WO2017103224A1 - Méthode de traitement de la sclérose latérale amyotrophique - Google Patents

Méthode de traitement de la sclérose latérale amyotrophique Download PDF

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WO2017103224A1
WO2017103224A1 PCT/EP2016/081602 EP2016081602W WO2017103224A1 WO 2017103224 A1 WO2017103224 A1 WO 2017103224A1 EP 2016081602 W EP2016081602 W EP 2016081602W WO 2017103224 A1 WO2017103224 A1 WO 2017103224A1
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mch
als
agonist
mch receptor
receptor
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Luc Dupuis
Albert LUDOLPH
Pauline VERCRUYSSE
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Universitaet Ulm
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Strasbourg
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Universitaet Ulm
Institut National de la Sante et de la Recherche Medicale INSERM
Universite de Strasbourg
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin
    • A61K38/34—Melanocyte stimulating hormone [MSH], e.g. alpha- or beta-melanotropin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention concerns the treatment of amyotrophic lateral sclerosis, in particular the prevention of weight loss in subjects suffering from amyotrophic lateral sclerosis.
  • ALS Amyotrophic lateral sclerosis
  • MNDs motor neuron diseases
  • ALS a neuroprotective agent commercialized in 1996. It increases the lifespan from 3 to 6 months only, illustrating the poverty and ineffectiveness of current therapeutic solutions.
  • Rilutek Apart from Rilutek, the arsenal is completed with more than 30 supplementary off-label drugs, prescribed in order to relieve the myriad of symptoms associated with ALS.
  • ALS-related weight loss is an early phenomenon, occurring before the onset of motor symptoms in patients. It has been established repeatedly that patients who lose the most weight are those who have shorter life. It was shown, for example, that patients with a body mass index indicative of denutrition exhibited very short survival (Desport, 1999). More recently the same group has shown that a modest weight loss of 5% of initial body weight (or 3-5 kgs) at diagnosis was strongly correlated with very poor prognosis (Marin, 201 1 ). Similar studies have been undertaken in many countries and are all converging. Based on those correlations, nutritional intervention to increase caloric intake has been tested in mouse models, and more recently in a specific population of ALS patients.
  • the present inventors discovered a mechanism causing unbalanced metabolism and weight loss in ALS. They observed that typical ALS pathology occurs in the lateral hypothalamus in both SOD1 mice and human ALS patients. By comparing SOD1 mice and ALS patients, the inventors identified that MCH (Melanin Concentrating Hormone), a hypothalamic neuropeptide known for its role in appetite stimulation, and weight control, was a potential target for treating weight loss in these patients. They showed that continuous intracerebroventricular (ICV) administration of the peptide leads to weight gain in SOD1 model and no effect on control mice.
  • MCH Mellanin Concentrating Hormone
  • the present invention thus concerns an agonist of a Melanin Concentrating Hormone (MCH) receptor for use for reestablishing a metabolic balance in a subject suffering from amyotrophic lateral sclerosis (ALS).
  • MCH Melanin Concentrating Hormone
  • the present invention also concerns an agonist of a MCH receptor for use in the treatment of ALS for reestablishing a metabolic balance in a subject suffering from ALS.
  • the present invention also concerns an agonist of a MCH receptor for use for preventing, slowing or stopping weight loss or promoting weight gain in a subject suffering from ALS.
  • the present invention further concerns an agonist of a MCH receptor for use in the treatment of ALS for preventing, slowing or stopping weight loss or promoting weight gain in a subject suffering from ALS.
  • Another object of the invention concerns an agonist of a MCH receptor for use for prolonging life expectancy of a subject suffering from ALS.
  • Still another object of the invention concerns an agonist of a MCH receptor for use in the treatment of ALS for prolonging life expectancy of a subject suffering from ALS.
  • the present invention further concerns an agonist of a MCH receptor for use in the treatment of ALS.
  • MCH receptor refers to an integral plasma membrane protein which binds melanin-concentrating hormone and agonists thereof. It encompasses MCHR1 and MCHR2.
  • MCHR1 is typically 353 amino acids long and it has the highest degree of homology to the somatostatin receptor family. The receptor is highly conserved between human and rodents. Database scanning revealed a second human high-affinity receptor for MCH (MCHR2). Human MCHR1 preferably consists of the sequence SEQ ID NO: 3
  • MCHR2 is typically 340 amino acids long and has low (38%) homology to the MCHR1 receptor.
  • Human MCHR2 preferably consists of the sequence SEQ ID NO: 4
  • MCHR2 mRNA In higher species, the expression pattern of MCHR2 mRNA is similar to MCHR1 with the highest expression observed in the brain, notably in the frontal cortex, amygdala and nucleus accumbens. Both MCH receptors are also expressed in areas associated with energy homeostasis such as the arcuate nucleus and the ventral medial hypothalamus.
  • the MCH receptor refers to MCHR1 or MCHR2.
  • the MCH receptor is MCHR1 .
  • agonist of MCH receptor refers to any compound that can directly or indirectly activate the signal transduction cascade related to an MCH receptor, in particular of MCHR1 or MCHR2, preferably of MCHR1 .
  • Tests and assays for determining whether a compound is an agonist of MCH receptor are well known by the skilled person in the art such as described in An et al. (2001 ).
  • binding of a candidate compound to MCHR1 and MCHR2 can be measured by filtration binding assay.
  • Membranes (10 mg protein) from transiently transfected HEK293-MCHR1 and HEK293-MCHR2 cells can be mixed with 0-9.8 nM 125 l- labeled candidate compound in a binding buffer (50 mM Hepes 10 mM MgCI 2 2 mM EGTA; protease inhibitors, 0.1 % BSA, pH 7.6).
  • membrane-bound 125 l-labeled candidate compound can be separated from the free 125 l-labeled candidate compound by filtration through a filter plate on a cell harvester and washed with ice-cold binding buffer supplemented with 80 mM NaCI. Eighty microliters of scintillation liquid can typically be added, and the radioactivity can be counted on a counter.
  • the agonist of MCH receptor used in the context of the invention may be a MCH peptide or a derivative thereof which retains the ability to activate a MCH receptor, small organic molecules, a nucleic acid encoding a MCH peptide or a derivative thereof which retains the ability to activate a MCH receptor and a MCH peptide expression activator.
  • the agonist of a MCH receptor is selected from the group consisting of a MCH peptide or a derivative thereof which retains the ability to activate a MCH receptor, small organic molecules, a nucleic acid encoding a MCH peptide or a derivative thereof which retains the ability to activate a MCH receptor and a MCH peptide expression activator.
  • the agonist of a MCH receptor is selected from the group consisting of a MCH peptide or a derivative thereof which retains the ability to activate a MCH receptor, a nucleic acid encoding a MCH peptide or a derivative thereof which retains the ability to activate a MCH receptor and a MCH peptide expression activator,
  • the agonist of a MCH receptor is a MCH peptide or a derivative thereof which retains the ability to activate a MCH receptor.
  • the agonist of a MCH receptor is a MCH peptide.
  • the agonist of a MCH receptor can pass through the Blood-Brain
  • polypeptide and peptide are used indifferently and refer to native peptides (either proteolysis products or synthetically synthesized peptides) and further to peptidomimetics, such as peptoids and semipeptoids which are peptide analogs, which may have, for example, modifications rendering the peptides more stable while in a body, or more immunogenic.
  • Methods for preparing peptidomimetic compounds are well known in the art and are specified in Quantitative Drug Design, CA. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992).
  • a peptide consists of less than 50 amino acids, preferably less than 40 amino acids, more preferably less than 30 amino acids, still preferably less 20 amino acids. More preferably, peptides according to the invention have a length of from about 5 to about 19 amino acids, from about 5 to about 18 amino acids, from about 5 to about 17 amino acids, from about 5 to about 16 amino acids, from about 5 to about 15 amino acids, from about 5 to about 14 amino acids, from about 5 to about 13 amino acids.
  • amino acid is understood to include: the 20 naturally occurring amino acids i.e. alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, praline, serine, threonine, tryptophan, tyrosine, and valine; amino acids harbouring the post-translational modifications which can be found in vivo such as hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acids including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine.
  • amino acid includes both D- and L-amino acids.
  • the peptides of the invention can be substituted by one or more atoms or groups.
  • MCH peptide Melanin Concentrating Hormone, a cyclic 19- amino acid orexigenic hypothalamic peptide of sequence DFDMLRCMLGRVYRPCWQV identified as SEQ ID NO: 1 that is a natural agonist of MCH receptors (MCHR1 and MCHR2) known for its role in appetite stimulation and weight control, Human and rodent MCH are identical.
  • MCH peptide is processed from proMCH.
  • proMCH consists typically of 165 amino acids.
  • proMCH consists of the sequence SEQ ID NO: 5
  • a MCH peptide By “derivative of a MCH peptide” is meant herein a molecule comprising the amino acid sequence of a MCH peptide subject to various changes, including, but not limited to, chemical modifications, substitutions, insertions, extensions and deletions where such changes do not destroy the ability of the peptide to activate a MCH receptor.
  • Derivatives of a MCH peptide thus include a protein comprising a MCH peptide, a fragment of a MCH peptide and a mutant of a MCH peptide, which retain the ability to activate a MCH receptor.
  • a protein comprising a MCH peptide may be proMCH.
  • a mutant of a MCH peptide is preferably a peptide having at least 80% identity with the peptide of sequence SEQ ID NO: 1 .
  • the percentage of identity relates to the number of identical amino- acids obtained for an optimal paired alignment (i.e. the alignment maximizing the number of identical amino-acids) of the sequence of a protein homologous to SEQ ID NO: 1 , divided by the total number of amino-acids in SEQ ID NO: 1 .
  • Alignment can be performed manually or using computer programs such as the EMBOSS-Needle program (Needleman and Wunsch (1970) J. Mol. Biol. 48:443 - 453).
  • the identity percentage may be computed by producing an overall pair wise alignment based on the Needleman- Wunsch alignment algorithm for finding the optimum alignment (including "holes” or "gaps") between two sequences over the whole of their length, for example by using Needle, and by using the BLOSUM62 matrix with a penalty for inserting « gaps » of 10 and a penalty of extension of « gaps » of 0.5.
  • the percentage of identity according to the invention is at least 85%, more preferably from at least 90%, and even more preferably from at least 95%.
  • a 'fragment of a MCH peptide' may be 1 1 , 12, 13, 14, 15, 16, 17, 18 contiguous amino acids.
  • retaining the ability of a MCH peptide to activate a MCH receptor is meant herein that the compound can be bound to MCHR1 or MCHR2 and activate the signaling pathway of these receptors, as defined above.
  • nucleic acid encoding a MCH peptide or a derivative thereof is meant herein an isolated nucleic acid comprising or consisting of a sequence encoding a MCH peptide, preferably, of SEQ ID NO:1 or encoding a derivative thereof.
  • SEQ ID NO: 2 shows the nucleotide sequence encoding the full-length MCH peptide.
  • nucleic acid herein refers to the phosphate ester polymeric form of ribonucleosides (also called “RNA molecule”), deoxyribonucleosides (also called “DNA molecule”) or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form or a double-stranded form.
  • the polynucleotide is selected from the group constituted of DNA, RNA, genomic DNA and cDNA.
  • Nucleic acids may be produced recombinantly, synthetically, or by any means available to those of skill in the art. They may also be cloned by standard techniques such as PCR (polymerase chain reaction).
  • the nucleic acid of the invention contains only a coding region for the MCH peptide of the invention or a derivative thereof.
  • the nucleic acid further comprises, in operable linkage, a portion of nucleic acid that allows for efficient translation of the coding sequence in the target cell.
  • the nucleic acid (when in a DNA form) further comprises a promoter in operable linkage which allows for the transcription of the coding region and the portion of nucleic acid that allows for efficient translation of the coding region in the target cell.
  • Variant nucleic acid sequences may include nucleic acid sequences that have at least about 80% nucleic acid sequence identity with a nucleic acid sequence disclosed herein.
  • a variant nucleic acid sequences will have at least about 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% nucleic acid sequence identity to a nucleic acid sequence or a fragment of a nucleic acid sequence as disclosed herein.
  • Nucleic acid sequence identity is defined as the percentage of nucleic acids in the variant sequence that are identical with the nucleic acids in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Sequence identity may be determined over the full length of both variant and reference nucleic acid sequences.
  • vector refers to vectors used in recombinant DNA techniques allowing entities, such as a nucleic acids, to be transferred into a target cell for the purpose of replicating the vectors comprising the nucleotide sequences used in the invention and/or expressing the proteins encoded by the nucleic acids of the invention.
  • vectors used in recombinant DNA techniques include but are not limited to plasmids, chromosomes, artificial chromosomes or viruses.
  • the vector is also a so-called "delivery system".
  • the vector is preferably an isolated vector.
  • the vector is an expression vector.
  • an "expression vector” herein refers to a vector as defined above designed for protein expression in cells.
  • the vector is engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. Those regulatory sequences may as well be called “expression signal” or "control sequences".
  • a nucleic acid in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the target cell.
  • operably linked means that the components described are in a relationship permitting them to function in their intended manner.
  • a regulatory sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences.
  • the vector preferably comprises a MCHR agonist expression cassette, i.e. a nucleic acid encoding a MCHR agonist of the invention operably linked to at least one expression signal allowing its expression.
  • a MCHR agonist expression cassette i.e. a nucleic acid encoding a MCHR agonist of the invention operably linked to at least one expression signal allowing its expression.
  • the at least one expression signal is particularly selected among a promoter, a terminator, an enhancer and their combinations, preferably among a promoter, a terminator.
  • Suitable promoters, terminators and enhancers are well-known by the skilled person and may be selected to be compatible with the target cell for which the expression vector is designed to be used in.
  • control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.
  • promoter is well-known in the art and encompasses nucleic acid regions ranging in size and complexity from minimal promoters to promoters including upstream elements and enhancers.
  • the promoter is typically selected from promoters which are functional in mammalian cells, although prokaryotic promoters and promoters functional in other eukaryotic cells may be used.
  • the promoter is typically derived from promoter sequences of viral or eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which expression is to occur.
  • eukaryotic promoters they may be promoters that function in a ubiquitous manner (such as promoters of a-actin, b-actin, tubulin, human elongation factorl -alpha (EF1 -alpha)) or, alternatively, a tissue-specific manner (such as promoters of the genes for pyruvate kinase).
  • Viral promoters may also be used, for example the moloney murine leukaemia virus long terminal repeat (MMLV LTR) promoter, the rous sarcoma virus (RSV) LTR promoter or the human cytomegalovirus (CMV) IE promoter.
  • MMLV LTR moloney murine leukaemia virus long terminal repeat
  • RSV rous sarcoma virus
  • CMV human cytomegalovirus
  • the promoters are inducible so that the levels of expression of the heterologous gene can be regulated during the life-time of the cell. Inducible means that the levels of expression obtained using the promoter can be regulated.
  • Chimeric promoters may also be used comprising sequence elements from two or more different promoters described above.
  • the vectors used in the present invention further comprise an origin of replication.
  • the vectors of the invention may be used, for example, to transfect or transform a target cell.
  • the vector comprising a nucleic acid encoding a nucleic acid of the invention is preferably a non-viral vector or a viral vector.
  • a non-viral vector may be selected in the group consisting of a plasmid, a liposomal nucleic acid complex and a carrier-associated nucleic acid.
  • plasmid it is herein meant a double-stranded circular DNA.
  • the plasmid may include a marker gene enabling to select the cells comprising said plasmid, an origin of replication to allow the cell to replicate the plasmid and/or a multiple cloning site allowing the insertion of a DNA fragment, in particular the nucleic acid encoding a nucleic acid of the invention.
  • Non-limitative examples of carrier-associated nucleic acids are polymer-carried DNA and cationic lipids.
  • liposomes used in liposomal DNA complexes are well-known in the art. Said liposomes may be cationic, anionic or neutral liposomes.
  • Cationic lipids are also known in the art and are commonly used for gene delivery.
  • Such lipids include Lipofectin Tm also known as DOTMA (N- [I- (2, 3-dioleyloxy) propyls N, N, N-trimethylammonium chloride), DOTAP (1 , 2-bis (oleyloxy)-3 (trimethylammonio) propane), DDAB (dimethyldioctadecyl- ammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Choi (3 beta- (N- ( ⁇ ', ⁇ '- dimethyl aminomethane)-carbamoyl) cholesterol).
  • Cationic lipids for gene delivery are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine).
  • DOPE dioleyl phosphatidylethanolamine
  • viral vector it is herein meant a recombinant viral vector.
  • the viral vector is preferably selected in the group consisting of a retrovirus vector, a lentivirus vector, Herpes simplex viral vector, an adenovirus vector and an adeno- associated virus vector, preferably a lentivirus vector.
  • a retrovirus vector herein refers to a genetically engineered a retrovirus based vector that cannot replicate and produce progeny infectious virus particles once the virus has entered the target cell.
  • a retrovirus vector genome consists in a single-stranded positive-sense RNA that encodes a transcriptase enabling to generate double-stranded DNA.
  • retroviruses that are widely used for delivery of genes both in tissue culture conditions and in living organisms. Examples include and are not limited to murine leukemia virus (MLV), human immunodeficiency virus (HIV-1 ), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), rous sarcoma virus (RSV), fujinami sarcoma virus (FuSV), moloney murine leukemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), moloney murine sarcoma virus (Mo-MSV), abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29), and avian erythroblastosis virus (AEV) and all other retroviridiae including lentiviruses.
  • MMV murine leukemia virus
  • HMV-1 human immunodeficiency virus
  • EIAV equine infectious ana
  • the basic structure of a retrovirus genome is a 5' LTR and a 3' LTR, between or within which are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a target cell genome and gag, pol and env genes encoding the structural and enzymatic components - these are polypeptides required for the assembly of viral particles.
  • More complex retroviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell.
  • LTRs long terminal repeats
  • the LTRs are responsible for pro viral integration, and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes. Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome.
  • the LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R and U5.
  • U3 is derived from the sequence unique to the 3' end of the RNA.
  • R is derived from a sequence repeated at both ends of the RNA and
  • U5 is derived from the sequence unique to the 5' end of the RNA.
  • the sizes of the three elements can vary considerably among different retroviruses.
  • the viral vector is a targeted vector that is it has a tissue tropism which is altered compared to the native virus, so that the vector is targeted to particular cells.
  • This may be achieved by modifying the retroviral Env protein.
  • the envelope protein is a non-toxic envelope or an envelope which may be produced in non- toxic amounts within the primary target cell, such as for example a MMLV amphotropic envelope or a modified amphotropic envelope.
  • the envelope is one which allows transduction of human cells.
  • env genes include, but are not limited to, VSV-G, a MLV amphotropic env such as the 4070 A env, the RD1 14 feline leukaemia virus env or haemagglutinin (HA) from an influenza virus.
  • the Env protein may be one which is capable of binding to a receptor on a limited number of human cell types and may be an engineered envelope containing targeting moieties.
  • the env and gag-pol coding sequences are transcribed from a promoter and optionally an enhancer active in the chosen packaging cell line and the transcription unit is terminated by a polyadenylation signal.
  • a suitable promoter-enhancer combination is that from the human cytomegalovirus major immediate early (hCMV-MIE) gene and a polyadenylation signal from SV40 virus may be used.
  • hCMV-MIE human cytomegalovirus major immediate early
  • polyadenylation signal from SV40 virus
  • the retrovirus vector is a murine leukemia virus (MLV) vector.
  • MLV murine leukemia virus
  • Retroviral vectors derived from the amphotropic Moloney murine leukemia virus (MLV-A) are commonly used in clinical protocols worldwide.
  • the "lentivirus vector” is a single-stranded RNA that integrates in the genome of the target cell, thereby allowing long term expression of the nucleic acid.
  • the lentivirus is a genus of viruses of the retroviridae family, characterized by a long incubation period.
  • a distinction between the lentivirus family and other types of retroviruses is that lentiviruses have the capability to infect both dividing and non-dividing cells (Lewis, 1992; Lewis and Emerman, 1994).
  • a detailed list of lentiviruses may be found in Coffin et al. ("Retroviruses" 1997 Cold Spring Harbour Laboratory Press Eds: JM Coffin, SM Hughes, HE Varmus pp 758-763).
  • lentiviruses can be divided into primate and non-primate groups.
  • primate lentiviruses include but are not limited to: the human immunodeficiency virus (HIV), the causative agent of human acquired-immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV).
  • the non-primate lentiviral group includes the prototype "slow virus” visna maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BTV).
  • the lentiviral vector is a HIV-based VSV-pseudotyped defective vector.
  • Herpes simplex virus is an enveloped, double-stranded (ds) DNA virus.
  • the mature virion consists of different components: an external envelope containing about 13 glycoproteins involved in different functions, among which the first steps of binding and entry into the target cell; an amorphous layer known as the tegument, containing some 20 different proteins with structural and regulatory roles; and an icosadeltahedral capsid containing a toroidal dsDNA.
  • the Herpes simplex virus vector is in particular a HSV-1 vector.
  • Different types of HSV-1 vectors a known to the skilled in the art, such as amplicons, replication-defective and replication-competent vectors.
  • amplicons are plasmid-derived vectors engineered to contain both the origin of HSV DNA replication (ori) and HSV cleavage-packaging recognition sequences (pad).
  • ori origin of HSV DNA replication
  • pad HSV cleavage-packaging recognition sequences
  • amplicons are transfected into mammalian cells with HSV helper functions, they are replicated, form head-to-tail linked concatamers and are then packaged into viral particles.
  • There are two major methods currently used for producing amplicon particles one based on infection with defective helper HSVs and the other based on transfection of HSV-1 genes, such as a set of pac-deleted overlapping cosmids or a pac-deleted and ICP27- deleted BAC-HSV-1 .
  • Replication-defective vectors are made of mutant viruses with deletions in one or more genes essential for the lytic cycle, whereas “replication-competent vectors” are composed of attenuated viruses where genes that are not essential for replication in cultured cells in vitro are either mutated or deleted (Roberto Manservigi et at. 2010, “HSV Recombinant Vectors for Gene Therapy", Open Virol J. 2010; 4: 123-156).
  • the "adenovirus” is a double-stranded, linear DNA virus that does not go through an RNA intermediate.
  • adenovirus There are over 50 different human serotypes of adenovirus divided into 6 subgroups based on the genetic sequence homology.
  • the natural target of adenovirus is the respiratory and gastrointestinal epithelia, generally giving rise to only mild symptoms.
  • Serotypes 2 and 5 are most commonly used in adenoviral vector systems and are normally associated with upper respiratory tract infections in the young.
  • Adenoviruses are non-enveloped, regular icosohedrons.
  • a typical adenovirus comprises a 140nm encapsidated DNA virus.
  • the icosahedral symmetry of the virus is composed of 152 capsomeres: 240 hexons and 12 pentons.
  • the core of the particle contains the 36kb linear duplex DNA which is covalently associated at the 5' ends with the Terminal Protein (TP) which acts as a primer for DNA replication.
  • TP Terminal Protein
  • the DNA has inverted terminal repeats (ITR) and the length of these varies with the serotype.
  • the adenovirus is a double stranded DNA non-enveloped virus that is capable of in vivo and in vitro transduction of a broad range of cell types of human and non-human origin. These cells include respiratory airway epithelial cells, hepatocytes, muscle cells, cardiac myocytes, synoviocytes, primary mammary epithelial cells and post-mitotically terminally differentiated cells such as neurons.
  • Adenoviral vectors are also capable of transducing non dividing cells. This is very important for diseases, such as cystic fibrosis, in which the affected cells in the lung epithelium have a slow turnover rate. In fact, several trials are underway utilizing adenovirus-mediated transfer of cystic fibrosis transporter (CFTR) into the lungs of afflicted adult cystic fibrosis patients.
  • CFTR cystic fibrosis transporter
  • Adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes.
  • the large (36 kilobase) genome can accommodate up to 8kb of foreign insert DNA and is able to replicate efficiently in complementing cell lines to produce very high titres of up to 10 12 .
  • Adenovirus is thus one of the best systems to study the expression of genes in primary non-replicative cells.
  • Adenoviral vectors enter cells by receptor mediated endocytosis. Once inside the cell, adenovirus vectors rarely integrate into the host chromosome.
  • AAV adeno-associated virus vector
  • ITRs inverted terminal repeats
  • ORFs open reading frames
  • the former is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and the latter contains overlapping nucleotide sequences of capsid proteins: VP1 , VP2 and VP3, which interact together to form a capsid of an icosahedral symmetry. Removal of the rep and cap from the DNA of the vector eliminates the above-mentioned integrative capacity.
  • an adeno-associated virus vector is selected from the group consisting of an AAV1 vector, AAV2 vector, AAV6 vector, AAV8 vector, AAV9 vector and a hybrid AAV vector.
  • a “hybrid AAV vector” is a vector comprising the rep gene of one AAV vector and the cap gene of another AAV vector.
  • Non-limitative examples of hybrid AAV vector are an AAV1/2 vector, i.e. a vector comprising AAV1 rep gene and AAV2 cap gene, or an AAV2/9 vector, i.e. a vector comprising with AAV2 rep gene and AAV9 cap gene.
  • AAV2/9 vector is pZac2.1 (for example from Penn Vector Core, Upenn).
  • target cells is meant herein the MCH expressing neurons, located within the lateral hypothalamus and zona incerta. Despite this restricted distribution MCH neurons project widely throughout the brain.
  • MCH peptide expression activator is meant herein a natural or synthetic compound that has a biological effect to activate the expression of a nucleic acid encoding an MCH peptide as defined above.
  • a gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA.
  • Gene products also include messenger RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins (e.g., calpain or calpastatin) modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, SUMOylation, ADP-ribosylation, myristilation, and glycosylation.
  • proteins e.g., calpain or calpastatin
  • agonists of a MCH receptor are cited for example in MacNeil et al., 2009 and include the peptide of sequence DFDMLRCMLGRVFRPCWQY (SEQ ID NO: 8), wherein the last amino-acid is preferably radiolabeled.
  • Examples of agonists of a MCH receptor also include the peptide of sequence of SEQ ID NO: 6 consisting of the 6-16 segment of MCH, which was demonstrated as being a full agonist of MCHR1 and MCHR2 of potency similar to those of the full-length MCH.
  • MCHR agonists also include the peptide of sequence SEQ ID NO: 7 which consists of the 6-17 segment of MCH.
  • MCHR agonists include compounds disclosed in EP2098242, WO200808357, US20080124319, US20040242487 and US5449766.
  • ALS Amyotrophic lateral sclerosis
  • MNDs motor neuron diseases
  • the disease occurs apparently at random with no clearly associated risk factors.
  • Individuals with this sporadic form of the disease do not have a family history of ALS, and their family members are not considered to be at increased risk for developing it.
  • About 5 to 10 percent of all ALS cases are inherited.
  • the familial form of ALS usually results from a pattern of inheritance that requires only one parent to carry the gene responsible for the disease.
  • Subsets of ALS cases are of familial origin and five major genes are currently associated with familial ALS (C90RF72, SOD1, FUS, TARDBP and TBK1).
  • the SOD1 gene was the first associated with ALS and most ALS mouse models currently used are based upon overexpression of mutant forms of SOD1.
  • ALS can be sporadic ALS or inherited ALS.
  • ALS is inherited ALS.
  • inherited ALS can result from at least one mutation in the C90RF72, SOD1, FUS, TARDBP and/or TBK1 gene.
  • inherited ALS results from at least one mutation in the SOD1 gene.
  • the term "subject” refers to any human or animal subject suffering from ALS.
  • subject refers to a human.
  • the present invention concerns an agonist of a MCH receptor, as defined above, for use for reestablishing a metabolic balance in a subject suffering from ALS, as defined above.
  • the present invention concerns an agonist of a MCH receptor, as defined above, for use in the treatment of ALS for reestablishing a metabolic balance in a subject suffering from ALS, as defined above.
  • the invention also concerns the use of an agonist of a MCH receptor, as defined above, for the manufacture of a medicament intended for reestablishing a metabolic balance in a subject suffering from ALS, as defined above.
  • the invention also concerns the use of an agonist of a MCH receptor, as defined above, for the manufacture of a medicament intended for the treatment of ALS for reestablishing a metabolic balance in a subject suffering from ALS, as defined above.
  • the invention further concerns a method for reestablishing a metabolic balance in a subject suffering from ALS, as defined above, comprising the step of administering to said subject a therapeutically effective amount of an agonist of a MCH receptor as defined above.
  • the invention further concerns a method of treatment of ALS for reestablishing a metabolic balance in a subject suffering from ALS, as defined above, comprising the step of administering to said subject a therapeutically effective amount of an agonist of a MCH receptor as defined above.
  • metabolic balance is meant herein equilibrium between the intake of nutrients and their eventual loss through absorption or excretion. In a positive balance the intake of a nutrient exceeds its loss; in a negative balance a nutrient is used or excreted faster than it is consumed in the diet. In case of ALS, the metabolic balance is importantly negative as ALS patients are reported to consume 15-16% fewer calories than required to meet their energetic needs, and are thus subject to hypermetabolism.
  • reestablishing a metabolic balance is meant herein reestablishing an equilibrium between food intakes and expenses in order to establish a metabolic balance corresponding to a healthy subject.
  • the present invention also concerns an agonist of a MCH receptor, as defined above, for use for preventing, slowing or stopping weight loss or promoting weight gain in a subject suffering from ALS, as defined above.
  • the present invention also concerns an agonist of a MCH receptor, as defined above, for use in the treatment of ALS for preventing, slowing or stopping weight loss or promoting weight gain in a subject suffering from ALS, as defined above.
  • the invention also concerns the use of an agonist of a MCH receptor, as defined above, for the manufacture of a medicament intended for preventing, slowing or stopping weight loss or promoting weight gain in a subject suffering from ALS, as defined above.
  • the invention also concerns the use of an agonist of a MCH receptor, as defined above, for the manufacture of a medicament intended for treating ALS by preventing, slowing or stopping weight loss or promoting weight gain in a subject suffering from ALS, as defined above.
  • the invention further concerns a method for preventing, slowing or stopping weight loss or promoting weight gain in a subject suffering from ALS, as defined above, comprising the step of administering to said subject a therapeutically effective amount of an agonist of a MCH receptor as defined above.
  • the invention further concerns a method of treatment of ALS for preventing, slowing or stopping weight loss or promoting weight gain in a subject suffering from ALS, as defined above, comprising the step of administering to said subject a therapeutically effective amount of an agonist of a MCH receptor as defined above.
  • weight loss is prevented, slowed or stopped or weight gain is promoted by the reestablishment of a metabolic balance in said subject.
  • weight loss is prevented, slowed or stopped, so that it is inferior to a loss of 3 to 5 kg. Still preferably, weight loss is prevented, slowed or stopped, so that the BMI of the subject is superior to 18.5 kg/m 2 .
  • the present invention also concerns an agonist of a MCH receptor, as defined above, for use for prolonging life expectancy of a subject suffering from ALS, as defined above.
  • the present invention also concerns an agonist of a MCH receptor, as defined above, for use in the treatment of ALS for prolonging life expectancy of a subject suffering from ALS, as defined above.
  • the invention also concerns the use of an agonist of a MCH receptor, as defined above, for the manufacture of a medicament intended for prolonging life expectancy of a subject suffering from ALS, as defined above.
  • the invention also concerns the use of an agonist of a MCH receptor, as defined above, for the manufacture of a medicament intended for treating ALS by prolonging life expectancy of a subject suffering from ALS, as defined above.
  • the invention further concerns a method for prolonging life expectancy of a subject suffering from ALS, as defined above, comprising the step of administering to said subject a therapeutically effective amount of an agonist of a MCH receptor as defined above.
  • the invention further concerns a method of treatment of ALS for prolonging life expectancy of a subject suffering from ALS, as defined above, comprising the step of administering to said subject a therapeutically effective amount of an agonist of a MCH receptor as defined above.
  • “Prolonging life expectancy” is also referred to and encompasses "long-term survival time” or “reducing mortality or morbidity”, which as used herein relates to the superiority of an agonist of a MCH receptor, in particular MCH, to placebo, in particular non-treatment, in subjects suffering from ALS, meaning that the subjects have a higher life span following an administration as described herein, in particular on a regular daily basis.
  • ALS leads to death within 3 to 5 years after onset in human subjects.
  • a prolonged life expectancy as intended herein is preferably a lengthening of the life expectancy of at least 5 years.
  • the present invention also concerns an agonist of a MCH receptor, as defined above, for use in the treatment of ALS, as defined above.
  • the invention also concerns the use of an agonist of a MCH receptor, as defined above, for the manufacture of a medicament intended for treating a subject suffering from ALS, as defined above.
  • the invention further concerns a method for the treatment of ALS, as defined above, comprising the step of administering to said subject a therapeutically effective amount of an agonist of a MCH receptor as defined above.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such disorder or condition.
  • ALS is treated by preventing, slowing or stopping weight loss or promoting weight gain, in particular by reestablishing a metabolic balance.
  • a “therapeutically effective amount” of a compound of the invention is meant a sufficient amount of the compound to treat ALS, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treating and the severity of the disorder, activity of the specific compounds employed, the specific combinations employed, the age, body weight, general health, sex and diet of the subject, the time of administration, route of administration and rate of excretion of the specific compounds employed, the duration of the treatment, drugs used in combination or coincidental with the specific compounds employed, and like factors well known in the medical arts.
  • the agonist of a MCH receptor in particular the MCH peptide, is administered at a low dose.
  • low dose is meant herein a dose lower than the one conventionally used to increase food intake.
  • Doses conventionally used to increase food intake in mice are typically 3 ⁇ g/day (Ito et al., 2003).
  • a low dose refers to a dose at least 2 fold lower, more preferably at least 3 fold lower, still preferably at least 4 fold lower, most preferably at least 5 fold lower than the dose conventionally used to increase food intake.
  • the agonist of a MCH receptor used in the context of the invention may be administered by any suitable route, such as intravenous, intradermal, intracerebroventricular, subcutaneous, intramuscular, intraperitoneal, oral (e.g., buccal, inhalation, nasal and pulmonary spray), intradermal, transdermal (topical), transmucosal or intraocular route.
  • suitable route such as intravenous, intradermal, intracerebroventricular, subcutaneous, intramuscular, intraperitoneal, oral (e.g., buccal, inhalation, nasal and pulmonary spray), intradermal, transdermal (topical), transmucosal or intraocular route.
  • the agonist of a MCH receptor in particular the MCH peptide, is administered by the intracerebroventricular route.
  • the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • vehicles which are pharmaceutically acceptable for a formulation capable of being injected.
  • These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.
  • the preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small tumor area.
  • the agonist of a MCH receptor may further be used in combination with another anti-ALS therapy.
  • anti-ALS therapy is meant herein any anti ALS treatment approved for use in a subject in need thereof.
  • Such anti-ALS therapies are well-known from the skilled person and include mostly glutamate antagonists (Rilutek), which provide protection from amino-acid-derived excitotoxicity intracellular processes, or free radical scavengers (Radicut).
  • Such therapies do not reverse the course of the disease and are purely supportive. They do not act on the metabolic disorders caused by ALS.
  • SEQ ID NO: 1 shows the amino acid sequence of full-length MCH peptide.
  • SEQ ID NO: 2 shows the nucleotide sequence encoding full-length MCH peptide.
  • SEQ ID NO: 3 shows the amino acid sequence of Human MCHR1 .
  • SEQ ID NO: 4 shows the amino acid sequence of Human MCHR2.
  • SEQ ID NO:5 shows the amino acid sequence of proMCH.
  • SEQ ID NO: 6 shows the amino acid sequence of the 6-16 fragment of peptide MCH RCMLGRVYRPC.
  • SEQ ID NO: 7 shows the amino acid sequence of the 6-17 fragment of peptide MCH RCMLGRVYRPCW.
  • SEQ ID NO: 8 shows the amino acid sequence of the MCH agonist (prot) DFDMLRCMLGRVFRPCWQY. See Table 1 for primer sequences identified as SEQ ID NO: 9 to SEQ ID NO: 44.
  • Figure 1 Effects of pioglitazone on peripheral biomarkers in ALS patients
  • A summary of metabolic effects of pioglitazone (and TZDs) in humans
  • B-E Changes in plasma adiponectin (% from the baseline) (B), glycaemia (% from the baseline, C), circulating aspartate amino-transferase (ASAT, changes in U/L from the baseline, D), and alanine amino-transferase (ALAT, changes in U/L from the baseline, E).
  • Pioglitazone treated patients are significantly different from placebo treated patients for these items as assessed using a mixed effects regression model analysis. Data are presented as mean and standard error.
  • Weight loss (kg loss per month, A, C, E, G) and changes in BMI from the baseline (B, D, F, H) in the whole ALS population (A, B), spinal onset patients (C, D), in patients with relatively preserved quality of life (E, F) and in patients with preserved bulbar function (G, H).
  • the inventors used the results from the EuroQoL questionnaire to identify patients that had no or only few problems with their everyday-life. Selected patients answered that they had either no or few problems in their everyday life during at least 6 months after their allocation to a group.
  • A Experimental scheme: pioglitazone was provided per os after 6 hours of fasting. Food was reintroduced 1 hour after gavage, and food intake recorded for the next 24 hours.
  • * p ⁇ 0.05 Paired Student's t-test for the drug. Data are presented as mean and standard error.
  • POMC Proopiomelanocortin
  • AgRP Agouti Related Peptide
  • MCH Melanin concentrating hormone
  • CART cocaine- and am p h etam i n e- reg u I ated transcript
  • NPY neuropeptide Y
  • AVP Arginine vasopressin
  • CRH Corticotropin-releasing hormone
  • TRH Thyrotropin-releasing hormone
  • SST Thyrotropin-releasing hormone
  • ORX orexin
  • NTS neurotensin.
  • A: pTDP43 pathological staining in the lateral hypothalamus of post-mortem patient tissues. Representative images are shown for ALS patients (a,b,c, scale bar 20 ⁇ ). pTDP43 staining in the lateral hypothalamus (LH) and Arcuate Nucleus (Arc) were scored by a blind observer in ALS post mortem patient tissues as compared with their control patient tissues (d, n 10).
  • mice p62 pathological aggregates in the lateral hypothalamus of mice. p62 immunohistochemistry was performed on selected sections after identification of lateral hypothalamus according to Paxinos Brain Atlas (identified regions in box of a and b). Representative images are shown for SOD1 (G86R) (b,d,e) mice and control littermates (a,c) at onset of symptoms at three magnifications (scale bar 1000 ⁇ (upper row), 250 ⁇ (middle row), 50 ⁇ (lower row)).
  • EXAMPLE 1 Alterations in the hypothalamic melanocortin pathway in amyotrophic lateral sclerosis
  • the two treatment groups were 100 mg riluzole plus 45 mg pioglitazone (pioglitazone group) and 100 mg riluzole plus placebo (placebo group). Patients were randomly assigned to one of the two treatment groups and both groups were matched for age, gender and site of onset (Dupuis et at., 2012).
  • Transgenic mice were housed in the animal facility of the medicine faculty of France University, with 12 h/12 h of light/dark and unrestricted access to food and water. In all experiments, littermates were used for comparison.
  • Transgenic SOD1 G86R
  • TDP43(A315T) were previously described and were maintained as heterozygous in their initial C57BI6/J background (Wegorzewska et al, 2009).
  • Heterozygous Fus? NLS/+ are knock-in mice expressing FUS protein deleted from its C-terminal NLS from one of the endogenous Fus gene.
  • Tph2-YFP mice were purchased from Jackson laboratories (Bar Harbor, ME, USA; strain 014555) and maintained in their initial genetic background. Female Tph2-YFP mice were crossed with male SOD1 (G86R) to generate compound transgenic mice.
  • mice were sacrificed at the ages indicated at 2PM, and tissues were quickly dissected, frozen in liquid nitrogen, and stored at -80 °C until use.
  • animals were anesthetized by intraperitoneal injection of ketamine (Imalgene 1000®, Merial, Lyon France; 90 mg/kg body weight) and xylazine (Rompun 2% ®, Bayer, Leverkusen, Germany; 10 mg/kg body weight) at the ages indicated at 2PM.
  • Pioglitazone (Actos®, Takeda, London, UK) was dissolved in 10% (v/v) dimethyl sulfoxide (DMSO, Fisher Scientific, lllkirch, France) and a single oral administration was given by gavage at a dose of 40 mg/kg body weight.
  • DMSO dimethyl sulfoxide
  • MCH (Sigma, St Louis, MO, USA) was administrated by Intracerebral Injection (ICV) using continous delivery through Alzet osmotic mini-pumps (see below). Measurements of food intake
  • mice were fasted from 9AM to 3PM, and pioglitazone was administrated at 3PM. Food was reintroduced 1 hour after gavage and food intake was recorded during 24h.
  • mice were fasted from 8AM to 3PM (7 hours fasting conditions) or from 2PM to 3PM (1 hour fasting conditions) and food was reintroduced after 7 hours or 1 hour of fasting. Food intake was measured one hour and 24 hours after refeeding.
  • ICV Surgical procedure 8AM to 3PM (7 hours fasting conditions) or from 2PM to 3PM (1 hour fasting conditions) and food was reintroduced after 7 hours or 1 hour of fasting. Food intake was measured one hour and 24 hours after refeeding.
  • ketamine and xylazine 75 days old-mice were anesthetized with ketamine and xylazine (respectively 90mg/kg from Imalgene 1000, Merial, Lyon, France and 10mg/kg from Rompun 2%, Bayer, Puteaux, France).
  • 50 ⁇ _ of lidocaine 21 .33mg/ml_, Xylovet, Ceva, Libourne, France
  • a sterile brain infusion cannula of Brain Infusion Kit 3 28-gauge; Alzet, Palo Alto, CA
  • the stereotaxic coordinates were 0.4 mm posterior to the bregma, 0.8 mm left lateral to the midline, and 2.0 mm from the surface of the skull.
  • the cannulas were fixed to the skull with instant adhesive gel (loctite 454).
  • the infusion cannula was connected to an osmotic minipump (Alzet, model no.
  • Body weight and daily food intake were measured daily between 1 1 am and 1 pm during 14 days.
  • Biotinylated donkey anti-Rabbit secondary antibody Jackson, West Grove, PA, USA; 1 :500
  • biotinylated donkey anti-Mouse secondary antibody Jackson, West Grove, PA, USA, 1 :500
  • Staining was performed using Vectastain Elite ABC kit (Vector, Burlingame, CA, USA). After revelation with 3,3'-Diaminobenzidine (DAB, Sigma, St Louis, MO, USA; 0.5mg/ml), sections were mounted and images of all sections were taken.
  • DAB 3,3'-Diaminobenzidine
  • White balance, gain, exposure, and light settings were kept the same when acquiring all images of a given staining.
  • Paraffin included human hypothalamus were sectioned in 70 ⁇ sections according to the method of Feldengut (2013, J. of Neurosciences Methods).
  • mRNA levels of genes of interest were normalized to expression levels of the 18S ribosomal, TBP and pol2 RNA housekeeping genes using GeNorm (Vandesompele et at., 2002). Primer sequences are shown in Table 1 .
  • Orexin 29 F-CTTCAGGCCAACGGTAACCA 30 R-GGTGCTAAAGCGGTGGTAGT
  • PC2 33 F- AAAATACCACCCACCGGCAA 34 R- CCAGGTAGCGGACGAAGTTT
  • the inventors took advantage of data collected during the pioglitazone GERP-ALS trial to investigate energy metabolism in ALS patients in response to pioglitazone (Dupuis et al., 2012).
  • thiazolinediones including pioglitazone
  • adiponectin an adipose-derived hormone through direct transcriptional activation of the adiponectin gene in adipocytes
  • pioglitazone treatment multiplied by four the levels of circulating adiponectin in ALS patients after six months of treatment, and this was maintained after 12 months of treatment (Fig. 1 B).
  • Pioglitazone decreases glycemia through hepatic and skeletal muscle PPARy.
  • pioglitazone decreased glycaemia (Fig. 1 C), although this effect was milder than observed in other populations, including non-diabetic patients (Belfort et al., 2006).
  • Fig. 1 C glycaemia
  • pioglitazone decreased levels of ASAT and more robustly levels of ALAT (Fig. 1 D- E).
  • pioglitazone displayed the expected metabolic effects on adipocytic, muscular, and hepatic biomarkers, and was likely able to activate PPARy in these tissues.
  • Table 2 Numbers of patients for Figure 1
  • Pioglitazone does not lead to weight gain in ALS patients
  • TZDs are also known to act in hypothalamic melanocortin neurons to promote feeding (Diano et at., 201 1 , Long et at., 2014), and this activation of PPARy in melanocortin neurons is responsible for the robust weight gain associated with TZDs treatment (Lu et at., 201 1 , Ryan et at., 201 1 , Long et al., 2014).
  • the evolution of body weight upon pioglitazone treatment represents a reliable proximal marker of PPARy action in hypothalamic melanocortin neurons.
  • pioglitazone had no effect on weight loss (Fig. 2 A), or BMI (Fig.
  • the inventors turned to transgenic mice expressing mutant SOD1 (G86R) (SOD1 m mice) as a model of ALS and set up an experimental protocol to study food intake in response to pioglitazone.
  • G86R mutant SOD1
  • pioglitazone has various effects on food intake depending on genetic background, dose, route and associated diet (Diano et al., 201 1 , Ryan et at., 201 1 , Long et at., 2014).
  • hypothalamic melanocortin neurons constitute the primary target of TZDs to promote food intake (Diano et al., 201 1 , Long et at., 2014).
  • the melanocortin system is mainly constituted of two antagonistic neuronal types located in the arcuate nucleus: pro- opiomelanocortin (POMC) neurons, that secrete the anorexigenic peptide ocMSH, and AgRP neurons, that promote food intake, mostly through production of AgRP, an endogenous ocMSH antagonist.
  • POMC mRNA levels were two-fold lower in SOD1 m mice, either at 75 days of age (Fig. 4 A a.) or at onset (Fig.
  • the inventors then asked whether the observed melanocortin defects translated into functional abnormalities. Indeed, the combination of decreased POMC with increased AgRP is usually found in situations of promotion of food intake, such as during fasting or in case of leptin deficiency (Mizuno et al., 1998, Mizuno et al., 1999, Ziotopoulou et al., 2000), and compromised melanocortin system is likely to affect food intake behaviour, in particular during refeeding (Perez-Tilve et at., 2010). Thus, the inventors measured food intake after short term fasting in SOD1 m mice.
  • TDP43 phosphorylated TDP43
  • LH lateral hypothalamic
  • Figure 6A Mice expressing mutant SOD1 do not develop TDP-43 positive inclusions, but rather inclusions positive for p62, a ubiquitin binding protein that accumulates upon UPS or autophagic dysfunction.
  • the LH is known to express two major neuropeptides, orexin and MCH, two orexigenic peptides.
  • the inventors observed that MCH, but not orexin expression was downregulated in mutant SOD1 mice ( Figure 4A).
  • the decrease of MCH expression was also observed using immunohistochemistry, with decreased numbers of MCH-positive neurons, and overall decreased staining ( Figure 4B).
  • the inventors then studied whether the loss of MCH could be responsible for weight loss in mutant SOD1 mice.
  • SOD1 (G86R) are already losing weight at 75 days and they are living between 105 and 1 15 days generally.
  • MCH delivery was at a constant flow of 1 ⁇ g/day during 13 days intra-cerebro ventricularly through a cannula stereotactically inserted in the lateral ventricle.
  • Dupuis L Oudart H, Rene F, Gonzalez de Aguilar JL, Loeffler JP. Evidence for defective energy homeostasis in amyotrophic lateral sclerosis: benefit of a high-energy diet in a transgenic mouse model. Proc Natl Acad Sci U S A. 2004;101 (30):1 1 159-64.
  • Dupuis L Dengler R, Heneka MT, Meyer T, Zierz S, Kassubek J, et al. A randomized, double blind, placebo-controlled trial of pioglitazone in combination with riluzole in amyotrophic lateral sclerosis. PLoS One. 2012;7(6):e37885.
  • PPARgamma ligands increase expression and plasma concentrations of adiponectin, an adipose-derived protein. Diabetes. 2001 ;50(9):2094-9.
  • Mizuno TM Kleopoulos SP, Bergen HT, Roberts JL, Priest CA, Mobbs CV.
  • Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin. Diabetes. 1998;47(2):294-7.
  • Mizuno TM Makimura H, Silverstein J, Roberts JL, Lopingco T, Mobbs CV. Fasting regulates hypothalamic neuropeptide Y, agouti-related peptide, and proopiomelanocortin in diabetic mice independent of changes in leptin or insulin.
  • Vandesompele J De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al.
  • ciliary neurotrophic factor does not reverse the starvation-induced changes of serum corticosterone and hypothalamic neuropeptide levels but induces expression of hypothalamic inhibitors of leptin signaling. Diabetes. 2000;49(1 1 ):1890-6.

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Abstract

La présente invention concerne un agoniste d'un récepteur de MCH (hormone de mélano-concentration) destiné à être utilisé pour prévenir, ralentir ou stopper la perte de poids ou pour favoriser la prise de poids chez un sujet atteint de sclérose latérale amyotrophique (SLA).
PCT/EP2016/081602 2015-12-16 2016-12-16 Méthode de traitement de la sclérose latérale amyotrophique Ceased WO2017103224A1 (fr)

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