U.S. patent application number 13/341028 was filed with the patent office on 2012-06-28 for anaplerotic therapy of huntington disease and other polyglutamine diseases.
This patent application is currently assigned to INSERM (Institut National de la Sante et de la Recherche Medicale). Invention is credited to Alexandra Durr, Fanny Mochel.
Application Number | 20120165405 13/341028 |
Document ID | / |
Family ID | 37733780 |
Filed Date | 2012-06-28 |
United States Patent
Application |
20120165405 |
Kind Code |
A1 |
Durr; Alexandra ; et
al. |
June 28, 2012 |
Anaplerotic Therapy of Huntington Disease and Other Polyglutamine
Diseases
Abstract
The present invention relates to a method for treating and/or
preventing Huntington disease and other polyglutamine diseases,
comprising the step of administering an effective amount of a
precursor of propionyl-CoA to an individual in need thereof.
Inventors: |
Durr; Alexandra; (Paris,
FR) ; Mochel; Fanny; (Saint Maur Des Fosses,
FR) |
Assignee: |
INSERM (Institut National de la
Sante et de la Recherche Medicale)
Paris
FR
|
Family ID: |
37733780 |
Appl. No.: |
13/341028 |
Filed: |
December 30, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12516486 |
May 27, 2009 |
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PCT/EP07/63181 |
Dec 3, 2007 |
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13341028 |
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Current U.S.
Class: |
514/547 ;
514/557; 514/558 |
Current CPC
Class: |
A23V 2002/00 20130101;
A23L 29/04 20160801; A23V 2200/322 20130101; A61P 25/28 20180101;
A23L 33/115 20160801; A61K 31/225 20130101; A23L 33/12 20160801;
A23V 2002/00 20130101 |
Class at
Publication: |
514/547 ;
514/557; 514/558 |
International
Class: |
A61K 31/225 20060101
A61K031/225; A61K 31/20 20060101 A61K031/20; A61P 25/00 20060101
A61P025/00; A61K 31/19 20060101 A61K031/19 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 4, 2006 |
EP |
06291873.5 |
Claims
1. A method of treating and/or preventing a polyglutamine disease
comprising administering a precursor of propionyl-CoA in an amount
effective for treating and/or preventing a polyglutamine
disease.
2. The method according to claim 1, wherein the polyglutamine
disease is Huntington disease.
3. The method according to claim 1, wherein the precursor of
propionyl-CoA is selected from the group consisting of
odd-medium-chain fatty acids, seven-carbon fatty acid,
triheptanoin, heptanoate, C5 ketone bodies, 3-ketovalerate and
3-hydroxyvalerate.
4. The method according to claim 1, wherein the precursor of
propionyl-CoA is triheptanoin, heptanoic acid or heptanoate.
5. The method according to claim 1, wherein the precursor of
propionyl-CoA is triheptanoin.
6. The use method according to any preceding claims claim 1,
wherein the precursor of propionyl-CoA is administered via
ingestion of a food substance containing said precursor.
7. The method according to claim 2, wherein the precursor of
propionyl-CoA is selected from the group consisting of
odd-medium-chain fatty acids, seven-carbon fatty acid,
triheptanoin, heptanoate, C5 ketone bodies, 3-ketovalerate and
3-hydroxyvalerate.
8. The method according to claim 2, wherein the precursor of
propionyl-CoA is triheptanoin, heptanoic acid or heptanoate.
9. The method according to claim 3, wherein the precursor of
propionyl-CoA is triheptanoin, heptanoic acid or heptanoate.
10. The method according to claim 7, wherein the precursor of
propionyl-CoA is triheptanoin, heptanoic acid or heptanoate.
11. The method according to claim 2, wherein the precursor of
propionyl-CoA is triheptanoin.
12. The method according to claim 3, wherein the precursor of
propionyl-CoA is triheptanoin.
13. The method according to claim 4, wherein the precursor of
propionyl-CoA is triheptanoin.
14. The method according claim 2, wherein the precursor of
propionyl-CoA is administered via ingestion of a food substance
containing said precursor.
15. The method according to claim 3, wherein the precursor of
propionyl-CoA is administered via ingestion of a food substance
containing said precursor.
16. The method according to claim 4, wherein the precursor of
propionyl-CoA is administered via ingestion of a food substance
containing said precursor.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the treatment and the
prevention of Huntington disease and other polyglutamine
diseases.
BACKGROUND OF THE INVENTION
[0002] Huntington disease (HD) is a devastating inherited
neurodegenerative disease without curative treatment. HD is the
founding member of a large group of diseases due to to
polyglutamine accumulation and toxicity. There is a critical need
for new insights in the pathophysiology of this disease, as well as
for the identification of relevant molecules for clinical
trials.
[0003] Several observations have led to the hypothesis that
mitochondrial dysfunction has a role in polyglutamine diseases, and
in Huntington disease in particular. Several lines of evidence
indicate abnormal energy metabolism, including reduced glucose
metabolism, elevated lactate levels and impaired
mitochondrial-complex activity (Di Prospero and Fischbeck 2005, Nat
Rev Genet 6(10): 756-65). To explain this abnormal energy
metabolism most studies favoured a secondary impairment of the
mitochondrial respiratory chain. An important decrease in complexes
II & Ill (55%) has been shown in the caudate of HD patients (Gu
1996, Ann Neural 39: 385-9), as well as a deficiency in complex I
in muscle (Arenas 1998, Ann Neural 43: 397-400), therefore
supporting the possibility of mitochondrial respiratory chain
defects in pathogenesis of HD (Shapira 1998, Biochem Biophys Acta
1366: 225-33, Grunewald 1999, Ann N Y Acad Sci 893: 203-13). These
findings correlated with HD models induced by 3-nitropropionic
acid, an irreversible complex II inhibitor (Beal 1993, J Neurosci
13: 4181-92). However, these data are controverted by the
demonstration of normal mitochondrial electron transport complexes
in transgenic mice at an early stage (Guidetty 2001, Exp Neural
169: 340-50), as well as in striatal cells in culture expressing
mutant huntinetin, despite the significant reduction in ATP
synthesis observed in those cells (Milakovic 2005, JBC 280:
30773-82). Additional indirect evidence for an energy defect in
polyglutamine diseases arise from the partial efficacy of energetic
therapies, such as dichloroacetate (Andreassen 2001, Ann Neurol 50:
112-9), pyruvate (Ryu 2004, Exp Neurol 187: 150-9), creatine
(Ferrante 2000, J Neurosci 20: 4389-97) and coenzyme Q10 (Schilling
2001, Neurosci Lett 315: 149-53) in mice models.
[0004] However, to date, effective pharmacotherapy for
neurodegenerative diseases associated with impaired energy
metabolism like polyglutamine diseases in particular, remains
rather elusive.
SUMMARY OF THE INVENTION
[0005] Therefore, it is an object of the present invention to
provide a method for treating and/or preventing polyglutamine
disease such as Huntington disease.
[0006] In fulfilling this object, there is provided a method for
treating and/or preventing a polyglutamine disease, comprising the
step of administering an effective amount of a precursor of
propionyl-CoA to an individual in need thereof.
[0007] Also provided is the use of a precursor of propionyl-CoA in
the manufacture of a medicament for treating and/or preventing
polyglutamine disease.
DETAILED DESCRIPTION OF THE INVENTION
[0008] The present invention provides a method for treating and/or
preventing a polyglutamine disease, comprising the step of
administering an effective amount of a precursor of propionyl-CoA
to an individual in need thereof.
[0009] By individual, it is meant animal or human being.
[0010] Also provided is a precursor of propionyl-CoA for treating
and/or preventing a polyglutamine disease.
[0011] Also provided is the use of a precursor of propionyl-CoA in
the manufacture of a medicament and/or a food substance for
treating and/or preventing a polyglutamine disease.
[0012] Po lyglutamine diseases constitute a class of nine
genetically distinct disorders that are caused by expansion of
translated CAG repeat. These include Huntington disease (HD),
dentatorubralpallidoluysian atrophy (DR PLA), spinal and bulbar
muscular atrophy (SBMA) and spinocerebellar ataxia 1, 2, 3, 6, 7
and 17. Although the disease causing proteins are expressed widely
in the central nervous system, specific populations of neurons arc
vulnerable in each disease, resulting in characteristic patterns of
neurodegeneration and clinical features.
[0013] In an embodiment of the present invention, the polyglutamine
disease is selected from the group consisting of Huntington disease
(HD), dentatorubralpallidoluysian atrophy (DRPLA), spinal and
bulbar muscular atrophy (SBMA), spinocerebellar ataxia 1, 2, 3, 6,
7 and 17.
[0014] In a preferred embodiment of the present invention, the
polyglutamine disease is Huntington disease.
[0015] By precursor of propionyl-CoA, it is meant a substance from
which propionyl-CoA can be formed by one or more metabolic
reactions taking place within the body.
[0016] Examples of precursors of propionyl-CoA are shoWn in FIG. 5.
Typical examples of precursors of propionyl-CoA are
odd-medium-chain fatty acids, in particular seven-carbon fatty
acid, triheptanoin (triheptanoyl-glycerol), heptanoate, C5 ketone
bodies (e.g. .beta.-ketopentanoate (3-ketovalerate), and
(.beta.-hydroxypentanoate (3-hydroxyvalerate)) (Kinman 2006, Am J
Physiol Endocrinol Metab 291 (4): E860-6, Bruneneraber and Roe
2006, J Inherit Metabol Dis 29 (2-3): 327-31).
[0017] The examples of precursors of propionyl-CoA described above
include the compounds themselves, as well as their salts, prodrugs,
solvates, if applicable. Examples of prodrugs include esters,
oligomers of hydroxyalkanoate such as oligo(3-hydroxyvalerate)
(Seebach 1999, Int J Biol Macromol 25 (1-3): 217-36) and other
pharmaceutically acceptable derivatives, which, upon administration
to a individual, are capable of providing propionyl-CoA. A solvate
refers to a complex formed between a precursor of propionyl-CoA
described above and a pharmaceutically acceptable solvent. Examples
of pharmaceutically acceptable solvents include water, ethanol,
isopropanol, ethyl acetate, acetic acid, and ethanolamine.
[0018] A very practical dietary source of propionyl-CoA is
triheptanoin (triheptanoyl-glycerol). After intestinal hydrolysis
of triheptanoin, heptanoate is absorbed in the portal vein. In the
liver, it is partially converted to the C5 ketone bodies
.beta.-ketopentanoate (3-ketovalerate), and P-hydroxypentanoate
(3-hydroxyvalerate). The C5-ketones bodies are also precursors of
propionyl-CoA in peripheral tissues. Thus, alter ingestion of
triheptanoin, peripheral tissues receive two precursors of
propionyl-CoA, i.e., heptanoate and C5-ketone bodies. Quite
interestingly, C5-, like C4-, ketone bodies are natural substrates
for the brain and can target physiological receptors at the surface
membrane of the blood brain barrier. The demonstration of the
transport of C5-ketone bodies across the blood-brain barrier was
recently provided by the treatment of a patient with pyruvate
carboxylase deficiency, where cerebral anaplerosis is primarily
impaired (Mochel 2005, Mol Genet Metab 84: 305-12). The
availability of C5-ketone bodies for cerebral anaplerosis was also
demonstrated by the normalisation of glutamine and GABA in the CSF
of this patient, as well as the absence of brain pathology.
[0019] The person skilled in the art is aware of standard methods
for production of precursors of propionyl-CoA.
[0020] In a preferred embodiment of the present invention, the
precursor of propionyl-CoA is triheptanoin, heptanoic acid or
heptanoate.
[0021] Triheptanoin has already been used in the anaplerotic
treatment of a few pathologies having in common a decrease in ATP
production in spite of ample supply of acetyl-CoAto the citric acid
cycle (CAC), and a normal respiratory chain. Such pathologies
include cardiac reperfusion injury (Reszko 2003, JBC 278:
34959-65), long-chain Fatty acid oxidation disorders (FOD) (Roe
2002, JCI 110: 259-69 and WO 0045649), pyruvate carboxylase
deficiency (Mochel 2005, Mol Genet Metab 84: 305-12) and glycogen
storage disease type II (Roe and Mochel 2006, J Inherit Metab Dis
29 (2-3): 332-40)
[0022] Triheptanoin is a triglyceride made by the esterification of
three n-heptanoic acid molecules and glycerol. In regard to
therapy, the terms heptanoic acid, heptanoate, and triheptanoin may
be used interchangeably in the following description. Also, it will
be understood by one skilled in the art that heptanoic acid,
heptanoate, and triheptanoin are exemplary precursors of
propionyl-CoA of the invention. Substituted, unsaturated, or
branched heptanoate, as well as other modified seven-carbon fatty
acids can be used without departing from the scope of the
invention.
[0023] Heptanoic acid is found in various fusel oils in appreciable
amounts and can be extracted by any means known in the art. It can
also be synthesized by oxidation of heptaldehyde with potassium
permanganate in dilute sulfuric acid (Ruhoff, Org Syn Coll. volll.
315 (1943)). Fleptanoic acid is also commercially available through
Sigma Chemical Co. (St. Louis, Mo.).
[0024] Triheptanoin can be obtained by the esterification of
heptanoic acid and glycerol by any means known in the art.
Triheptanoin is also commercially available through CondeaChemie
GmbH (Witten, Germany) as Special Oil 107.
[0025] Unsaturated heptanoate can also be utilized in the present
invention. In addition, substituted, unsaturated, and/or branched
seven-carbon fatty acids which readily enter the mitochondrion
without special transport enzymes can be utilized in the present
invention. For example. 4-methylhexanoate, 4-methylhexenoate, and
3-hydroxy-4-methylhexanoate are broken down by normal b-oxidation
to 2-methylbutyric acid with final degradation accomplished via the
isoleucine pathway. Likewise, 5-methylhexanoate, 5-methylhexenoate,
and 3-hydroxy-5-methylhexanoate are broken down by normal
b-oxidation to isovaleric acid with final degradation accomplished
via the leucine pathway.
[0026] Precursors of propionyl-CoA of the present invention can he
administered orally, parenterally, or intraperitoneally.
Preferably, it can be administered via ingestion of a food
substance containing a precursor of propionyl-CoA such as
triheptanoin at a concentration effective to achieve therapeutic
levels. Alternatively, it can be administered as a capsule or
entrapped in liposomes, in solution or suspension, alone or in
combination with other nutrients, additional sweetening and/or
flavoring agents. Capsules and tablets can be coated with sugar,
shellac and other enteric agents as is known. Typically medicaments
according to the invention comprise a precursor of propionyl-CoA,
together with a pharmaceutically-acceptable carrier. A person
skilled in the art will be aware of suitable carriers. Suitable
formulations for administration by any desired route may be
prepared by standard methods, for example by reference to
well-known text such as Remington; The Science and Practice of
Pharmacy.
[0027] In the following, the invention will be illustrated by means
of the following examples as well as the figures.
FIGURE LEGENDS
[0028] FIG. 1: Partial least square (PLS) analyses of NMR spectra
of plasma samples from HD patients with no or little signs of the
disease and controls. Three groups of premanifest, early and mildly
affected HD patients were constituted on the basis of their UHDRS
scores, as described in the methods. The first and second
components in the X space (NMR spectrum) are denoted PC[1] and
PC[2] respectively. PLS score plots (PC[1]/PC[2]) of pair-wise
compared groups show the greater variation within the NMR spectrum
according to a priori classification with UHDRS. Their is a clear
separation between premanifest and early HD patients (a), as well
as between early and mildly affected HD patients (h). Therefore,
plasma NMR spectroscopy allows separation of HD patients at
different stages of the disease. Despite some overlap,
differentiation between controls and premanifest individuals is
also observed (c).
[0029] FIG. 2: Plasma relative concentrations of branched chain
amino acids are responsible for separation between HD groups.
PLS-contribution plot allows comparison between plasma metabolic
profiles from early affected HD patients to premanifest carriers.
NMR variables that have the greatest weight (w*.sub.1, scaled in
units of standard deviation), therefore contributing most to the
separation between HD groups, are decreased concentrations
(>2SD) of metabolites located between 0.9 and 1.05 ppm: valine,
leucine and isoleucine. The same contribution plot was obtained
when comparing plasma metabolic profiles from mildly to early
affected HD patients (data not shown).
[0030] FIG. 3: The levels of branched chain amino acids are
significantly different in HD patients and controls. The
concentrations of valine, leucine and isoleucine in plasma were
determined by ion exchange chromatography. Comparisons of means
(ANOVA) were made between men or women with HD and their respective
controls. In men, there is a significant decrease of valine,
leucine and isoleucine in the HD group. In women, similar results
are observed for leucine and isoleucine. Of note, in both men and
women, the comparison of the standard deviations of valine, leucine
and isoleucine values shows almost no overlap between the control
and the HD groups.
[0031] FIG. 4: Plasma branched chain amino acids are negatively
correlated with disease progression in HD. Principal component
analysis (PCA) loading plot shows the relative importance of each
variable from the study and the correlation between these
variables. The more the loading (p) of each variable diverges from
zero, the .more this variable is important in the explained
variance (tithe given component (expressed by R2x). The explained
variance of all data reach 44% in the first component and 22% in
the second component. There is strong negative correlation between
clinical markers (the size of the abnormal CAG repeat expansion,
disease severity measured by the UHDRS and depression scores) and
parameters associated with weight (weight, BMI, LBM and FBM for
lean and fat body mass respectively). The BCAA, valine, leucine and
isoleucine, are negatively correlated with disease progression and
positively correlated with weight loss. Note that the number. of
CAG repeats are negatively correlated with BCAA values: the larger
the repeat the lower the BCAA values (p=0.015 for valine, 0.018 for
leucine and 0.020 for isoleucine).
[0032] FIG. 5: Diagram depicting the metabolic pathway of
triheptanoin.
[0033] FIG. 6: Purkinje cell survival 12 days and 20 days following
infection with the lentiviral vectors. 10.0Q: ATXN7T-100Q-GFP. 10Q:
ATXN7T-10Q-GFP. GFP: control vector expressing GFP alone. The
Purkinje cell survival is expressed as the percentage of
Calbindin-positive cells in infected cultures compared to
non-infected cultures. 12 days after infection by ATXN7T-100Q-GFP,
Purkinje cell survival is reduced to .about.30% and further
decreases to .about.15% after 20 days demonstrating the high and
progressive neurotoxicity of the mutant protein, which is clearly
distinct from the toxicity of the viral vector alone (GFP
condition).
[0034] FIG. 7: Weight and food intake evolution of female knock-in
mice (n=3) and female wild-type mice (n=3) from 7 to 11 weeks of
age.
EXAMPLES
[0035] In the following description, all molecular biology
experiments for which no detailed protocol is given are performed
according to standard protocol.
Example 1
[0036] Identification of a plasma biomarker in premanifest carriers
of Huntington disease indicating early energy imbalance
[0037] Abbreviations: HD (Huntington disease), UHDRS (Unified
Huntington disease rating scale), ppm (parts per million); PCA
(principal components analysis), PLS (partial least square).
[0038] Abstract
[0039] Huntington disease (HD) is an autosomal dominant
neurodegenerative disorder in which an energy deficiency is thought
to play a role. Patients consistently lose weight, although the
reason for this is unknown. In view of the specific access to
premanifest carriers in HD, we performed a multiparametric study in
a group of 32 individuals with no sign or little of the disease
compared to 21 controls. Weight loss was observed even in
premanifest carriers in the HD group, although their caloric intake
was higher. Inflammatory processes were ruled out, as well as
primary hormonal dysfunction, including ghrelin and leptin balance.
Proton nuclear magnetic resonance spectroscopy on plasma did,
however, distinguish HD patients at different stages of the disease
and .premanifest carriers from controls. Differences between groups
were attributable to low levels of the branched chain amino acids
(BCAA), valine, leucine and isoleucine. We confirmed that BCAA
levels were negatively correlated with weight loss and more
importantly with disease progression. Levels of insulin growth
factor type 1 (IGF1), which is regulated by BCAA, were also lower
in the HD group than in controls. BCAA are, therefore, the first
biomarkers identified in HD and offer new insights into an
underlying early energy deficiency.
[0040] Results
[0041] Evidence of Early Hypercatabolism in HD
[0042] Three groups were defined according to their UHDRS scores.
There were 15 carriers of the mutation without any motor or
cognitive signs of HD (UHDRS 0.5.+-.1.0), 10 patients in an early
stage of the disease (UHDRS 11.9.+-.4.9) and 7 patients mildly
affected (UHDRS 44.4.+-.14.1)
[0043] Weight loss during the last 5 years was significantly
greater in the HD group than in controls (p<0.000 I). The
difference remained significant when men (p=0.002) and women
(p=0.003) were analyzed separately. In spite of higher intake of
calories, HD individuals and controls showed no different BMI.
Importantly, in HD men. BMI was significantly lower than in
controls, and total calories were even inversely correlated with
weight (p=0,029) and lean body mass (p=0.004).
[0044] These observations confirm that weight balance is abnormal
early in HD. Even in premanifest carriers, the nutritional pattern
of HD patients differed from that of controls; they had
significantly higher caloric intake (2195.+-.495, n=15 versus
1665.+-.305, n=21, p<0.001) and greater protein intake
(85.+-.24, n=15 versus 70.+-.14, n=21, p=0.025). These observations
clearly show hypercatabolism in the HD group, even in the very
early stages of the disease. Relationship to the disease was not
explained by common causes of hypercatabolism such as inflammation
or classical endocrine dysfunctions. Indeed, ERS, CRP, serum
interleukins 1.beta. and 6 and serum fasting cortisol, T41.: and
TSH were similar in the HD and control groups. There was no
glycosuria, and fasting blood glucose and insulin levels were in
the normal range in the HD group.
[0045] Identification of Candidate Biomarkers by Plasma .sup.1H NMR
Spectroscopy (FIGS. 1 and 2)
[0046] PCA on plasma NMR spectra identified no outliers in both the
control and HD dataset. PLS analyses could distinguish HD
individuals at different stages of the disease, meaning that
underlying plasma metabolites behaved differently. The difference
between premanifest carriers and early HD was evident (FIG. 1a),
and extended to more advanced stages of the disease (FIG. 1b). In
addition, controls and premanifest carriers did not have the same
metabolic profile, despite some overlap (FIG. 1c).
[0047] The spectral region that contributed to differences among
the HD groups determined from PLS contribution plots is shown in
FIG. 2. Plasma metabolic profile from early affected HD patients to
premanifest carriers was compared, as well as from mildly to early
affected HD patients. There was a significant (>2SD) decrease
along with disease progression in the plasma concentrations of a
group of variables from the buckets located between 0.9 to 1.05 ppm
on the NMR spectrum. These peaks correspond to the branched chain
amino acids (BCAA), valine, leucine and isoleucine. No other
significant differences among the groups were detected in the
spectra even though very small buckets (0.02 ppm) were analyzed.
This indicates that a selective decrease in BCAA concentrations
accompanies the progression of the disease, and even distinguishes
premanifest carriers from both controls and early HD patients.
Plasma BCAA levels appear, therefore, to be relevant biomarkers of
HD.
[0048] Confirmation that BCAA are Affected in HD (FIGS. 3 and
4)
[0049] To confirm that BCAA are affected in HD, we also measured
their concentrations in plasma by ion exchange chromatography.
Valine, leucine and isoleucine levels were significantly lower in
the HD group compared to controls (p=0.009, p<0.00I and p=0.002,
respectively). In addition, the levels of each BCAA were
significantly correlated with the observed weight loss in the
patients (p=0.005, 0.002 and 0.014 respectively). More importantly,
BCAA levels were negatively correlated with UHDRS values (p=0.017,
<0.0001 and 0.003 respectively) in both men (p=0.035, 0.019 and
0.036 respectively) and women (p=0.007 for leucine and 0.01 for
isoleucine). Although the BMI values of women with HD were similar
to the controls, they had significantly lower leucine (p=0.002) and
isoleucine (p=0.014) levels (FIG. 3). This indicates that lower
BCAA levels are not only associated with weight balance. but more
importantly with Huntington disease itself. Interestingly, the
plasma levels of the three BCAA were significantly lower in
patients at an early stage of the disease compared to premanifest
carriers (p=0.042, 0.019 and 0.024 respectively). BCAA levels are,
therefore, associated with disease onset, emphasizing that they can
be used as reliable biomarkers in HD. When comparing premanifest
carriers to controls, the plasma levels of valine (228.+-.50 versus
245.+-.44), leucine (130.+-.24 versus 144.+-.23) and isoleucine
(62.+-.12 versus 68.+-.15) were lower in the former group, although
not significantly. This is likely due to the heterogeneity of the
premanifest group in which the estimated time to disease onset is
expected to vary between individuals, so that the metabolic profile
of some premanifest carriers can be similar to controls.
[0050] A multivariate PCA confirms that there is a strong negative
correlation between clinical markers (abnormal CAG repeat expansion
size, UHDRS scores, depression scores) and weight parameters (FIG.
4). Low BCAA values appear to be the strongest variables that are
negatively correlated with HD and positively correlated with weight
loss. The numbers of CAG repeats are also negatively correlated
with BCAA values: the larger the repeat the lower the values
(p=0.015 for valine, 0.018 for leucine and 0.020 for
isoleucine).
[0051] The other metabolic markers (serum cholesterol and
triglycerides, remaining amino acids and acylcarnitines in plasma,
organic acids in urine) were similar in the HD group and in
controls. IGF1 levels, however, were significantly lower in the HD
group (p32 0.011) and negatively correlated with UHDRS scores
(p=0.004). IGF1 levels were also correlated with leucine (p=0.04)
and isoleucine (p=0.02) levels, which was expected since IGF1 is
regulated by BCAA. However, this has to be modulated by the tact
that IGF1 levels are known to decrease with age, as observed in our
HD cohort (p=0.002). The decrease in IGF1 levels was not associated
with significant changes in other nutritional parameters (albumin,
prealbumin, orosomucoid). There was no correlation either between
IGF1 levels and parameters associated with weight (BMI, lean and
fat body mass) or food intake.
[0052] Discussion
[0053] This is the first extended investigation of weight disorder
in HD. We have shown that weight loss begins early in the disease,
despite higher caloric intake, and is evident even in premanifest
mutation carriers and those with little or no chorea. This
hypercatabolism cannot be explained by common mechanisms like
inflammation or altered endocrine functions, both of which have
been incriminated in the pathophysiology of HD (Kremer et al. 1989;
Pavese et al. 2006). Hypercatabolism in HD seems, therefore, to be
part of the pathological process induced by the disease. Our
.sup.1H NMR spectroscopy analysis shows that patients at different
stages of HD can be distinguished from each other, and premanifest
mutation carriers can be to distinguished from controls, on the
basis of their plasma levels of branched chain amino acids. The
decrease in the levels of these amino acids correlated with weight
loss in HD patients, but more importantly with the severity of the
clinical impairment, i.e., with Huntington disease itself. This
finding is supported by previous studies in which a decrease in
plasma BCAA was documented in more severely affected HD patients
(Perry et al. 1969; Phillipson and Bird 1977; Reilmann et al.
1995). The extensive metabolic screening we performed in
combination with an independent technique confirmed that plasma
BCAA were the only metabolites that differed between the HD group
and controls. This difference existed regardless of sex and between
patients at an early stage of the disease and premanifest carriers.
Consequently, plasma BCAA can be considered as relevant biomarkers
for Huntington disease. They should help to detect the onset of the
disease and to monitor its progression in view of therapeutic
trials. To our knowledge, this is the first accessible biomarker
identified in Huntington disease and, more widely, the first
peripheral biomarker evidenced in a neurodegenerative disorder.
[0054] Only few metabonomic studies have led to the identification
of biomarkers that can be used routinely for the follow up of
patients (Sabatine et al. 2005). Inter-individual variability is
known to complicate such analyses in human body fluids, thus
restricting metahonomic studies essentially to animal models
(Wagner et al. 2006). Common experimental and analytical biases in
humans include dietary intake, time of sample collection, sample
conditioning and chemical shifts due to chances in pH (Cloarec et
al. 2005; Teahan et al. 2006; Walsh et al. 2006). In the present
study, each of these parameters was rigorously controlled. This
probably explains the accuracy of our NMR findings.
[0055] The implication of BCAA in mitochondrial intermediary
metabolism, both in brain and peripheral tissues, further supports
an important role for energy deficit in HD. A reduction in ATP
production was shown in brain of HD mice, including presymptomatic
mice (Gines et al. 2003). A significant reduction in ATP levels and
mitochondrial respiration was also evidenced in striatal cells of
HD mice, although the respiratory chain complexes were not impaired
(Milakovic and Johnson 2005). In HD patients, there is strong
evidence for hypometabolism in the brain where glucose consumption
is reduced, especially in the basal ganglia, even in presymptomatic
mutation carriers (Grafton et al. 1992; Kuwert et al. 1993;
Antonini et al. 1996). The underlying cause of this early energy
deficit in HD brain is currently unknown, but impaired glycolysis
(Browne and Beal 2004), citric acid cycle (Tabrizi et al. 1999)
and/or oxidative phosphorylation (Milakovic and Johnson 2005) may
be involved. Recently, mutated huntingtin was shown to decrease the
expression of PGC-1.alpha. (peroxisome proliferators-activated
receptor gamma coactivator-1.alpha.) in the striatum of HD mice and
patients, through a CREB-dependent transcriptional inhibition (Cui
et al. 2006). PGC-la is a transcriptional coactivator that
regulates key energetic metabolic pathways, both in the brain and
peripheral tissues (Lin et al. 2005). The possible role of
PGC-1.alpha. in HD was initially suspected from the observation of
selective striatal lesions in the PGC-Ia knockout mouse (Lin et al.
2004). Down-regulation of PGC-1.alpha. in HD striatum was then
shown to affect mitochondrial energy metabolism, possibly by
impairing oxidative phosphorylation (Cui et al. 2006). In addition,
the inhibition of succinate dehydrogenase, by 3-nitropropionic acid
or malonate, mimics HD neuropathology in mice (Klivenyi et al.
2004), indicating that a lack of substrates for the citric acid
cycle and the respiratory chain is implicated in the energy deficit
in HD brain. Importantly, mitochondrial oxidation of BCAA leads to
the production of acetyl-CoA and succinyl-CoA, two key
intermediates of the citric acid cycle. Insufficient caloric or
protein intake was excluded in our study, as well as impairment of
the BCAA oxidation pathway since organic acid levels in urine were
normal. Therefore, the decrease in plasma BCAA observed in the. FED
group probably results from the activation of a compensatory
mechanism to provide energy substrates to the citric acid cycle, as
described in various cachexia-producing illnesses (Szpetnar et al.
2004; De Bandt and Cynober 2006). The correlation between decreased
BCAA levels and weight loss in our study suggests that excessive
mobilization and oxidation of BCAA to produce energy in muscle is
associated with weight loss and reduced lean body mass. The
observation of weight loss prior to neurocognitive decline suggests
that neurological symptoms are exacerbated when substrates from the
periphery become insufficient to compensate for the energy deficit
in the HD brain. The normal rate of oxygen consumption recently
observed after striatal infusion of succinate (Weydt et al. 2006)
supports the idea that providing energy through an increase in both
systemic and cerebral citric acid cycle intermediates may be a
promising therapeutic approach in HD.
[0056] On the pathophysiological level, our study also showed that
low plasma BCAA levels result in low IGF I levels in the HD group
compared to controls although they have a higher protein and
caloric intake. The tight connection between IGF1 and essential
amino acids has been extensively studied (Straus and Takemoto 1988;
Harp et al. 1991; Thissen et al. 1994; Gomez-Merino et al. 2004).
The availability of essential amino acids seems to have a greater
effect on IGF1 gene expression than hormonal stimuli such as serum
insulin concentrations (Maiter et al. 1989). IGF1 is also a more
sensitive indicator of nutrient repletion than albumin, prealbumin
or orosomucoide, as observed in our study. Interestingly,
huntingtin is a substrate of the serine-threonine Akt pathway,
which is activated by IGF1 (Humbert et al. 2002). Altered
activation of the Akt pathway has been shown to decrease
phosphorylation of the mutated huntingtin, resulting in an
increased neuronal toxicity (Rangone et al. 2005). Low levels of
IGF1 in HD patients might therefore provide an explanation of the
alteration in Akt activation observed in HD cellular models.
Consequently, increasing BCAA levels to correct the deficit in
IGF1, should favor the phosphorylation of mutated huntingtin,
thereby decreasing its toxicity.
[0057] In conclusion, the combination of a rigorous nutritional
assessment and metabonomic tools has provided new insight into HD.
We have demonstrated the existence of an early energy deficiency in
this neurodegenerative disorder, reflected by weight loss. We have
also identified the first reliable biomarker in Huntington disease.
The evidence of decreased plasma BCAA levels in very early stages
of HD highlights the io possibility for therapies aimed at
supplying a sufficient pool of acetyl-CoA to compensate for the
early energy deficit.
[0058] Altogether, these data are supportive of a causative role
for energy delidiency in Huntington's disease. Rather than a detect
in the respiratory chain, low plasma BCAA levels indicate an
energetic impairMent in the Krebs cycle. Therefore, molecules
selected for their ability to reverse this deficiency in
mitochondrial ATP production should refill the pool of catalytic
intermediates of the Krebs cycle. Dietary compounds such as
triheptanoin have recently been used in human therapeutic trials
for their ability to refill the pools of Catalytic intermediates of
the Kreb's cycle, a key energetic process called anaplerosis.
Because of additional evidence for triheptanoin metabolites to
cross the blood brain barrier, anaplerotic therapies represent
promising molecules for reversing the energy deficiency associated
with neurodegenerative diseases and thereby correcting some if not
all clinical manifestations of these diseases.
[0059] Material and methods
[0060] HD Patients, Premanifest Carriers and Controls
[0061] We included 32 individuals with abnormal CAG repeats
expansions (>36) in the HDl gene (19 women and 13 men) and 21
controls (13 women and 8 men). In the HD group, 15 were premanifest
carriers who had before applied for predictive testing clue to
their risk tbr HD, 10 were at a very early stage of the disease and
7 had moderate signs of the disease. Controls were healthy
volunteers in the same age range, unrelated to HD individuals. All
participants were examined and blood samples were taken during a
single visit to the reference centre for HD at the Salpetriere
Hospital (Paris). Patients, premanifest carriers and controls were
enrolled in a clinical protocol authorized by the Assistance
Publique des Hopitaux de Paris (CRC 05129), and approved by the
local ethics committee. Informed consent was obtained for all
participants.
[0062] Determination of Weight Balance and Food Intake
[0063] Height and weight were recorded the day of the clinical
examination. Weight loss was calculated by subtracting current
weight from the weight of the patient 5 years before inclusion in
the study. This information was obtained during the interview and
was verified retrospectively from the patients' medical files. The
body mass index (BMI) was obtained by dividing weight (in
kilograms) by height (in meters) squared. Bioelectrical impedance
(Tanita.RTM.) was measured to evaluate the lean mass and fat mass
of all participants (Segal et al. 1988).
[0064] To determine food intake, HD patients at early stages,
premanifest carriers and controls prospectively recorded their
normal food consumption during 3 days preceding their examination.
The accuracy of the 3-days record was verified one month later with
a questionnaire assessing food intake over a 24-hours period that
was sent to the homes of all participants. A professional dietician
(CG) used these two documents to calcidate mean total calories, and
protein, lipid and sugar intake for both the HD and control groups
using an automated system (Diaeta software.RTM.).
[0065] Multiparametric Evaluation of Weight Balance
[0066] A standardized protocol was designed to thorouuhly evaluate
all possible causes of weight loss and to avoid biases related to
food intake and circadian chanues. It included sequentially: (i) a
minimal 12 hours fast the night preceding the examination, (ii) and
morning blood and urine collection at the same hour (9 am) Samples
were stored on ice for immediate analyses or frozen at -80.degree.
C. for further analyses.
[0067] Standard analyses included blood cell count, blood and urine
glucose, serum electrolytes, and basic nutritional parameters, such
as serum cholesterol, triglycerides, albumin, prealbumin and
orosomucoid. To refine the evaluation of nutrient repletion, serum
insulin growth factor type I (IGF1) concentrations were measured
using a specific immunoradiometric assay (IGF1 RIACT, Cis-Bio
International, Gif- sur-Yvette. France). The three main axes
involved in the regulation of Weight balance were explored:
inflammation, endocrine function and intermediary metabolism. The
evaluation of inflammation included determination of the
erythrocyte sedimentation rate (ESR) and quantification of
C-reactive protein (CRP) and the serum interleukins IL1 .beta. and
IL6 by ELISA (Diaclone, Besancon. France). Besides serum IGF1, the
basic endocrine evaluation included measurements of fasting serum
cortisol (at 9am), tetraiodothyronine (T4L), thyroid stimulating
hormone (TSH) and insulin (Elisa Access ultrasensitive insulin,
Beckman Coulter, Roissy, France). We explored intermediary
metabolism through analysis of (i) plasma amino acids. using ion
exchange chromatography after coloration by ninhydrine (Aminotag,
Geol), (ii) organic acids in urine by gas chromatography (GS
Variant 3400) coupled to mass spectrometry (Ion trap, Saturn 2000,
Variant) after extraction with ethyl acetate. and derivation by
silylation, (iii) the plasma acylcarnitines profile by tandem mass
spectrometry (Applied Biosystem) with electrospray ionization (ESI)
and FIA (flow injection analysis). Acylcarnitines were identified
by using a precursor ion m/z 85 scan and quantified in MRM
(multiple reaction monitoring) mode. Acetylcarnitine-(C2-carnitine)
levels were used to survey the fasting status of both HD
individuals and controls (Costa et al. 1999).
[0068] .sup.1H Nuclear Magnetic Resonance Spectroscopy (NMR) on
Plasma
[0069] Plasma samples were prepared for .sup.1H NMR spectroscopy
with minimal handling. Plasma samples were deproteinized using a 10
kDa filter (Nanosep, Omega) to avoid interference from high
molecular weight species such as lipoproteins. Before use, the
filter was washed twice with water by centrifugation to remove
glycerol. A 100 .mu.l aliquot of 3.89 mM
[trimethylsilyl]-2,2,3,3-tetradeuteropropiOnic acid in
.sup.2H.sub.2O (TSP-.sup.2H.sub.2O, Aldrich) was added to 500 .mu.l
of the ultrafiltrate, providing a chemical shift reference
(.delta.=0.00 ppm), a concentration reference and a deuterium lock
signal. The pH of the ultrafiltrate was adjusted to 2.50.+-.0.05
with concentrated HCl. Finally 500 .mu.l of the sample was placed
in a 5 mm NMR tube (Wilmad Royal Imperial). The .sup.1H NMR spectra
were determined on an Avance-500 SB spectrometer (Broker, France)
eqUipped with a 5 mm BBI (broadband inverse) probe; samples were
not spun. Spectra were collected at 25.degree. C. and consisted in
32K data points with a spectral width of 6,000 Hz and a total
acquisition times of 27 min. A 90.degree. radiofrequency pulse,
following a water signal presaturation of 10s, was used for each
128 scans. Shimming of the sample was performed automatically on
the deuterium signal. The resonance line widths for TSP and
metabolites were <1 Hz. Before a Fourier transformation into 64K
data points, a sine-bell squared filter (SSB=2) was used to reduce
noise. The phase and the baseline were corrected manually using the
spectrometer software (X-Win NMR 3:5, Broker, France). NMR spectra
were first analyzed individually in order to detect abnormal
signals--i.e. treatment or special food--that could further
interfere with global analyses. For statistical analyses, spectra
were data reduced in numerical format by integrating spectral
regions (buckets) every 0.02 ppm and scaled to the total intensity
of the spectruM with Amix 3.6.8 software (Broker Analytische
Messtechnik, Germany) from 0.8 to 8.6 ppm, the water peak area
being excluded from each spectrum (4.4 to 5.2 ppm). Accordingly,
each bucket from the NMR spectrum corresponded to a single
variable.
[0070] Statistical Analyses
[0071] Metabonomic studies consist in multivariate statistical
analyses, e.g. principal components analysis (PCA) and partial
least squares analysis (PLS), with as many components as variables.
Multivariate analyses of the data obtained by NMR spectroscopy were
performed with Simca-P.RTM. 11.0 software (Umetrics, Sweden). For
PCA and PLS, unit variance scaled data were used to ensure the
inclusion of metabolites present in both high and low
concentrations. Each variable was mean centered and computed as
I/SD.sub.j, standard deviation of variable j computed around the
mean. PCA considers each bucket from the NMR spectrum as an X
variable and was therefore used to discern the presence of inherent
similarities between spectral profiles and to identify outliers.
PLS is a regression extension of PCA and best describe the
variation within the data according to a priori classification,
corresponding to a Y variable, which was the UHDRS score in our
study. PLS was used to identify principal components maximizing the
covariance between all X (NMR spectrum) and Y (UHDRS) variables.
The greatest dispersion of the spectral profiles is usually best
observed in the two first components of the analyses. The first and
second components in the X space (NMR spectrum) were denoted PC[1]
and PC[2] respectively. Therefore, PLS score plot (PC[1]/PC[2]) of
pair-wise compared groups displayed the greater variation within
the NMR spectrum according to UHDRS. The validity of each component
was obtained by cross validation. Contribution plot was then
analyzed in order to determine the respective weight of variables
contributing most to the separation between groups.
[0072] For comparison of means, ANOVA or non-parametric tests when
appropriate, were used (SPSS.RTM. software). Since our study was
based on a multiparametric approach, we also performed PCA to
search for possible correlations between the different parameters
that were analyzed (Simca-PC) software).
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Example 2
[0106] Triheptanoin Therapy of HD Mouse Models
[0107] Aims of the Study
[0108] A pilot study is conducted to test the effect of dietary
triheptanoin therapy versus control diet on selected strains of HD
R6/2 mice (Mangariani 1996, Cell 87: 493-506, Kosinski 1999,
Neuroreport 10: 3891-6). The study includes (i) measuring rates of
cerebral anaplerosis from heptanoate and brain ATP in R6/2 mice of
different ages and in control mice in order to demonstrate the
.ability of triheptanoin metabolites to cross the blood brain
barrier of R6/2 mice and to reverse central energy deficit; (ii)
assessing the therapeutic efficacy of triheptanoin by accurate
behavioral testing, in vivo brain microdialysis (to assay
neurotransmitters, triheptanoin metabolites, and BCAA) and
neuropathological examination (iii) metabolornic analyses on mouse
plasma and urine.
[0109] Research Methods
[0110] R6/2 and control mice, on triheptanoin-enriched and control
diets, are infused sequentially--at 4, 8 and 12 weeks--with various
doses of [5,6,7-.sup.13C.sub.3] heptanoate (by gavage or by
intravenous infusion) for 1 hr before brain sampling, in order to
follow the kinetics of anaplerosis in the brain (using the assay of
anaplerotic CoA esters). The concentrations of ATP, ADP, and AMP
are assayed in all brain samples, as well as in muscle of R6/2
mice. We arc also assessing the concentration and labeling pattern
of neurotransmitters ttenerated by anaplerosis (GABA, glutamate) to
determine whether the brain is deficient in these compounds and
whether anaplerotic therapy improves these parameters. Brain
tissues are be stored at -70.degree. C. in view of possible
additional analyses (like measurement of oxidative stress).
Behavioral analyses include (i) open field activity monitoring
using the TruScan system at 4, 8 and 12 weeks: (ii) RotaRod
analysis performed using the AccuScan system equipped with a
shockable floor at 6 and 12 weeks; (iii) and the Morris Water Maze,
the most popular task in behavioral neuroscience used to assess
spatial learning and memory at 12 weeks. Other primary endpoints
from the study are weight loss and survival.
[0111] As both HD patients and genetic mouse models of HD manifest
a presymptomatic loss of DA receptors, a dysfunctional dopaminergic
neurotransmission may be involved in early HD presentation. More
recent studies have shown that DA release is severely compromised
in R6/2 mice (Johnson 2006, J Neurochem 97: 737-46). Accordingly, a
first group of animals is sacrificed at 12 weeks by cervical
dislocation for neurotransmitters analyses. Blood are collected and
brain tissue arc rapidly removed and dissected on ice into the
following regions: striatum, hippocampuS, frontal cortex, posterior
cortex, cerebellum, and midbrain. These regions are dissected for
both la and right hemispheres of the brain. All right side regions
are processed for analysis of DA, 5-hydroxytryptophane (5-HT) and
norepinephrine (NE) and related metabolites (3-d ihydrophenylacetic
acid, homovalinic acid, 3-methoxytyramine and 5-hydroxyindolacetic
acid) by HPLC with electrochemical detection. Acetylcholine (Ach)
is also measured in frontal posterior cortex tissue by HPLC with
electrochemical detection. Branched chain amino acids is measured
by HPLC and triheptanoin metabolites by mass spectrometry.
Neuropathology, and especially nuclear neuronal inclusions, is
performed on the left side regions. At 12 weeks, a second group of
animals is prepared for in vivo microdialysis to monitor in-vivo
release of DA, 5-HT by potassium and Ach release by scopolamine in
the striatum. Changes in the extracellular concentrations of DA,
5-HT and Ach is compared by three ways ANOVA
(time.times.genotype.times.diet) with repeated measures. Sequential
analyses of plasma and urine samples from R6/2 and control mice at
4, 8 and 12 weeks are also perfomed with both NMR spectroscopy and
mass spectrometry. This aims the detection of triheptanoin
metabolites (propionyl and pentanoyl-CoA derivatives) in body
fluids from treated mice. The comparison of the metabolic profile
from R6/2 and control mice on control diet, by multivariate data
statistical analyses, can possibly confirm the implication of BCAA
in the pathophysiology of R6/2 mice, as evidenced in HD piemani
fest carriers and patients, and partially suggested in previous
metabolomic study in R6/2 mice (Underwood 2006, Brain 129: 877-8).
More importantly, the comparison of the metabolic profile from
treated versus non -treated R6/2 mice can assess whether
triheptanoin can lead to the correction of such hypercatabolic
profiles.
Example 3
[0112] Therapy of Spinocerebellar Ataxia 7 (SCA7)
[0113] 1/In vitro Trial
[0114] To create a simplified model of SCA7 in vitro we used
primary cultures of dissociated cerebellar cells because lesion of
the cerebellum, particularly the Purkinje cell (PC) layer, accounts
for the ataxia phenotype in patients with SCA7. Our cerebellar cell
cultures were composed of glial cells and neurons, 5 to 10% of
which expressed calbindin (CaBP) identifying them as PC. To examine
the effects of mutant. ATXNT7 on PC survival, the cells were
infected at DIV1 (1st Day In Vitro) with lentiviral vectors
carrying truncated forms of normal and mutant ataxia 7 (ATXN7T:
amino acids 1-232) fused to GUT (ATXN7T-10Q-GFP, ATXN7T-100Q-GFP).
These lentiviral vectors allowed efficient expression of these
proteins in about 90% of neurons, including Purkinje cells.
Infection by ATXN7T-I 00Q-GFP led to massive neuronal loss, almost
exclusively in Purkinje neurons (-85% of Purkinje cell death versus
.about.20% loss of other neurons), thus reproducing one of the
major features of the human disease (FIG. 6).
[0115] This model is used to assess the ability of anaploretic
molecules to rescue Purkinje cells infected by ATXN7T-100Q-GFP. Two
compounds are tested: the 3-ketovalerate and the 3-hydroxyvalerate,
which are both be directly incorporated by the cells in culture.
These molecules are added in the culture medium on the same day
when the cells are infected and half of the medium is replaced
every 4 days. The cultures are maintained for 20 days and the
potential rescue of Purkinje cells is quantified as described
above.
[0116] 2/In Vivo Experiments
[0117] We chose to use the SCA7 knock-in mouse model developed in
the group of H. Y. Zoghbi (Yoo 2003 Neuron, 37: 383-401), which
expresses ATXN7 with 266 glutamines at endogenous levels in the
proper spatio-temporal pattern. Mouse Sca7 is highly homologous to
human SCA7, with 88.7% identity at the protein level.
Sca7.sup.266Q/5Q mice reproduce features of infantile SCA7, which
is characterized by a more rapid progression and a broader spectrum
of phenotypes than the adult-onset disease. From 5 weeks of age,
these mice develop progressive weight loss, ptosis, visual
impairment, tremor, ataxia, muscle wasting, kyphosis and finally
die at around 14-19 weeks of age. Sca7.sup.266Q/5Q mice manifested
coordination impairment in the rotarod test by 5 weeks. By 8-9
weeks, gait ataxia is apparent and motor coordination further
deteriorates. As in patients, neuropathological studies revealed
progressive Nls formation in many brain regions. Although no
neuronal loss is observed in the brain, Purkinje cells that are one
of the most commonly affected cells in SCA7, have a decreased body
cell size. [0118] a--Metabolic Study of the SCA7 Knock-in Mice
[0119] Similarly to patients with HD or SCA7, the SCA7 knock-in
mice show a severe progressive weight loss already significant at
the onset of the motor phenotype. A protocol has been set up to
measure lbod and beverage intake in correlation with weight
evolution of these mutant mice compared to wild-type ones. This
protocol is tested on a group of 3 knock-in females and 3 wild-type
females from the same litters. The animals are kept one per cage
and they are given a definite amount of food and beverage. Then,
their intake and their weight are measured four times a week.
Preliminary results show that this procedure is efficient to
evidence progressiVe weight loss and food intake evolution (FIG.
7). Soon after onset (7-8 weeks) but before serious motor
deterioration (8-9 weeks) food intake from mutant animals is higher
than in wild-type although there are already lighter. These data
are in favour of the hypothesis of a hypermetabolic state during
the early phases of the phenotype. to After 8-9 weeks, the
locomotor impairment is already so disabling that the mutant mice
probably can't reach the food as easily as the wild-type ones.
[0120] b--Microarray analysis of early transcriptional
modifications
[0121] Metabolic impairment in HD and other related diseases have
been proposed to result from dysregulation of major metabolic
pathways at the transcriptional level (Mothel 2007 PLoS ONE, 2(7):
e647: Cui 2006 Cell 127: 59-69). Considering the function of the
ATXN7 protein and the early transcriptional abnormalities
previously evidenced in SCA 7 and other polyglutaminc disease, the
transeriptome of the cerebellum 014-5 knock-in mice versus 4-5
wild-type mice is analysed at two early stages betbre onset
(post-natal day 10 and post-natal day 22) and one late symptomatic
stage (11 weeks of age).
Example 4
[0122] Evaluation of the Potential Benefit and Safety of
Anaplerotic Therapy in Huntington Disease (HD)
[0123] A 5-days preclinical trial with triheptanoin in 6 HD
affected patients is conducted. This short-term protocol is as
follows:
[0124] 1. Day 1: (i) an extended neurological and general clinical
examination; (ii) a global metabolic workup (blood and urine
samples) to have an overview of the metabolic profile of HD
patients at baseline: (iii) a skin biopsy to test in vitro the
ability of triheptanoin to generate energy from the Krebs cycle and
the respiratory chain; (iv) the measurement of 5'AMP-activated
protein kinase (5'AMPK) activity in patients' fibroblasts, as a
reflection of the levels of intracellular energy metabolism; and
(v) a .sup.31P-NMR spectroscopy on patients' muscle in order to
assess their skeletal muscle ATP production.
[0125] 2. Day 2: an oral loading test of a meal enriched with
triheptanoin, together with urine and blood samples before and
after meal to determine: [0126] measurements of triheptanoin
metabolites, through plasma acylcarnitines profile and urine
organic acids (Roe et al. 2002), to ensure that triheptanoin is to
properly metabolized in HD patients: [0127] analyses of
mitochondrial redox status, through the ratio of lactate to
pyruvate and 3-hydroxybutyrate to acetoacetate (Mochel et al.
2005), to assess. in vivo the ability of triheptanoin to generate
energy from the Krebs cycle without overloading the respiratory
chain.
[0128] 3. On days 3, 4 and 5: the pursuit of a diet enriched with
triheptanoin to determine if a protein sparing effect occurs, i.e.
the normalization of the plasma branched chain amino acids (BCAA)
and serum IGF1 levels, and/or the elevation of urinary urea.
Clinical examination attempts to identify acute effects on the
systemic energy deficiency (muscle strength, motor function)
associated with HD. In addition, patients undergo a second muscle
.sup.31P-NMR spectroscopy in order to evidence. a possible
short-term effect of triheptanoin on patients' peripheral energy
metabolism.
[0129] Study Design
[0130] On the first day of admission, HD patients are examined.
Motor dysfunction is evaluated with the Unified Huntington disease
rating scale, UHDRS (Siesling et al. 1998), and a total functional
capacity score, TFC (harder et al. 2000). General health condition
is also recorded, in particular history of dysfunction of the
digestive tract. Before lunch, blood and urine samples are
collected. Standard analysesare performed (blood cell count, blood
clotting factors, blood and urine glucose, serum electrolytes), as
well as a global metabolic workup including plasma redox status
(lactate, pyruvate, acetoacetate, 3-hydroxybutyrate), plasma amino
acids and acylcarnitines, and urine organic acids as described
(Mochel et al. 2005). In the absence of blood clotting dysfunction.
a skin biopsy is performed. A simple functional test using
propionate labelled with C.sup.P is further performed in cultured
fibroblasts (Benoist et al. 2001). Propionate is one of the main
anaplerotic products of triheptanoin, and is incorporated into
protein providing that enough ATP is produced from the Krebs cycle
and the respiratory chain. The normal rate of protein synthesis,
after incorporation of C.sup.14-propionate, in HD cells therefore
reflect the integrity of the respiratory chain in HD, as well as
the possibility to generate energy from the Krebs cycle through the
anaplerotic pathway. The activity of 5'-AMPK, which senses changes
in the cellular energy state, is also determined in patients'
fibroblasts (Chou et al. 2005), in order to evidence a peripheral
deficit in intracellular energy metabolism. In addition, oxidative
mitochondrial metabolism is specifically assessed by muscle
.sup.31P-NMR spectroscopy using data collected at the end of a
given exercise and during the following recovery (Lodi et al.
2000).
[0131] On the second day, HD patients ingest a loading dose of
triheptanoin (1 g/Kg). For convenience, and better digestive
tolerance, triheptanoin is usually administrated together with a
dairy product. Repeated blood samples are collected before and,
sequentially. after meal (30, 60, 90, 120 and 180 minutes after
triheptanoin ingestion) for assessment of redox status and
acylcarnitines profile. Urine is also collected before and alter
the triheptanoin load (90 and 180 minutes) for analyses of organic
acids.
[0132] On the next 3 following days, HD patients pursue an
isocaloric diet enriched with triheptanoin (1 g/Kg/ day divided in
3 to 4 meals). Fasting plasma BCAA, serum IGF1 and urinary urea are
analyzed daily and neurological examination is repeated with UHDRS
and TFC scoring. On day 5, muscle .sup.31P-NMR spectroscopy is
repeated in order to determine the relative concentrations of
inorganic phosphate, phosphocreatine and ATP levels after
triheptanoin administration.
[0133] Patient Selection Criteria
[0134] This study involves 6 patients with abnormal CAG repeats
expansions (>36) in the HDI gene, with regular medical and
psychological follow-up. The selection of patients is based on:
[0135] UHDRS score ranging 15 and 50, corresponding to patients at
an early to moderate stage of the disease, in order to facilitate
the compliance of patients to dietary treatment; [0136] low levels
of plasma BCAA, in order to search for a raise in these amino acids
under triheptanoin treatment.
[0137] Informed consent is obtained for all participants.
REFERENCES
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T T (2000). "Abnormal in vivo skeletal muscle energy metabolism in
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(2000). "Rate of functional decline in Huntington's disease.
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DeLonlay P. Touati G. et al. (2005). "Pyruvate carboxylase
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[0145] Throughout this application, various references describe the
state of the art to which this invention pertains. The disclosures
of these references are hereby incorporated by reference into the
present disclosure.
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