U.S. patent application number 15/928124 was filed with the patent office on 2018-07-26 for diagnostic tools for charcot-marie-tooth disease.
The applicant listed for this patent is PHARNEXT. Invention is credited to ILYA CHUMAKOV, DANIEL COHEN, SERGUEI NABIROCHKIN.
Application Number | 20180209997 15/928124 |
Document ID | / |
Family ID | 41698035 |
Filed Date | 2018-07-26 |
United States Patent
Application |
20180209997 |
Kind Code |
A1 |
COHEN; DANIEL ; et
al. |
July 26, 2018 |
DIAGNOSTIC TOOLS FOR CHARCOT-MARIE-TOOTH DISEASE
Abstract
The present invention relates in particular to methods of
detecting predisposition to or diagnosis and/or prognosis of
Charcot-Marie-Tooth disease (CMT) and related disorders. More
specifically, the invention relates to development, validation and
application of new biomarkers which can be used for detecting the
presence or risk of CMT disease and related disorders. In
particular, the present invention relates to metabolite, lipid,
carbohydrate and proteinaceous biomarkers that can be measured in
biological body fluids and easily available extracts of biopsies,
which can be used to aid in the detection, prediction of drug
treatment and follow-up of this treatment of neurodegenerative
disorders, including CMT disease. The present invention also
relates to methods for identification of CMT disease subtypes and
assessing the responsiveness to treatments and the efficacy of
treatments in subjects having CMT or a related disorder.
Inventors: |
COHEN; DANIEL; (SAINT CLOUD,
FR) ; CHUMAKOV; ILYA; (VAUX-LE-PENIL, FR) ;
NABIROCHKIN; SERGUEI; (CHATENAY-MALABRY, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PHARNEXT |
Issy Les Moulineaux |
|
FR |
|
|
Family ID: |
41698035 |
Appl. No.: |
15/928124 |
Filed: |
March 22, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14755466 |
Jun 30, 2015 |
9945876 |
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15928124 |
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13510700 |
Dec 5, 2012 |
9494605 |
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PCT/EP2010/067855 |
Nov 19, 2010 |
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14755466 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/92 20130101;
G01N 33/9433 20130101; G01N 2800/52 20130101; G01N 33/942 20130101;
G01N 33/6812 20130101; A61K 31/485 20130101; G01N 33/50 20130101;
A61K 31/047 20130101; G01N 2800/285 20130101; A61K 31/197 20130101;
G01N 33/6896 20130101; G01N 33/88 20130101; G01N 33/78 20130101;
G01N 33/743 20130101; A61K 31/047 20130101; A61K 2300/00 20130101;
A61K 31/197 20130101; A61K 2300/00 20130101; A61K 31/485 20130101;
A61K 2300/00 20130101 |
International
Class: |
G01N 33/92 20060101
G01N033/92; A61K 31/047 20060101 A61K031/047; A61K 31/197 20060101
A61K031/197; A61K 31/485 20060101 A61K031/485 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 20, 2009 |
EP |
09306121.6 |
Claims
1. A method for predicting the responsiveness to a treatment of CMT
disease and for treating an individual suffering from CMT, the
method comprising: i) determining in vitro the free cholesterol
level in a biological sample from said individual, ii) predicting
the responsiveness of said individual to said treatment by
comparing the free cholesterol level obtained in i) to a reference
value of a responder or non-responder group, and iii) treating said
individual predicted to be responsive with said treatment.
2. The method of claim 1, wherein said reference value is a
reference value of a non-responder group and wherein a lower free
cholesterol level in the individual as compared to the reference
value is indicative that the individual shall respond to the
treatment.
3. The method of claim 2, wherein said lower free cholesterol level
is at least 5% lower than said reference value.
4. The method of claim 1, wherein said biological sample is a
plasma sample and wherein said reference value of a responder or
non-responder group is the free cholesterol level in plasma of a
responder or non-responder group.
5. The method of claim 4, wherein the reference value of free
cholesterol level in plasma in a responder group is less than 483
.mu.g/ml.
6. The method of claim 4, wherein the reference value of free
cholesterol level in plasma in a non-responder group is superior to
520 .mu.g/ml.
7. The method of claim 4, wherein a free cholesterol level in said
plasma sample in said individual superior to 520 .mu.g/ml is
indicative that the individual shall not respond to said
treatment.
8. The method of claim 4, wherein a free cholesterol level in said
plasma sample in said individual below or equal to 483 .mu.g/ml is
indicative that the individual shall respond to said treatment.
9. The method of claim 1, wherein said treatment of CMT disease
comprises administering baclofen, naltrexone and sorbitol, or salts
thereof, to said individual.
10. The method of claim 1, wherein said CMT disease is CMT1A.
11. The method of claim 1, wherein said reference value is a
reference value of a responder group with similar age, sex,
condition, and/or any ongoing treatment.
12. An in vitro method for assessing efficacy of a treatment
against CMT in a subject, the method comprising determining in a
fluid biological sample from the subject, during the treatment, the
(relative) amount or alteration of free cholesterol level and
comparing said amount or alteration to a level of free cholesterol
determined before treatment or at an earlier stage of treatment in
said individual, wherein a deviation is indicative of the efficacy
of the treatment.
13. The method of claim 12, wherein said amount or alteration is a
decrease of free cholesterol in regard of total cholesterol.
14. The method of claim 12, wherein the relative amount or the
presence of at least a second biomarker is determined,
simultaneously or sequentially.
15. The method of claim 14, wherein said at least one second
biomarker is selected from LDL cholesterol, alanine,
.alpha.-aminobutyric acid, citrulline, cystine, glutamine,
hydroxyproline, lysine, methionine, proline, threonine, tryptophan,
tyrosine, T4 thyroid hormone, testosterone, iron, LTB4, adrenaline,
dopamine or serotonin.
16. The method of claim 14, wherein the level of said biomarker(s)
is compared to a reference value and wherein the deviation from
said value is indicative of the efficacy of said treatment.
17. The method of claim 12, wherein CMT disease is CMT1A.
18. The method of claim 12, wherein the treatment is continued in
an individual in whom a deviation of the (relative) amount or
alteration of free cholesterol level is determined.
19. The method of claim 12, wherein said treatment against CMT
comprises administering baclofen, naltrexone and sorbitol, or salts
thereof, to said individual.
20. The method of claim 12, wherein the time interval between the
measures of free cholesterol level is at least 2 months, preferably
3 or 4 months.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/755,466, filed Jun. 30, 2015, which is a
continuation-in-part of co-pending application U.S. Ser. No.
13/510,700, filed Dec. 5, 2012, now U.S. Pat. No. 9,494,605, which
is the U.S. national stage of International Patent Application No.
PCT/EP2010/067855, filed Nov. 19, 2010.
[0002] The present invention relates generally to the field of
medicine. The present invention relates in particular to methods of
detecting predisposition to or diagnosis and/or prognosis of
Charcot-Marie-Tooth (CMT) and related disorders. More specifically,
the invention relates to development, validation and application of
new biomarkers, which can be used for detecting the presence or
risk of CMT disease and related disorders. In particular, the
present invention relates to metabolite, lipid, carbohydrate and
proteinaceous biomarkers that can be measured in biological body
fluids and easily available extracts of biopsies, which can be used
to aid in the detection, prediction of drug treatment and follow up
of this treatment of neurodegenerative disorders, including CMT
disease. The present invention also relates to methods for
identification of CMT disease subtypes, and assessing the
responsiveness to the treatments and the efficacy of treatments in
subjects having CMT or a related disorder.
[0003] Charcot-Marie-Tooth disease ("CMT") is an orphan genetic
peripheral polyneuropathy. 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. The course of the 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), more than two dozen
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 clustered around tightly connected molecular
pathways affecting differentiation of Schwann cells or neurons or
changing interplay of these cells in peripheral nerves.
[0004] At present, the diagnosis of CMT disease is based on
clinical criteria and electrophysiology data that distinguish only
few subtypes of this disease. More precise classification relies on
mutation analysis in relevant genes if known.
[0005] The multiple mutations leading to CMT disease occur in more
than 25 different genes. They are not identified for all cases of
CMT disease and cannot be exhaustingly classified by genetic typing
(Suter & Scherer, 2003; Berger et al., 2006; Niemann et al.,
2006; Nave et al., 2007). Moreover, clinical heterogeneity does
occur and is not only important for clinical characterization but
provides further implication of specific management/treatment for
functionally different forms (Sereda et al., 2003; Passage et al.,
2004; Sahenk et al., 2005; Young et al., 2008).
[0006] For the moment, no drug treatment exists for this disease
but some clinical management procedures have been described
(Grandis & Shy, 2005; Kapur et al., 2007; Weiner et al., 2008)
and clinical trials with ascorbic acid for the treatment of the
CMT1A form of this disease are under way (Burns et al., 2009).
[0007] A specific tool to measure the stage of the disease is CMT
Neuropathy Score (CMTNS, Shy et al. 2005). CMTNS is used to measure
patient disability in CMT patients and as an outcome measurement in
treatment trials. It is a composite score gathering the results of
symptoms, signs, and neurophysiological tests. Patients are
classified as mild (CMTNS.ltoreq.10), moderate (CMTNS 11-20), or
severe (CMTNS>20) depending of their performances assessed in
nine tests.
[0008] The Overall Neuropathy Limitations Scale (ONLS), though not
specifically designed for CMT, is used to record serial changes in
limitations in a clinical environment and as an outcome measure in
clinical trials for patients suffering from neuropathies. It
measures limitations in the everyday activities of the upper and
lower limbs (Graham and Hughes, 2006).
[0009] The progression of this disease measured by CMTNS or ONLS is
rather slow and necessitates long clinical trials with hundreds of
patients.
[0010] The protein and RNA levels of PMP22 have been recently
proposed as biological markers to follow up such trials
pharmacodynamically as a substitute for CMTNS endpoint in a case of
CMT1A (Li et al., 2005; Meyer zu Horste et al., 2007). Still, such
analysis is tedious and requires invasive procedures. Moreover,
expression of PMP22 in such biopsies is not correlated with
severity of disease (Katona et al., 2009).
[0011] Patrocolo et al., 2009 reports elevated total cholesterol
and triglyceride levels in a patient with Autosomal Dominant
Hereditary Motor Sensory Neuropathy with Proximal Dominant
Involvement (HMSN-P).
[0012] Yao et al., 1978 studies the distribution of specific
fractions of cholesteryl esters in patients having hereditary
neuropathies. This document provides no information regarding free
cholesterol or LDL cholesterol levels.
[0013] Swartz et al., 1988 concerns immunogenicity of cholesterol
and production of monoclonal IgM complement-fixing antibodies to
cholesterol.
[0014] Niebroj-Dobosz et al., 1976 concerns patterns of different
lipid fractions in neuropathic patients (such as total lipids,
total phospholipids, free fatty acids or cholesterol esters). The
authors conclude that there is no correlation between the type of
lipid pattern changes and the clinical syndrome.
[0015] The availability of easily detectable biological markers
would permit rapid diagnosis of functionally relevant forms of CMT
and related diseases, clinical testing of efficacy of new
medications and monitoring the individual response of patients to
drug treatment and disease management.
SUMMARY OF INVENTION
[0016] The purpose of the present invention is to provide novel
methods for detecting predisposition to, or diagnosis and/or
prognosis of CMT disease and related disorders, as well as for
assessing the responsiveness to the treatments and/or the efficacy
of treatments in subjects having CMT or a related disorder.
[0017] As indicated herein, the present invention provides a method
for the diagnosis of CMT and CMT-related diseases. The present
invention also provides methods for aiding in the diagnosis and
sub-classification of neurological disorders, or in patient
stratification steps in clinical trials, including CMT and
CMT-related diseases, by quantifying the amount of lipids, amino
acids, steroid hormones, carbohydrates, metals, arachidonic acid
metabolites, biogenic amines, nucleosides, nucleotides, small
peptides and proteins in a biological fluid sample of the subject,
such as cerebrospinal fluid, serum, saliva, urine, etc. and
comparing the measured amount with a reference value for the
biomarker. These methods can also be applied to quantification of
biomarkers in extracts of biopsies including skin biopsies. The
information thus obtained may be used to aid in the diagnosis, to
diagnose the disease, or to predict potential drug response in the
individual. The biomarkers are differentially present in subjects
having a neurological disease, including CMT and CMT-related
diseases, versus subjects free of the disease.
[0018] One embodiment of the present invention is a method of
diagnosing or assessing the likelihood that a patient is afflicted
with a neurological disease, including CMT, preferably CMT1A, and
CMT-related diseases, the method comprising measuring a level of
complex combination biomarkers of the present invention.
[0019] The present invention more specifically relates to an in
vitro method for detecting the presence or risk of CMT disease in a
mammal, or for aiding in the diagnosis, prognosis or
sub-classification of CMT disease, or in patient stratification
steps in clinical trials, the method comprising determining the
(relative) amount or the presence, absence or alteration of a
target biomarker in a biological fluid sample from the subject,
wherein said amount or alteration is indicative of the presence,
risk, progression or severity of said disease, and wherein said
biomarker is selected from lipids, amino acids, steroid hormones,
metals, metabolites of arachidonic acid, biogenic amines,
carbohydrates, peptides, nucleosides and nucleotides.
[0020] The present invention also relates to an in vitro method for
assessing efficacy of a treatment against CMT in a mammal, the
method comprising determining in a biological fluid sample from the
subject, during the treatment, the (relative) amount or the
presence, absence or alteration of a target biomarker selected from
lipids, amino acids, steroid hormones, metals, metabolites of
arachidonic acid, biogenic amines, carbohydrates, peptides,
nucleosides and nucleotides, and comparing said amount or
alteration to a level of said biomarker determined before treatment
or at an earlier stage of treatment in said mammal, wherein a
deviation is indicative of the efficacy of the treatment.
[0021] The methods of the invention may use one or more target
biomarker(s). In a preferred embodiment, said target biomarker(s)
are selected from cholesterol, alanine, .alpha.-aminobutyric acid,
citrulline, cystine, glutamine, hydroxyproline, lysine, methionine,
proline, threonine, tryptophan, tyrosine, T4 thyroid hormone,
testosterone, iron, LTB4, adrenaline, dopamine and serotonin, or
combinations thereof.
[0022] In another embodiment, said one or more biomarkers are used
in conjunction with at least one additional diagnostic test or
marker for CMT, preferably selected from nucleic acids, proteinous,
physiological, neurophysiological, genetic, behavioral,
electrophysiological, clinical and phenotypical tests or
markers.
[0023] The present invention also relates to a use of one or more
biomarker(s) of the present invention in a method of detecting
predisposition to or diagnosis and/or prognosis of CMT disease in a
mammalian subject.
[0024] A further particular object of the invention is to provide
an in vitro method for predicting the responsiveness to a treatment
of CMT disease of an individual suffering from CMT, the method
comprising: [0025] i) determining the free cholesterol level in a
biological sample from said individual, and [0026] ii) predicting
the responsiveness of said individual to said treatment by
comparing the free cholesterol level obtained in i) to a reference
value of a responder or non-responder group.
[0027] The invention also provides, in a specific embodiment, an in
vitro method for determining the progression of CMT disease in an
individual having CMT, the method comprising: [0028] i) determining
the level of alanine or tryptophan, or both, in a biological sample
from said individual, and [0029] ii) comparing the level of alanine
or tryptophan, or both, obtained in i) to level(s) of alanine
and/or tryptophan, respectively, determined previously in the same
individual, [0030] wherein a change in the level of alanine and/or
tryptophan is indicative of the progression of CMT disease in said
individual.
[0031] The invention also provides a method of partitioning a group
of patients suffering from CMT disease comprising determining the
levels of alanine or tryptophan, or both, in a biological sample
from said patients, wherein said level(s) is/are used to partition
said group of patients as a function of the severity of the
disease.
BRIEF DESCRIPTION OF THE FIGURE
[0032] FIG. 1: Free cholesterol levels in CMT1A patients at
baseline. Before the beginning of treatment with a mix of baclofen,
naltrexone and sorbitol, free cholesterol levels were found to be
significantly lower in patients who turned out to respond to the
treatment, i.e., whose condition was stabilized or improved
(responders, white box) in comparison with those who turned out to
not respond to the treatment, i.e., whose condition worsened after
one year of treatment (non-responders, grey box). (p<0.034,
Welch's t-test, free cholesterol levels of responders significantly
different from non-responders).
DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention provides new diagnostic methods and
tools for CMT and related disorders.
[0034] Within the context of this invention, CMT includes CMT1A,
CMT1B, CMT1C, CMT1D, CMT1X, CMT2A, CMT2B, CMT2D, CMT2E, CMT2F,
CMT2I, CMT2J, CMT2-P0, CMT2K, CMT4A, CMT4B1, CMT4B2, CMT4C, CMT4D,
CMT4F, CMT4, AR-CMT2A, CMT4J or other forms of Charcot-Marie-Tooth
disease. In the most preferred embodiment, CMT is CMT1A.
[0035] Within the context of the present invention, the term
"CMT-related disorder" designates other diseases associated with
neurological symptoms. 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 problems associated with aging,
post-encephalitic Parkinsonism, schizophrenia, depression, bipolar
disease and other mood disorders, Huntington's disease, motor
neuron diseases including amyotrophic lateral sclerosis (ALS),
multiple sclerosis, idiopathic neuropathies, diabetic neuropathy,
toxic neuropathies including neuropathy induced by drug treatments,
neuropathies provoked by HIV, radiation, heavy metal and vitamin
deficiency states, prion-based neurodegeneration, including
Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy
(BSE), GSS, FFI, Kuru and Alper's syndrome.
[0036] The purpose of the present invention is to provide new body
fluid biomarkers for diagnosing or monitoring CMT and related
disorders, and for assessing the responsiveness of subjects or the
efficacy of therapeutic treatments in subjects having CMT or a
related disorder. Thus, according to a preferred embodiment, the
method of the invention comprises the detection of the presence or
absence or (relative) quantity of metabolites in body fluids.
[0037] An object of the invention resides in detecting (in vitro or
ex vivo) the presence of or risk of developing CMT or a related
disorder in a mammal, comprising the determination of the presence,
in a biological sample of the mammal, of an alteration in one or
more selected body fluid biomarkers.
[0038] Another object of the invention resides in a method for
detecting (in vitro or ex vivo) the presence of or risk of
developing CMT or a related disorder in a mammal, comprising the
determination of the presence, in a biological sample of the
mammal, of an alteration of the level in one or more markers, the
presence of such an alteration being indicative of the presence of
or risk of developing CMT in said mammal.
[0039] In a preferred embodiment, a method of the invention is an
in vitro method for detecting the presence or risk of CMT disease
in a mammal, or for aiding in the diagnosis, prognosis or
sub-classification of CMT disease, or aiding in patient
stratification steps in clinical trials, the method comprising
determining the (relative) amount or the presence, absence or
alteration of a target biomarker in a biological fluid sample from
the subject, wherein said amount or alteration is indicative of the
presence, risk, progression or severity of said disease, and
wherein said biomarker is selected from lipids, amino acids,
steroid hormones, metals, metabolites of arachidonic acid, biogenic
amines, carbohydrates, peptides, nucleosides and nucleotides.
[0040] Within the context of the present invention, the term
"alteration" of a target biomarker may designate an increase or a
decrease of the target biomarker quantity in a fluid biological
sample from the subject, in comparison with a control sample or
reference value. Typically, the term "decrease" in relation to a
biomarker level designates a reduction of the concentration or
level of the biomarker in a biological sample from the subject of
at least 5% or 10% or 15% in comparison with a control sample or
reference or mean value. Decreases may be more substantial, such as
a reduction by at least 20% or 30% or 40% or even more. Similarly,
the term "increase" in relation to the biomarker level designates
an augmentation of the concentration or level of the biomarker in a
biological sample from the subject of at least 5% or 10% or 15% in
comparison with a control sample or reference or mean value.
Increases may be more substantial, such as increases by at least
20% or 30% or 40% or even more.
[0041] Preferred types of alterations are disclosed below for each
biomarker in Table A. This table indicates, for each biomarker,
whether an increase or a decrease is indicative of CMT in human
subjects. A distinction between male and female patients is also
provided.
TABLE-US-00001 TABLE A Increase (+) or decrease (-) of a biomarker
concentration in CMT patients total female male Adrenaline -
Alanine - - Alpha Amino Butyric acid - Citrulline - Cystine -
Dopamine + Free cholesterol - - - Glutamine - - Hydroxyproline - -
Iron + LDL cholesterol - LTB4 - Lysine - Methionine + Proline +
Serotonin + T4 - - Testosterone + Threonine - Tryptophan + +
Tyrosine -
[0042] Specific examples of alterations of each target biomarker(s)
according to the invention are shown in Tables 1-4 of the
experimental part.
[0043] Another embodiment of the present invention comprises
qualifying and sub-classifying a CMT disease, for example CMT1A,
CMT1B, CMT1C, CMT1D, CMT1X, CMT2A, CMT2B, CMT2D, CMT2E, CMT2F,
CMT2I, CMT2J, CMT2-P0, CMT2K, CMT4A, CMT4B1, CMT4B2, CMT4C, CMT4D,
CMT4F, CMT4, AR-CMT2A, CMT4J or other forms of Charcot-Marie-Tooth
disease or CMT-related disorders in a subject, comprising measuring
sets of complex biomarkers of the present invention.
[0044] In other aspects, methods of the present invention further
comprise the step of managing the individual treatment. For
example, if measurement of the set of biomarkers correlates with
the presence of clinical subtype CMT disease, then managing
treatment comprises administering a matched drug or drug
combination to slow or revert the progression of the disease.
Further measurements can be compared to the previous measurements
or the standard to monitor the progression of the disease.
[0045] In another aspect of the invention, the method further
comprises measuring the biomarker after treatment has begun, to
monitor the progression of the disease.
[0046] In another embodiment, the method of the present invention
comprises monitoring the progression of CMT, preferably CMT1A, and
measuring a level of sets of biomarkers of the present
invention.
[0047] Another object of the invention relates to a method to
evaluate or follow the response to a treatment for CMT in a
subject, the method comprising a step of measuring the level of one
or more markers, the presence of such an alteration before and/or
during the treatment, and a comparison of the level thus measured
with that measured at a former stage of the treatment or before
treatment.
[0048] Another object of the invention relates to a method to
evaluate or follow the response to a treatment of CMT in a subject,
the method comprising a step of measuring the amount of one or more
selected body fluid biomarkers before and/or during the treatment,
and a comparison of the amount thus measured with that measured at
a former stage of the treatment or before treatment.
[0049] The level of the biomarker(s), measured according to the
method of the present invention, is correlated with neurological
disease, preferably CMT disease. In preferred embodiments, this may
be accomplished by comparing the measured amount to a reference
value for the biomarker(s). The reference value can be obtained by
measuring an amount of the biomarker(s) in age-matched control
subjects that are not affected by the disease, or that are free of
the disease.
[0050] Another object of the invention relates to an in vitro
method for assessing efficacy of a treatment against CMT in a
mammal, the method comprising determining in a biological fluid
sample from the subject, during the treatment, the (relative)
amount or the presence, absence or alteration of a target biomarker
selected from lipids, amino acids, steroid hormones, metals,
metabolites of arachidonic acid, biogenic amines, carbohydrates,
peptides, nucleosides and nucleotides, and comparing said amount or
alteration to a level of said biomarker determined before treatment
or at an earlier stage of treatment in said mammal, wherein a
deviation is indicative of the efficacy of the treatment.
[0051] Another embodiment of the present invention comprises
monitoring the efficacy of a treatment method of CMT, comprising
measuring a level of a complex set of biomarkers of the present
invention. In embodiments, the efficacy of treatment is measured by
monitoring levels of the biomarkers in the subject compared to a
reference, and/or compared to other previous tests of the subject
or to an earlier stage of treatment/disease in the subject.
[0052] Another object of the invention relates to an improvement in
methods of treating CMT or related disorders, the improvement
consisting of measuring the level of expression of one or,
preferably, several biomarkers before and/or during the treatment.
The measurement of the level of biomarker expression makes it
possible to adapt the treatment according to the evolution of
pathology and/or efficacy of the treatment.
[0053] In a preferred embodiment, diagnosing or monitoring CMT and
related disorders comprises the determination of the quantity (or
the presence or absence), in a biological sample of the mammal, of
said body fluid biomarker(s) selected from lipids, amino acids,
steroid hormones, carbohydrates, metals, metabolites of arachidonic
acid, biogenic amines, nucleosides, nucleotides, small peptides and
proteins.
[0054] In a preferred embodiment, the method of the invention
comprises the determination of the quantity (or the presence or
absence), in a biological fluid sample of the mammal, of one or
more body fluid biomarkers, wherein said body fluid biomarkers are
selected from: [0055] lipids, preferably cholesterol and its
metabolites, including dehydroepiandrosterone (DHEA), and including
more preferably free cholesterol or LDL cholesterol, or their
amount in regard to total cholesterol, [0056] amino acids or their
derivatives, preferably including alanine, a amino butyric acid,
citrulline, cystine, glutamine, hydroxyproline, lysine, methionine,
proline, threonine, tryptophan, tyrosine, arginine, asparagine,
aspartic acid, glutamic acid, glycine, histidine, 1-methyl
histidine, isoleucine, leucine, ornithine, phenylalanine, serine,
taurine and valine, [0057] steroid hormones and their precursors or
derivatives, preferably including T3 and T4 thyroid hormones,
testosterone, 5.alpha.-dihydroprogesterone, allopregnanolone and
corticosterone, [0058] metals, preferably iron and zinc, [0059]
metabolites of arachidonic acid, preferably including leukotrienes
(e.g., LTB4/5), prostaglandin PGE2, prostacyclin PGI2 and
tromboxanes TXA2 and TXB2, [0060] biogenic amines, preferably
including adrenaline, dopamine and serotonin, [0061] carbohydrates,
preferably sorbitol, [0062] nucleotides, preferably 3'-5'-cyclic
adenosine monophosphate (cAMP), and [0063] any combination
thereof.
[0064] More preferably, said body fluid biomarkers are selected
from: [0065] lipids, preferably cholesterol and its metabolites,
including free cholesterol or LDL cholesterol, or their amount in
regard to total cholesterol, [0066] amino acids or their
derivatives, preferably including alanine, a amino butyric acid,
citrulline, cystine, glutamine, hydroxyproline, lysine, methionine,
proline, threonine, tryptophan and tyrosine, [0067] steroid
hormones, preferably including T4 thyroid hormone and testosterone,
[0068] metals, preferably iron, [0069] metabolites of arachidonic
acid, preferably including leukotrienes (e.g., LTB4/5), [0070]
biogenic amines, preferably including adrenaline, dopamine and
serotonin, and [0071] any combination thereof.
[0072] In another preferred embodiment, the method of the invention
comprises the determination of the quantity (or the presence or
absence), in a biological fluid sample of the mammal, of one or
more body fluid biomarkers, wherein said body fluid biomarkers are
selected from: [0073] cholesterol metabolites, preferably including
an ester of cholesterol, 27-hydroxycholesterol, pregnenolone,
pregnenolone sulfate, and dehydroepiandrosterone sulfate (DHEAS),
[0074] steroid hormones and their precursors or derivatives,
preferably including cortisol, cortisone, aldosterone,
androstanediol, androstenedione, estradiol and estrone, [0075]
metabolites of arachidonic acid, preferably including
prostaglandins PGD2 and PGF2.alpha., 12-hydroxyeicosatetraenoic
acid (12-HETE) and lipoxins (LXA4 and LXB4), [0076] inositol and
its derivatives, preferably including inositol monophosphates,
phosphatidylinositol 3-phosphate [PI3P] and phosphatidylinositol
(3,5)-bi-phosphate [PI(4,5)P2], [0077] sphingolipids or
phospholipids or their derivatives, preferably including
lysophosphatidic acid, phosphatidic acid and
sphingosine-1-phosphate (SIP), [0078] endocannabinoids, preferably
including arachidonoylethanolamine, 2-arachidonoyl glycerol,
2-arachidonyl glyceryl ether, N-arachidonoyl-dopamine and
virodhamine, and [0079] any combinations thereof.
[0080] In another preferred embodiment, a method of the invention
comprises determining in a biological fluid sample from the subject
the (relative) amount or the presence, absence or alteration of a
target biomarker selected from cholesterol, alanine,
.alpha.-aminobutyric acid, citrulline, cystine, glutamine,
hydroxyproline, lysine, methionine, proline, threonine, tryptophan,
tyrosine, T4 thyroid hormone, testosterone, iron, LTB4, adrenaline,
dopamine and serotonin, as well as any combinations thereof.
[0081] In a preferred embodiment, the biomarker used in the
invention is or comprises at least cholesterol, more preferably
free cholesterol, and/or LDL cholesterol and/or their amount in
regard to total cholesterol. Within the context of the present
invention the term "LDL cholesterol" designates all forms of
cholesterol contained in LDL, including non-esterified
cholesterol.
[0082] As shown in the experimental part, the inventors have
surprisingly discovered that the level of free cholesterol or the
level of LDL cholesterol decreases in diseased animals.
[0083] Thus, in the most preferred embodiment, the method of the
invention comprises determining a decrease of free cholesterol
and/or LDL cholesterol and/or their amount in regard to total
cholesterol in a biological fluid sample from the subject, wherein
said decrease of free cholesterol and/or LDL cholesterol and/or
their amount in regard to total cholesterol is indicative of the
presence, risk, progression or severity of the disease.
[0084] In a particular embodiment, the method of the invention
comprises determining in a biological fluid sample from the subject
a decrease of the ratio of free cholesterol to total
cholesterol.
[0085] In another particular embodiment, the method of the
invention comprises determining a decrease of the ratio of LDL
cholesterol to total cholesterol.
[0086] As indicated, the method may comprise the determination of
several biomarkers, e.g., 2, 3, 4, 5 or even more. These may be
determined simultaneously or sequentially in a biological fluid
sample.
[0087] In a particular variant, the presence or the absence or the
(relative) quantity of at least three biomarkers is determined
simultaneously or sequentially in a biological fluid sample from
the mammalian subject.
[0088] In another embodiment, the method of the invention comprises
the determination of the presence or the absence or the (relative)
quantity, in a biological sample of the mammal, of at least four
distinct biomarkers.
[0089] In another embodiment, the sets of biomarkers used in
methods of the invention are selected from Table 5.
[0090] In a preferred embodiment, the sets of biomarkers comprise:
[0091] free cholesterol and alanine; [0092] free cholesterol and T4
and tryptophan and hydroxyproline; [0093] free cholesterol and
hydroxyproline; [0094] free cholesterol and T4 and tryptophan;
[0095] free cholesterol and T4 and serotonin; [0096] free
cholesterol and T4 and hydroxyproline; [0097] free cholesterol and
T4; or [0098] free cholesterol and serotonin.
[0099] As illustrated in the examples, such sets of biomarkers are
particularly efficient in predicting the presence of CMT disease.
In particular, the results depicted in the examples show
performances of 100% in training tests and between 78% and 100% in
validation tests for these sets of biomarkers.
[0100] The level of said biomarker(s) may be determined by any
methods known per se in the art, such as, without limitation,
immunological methods, biochemical methods, chromatographic
methods, enzymatic methods, cell-based assays, in vitro tests, etc.
Examples of suitable methods are disclosed in the experimental
section. The level of biomarker(s) determined may be compared to a
reference value, a control, or a mean value, wherein a deviation
from said value is indicative of the presence, risk, progression or
severity of CMT. The deviation should typically be greater than 5%,
more preferably 10%, even more preferably 15%.
[0101] Another aspect of the invention relates to a use of one or
more biomarker(s) selected from cholesterol, alanine,
.alpha.-aminobutyric acid, citrulline, cystine, glutamine,
hydroxyproline, lysine, methionine, proline, threonine, tryptophan,
tyrosine, T4 thyroid hormone, testosterone, iron, LTB4, adrenaline,
dopamine and serotonin in a method of detecting predisposition to
or diagnosis and/or prognosis of CMT disease in a mammalian
subject.
[0102] Also, as illustrated in the experimental part, the inventors
have found that within a diseased population suffering from CMT,
free cholesterol levels are also predictive of the responsiveness
of a patient to a treatment for CMT. More particularly, the
invention shows that, in a population of CMT patients that responds
to a treatment with baclofen, naltrexone and sorbitol, free
cholesterol levels are significantly lower than in a population
that does not respond to such treatment.
[0103] Thus, in another preferred embodiment, the invention relates
to an vitro method comprising identifying a patient more likely to
respond to a treatment of CMT by measuring the free cholesterol
level in said patient. Preferably, the method comprises comparing
said free cholesterol level determined in a patient to a reference
value of a non-responder or responder group, wherein said
comparison allows determining the likelihood that the patient is a
responder or non-responder. In a more particular embodiment, the
method comprises comparing said free cholesterol level determined
in a patient to a reference value of a non-responder group, wherein
a significantly lower level of free cholesterol when compared to
said reference value is indicative that the patient shall respond
to the treatment.
[0104] As shown in the experimental part, the reference value of
free cholesterol (in plasma) in a responder group is typically
comprised between 446 and 520 .mu.g/mL, more particularly between
446 and 483 .mu.g/mL, even more particularly less than 483
.mu.g/mL.
[0105] Accordingly, in a particular embodiment, the in vitro method
comprises determining the free cholesterol level of said individual
from a plasma sample obtained from the individual, wherein a free
cholesterol level in said patient comprised between 446 and 520
.mu.g/mL, more preferably between 446 and 483 .mu.g/mL, even more
preferably below 483 .mu.g/mL, is indicative that the patient shall
respond to said treatment. In a further embodiment, the in vitro
method comprises comparing the plasma free cholesterol level of
said patient with a reference value of a responder group with
similar age, sex, condition, and/or any ongoing treatment (e.g.,
statin treatment).
[0106] As shown in the experimental part, the reference value of
free cholesterol (plasma free cholesterol) in a non-responder group
is typically greater than 520 .mu.g/mL, even more particularly
greater than 578 .mu.g/mL.
[0107] Thus, in another embodiment, the in vitro method comprises
determining the free cholesterol level in the plasma of said
patient, wherein a free cholesterol level greater than 520
.mu.g/mL, even more preferably greater than 578 .mu.g/mL, is
indicative that the patient will not respond to said treatment.
[0108] In a particular embodiment, the method allows determining
responsiveness to a treatment of CMT comprising co-administering
baclofen, naltrexone and sorbitol, or salts thereof, to said
patient.
[0109] In a particular embodiment, the term "significantly lower"
designates a level that is at least 5% lower, more preferably at
least 10% lower, even more preferably at least 15% or more lower
than the reference value.
[0110] In a particular embodiment, the biological fluid sample is a
blood sample of the subject, preferably a plasmatic fraction from
said subject.
[0111] In a particular embodiment, the above biomarkers are used in
a method of detecting predisposition to, diagnosis and/or prognosis
of CMT disease, or aiding in patient stratification steps in
clinical trials, in conjunction with at least one additional
diagnostic test or marker for CMT, selected preferably from
proteinous, physiological, neurophysiological, genetic, behavioral,
electrophysiological, clinical and phenotypical tests or
markers.
[0112] In another particular embodiment, the level of said
biomarker(s) used in a method of detecting predisposition to or
diagnosis and/or prognosis of CMT disease, or aiding in patient
stratification steps in clinical trials, is compared to a reference
value wherein the deviation from said value is indicative of the
presence, risk, progression or severity of CMT.
[0113] In more particular embodiments, said biomarkers used in a
method for aiding in patient stratification steps in clinical
trials or evaluating progression or severity of CMT comprise at
least tryptophan (trp) and/or alanine (ala).
[0114] As shown in the experimental part, the inventors have
surprisingly discovered that the variation of trp and/or alanine
level(s) in CMT patients is correlated with the severity of the
disease as determined by electrophysiological clinical tests or
functional measures.
[0115] Accordingly, in a particular embodiment, the invention
resides in an in vitro method for determining the progression of
CMT disease in an individual having CMT, the method comprising:
[0116] i) determining the level of alanine or tryptophan, or both,
in a biological sample from said individual, and [0117] ii)
comparing the level of alanine or tryptophan, or both, obtained in
i) to level(s) of alanine or tryptophan, respectively, determined
previously in the same individual, [0118] wherein a change in the
level of alanine and/or tryptophan is indicative of the progression
of CMT disease in said individual.
[0119] In a preferred embodiment, the method of the invention
comprises determining an increase of trp and/or ala level(s) in a
biological fluid sample from the subject in regard with previously
determined level(s) in said patient, wherein said increase of trp
and/or ala level(s) is indicative of an improvement of CMT
disease.
[0120] In a particular embodiment, determining a level of trp
and/or ala levels in a biological fluid sample from the subject is
made in conjunction with at least one additional diagnostic test or
marker for CMT, selected preferably from proteinous, physiological,
neurophysiological, genetic, behavioral, electrophysiological,
clinical and phenotypical tests.
[0121] In a preferred embodiment, determining the level of trp
and/or ala level(s) in a biological fluid sample from the subject
is made in conjunction with the determination of CMTNS and/or ONLS
which are known to those skilled in the art.
[0122] In another preferred embodiment, determining the level of
trp and/or ala level(s) in a biological fluid sample from the
sample is made in conjunction with one or more of the assessments
that compose CMTNS and/or ONLS which are well known to those
skilled in the art.
[0123] In another preferred embodiment the method of the invention
comprises determining the level(s) of trp and/or ala in biological
fluid samples from a group of patients wherein said level(s) is/are
used to partition said group of patients as a function of the
severity of the disease.
[0124] In a particular embodiment the biological fluid sample is a
blood sample of the subject, preferably plasma or a plasma fraction
from said subject.
[0125] As shown in the experimental section, trp and/or ala
level(s) is/are surprisingly found to vary as a function of the
presence of a treatment of the disease which has been found to be
effective in treating CMT. Indeed, in the course of a clinical
trial for evaluating a mix of baclofen, naltrexone and sorbitol as
a treatment for CMT disease, the inventors found that the level(s)
of trp and/or ala were significantly higher in the treated
population of patients when compared to patients administered with
placebo, and this as soon as 3 months after the beginning of the
treatment, whereas a disease evolution on such a short period
cannot be determined using either CMTNS or ONLS.
[0126] In this regard a preferred embodiment of the invention is a
method for assessing the response or responsiveness to a treatment
of CMT, the method comprising measuring an increase of the level(s)
of trp and/or ala in a biological fluid sample from a subject
undergoing a treatment for CMT in regard to previously determined
levels in said subject before the beginning of, or at an earlier
stage of, said treatment, wherein said increase is indicative of a
response to said treatment.
[0127] In a particular embodiment the time interval between the two
measures is 2 months or more, preferably 2, 3, or 4 months.
[0128] In a more particular embodiment of the invention the method
for assessing the response to a treatment of CMT comprises
measuring an increase of level(s) of trp and/or ala in a biological
fluid sample from a subject undergoing a treatment for CMT for 3
months, in regard to previously determined levels before the
beginning of said treatment.
[0129] Typically, an increase in trp and/or ala level(s) greater
than 5%, more preferably 10%, even more preferably 15% is
indicative of a response to a treatment.
[0130] In an even more preferred embodiment the above method is
used to assess the response to a combination treatment for CMT
comprising the administration of baclofen, naltrexone and
sorbitol.
[0131] In particular embodiment, any of the above mentioned body
fluid biomarkers, or their combinations, can be used in conjunction
with at least one additional diagnostic test or marker for CMT,
preferably selected from nucleic acids, proteinous, physiological,
neurophysiological, genetic, behavioral, electrophysiological,
clinical and phenotypical tests or markers.
[0132] Said proteinous biomarkers, detectable in body fluids, which
can be used for diagnosis of CMT, for monitoring the progression of
CMT, or for monitoring the efficacy of CMT-relevant drugs, include
NEFH neurofilament, p75/LNGFR nerve growth factor receptor, NTRK3
receptor, SCIP transcription factor, cyclin D1,
lysosomal-associated membrane protein LAMP1, ATG7 autophagy related
7 homolog, proteasome activator subunits PSME1/2, PSMA1 proteasome
subunit, ITGB1/4 integrins, insulin-like growth factor 1 (IGF1),
insulin-like growth factor binding proteins 1/2/5 (IGFBP1/2/5),
vitronectin (VTN), tenascins (TNC/R/XB), SCN10A voltage-gated
sodium channel, KCNC1 potassium voltage-gated channel, aldose
reductases including AKR1B1, sorbitol dehydrogenase (SORD),
inositol(myo)-1(or 4)-monophosphatases IMPA1/2, ADP-ribosylation
factor 6 (ARF6), calnexin (CANX), growth factors FGF2, PDGFA/B/C,
VEGFA/B/C and TGFB1/2, neuregulins including NRG1, matrix
metallopeptidase 2/9, tissue and urokinase plasminogen activators
PLAT and PLAU, monocyte chemoattractant protein-1 (CCL2), leukemia
inhibitory factor (LIF), interleukin 6, transferrin, and endogenous
opioids POMC, PENK and PDYN as well as smaller peptides and other
derivatives produced by metabolism of the above-mentioned
molecules.
[0133] Additional protein biomarkers useful for diagnosis of
Charcot-Marie-Tooth disease (CMT), for monitoring the progression
of CMT, or for monitoring the efficacy of CMT-relevant drugs can be
selected from peripheral myelin protein 22 PMP22, ciliary
neurotrophic factor CNTF, fatty acid elongase ELOV16, glypican
GPC3, myosins MYO1B/1G, phosphoprotein enriched in astrocyte PEA15,
calcium binding proteins S100A3/4, troponins TNNT1/3 and ferritin
FTH1 as well as smaller peptides and other derivatives produced by
their metabolism.
[0134] Further protein biomarkers detectable in body fluids and
useful for diagnosis of Charcot-Marie-Tooth disease, for monitoring
the progression of CMT, or for monitoring the efficacy of
CMT-relevant drugs can be selected from proteins or smaller
peptides and their derivatives encoded by ATP1A1, FGL2, ACAT2,
ACTN2, AK1, ANK3, ANXA1, APOD, CD151, CD24A, CD9, CD99, CETN2,
CHN1, CLIC4, COL1A1/2, COL2A1, COL3A1, COL4A1, CRYAB, CTSC, CYBSB,
CYB561, DEAF1, EMID1, EPB4.1L2, EZR, FASN, FBLN2, FDFT1, FHL1, FOS,
GAPD, GATM, HBA1, HBB, IGF2, ITIH5, KIT, LGALS1, LPL, LXN, MAPK3,
MFGE8, MGLL, MMP12, MRAS, MSLN, MTAP1B, NECL1, NPR3, ODF2, OGN,
OLFM1, PCOLCE, PMM1, PROS1, PYGM, RAB2, RAP1GDS1, SERPINE2, SH3GL3,
SIRT2, SPP1, TPM1/2, TUBA2 and UCHL1 genes.
[0135] The above groups of genes (or the corresponding proteins or
ligands) represent valuable biomarkers which may be used, alone or
in various combinations, to diagnose CMT or related disorders.
[0136] In still another aspect, the present invention provides a
kit comprising a solid support comprising at least one capture
agent attached thereto, wherein the capture agent binds or reacts
with one or more component(s) of the biomarker protein complex of
the present invention.
[0137] In a preferred embodiment, the kit of the invention
comprises a solid support comprising at least one capture agent
attached thereto, wherein the capture agent binds or reacts with at
least one biomarker selected from cholesterol, alanine,
.alpha.-aminobutyric acid, citrulline, cystine, glutamine,
hydroxyproline, lysine, methionine, proline, threonine, tryptophan,
tyrosine, T4 thyroid hormone, testosterone, iron, LTB4, adrenaline,
dopamine and serotonin. In a preferred embodiment, the kit of the
invention comprises at least one compound binding to or reacting
with at least one biomarker selected from cholesterol, alanine,
.alpha.-aminobutyric acid, citrulline, cystine, glutamine,
hydroxyproline, lysine, methionine, proline, threonine, tryptophan,
tyrosine, T4 thyroid hormone, testosterone, iron, LTB4, adrenaline,
dopamine and serotonin for the diagnosis, prognosis and/or for
assessing the efficacy of a treatment or following the evolution of
CMT1A disease.
[0138] The method of the invention is applicable to any biological
sample of the mammal to be tested, in particular any sample
comprising metabolites. Examples of such samples include blood,
plasma, serum, saliva, urine, feces, tissue biopsy, etc. The sample
can be obtained by any technique known in the art, for example by
collection using, e.g., non-invasive techniques, or from
collections or banks of samples, etc. The sample can in addition be
pretreated to facilitate the accessibility of the target molecules,
for example by lysis (mechanical, chemical, enzymatic, etc.),
purification, centrifugation, separation, etc.
[0139] The invention is applicable to any mammal, preferably to a
human.
[0140] Further aspects and advantages of this invention will be
disclosed in the following experimental section, which shall be
considered as illustrative only.
EXAMPLES
[0141] I. Identification of New Markers and Quantitation of
Biomarkers
[0142] The invention discloses biomarkers of body fluids useful for
the diagnosis, prognosis and/or for assessing the efficacy of a
treatment or following the evolution of CMT disease.
I.1 CMT1A Transgenic Rat Model and Serum Sample Collection
[0143] The CMT transgenic rat model is a hemizygous PMP22
transgenic rat bearing three additional copies of mouse PMP22 gene
and showing signs of demyelination in peripheral and cranial nerves
(Sereda et al., 1996; Grandis et al., 2004). This CMT rat model is
a good approximation of human CMT1A disease from a clinical point
of view. Furthermore, the CMT rats already served as a model for an
experimental CMT1A therapy (Meyer zu Horste et al., 2007).
[0144] Inventors have looked for small molecules showing
differential levels in wild-type and transgenic rats, thus
constituting relevant biomarkers for CMT disease.
[0145] Except when otherwise specified, CMT1A model rats, four
months old, are anaesthetized with ketamine (Imalgene) 100 mg/kg,
ip. Blood is collected by cardiac puncture in two different tubes:
[0146] in one sterilized blood collection tube for coagulation;
serum is collected and stored at -80.degree. C.; and [0147] in one
EDTA RNAse free tube; after centrifugation (+4.degree. C.; 1260 g;
10 min), plasma is stored at -80.degree. C.
1.2 Quantitation Methods
[0148] Free Cholesterol
[0149] Cholesterol was first extracted from samples with heptanes.
Free cholesterol was further analyzed using a method adapted from
Dong et al. (2007): cholest-4-en-3,6-dione formed from the
oxidation of non-esterified cholesterol by the Jones oxidation was
measured by HPLC/UV analyses. Stigmasterol was used as an internal
standard.
[0150] Sorbitol
[0151] Proteins are first precipitated with ethanol. Sorbitol is
analyzed mainly according the Dionex No. 20 technical note, using
anion exchange chromatography coupled with an electrochemical
detector.
[0152] Metals
[0153] Quantitation of iron and zinc was performed by ICP/MS after
mineralization of serum samples.
[0154] Arachidonic Acid Metabolites
[0155] Enzyme ImmunoAssays (ETA) kits from Cayman Chemical were
used to analyze:
Prostaglandin E.sub.2 (ref 514010)
Leukotriene B.sub.4 (ref. 520111)
Thromboxane B.sub.2 (ref. 519031)
[0156] 6-keto Prostaglandine F.sub.1 (ref. 515211)
[0157] Cyclic Adenosine Monophosphate
[0158] After precipitation of plasma proteins with ethanol, cAMP is
analyzed with an EIA kit from Cayman Chemical (ref 581001)
according the manufacturer's instructions.
[0159] Catecholamines
[0160] A solid phase extraction (SPE) was performed to concentrate
and purify the samples. Adrenaline, dopamine, and
serotoninserotonin were further analyzed by ion pair
chromatography.
[0161] Amino Acids
[0162] Plasma proteins were precipitated with sulfosalicylic acid
prior to analyzes. Derivatized amino acid quantitation was
performed with a spectrophotometer after an automated cation
exchange chromatography process.
[0163] Thyroid Hormones
[0164] Prior to the precipitation of plasma proteins with methanol,
an internal standard was added to samples. Triiodothyronine (T3)
and thyroxine (T4) were then quantified by an HPLC coupled to
LC-MS/MS mainly according to Soukhova et al. (2004).
[0165] Neurosteroids
[0166] CMT1A model rats, four months old, were decapitated and
blood was collected in two different tubes: [0167] in one
sterilized blood collection tube for coagulation; serum was
collected and stored at -80.degree. C.; and [0168] in one EDTA
RNAse free tube; after centrifugation (+4.degree. C.; 1260 g; 10
min), plasma was stored at -80.degree. C.
[0169] Prior to analysis, plasma proteins were precipitated and
neurosteroids were then purified and concentrated by SPE.
Neurosteroids were further chemically derivatized with either
2-hydrozino-1-methylpyridine (to lower the detection threshold)
(Higashi et al., 2005) or picolinic acid (Yamashita et al., 2007).
According to the derivatization method, internal standard is
.sup.2H testosterone or .sup.2H 3.alpha.-androstanediol. HPLC
analysis of neurosteroids was coupled to a mass spectrometer.
Searched neurosteroids and derivatives were aldosterone,
pregnenolone sulfate, allopregnanolone, progesterone,
5.alpha.-dihydroprogesterone (DHP), 3.alpha.-androstanediol,
testosterone, 5a dihydrotestosterone, DHEA and corticosterone.
[0170] Estrogens
[0171] Blood is collected as above. As for neurosteroids, estrone
and estradiol are extracted from the sample with ethyl acetate
prior to derivatization with picolinic acid purification and a
concentration and purification step by SPE. .sup.2H-estrone and
.sup.2H-17.beta.-estradiol are used as internal standards.
1.3 Results
[0172] Duplicate samples were analyzed for each biomarker.
Statistical analysis (Student's t-test, bilateral, type 3)
comparing WT rats versus CMT1A (transgenic) rats was performed.
Results are summarized in the three tables below. These tables
report the mean level of biomarkers which present a notable
difference (P<0.2) between WT and CMT1A rats in male and female
(Table 1), in only male (Table 2) and in only female (Table 3).
[0173] The analysis of biomarkers has revealed that plasmatic free
cholesterol level is significantly decreased in CMT1A male (P=0.05)
and female (P=0.06) rats compared to WT rats. In females, our
results displayed a significant decrease of alpha-aminobutyric acid
(P=0.019), glutamine (P=0.025) and tyrosine (P=0.03) plasmatic
levels versus controls.
[0174] Our results also show a significant decrease of the
following biomarkers: alanine, cystine, glutamine, hydroxyproline,
threonine, T4 thyroid hormone, citrulline, LTB4, adrenaline, and
lysine; and a significant increase of the following biomarkers:
tryptophan, testosterone, dopamine, serotonin, iron, methionine,
and proline (Tables 1, 2 and 3).
1.4 Fluid Biological Samples Collection and Quantification
Methods
[0175] Biomarkers of the invention can be easily quantified from
other biological fluids. As an example quantitation from saliva
samples is described by Karjalainen et al. (2007) for cholesterol,
and by Syrjanen et al. (1990) for glutamine and tyrosine. Likewise,
those small molecules can be quantified in urine as described
elsewhere for cholesterol (Cenedella et al., 1981) and for amino
acids (Venta et al., 2001).
TABLE-US-00002 TABLE 1 WT TG biomarkers MEAN s.e.m. MEAN s.e.m. P
Free cholesterol (.mu.g/ml) 144.82 4.18 118.45 3.96 0.0004 Alanine
(.mu.mol/l) 659.67 44.19 523.50 46.31 0.059 Cystine (.mu.mol/l)
15.50 2.85 8.67 2.51 0.103 Glutamine (.mu.mol/l) 792.00 60.46
694.50 18.11 0.174 Hydroxyproline (.mu.mol/l) 54.00 4.41 41.33 6.31
0.135 Threonine (.mu.mol/l) 295.00 20.30 258.17 15.76 0.184
Tryptophan (.mu.mol/l) 79.83 4.76 93.00 3.94 0.060 Dopamine (ng/ml)
0.30 0.02 0.41 0.07 0.195 Serotonin (ng/ml) 112.50 22.82 406.05
133.26 0.079 T4 (ng/ml) 52.57 4.26 42.64 3.11 0.092 Iron (.mu.g/ml)
4.77 0.45 7.03 1.24 0.134
TABLE-US-00003 TABLE 2 male WT TG biomarkers MEAN s.e.m. MEAN
s.e.m. P Free cholesterol (.mu.g/ml) 146.11 7.80 120.74 6.78 0.050
Citrulline (.mu.mol/l) 109.00 9.29 92.00 3.61 0.201 Methionine
(.mu.mol/l) 49.67 2.73 55.67 1.76 0.150 Proline (.mu.mol/l) 224.67
19.54 265.00 14.50 0.179 Tryptophan (.mu.mol/l) 84.33 5.24 99.00
4.93 0.111 Tyrosine (.mu.mol/l) 87.00 5.13 98.67 3.93 0.150
Testosterone (ng/ml) 1.81 0.40 3.37 0.85 0.201 T4 (ng/ml) 59.23
3.21 48.84 2.00 0.063
TABLE-US-00004 TABLE 3 female WT TG biomarkers MEAN s.e.m. MEAN
s.e.m. P Free cholesterol (.mu.g/ml) 143.54 4.41 116.16 4.88 0.006
LTB4 (pg/ml) 421.46 43.75 335.67 26.36 0.184 Alanine (.mu.mol/l)
660.33 46.83 551.33 44.86 0.168 Alpha Amino Butyric acid 13.33 1.33
6.67 0.88 0.019 (.mu.mol/l) Glutamine (.mu.mol/l) 905.33 46.94
687.33 38.84 0.025 Hydroxyproline (.mu.mol/l) 49.00 6.66 29.00 2.52
0.081 Lysine (.mu.mol/l) 510.67 25.96 419.00 22.50 0.057 Tyrosine
(.mu.mol/l) 80.00 2.65 56.33 5.36 0.030 Adrenaline (ng/ml) 7.99
0.76 5.97 0.58 0.107
[0176] II. Identification and Quantitation of Other
Cholesterol-Related Biomarkers
II.1 CMT1A Transgenic Rat Model and Serum Sample Collection
[0177] The CMT transgenic rat model and sample collection are the
same as described above (see section I.1).
II.2 Cholesterol Quantitation Methods
[0178] Total Cholesterol
[0179] Total cholesterol has been determined by an enzymatic assay
with ABX Pentra
[0180] Cholesterol CP kit (Horiba). The cholesterol is consumed by
cholesterol esterase and cholesterol oxidase in a color-forming
reaction where the color produced is proportional to the amount of
the total cholesterol present in the sample.
[0181] LDL Cholesterol
[0182] LDL cholesterol has been determined by an enzymatic assay
with ABX Pentra LDL Direct CP kit (Horiba). The method is in a
two-reagent format and depends on the properties of the detergents
used. The first detergent solubilizes all the non-LDL lipoprotein
particles. The cholesterol released is consumed by cholesterol
esterase and cholesterol oxidase in a non-color-forming reaction.
The second detergent solubilizes the remaining LDL particles and a
chromogenic coupler allows for color formation. The enzyme reaction
in the presence of the coupler produces color that is specifically
proportional to the amount of LDL cholesterol present in the
sample.
II.3 Results
[0183] Results presented in Table 4 below were extracted from
independent assays and analysed with a bilateral Student's t-test
comparing 20 WT rats versus 19 CMT1A (transgenic) rats.
TABLE-US-00005 TABLE 4 WT TG Biomarkers MEAN s.e.m MEAN s.e.m P
Total cholesterol 1.81 0.07 1.77 0.05 0.66129 LDL cholesterol 0.26
0.02 0.21 0.01 0.03951
[0184] Our results show that LDL-cholesterol level is significantly
decreased (P=0.039) in CMT1A rats (TG) compared to WT rats while
total cholesterol level isn't significantly modified.
LDL-cholesterol level is very easily quantifiable with commonly
used detection kits.
Correlation of Biomarker Concentration with Results from Behavioral
Tests, Histology, Gene Expression and Electrophysiology
[0185] Motor performance and muscular strength, Sensory Nerve
Action Potentials (SNAP), axonal diameter distribution and myelin
sheath of fixed sciatic nerve, fiber content in fixed muscles and
pmp22 mRNA expression were compared with biomarker amounts measured
in biological fluids. The test used is a test of linear association
between paired samples using Pearson's product moment correlation.
It is a unilateral test and a significance threshold of 0.05 is
applied on p-values.
[0186] Such analysis demonstrates that the levels of the biomarkers
of the invention are correlated with some of the above-mentioned
behavioral tests, histology, PMP22 gene expression and
electrophysiology, confirming thereby the significance of those
biomarkers in CMT1A physiology and the pertinence of the use of
these biomarkers in the diagnosis and follow-up of CMT1A.
[0187] III. Identification of Disease Predictors from Biomarkers of
the Invention
[0188] Statistical analysis of the level of biomarkers of the
invention obtained in the above experiments shows that said
biomarkers can also be used in different sets of grouped biomarkers
to predict the presence of disease with a good score.
Predictability scores are shown for some of the possible sets
comprising several of the molecules identified herein as biomarkers
for CMT disease Table 5).
[0189] Briefly, diagnosis of the disease was performed by applying
a Linear Discriminant Analysis (LDA), commonly used in statistics,
pattern recognition and machine learning to find a linear
combination of features which characterize or separate two or more
classes of objects. The LDA was implemented in R
(r-project.org/).
[0190] The LDA algorithm was applied on several sets of biomarkers
selected on the basis of their correlation to the trait of interest
(here transgenic versus wild-type). In order to properly assess the
performances of each set of biomarkers, groups of rats were split
into an independent "training set" (75% of rats) on which LDA was
trained, and a "validation set" (25% of rats), on which the trained
algorithm was validated. To be homogeneous, training and validation
sets were made of equal proportions of transgenic/wild-type and
male/female rats. Since the level of biomarkers differs between
males and females, for a given set of biomarkers, LDA was trained
and validated separately on males and females. Finally the trained
LDA was used to classify each rat of the training and validation
sets into "wild-type" and "transgenic", and the proportion of rats
that were well-classified allowed assessment of the performances of
the algorithm. This procedure was reapplied iteratively in order to
average the performances over all the possible samplings.
TABLE-US-00006 TABLE 5 Training Validation Male Male and and
Biomarkers Male Female Female Male Female Female Hydroxyproline and
Alanine 68% 100% 84% 45% 83% 64% Tryptophan and Hydroxyproline 86%
100% 93% 56% 84% 70% T4 and Tryptophan and Hydroxyproline 92% 100%
96% 60% 89% 75% and Alanine T4 and Hydroxyproline 92% 100% 96% 60%
79% 69% T4 and Hydroxyproline and Alanine 92% 100% 96% 61% 83% 72%
Hydroxyproline and SerotoninSerotonin 81% 100% 90% 61% 84% 72% and
Alanine Hydroxyproline and SerotoninSerotonin 74% 100% 87% 61% 72%
67% T4 and Tryptophan and Hydroxyproline 94% 100% 97% 61% 89% 75%
Tryptophan and Hydroxyproline and 89% 100% 94% 61% 73% 67% Alanine
Total of 20 biomarkers* 100% 100% 100% 62% 72% 67% T4 and
Tryptophan and Hydroxyproline 95% 100% 97% 67% 77% 72% and
SerotoninSerotonin Tryptophan and Hydroxyproline and 86% 100% 93%
67% 72% 69% SerotoninSerotonin and Alanine T4 and Serotonin 95% 74%
84% 71% 62% 67% Tryptophan and Hydroxyproline and 89% 100% 95% 72%
72% 72% Serotonin T4 and Tryptophan and Serotonin and 92% 95% 93%
72% 77% 75% Alanine T4 and Tryptophan 95% 97% 96% 72% 77% 75% T4
and Hydroxyproline and Serotonin 85% 95% 90% 72% 83% 78% and
Alanine T4 and Tryptophan and Alanine 94% 97% 96% 72% 78% 75% T4
and Hydroxyproline and Serotonin 91% 100% 96% 73% 71% 72% T4 and
Alanine 89% 83% 86% 73% 73% 73% T4 and Tryptophan and
Hydroxyproline 94% 100% 97% 73% 83% 78% and Serotonin and Alanine
Serotonin and Alanine 78% 78% 78% 73% 71% 72% T4 and Serotonin and
Alanine 86% 78% 82% 73% 71% 72% T4 and Tryptophan and Serotonin 94%
97% 96% 73% 77% 75% Tryptophan and Alanine 92% 83% 87% 77% 61% 69%
Free cholesterol and Hydroxyproline 100% 100% 100% 78% 78% 78% and
Serotonin and Alanine Free cholesterol and Tryptophan and 100% 100%
100% 78% 84% 81% Hydroxyproline and Alanine Free cholesterol and
Tryptophan and 100% 100% 100% 78% 72% 75% Hydroxyproline and
Serotonin and Alanine Free cholesterol and Hydroxyproline 100% 100%
100% 78% 88% 83% and Alanine Tryptophan and Serotonin 86% 82% 84%
78% 66% 72% Free cholesterol and Tryptophan and 100% 100% 100% 83%
83% 83% Hydroxyproline Free cholesterol and Tryptophan and 100%
100% 100% 84% 72% 78% Alanine Tryptophan and Serotonin and Alanine
83% 84% 83% 84% 72% 78% Free cholesterol and Tryptophan and 100%
100% 100% 84% 72% 78% Serotonin and Alanine Free cholesterol and
Hydroxyproline 100% 100% 100% 84% 77% 80% and Serotonin Free
cholesterol and Tryptophan and 100% 100% 100% 84% 73% 79%
Hydroxyproline and Serotonin Free cholesterol and Serotonin and
100% 100% 100% 84% 79% 81% Alanine Free cholesterol and T4 and
Serotonin 100% 100% 100% 88% 78% 83% and Alanine Free cholesterol
and T4 and Tryptophan 100% 100% 100% 89% 88% 88% and Alanine Free
cholesterol and T4 and Hydroxyproline 100% 100% 100% 89% 90% 89%
and Alanine Free cholesterol and T4 and Tryptophan 100% 100% 100%
89% 77% 83% and Serotonin and Alanine Free cholesterol and T4 and
Tryptophan 100% 100% 100% 89% 78% 84% and Hydroxyproline and
Serotonin and Alanine Free cholesterol and T4 and Tryptophan 100%
100% 100% 89% 88% 89% and Hydroxyproline and Alanine Free
cholesterol and T4 and Hydroxyproline 100% 100% 100% 89% 78% 84%
and Serotonin and Alanine Free cholesterol and T4 and Tryptophan
100% 100% 100% 94% 94% 94% and Hydroxyproline Free cholesterol and
T4 and Hydroxyproline 100% 100% 100% 94% 77% 86% and Serotonin Free
cholesterol and T4 and Tryptophan 100% 100% 100% 94% 79% 87% and
Serotonin Free cholesterol and T4 and Alanine 100% 100% 100% 94%
83% 89% Free cholesterol and T4 and Tryptophan 100% 100% 100% 94%
77% 86% and Hydroxyproline and Serotonin Free cholesterol and
Hydroxyproline 100% 100% 100% 94% 94% 94% Free cholesterol and
Tryptophan and 100% 100% 100% 95% 67% 81% Serotonin Free
cholesterol and Alanine 100% 100% 100% 95% 89% 92% Free cholesterol
and T4 and Tryptophan 100% 100% 100% 95% 89% 92% Free cholesterol
and Tryptophan 100% 100% 100% 95% 78% 86% Free cholesterol and T4
and Serotonin 100% 100% 100% 100% 78% 89% Free cholesterol and
Serotonin 100% 100% 100% 100% 79% 89% Free cholesterol and T4 and
Hydroxyproline 100% 100% 100% 100% 95% 97% Free cholesterol and T4
100% 100% 100% 100% 95% 97% *Total of 20 biomarkers: Free
Cholesterol, T4, Tryptophan, Hydroxyproline, Serotonin, Alanine,
alpha-Aminobutyric acid, Citrulline, Cystine, Glutamine, Lysine,
Methionine, Proline, Threonine, Tyrosine, Testosterone, Iron, LTB4,
Adrenaline, and Dopamine.
[0191] IV. Biomarker Analysis in CMT1A Patients
[0192] An analysis of blood biomarkers was performed as a secondary
objective of a double-blind, randomized, placebo-controlled Phase 2
study (ClinicalTrials.gov Identifier: NCT01401257) of which the
primary objective was to assess the clinical and laboratory safety
and tolerability of three doses of a mix of baclofen, naltrexone
and sorbitol (MIX), a candidate treatment administered orally for
12 months to CMT1A patients versus placebo.
[0193] Patients aged 18-65 years were included with CMT1A diagnosis
based on clinical examination and confirmation by genotyping
(duplication in 17p11.2), weakness in at least foot dorsiflexion,
and a Charcot-Marie-Tooth Neuropathy Score (CMTNS)<20, i.e.,
mild to moderate severity. Eligible patients were randomly assigned
in a 1:1:1:1 ratio to receive daily for one year Placebo, Low dose
(LD=0.6 mg baclofen, 0.07 mg naltrexone and 21 mg sorbitol),
Intermediate dose (ID=1.2 mg baclofen, 0.14 mg naltrexone and 42 mg
sorbitol) or High dose (HD=6 mg baclofen, 0.7 mg naltrexone and 210
mg sorbitol) of the mix.
[0194] The results regarding the efficacy of the treatment of this
Phase 2 study have been published (Attarian et al., 2014). Patients
treated with the highest dose show consistent evidence of
improvement beyond stabilization of the disease after one year of
treatment.
IV.1 Biomarker Collection
[0195] Blood samples were taken both at randomization and after 3
months of treatment with the mix (3 doses tested) or placebo.
Following sample collection in lithium-heparin, tubes were
centrifuged for 10 min at 1300 g at room temperature, and plasma
samples were stored at -80.degree. C. until analysis.
[0196] Plasma concentrations were determined using HPLC coupled
with Mass Spectrometry detection (LC-MS/MS) after protein
precipitation for L-alanine and L-tryptophan and Liquid/Liquid
extraction for free cholesterol.
[0197] The Lower Limits of Quantification (LLOQ) were 20 .mu.g/mL,
10 .mu.g/mL, and 5 .mu.g/mL for free cholesterol, L-alanine, and
L-tryptophan respectively. The results are expressed as .mu.g/mL
for L-alanine, free cholesterol and L-tryptophan.
IV.2 Efficacy Endpoints
[0198] The clinical endpoints considered in the trial are listed in
Table 6:
TABLE-US-00007 TABLE 6 Improvement (direction of variation for an
Endpoints improvement) Reference/comments Clinical scales CMTNS
.dwnarw. Shy et al. (2005) ONLS .dwnarw. Graham and Hughes (2006)
Functional measures 6MWT (m) .uparw. Guyatt et al. (1985) 9HPT (s)
.dwnarw. Hogrel et al. (2007) (non-dominant hand considered) Ankle
.uparw. Hogrel et al. (2007) Dorsiflexion (mean of left and right
side considered) (Nm) Grip (kg) .uparw. Hogrel et al. (2007)
(non-dominant hand considered) Electrophysiological parameters CMAP
(mV) .uparw. measured from the mean sensory MCV (m/s) .uparw.
responses of the median and ulnar nerves (non-dominant side)
[0199] Compound muscle action potential (CMAP) is an
electromyography investigation which represents the summation of a
group of almost simultaneous action potentials from several muscle
fibers in the same area which are evoked by stimulation of the
motor nerve. Patients with impaired peripheral nerves show a
decreased CMAP.
[0200] Motor conduction velocity (MCV) is the speed at which an
electrical stimulation of a nerve propagates down to a muscle
supplied by this nerve. Patients suffering from motor neuropathies
display reduced speeds.
IV.3 Statistical Analysis
[0201] All analyses were performed on the Full Analysis Set (all
randomized patients) using R version 3.1.2 (cran.r-project.org).
Considering the exploratory nature of the study, statistical tests
were conducted at a two-sided 5% level. Correlations were assessed
using a Spearman's rank test. When specified, correlations were
assessed based on data adjusted on Gender, Age and Centre with a
linear model. Comparisons of two groups were performed using a
Welch's t-test; comparisons of more than two groups were performed
using an Analysis of Variance (ANOVA).
Effect of Treatment Analysis
[0202] Differences at 3 months between patients under baclofen,
naltrexone and sorbitol mix treatment and patients under placebo
were assessed by Analysis of Covariance (ANCOVA) adjusted on
baseline values and also including gender, age and clinical centre
as fixed covariates. The significance of the treatment effect on
the combination of two biomarkers was assessed through the
O'Brien's OLS test.
Identification of Responders
[0203] Biomarker levels at baseline between non-deteriorated
patients (e.g., exhibiting improved or stabilized symptoms) and
deteriorated (exhibiting a worsened condition) at 12 months
following the approach of Attarian et al. (2014) on efficacy
endpoints have been compared.
IV. 4 Results
Correlation Between Disease State or Evolution and Biomarkers
[0204] A baseline correlation analysis has been performed between
efficacy endpoints and biomarkers by adjusting for gender, age and
clinical centre in order to take into account any variation not
related to the disease state.
[0205] The level of tryptophan is found to correlate significantly
with all efficacy endpoints (Table 7). This correlation is positive
with endpoints for which an increase means improvement and negative
with endpoints for which a decrease means improvement.
Consequently, higher tryptophan levels are found to be associated
with less severe disease profiles.
[0206] Alanine also shows a correlation with some of the endpoints
(Table 7).
[0207] Noteworthy, a significant and positive correlation between
the two biomarkers (R=0.44, p=7e-05) is observed, which confirms
the correlation to efficacy endpoints observed for both tryptophan
and alanine.
TABLE-US-00008 TABLE 7 Spearman's coefficient Improvement
correlation (direction of variation (p < 0.05) Endpoints for an
improvement) L-Trp L-Ala Clinical scales CMTNS .dwnarw. -0.27 ns
ONLS .dwnarw. -0.29 ns Functional measures 6MWT (m) .uparw. +0.4
+0.3 9HPT (s) .dwnarw. -0.25 ns Ankle Dorsiflexion (Nm) .uparw.
+0.36 ns Grip (kg) .uparw. +0.26 ns Electrophysiological parameters
CMAP (mV) .uparw. +0.34 +0.36 MCV (m/s) .uparw. +0.28 +0.24
[0208] Of note, the efficacy endpoints correlate well and
significantly with each other and in a coherent way with regard to
the severity of the disease. Then, even if moderate, due to the
multidimensional nature of the disease, efficacy endpoints and also
variation of tryptophan and alanine bear a predictable relationship
to the overall disease severity.
[0209] Hence, tryptophan and/or alanine level(s) can be used to
assess the evolution and the severity of CMT1A in the patient
population.
Biomarker Levels as Early Markers of the Efficacy of a
Treatment
[0210] The effect of 3 months' treatment with baclofen, naltrexone
and sorbitol on the biomarker levels was assessed (Table 8). A
significant increase in tryptophan (p=0.018) and alanine (p=0.04)
in patients treated for 3 months with the mix (1.56.+-.2.98
.mu.g/mL and 3.05.+-.8.16 .mu.g/mL respectively) compared to
placebo (0.43.+-.1.45 .mu.g/mL and 0.75.+-.6.75 .mu.g/mL
respectively) is found. When these two markers were considered
jointly, the significance of the effect of treatment was even
greater (p=0.0086). Noteworthy, after 3 months of treatment no
symptomatic change is evidenced in the treated population, because
of the slow and progressive nature of the disease. Biomarkers of
the invention thus provide efficient tools to determine the
response to a treatment.
TABLE-US-00009 TABLE 8 Placebo (n = 19) MIX HD (n = 19) Change from
Change from MIX HD vs Placebo Biomarker Baseline Baseline Baseline
Baseline Estimate p Alanine (.mu.g/ml) 30.17 (6.80) 0.75 (6.75)
32.77 (11.55) 3.05 (8.16) 4.0 (0.19; 7.87) 0.04* Tryptophan
(.mu.g/ml) 10.26 (2.19) 0.43 (1.45) 10.69 (1.75) 1.56 (2.98) 1.6
(0.29; 2.86) 0.018* MIX: mix of baclofen, naltrexone and sorbitol;
HD: High Dose of MIX (6 mg baclofen, 0.7 mg naltrexone and 210 mg
sorbitol). *data are mean changes from baseline (s.d.); Estimate:
differences of change from baseline adjusted on Gender, Age and
Centre, least squares mean (95% confidence interval). (ANCOVA, *p
< 0.05).
Free Cholesterol Levels to Discriminate Responders from
Non-Responders to Treatments of CMT
[0211] Among patients treated with baclofen, naltrexone and
sorbitol, those for which a worsening in their condition
(non-responders) was observed after one year of treatment were
found to have a significantly higher plasma concentration in free
cholesterol than the responders (p=0.034) at the beginning of the
study (FIG. 1). Hence biomarkers of the invention can be used as a
predictor of the response or responsiveness to baclofen, naltrexone
and sorbitol combination treatment.
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