9). [0242] The
performances of pmp22 rats treated with Mix7 (dose 2 and dose 3)
are improved in the inclined plane test at 35.degree. (FIG. 10).
More specifically, 29 and 33% of rats belong to the good
performance group, compared to 5% for TG placebo group, and 29 and
11% of rats belong to the poor performance group compared to 60%
for the TG placebo group. P-value (versus the TG placebo) is equal
to 0.0152 for the TG rats treated with Mix7-dose 2 and p-value is
equal to 0.002 for the TG rats treated with Mix7-dose 3 versus the
TG placebo). [0243] Mix7-dose 3 significantly increases the fall
latency time of pmp22 rats in bar test after 9 weeks of treatment
(FIG. 11): black dashed line, p=4,56.10-2, n=18. Significant
difference between TG placebo rats (black plain line, n=20) and WT
placebo rats (grey plain line, p=3,82.10-7, n=18) is also observed.
[0244] The FIG. 12 illustrates the improvement of grip strength of
the pmp22 rats treated with the Mix7-dose 3. [0245] The FIG. 13
shows the significant correlation between the bar test latency time
(after 9 Weeks of treatment with the Mix7-dose 3) and the
expression level of pmp22 RNA. [0246] The FIG. 14 displays the
significant correlation between the bar test latency time after 9
weeks of treatment with Mix7-dose 3 and the conduction velocity of
the sensitive nerve (tail). [0247] Similar results are obtained for
other combinations (see Table 3).
TABLE-US-00011 [0247] TABLE 3 Combination PMP22 rat disease
phenotype Mix1 improvement Mix2 improvement Mix3 improvement Mix4
improvement Mix5 improvement Mix6 improvement
[0248] These data show that, in vivo, the combinations and regimens
of this invention allow effective treatment of CMT.
D. In Vivo Effect in a Model of Toxic Neuropathy
[0248] [0249] The drug treatments or regimens are orally
administered from the day before the first intraperitoneal
injection of Oxaliplatin 3 mg/kg (D-1) until the day before the
last testing day (D16). Animals belonging to the
Oxaliplatin-treated group are dosed daily with distilled water (10
ml/kg). Animals are dosed with the tested treatment and distilled
water daily during the morning whereas Oxaliplatin is administered
on the afternoon. [0250] During the testing days (i.e. D1, D4,
D10), the treatment and distilled water are administered after the
test. Regarding, the testing day (D4), including compounds and
vehicle administrations and Oxaliplatin injection, the treatment
and distilled water are administered prior to the injection of
Oxaliplatin after the test. Animals from the reference-treated
group are dosed only during the testing days (i.e. D1, D4, D10 and
D17). [0251] Cold allodynia is assessed b measuring the responses
to thermal non-nociceptive stimulation (acetone test) on D1 (around
24 h after the first injection of Oxaliplatin 3 mg/kg (acute effect
of Oxaliplatin), on D4, D10 and (chronic effect of Oxaliplatin) and
on D17 (residual effect of Oxaliplatin one week after completion of
treatment). [0252] Testing is done using the acetone test 2 h
post-administration of the reference. The reference substance is
Gabapentin, 100 mg/kg, per os (once a day .times.4 testing
days).
[0253] Acetone Test [0254] Cold allodynia is assessed using the
acetone test. In this test, latency of hindpaw withdrawal is
measured after application of a drop of acetone to the plantar
surface of both hindpaws (reaction time) and the intensity of the
response is scored (cold score). [0255] Reaction time to the
cooling effect of is measured within 20 sec (cut-off) after acetone
application. Responses to acetone are also graded to the following
4-point scale: 0 (no response); 1 (quick withdrawal, flick of the
paw); 2 (prolonged withdrawal or marked flicking of the paw); 3
(repeated flicking of the pa with licking or biting). [0256] Six
trials by rat are performed. For each experimental group, the
results are expressed as the cumulative cold score defined as the
sum of the 6 scores for each tat together .+-. SEM. The minimum
score being 0 (no response to any of the 6 trials) and the maximum
possible score being 18 (repeated flicking and licking or biting of
paws on each of the six trials).
Gabapentin Source: Zhejiang Chiral Medicine Chemicals, China
Oxaliplatin Source: Sigma, France
[0256] [0257] The results are depicted on FIG. 8. They clearly show
a protective effect of the composition of this invention on
oxaliplatin-induced neuropathy.
E. In Vivo Effect in a Model of ALS
Animal Model
[0257] [0258] We have chosen the SOD1.sup.G93A rat model (generated
by Howland et al) to mimic the Amyotrophic Lateral Sclerosis
pathology. This model overexpresses the mutated SOD1 gene in spinal
cord, many brain regions as well as peripheral tissues. The onset
of the motor neuron disease of this model is about at 115 days; it
appears as hind limb abnormal gait. In few days, the paralysis of
hind limb arises.
Experimental Procedures
[0258] [0259] We obtained colonies by crossing breeder
SOD1.sup.G93A rats with Sprague Dawley female rats. Heterozygous
SOD1.sup.G93A rats are identified with polymerase chain reaction
(PCR) of tail DNA with primers specific for hSOD1 [1]. Animals are
maintained in a room with controlled illumination (lights on
0500-1900 h) and temperature (23.+-.1.degree. C.), and given free
access to food and water. All the animal procedures in the present
study are carried out in accordance With the guidelines standards
of animal care. [0260] Body weight measurement was performed every
week and behavioural tests began at an age of 60 days and continued
until endpoint. The treatments are administered every day per oral
or subcutaneous way from the age of 5 weeks.
[0261] 1. Observation Test: Characterization of the General Aspect
[0262] Each rat was observed in a novel rat cage (dimensions
55.times.33.times.18 cm) without litter for five minutes. 5
different parameters are recorded:
The Gait
[0262] [0263] score 0: normal gait (fluid) [0264] score 1: abnormal
gait (not fluid or the rat has a slight limp) [0265] score 2:
moderate incapacity the rat drags one's leg and is able to put it
right and walk) [0266] score 3: serious incapacity (the rat drags
its ones or both hindpaws but is unable to put it/them right)
The Coat Aspect
[0266] [0267] score 0: clean and silky coat [0268] score 1:
piloerection or dirty coat
The Tremor
[0268] [0269] score 0: no tremor [0270] score 1: tremor
The Body Position
[0270] [0271] score 0: normal [0272] score 1: abnormal (flattened
or archering its back)
The Hindpaws Position
[0272] [0273] score 0: normal [0274] score 1: spread hindpaws
[0275] 2. The Motor Score Test: Characterization of the Motor
Deficit [0276] This test evaluates the ability of rats to right
themselves within 30 sec of being turned on either side (righting
reflex) (Gale et al). [0277] A non-parametrical scoring system Was
used following these criteria (Matsumoto et al, Thonhoff et al):
[0278] score 0: the rat is unable to right itself from either side
within 30 sec [0279] score 1: the rat is unable to right itself
from only one side within 30 sec [0280] score 2: the rat is able to
right itself from both sides within 30 sec but is unable to stand
in the cage; it is always dragging some parts of body [0281] score
3: the rat is able to right itself from both sides within 30 sec,
is unable to stand in the cage but is not dragging some parts of
body [0282] score 4: the rat is able to right itself from both
sides within 30 sec, is able to stand in the cage but has visible
functional deficits [0283] score 5: the rat is able to right itself
from both sides within 30 sec, is able to stand in the cage and no
visible functional deficits. [0284] The end-point of disease is
fixed at score 0: the rat is then euthanized.
[0285] 3. Inclined Plane Test: Characterization of the Motor
Deficit [0286] The sliding apparatus had a 30.times.50 cm plexiglas
plane that could be inclined at an angle of 0.degree. (horizontal)
to 60.degree.. Each rat was initially placed on the
25.degree.-angled inclined plane in the up-headed position (head-up
orientation); two trials separated by 1 min are performed. 30 min
later, the same experiment is realized on a 35.degree.-angled
inclined plane then on 40.DELTA.-angled inclined plane. During this
time the rat was returned to its cage. The plane is cleaned after
each trial. [0287] The performances of rats are evaluated by 4
different scores: [0288] score 0: no slide [0289] score 1: a little
slide (one or two paws) [0290] score 2: a moderate slide (4 paws)
but not until the end of the plane [0291] score 3: the rat is
sliding until the very bottom of the plane.
[0292] 4. The Wire Mesh Test: Characterization of the Motor Ability
in Difficult Situation [0293] A wire mesh was placed in contact
with a box at the top (at an angle of 70.degree.) and the edge of a
table at the bottom. Each rat was placed on the bottom of the wire
mesh and motivated to ascend by placing their littermates in the
box at the top. Each rat was trained once a week (3 trials). [0294]
The recorded parameter was the latency time to reach the top of the
wire mesh.
[0295] 5. The Open Field Test Characterization of the Locomotor
Activity [0296] The locomotor activity was measured in a Plexiglas
box (45.times.45.times.30 cm, Acti-Track by BIOSEB, Lyon, France)
with 16 photo-cell beams following the two axes, 1 and 5 cm above
the floor. [0297] The spontaneous and exploratory activity of each
rat was evaluated dining 3 hours. [0298] 4 parameters are recorded
(the total travelled distance, the number of rearings, the
percentage of travelled distance and of time spent in the center of
the openfield).
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Sequence CWU 1
1
14117DNAartificial sequenceforward primer_rat PMP22 1ggaaacgcga
atgaggc 17219DNAArtificial sequencereverse primer_rat PMP22
2gttctgtttg gtttggctt 19317DNAArtificial sequenceforward
primer_RPL13A 3ctgccctcaa ggttgtg 17421DNAArtificial
sequencereverse primer_RPL13A 4cttcttcttc cggtaatgga t
21522DNAArtificial sequenceforward primer_Pmp22-FL 5gctctgagcg
tgcatagggt ac 22619DNAArtificial sequenceforward primer_ Rpl13A-FL
6tcgggtggaa gtaccagcc 19725DNAArtificial sequencereverse
primer_Pmp22-red 7agggagggag gaaggaaacc agaaa 25828DNAArtificial
sequencereverse primer_Rpl13A-red 8tgacagctac tctggaggag aaacggaa
28919DNAartificial sequenceSRY - Forward 9gagagaggca caagttggc
191019DNAartificial sequenceSRY - reverse 10gcctcctgga aaaagggcc
191119DNAartificial sequencePMP22-forward 11tgtaccacat ccgccttgg
191222DNAartificial sequencePMP22 - reverse 12gagctggcag aagaacagga
ac 221320DNAartificial sequenceMPZ - forward 13tgttgctgct
gttgctcttc 201421DNAartificial sequenceMPZ - reverse 14ttgtgaaatt
tccccttctc c 21
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