WO2014141124A1 - Composé d'activation de voie erk pour prévenir ou traiter une résistance à la leptine - Google Patents

Composé d'activation de voie erk pour prévenir ou traiter une résistance à la leptine Download PDF

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WO2014141124A1
WO2014141124A1 PCT/IB2014/059728 IB2014059728W WO2014141124A1 WO 2014141124 A1 WO2014141124 A1 WO 2014141124A1 IB 2014059728 W IB2014059728 W IB 2014059728W WO 2014141124 A1 WO2014141124 A1 WO 2014141124A1
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leptin
erk
tanycytes
compound
pathway activating
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PCT/IB2014/059728
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English (en)
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Vincent PREVOT
Eric Trinquet
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Institut National De La Sante Et De La Recherche Medicale
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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
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the present invention relates to the field of preventing or treating disorders or diseases caused by the occurrence of a leptin resistance in an individual.
  • Leptin is a 16 kDa hormone discovered in 1994, which is predominantly secreted by adipocytes and encoded by the obese (ob) gene. Leptin is able to interact with a variety of peripheral tissues as well as regions of the brain through specific receptors.
  • leptin is known to act on the brain to reduce food intake by regulating the activity of neurons in the mediobasal hypothalamus (Ahima, R.S. & Flier, 2000, J.S. Leptin. Annu Rev Physiol 62, 413-437).
  • Leptin is well-known for its appetite-suppressing effect. Indeed, obesity is associated with high circulating leptin levels that fail to trigger neuronal responses (Considine, R.V., et al, 1996, N.Engl.J.Med. 334, 292-295 ; Frederich, R.C., et al, 1995, Nat Med 1, 1311-1314).
  • deficient leptin signalling in humans or in murine models for instance models bearing a mutation of leptin (ob/ob mice) or the leptin receptor (db/db mice), is associated with increased food intake and reduced energy expenditure.
  • the main consequence is the development of imbalance between intake and expenditure, which may be associated with the development of various disorders.
  • leptin is known to regulate insulin sensitivity and glucose homeostasis, and is thus involved in a wide variety of other disorders associated with energy imbalance, apart from obesity, such as diabetes or cardiovascular diseases (Roth, 1998, Diabetes Metab. Rev., 13, 1-2). Apart from its role in food intake and glucose homeostasis, leptin has also been implicated in reward behaviour, hippocampal dependent learning and memory, and is further known for its anti-depressant and anti-convulsivant effects. Many of those actions of leptin can be linked to its effect in the central nervous system (or CNS), and more particularly to its effect in the basomedial hypothalamus. For a more complete review of the effects of leptin as a regulator of neuronal functions, one may for instance refer to Harvey (Harvey, 2007, Current Opinion in Pharmacology, 7 :643-647).
  • leptin is a particularly interesting hormone due to its diverse effects in health and disease, as an active agent, and for promising new therapies in a wide spectrum of conditions.
  • leptin resistance » negatively impacts the prospects of treating an individual suffering from a leptin resistance, and more particularly individuals suffering from obesity.
  • the mechanisms underlying resistance to leptin remain, however, poorly understood, and different models have been proposed (El-Haschimi et al, 2000, J Clin Invest 105, 1827- 1832).
  • leptin transport into the brain appears to be a crucial limiting step in the modulation of its central effects, and participates in leptin resistance both in humans and rodents (Caro et al, 1996, Lancet 348, 159-161; Schwartz et al, 1996, Nat Med, 2, 589- 593).
  • exogenous leptin administered intraperitoneally triggers LepR signalling in specialized neurons in the arcuate nucleus, a major hypothalamic leptin- sensing site adjacent to the median eminence (ME), within 15 minutes, and in more dorsal hypothalamic sites involved in energy homeostasis 1-2 h later.
  • This delay is reduced to minutes when leptin is administered intracerebroventricularly, suggesting that its transport from the periphery to the cerebrospinal fluid (CSF) is a limiting step in the propagation of leptinergic signaling (Faouzi et a/., 2007, Endocrinology, 148, 5414-5423).
  • CSF cerebrospinal fluid
  • leptin may be transported across the blood-brain barrier by a saturable transport system (Banks, W.A., 2004, Peptides, 25, 331-338).
  • leptin resistance occurs due to an inhibition of one or more intracellular signaling cascade associated with isoforms of the leptin receptor (LepR or LR), in particular the long isoform (LepRb or LRb).
  • LepR or LR isoforms of the leptin receptor
  • LepRb or LRb long isoform
  • Leptin receptor one may for instance refer to Tartaglia (Tartaglia L.A., 1997, J.Biol.Chem, 272, 6093-6096).
  • This signaling involves intracellular pathways, including STAT3, ERK and PI3K ⁇ Akt, which, up to now, had not been precisely defined (Robertson et al, 2008, Physiol Behav, 94, 637-642).
  • the invention relates to new strategies for treating and/or preventing the occurrence of leptin resistance in an individual.
  • the invention relates to ERK- pathway activating compounds for use for treating and/or preventing leptin resistance in an individual.
  • the development of a leptin resistance may be particularly detrimental for individuals suffering from diabetes, obesity and/or cognitive disorders, and/or any other leptin-related disorder, as it may reduce the efficacy of a treatment in which the administration of leptin is involved, and/or which is aimed at modulating deficient leptin signaling.
  • the invention relates to ERK-pathway activating compounds for treating or preventing leptin resistance in an individual.
  • compositions comprising an ERK-pathway activating compound in combination with leptin.
  • the invention further relates to methods for the screening of compounds for treating and/or preventing leptin resistance.
  • the present invention relates ERK-pathway activating compounds for use for treating and/or preventing leptin resistance in an individual in need thereof.
  • leptin resistance refers to a lack of leptin activity in the brain, which occurs because of a reduction or failure of sensitivity to leptin.
  • a reduction or failure of sensitivity to leptin may be due to a defective leptin transport to the cerebrospinal fluid, and/or defective signalling pathway, downstream of leptin receptor.
  • the present invention further relates to ERK-pathway activating compounds for treating or preventing diabetes, obesity and/or cognitive disorders associated with leptin resistance.
  • the invention relates to pharmaceutical compositions comprising an ERK-pathway activating compound in combination with leptin.
  • the invention relates to methods for the screening of compounds for treating and/or preventing leptin resistance in an individual in need thereof.
  • RT-PCR analysis of the expression of mRNAs for LepR isoforms (from up to bottom: LepRa; LepRb; LepRc; LepRe) in primary cultures of tanycytes (from left to right: tanycytes from the hypothalamus; tanycytes from the ME, tanycytes from the ME, as obtained from Prevot et al. , 2003.
  • the quantitative comparison of phosphorylated STAT3, Akt and ERK is assessed by determining (from left to right; y axis) the ratio pSTAT3/STAT3; pAkt/Akt; pERK/ERK, for three different leptin concentrations (x axis; from left to right: 0, 1 and 10
  • FIG. 3 Tanycytes of the ME release captured leptin via an ERK-dependent signalling pathway.
  • C Representative western blots and quantitative comparison of leptin in cell lysates from leptin-loaded tanycytes and in the medium (released leptin) over 15 min, in the presence or absence of the pharmacological inhibitors of LepR signalling pathways used in Figure 2B.
  • STAT3 was used as a loading control for cell lysate samples.
  • D not detected (two-tailed one-way ANOVA, followed by Tukey's multiple comparison test).
  • TCM tanycyte-cultured medium
  • CL cell lysate
  • the corresponding TCM/CL leptin ratio is indicated in the y axis.
  • FIG. 4 EGF-mediated activation of ERK signaling in the ME restores leptin transport into the hypothalamus.
  • the pERK/ERK ratio in median eminence (ME) tanycytes is indicated in the y axis.
  • the experiment is done on (from left to right) wild-type mice, diet-induced obesity (DIO) mice and db/db mutant mice.
  • FIG. 5 EGF-mediated activation of ERK signaling accelerates the restoration of leptin sensitivity in obese mice once they are replaced on a normal diet.
  • FIG. 6 EGF treatment affects glucose metabolism and restores leptin sensitivity and leptin transport.
  • Body weight (g) is indicated in the y axis, as a function of time (weeks).
  • Blood glucose (mg/dl) is indicated in the y axis, as a function of time
  • the molar ratio leptin/STAT3 is indicated in the y axis of the figure.
  • bottom panel sensitivity to leptin is assessed by measuring by measuring body weight loss after 3 days of peripheral leptin administration (3mg/kg) in control and DIO- mice, with and without long-term EGF treatment (8 weeks); BW decrease does not differ between control mice and DIO mice receiving long-term EGF treatment; T-test, * p ⁇ 0.05
  • the leptin-induced body weight change is indicated in the y axis of the figure.
  • ERK in tanycytes, which express its receptor, integrin ⁇ , in vitro.
  • the y axis indicates the corresponding pERK/ERK ratio in cultured tanycytes, and the x axis indicates the concentration of ghrelin which is present within the medium culture.
  • LepR leptin receptor
  • Tanycytes are specialized ependymal cells which line the surface of the third ventricle. They tend to be enriched, among other ependymal cells, at the ventral part of the third ventricle, and more specifically a region of the third ventricle called the median eminence (ME). So far, they have been involved in the control of feeding and energy balance. More generally, they are emerging as chemosensors in contact with the cerebrospinal fluid (Bolborea & Dale, 2013, Trends in Neurosciences, 36(2), pp 91-100).
  • the inventors have assessed the activation of LepR signalling in the median eminence of adult wild-type C57B1/6 mice by immunohistochemically detecting phosphorylated STAT3 (pSTAT3) at various times after peripheral leptin administration (3 mg/kg). Strikingly, five minutes after leptin injection, pSTAT3 immunoreactivity (IR) was detected both in tanycytic processes, which contact the plexus of permeable fenestrated vessels at the pial surface of the brain (Mullier et al, 2010, J Comp Neurol 518, 943-962), and in their nuclei, whereas pSTAT3-IR in neurons was both infrequent and weak. By 10-15 min after leptin injection, although some tanycytes were still labelled, pSTAT3-IR in neurons dramatically increased, while at longer intervals, pSTAT3-IR occurred almost exclusively in neurons.
  • leptin receptor lepR
  • ME median eminence
  • mice deficient in LepR or with diet-induced obesity DIO
  • blood-borne leptin that is taken up by tanycytes accumulates in the median eminence and fails to gain access to the mediobasal hypothalamus.
  • leptin resistance occurs due to the accumulation of leptin in the median eminence.
  • leptin which has been taken up by tanycytes cannot reach the mediobasal hypothalamus.
  • the invention relates to ERK-pathway activating compounds for use for treating and/or preventing leptin resistance in an individual in need thereof.
  • the inventors have shown that the activation of ERK signaling, in particular by means of a pharmacological treatment, is required for the release of the exogenous leptin captured by tanycytes, and for restoring leptin transport in the mediobasal hypothalamus (MBH) of obese mice (see example 4 herein).
  • MSH mediobasal hypothalamus
  • ERK and/or LepR-ERK signaling are thus shown to be an efficient therapeutic target in leptin-related diseases such as obesity or diabetes (see examples 5 and 6 herein).
  • the present invention relates to ERK-pathway activating compounds for treating and/or preventing leptin resistance in an individual affected by diabetes, obesity, or a cognitive disorder.
  • the invention further relates to ERK-pathway activating compounds for treating and/or preventing diabetes, obesity or a cognitive disorder.
  • This invention also pertains to ERK-pathway activating compounds for treating and/or preventing leptin resistance in an individual affected by obesity.
  • “obesity” comprises diet-induced obesity and especially diet-induced leptin-resistant obesity.
  • This invention also concerns ERK-pathway activating compounds for treating and/or preventing leptin resistance in an individual affected by diabetes, more particularly type II diabetes.
  • diabetes comprises type II diabetes.
  • This invention further relates to ERK-pathway activating compounds for treating and/or preventing leptin resistance in an individual affected with cognitive disorders.
  • a “cognitive disorder” comprises a cognitive disorder associated with leptin resistance, and is selected in the group comprising or consisting in major depression, adjustment disorder, schizoaffective disorder, schizophrenia, bipolar disorder, seizure, Alzheimer's disease, autism, anorexia, Binge- eating, bulimia nervosa, Prader-Willy syndrome.
  • the invention relates to individuals suffering from a cognitive disorder associated with eating disorders such as Binge-eating, bulimia nervosa, Prader-Willy and anorexia.
  • ERK-pathway activating compound » refers to any compound which is able to induce, at a given concentration, the phosphorylation of the intracellular protein ERK within tanycytes, or alternatively to increase the ratio of intracellular pERK/ERK.
  • ERK is the acronym for « Extracellular signal-regulated kinase
  • the protein ERK refers both to ERK-1 and ERK-2, which may be also referred in the literature as MAP kinases 1 and 2 (MAPKl and ⁇ 2
  • the phosphorylation of the intracellular protein ERK may be specifically detected by western blot and immunohistochemistry using the rabbit polyclonal anti-phospho-p44/42 ERK primary antibody (1 : 1000; #9101, Cell Signaling Technology), as it is disclosed in the examples herein.
  • the inventors provide an experimental framework to assess the phosphorylation of ERK on tanycytes, such as the protocols followed for assessing ERK- pathway activating properties of EGF, ghrelin, and semaphorin-7a (also referred herein as Sema7a), especially in examples 4A, 7, 8 or 9.
  • an "ERK-pathway activating compound” may be determined by culturing 90% confluent tanycytes from the median eminence in a suitable medium (tanycyte-defined medium or TDM) consisting of DMEM/F12 (devoid of phenol red; Invitrogen) supplemented with 1% L-glutamine, 2% penicillin/streptomycin, 5 ⁇ g/ml insulin (Sigma, Saint Quentin Fallavier, France), and 100 ⁇ putrescine (Sigma)) for two days, by incubating the said tanycytes in the said medium comprising a candidate compound for 15 minutes at a given concentration, and then by assessing the phosphorylation of ERK by western blot, relatively to control untreated tanycytes, and then selecting the said candidate compound as consisting of an "ERK- pathway activating compound” if an increased phosphorylation of ERK is measured.
  • TDM tanycyte-defined medium or T
  • the ERK-pathway activating compound may be obtained by chemical synthesis, by genetic engineering, by purification form natural sources or by a combination of two or more of these methods.
  • the ERK-pathway activating compound may be a protein, which comprises a protein obtained by chemical synthesis, a protein obtained by purification from a natural source and a recombinant protein.
  • An ERK-pathway activating compound, or a composition comprising or consisting in an ERK-pathway activating compound, as defined herein may be suitable for respiratory, enteral or parenteral administration.
  • an ERK-pathway activating compound, or a composition comprising or consisting in an ERK-pathway activating compound, as defined herein may be suitable for enteral or parenteral administration, and preferably suitable for parenteral administration.
  • An ERK-pathway activating compound, or a composition comprising or consisting in an ERK-pathway activating compound, as defined herein may be also suitable for administration by the respiratory route, in particular intranasal administration.
  • an ERK-pathway activating compound as defined herein may be suitable for intravenous, intranasal, intraperitoneal, intracerebral or intracerebroventicular administration.
  • the ERK-pathway activating compound as defined herein may be suitable for intravenous, intraperitoneal, intracerebral or intracerebroventicular administration.
  • an ERK-pathway activating compound as defined herein is suitable for intraperitoneal and intravenous administration.
  • an ERK-pathway activating compound as defined herein is suitable for enteral administration, and especially for oral administration.
  • An « ERK-pathway activating » compound as defined herein may be selected in a group comprising or consisting in VEGF, fluoxetin, B-Raf, integrin ⁇ -l ligands, GHS-R-la ligands, and EGF receptor ligands.
  • the « ERK-pathway activating compound is selected from a group comprising: EGF, VEGF, fluoxetin, Sema7a, B-Raf, ghrelin, TGFa, neuregulins, betacellulin, FIB-EGF, amphiregulin, epigen and epiregulin.
  • the GHS-R-la ligand may be ghrelin.
  • the integrin ⁇ -ligand may be Sema7a.
  • the EGF receptor ligand may be selected in a group comprising EGF, TGFa, neuregulins, betacellulin, HB-EGF, amphiregulin, epigen and epiregulin.
  • the ERK-pathway activating compound is chosen in the group comprising EGF, ghrelin and Sema7a, and preferably EGF.
  • epidermal growth factor is a preferred compound, because of the abundant expression of erbBl (an EGF receptor) in tanycytes.
  • ERK-pathway activating compounds and/or “leptin” compounds include derivatives of the compounds listed previously and having the same biological activity.
  • the invention relates to any ERK-pathway activating compound or leptin derivative having the same biological activity regarding respectively the phosphorylation of ERK within tanycytes and the activation of leptin signaling within tanycytes.
  • derivatives of an "ERK-pathway activating compound” encompass compounds that are covalently linked to, or non-covalently linked to, a functional molecule such as a detectable molecule and/or a targeting-ligand, e.g. a cell penetrating peptide.
  • a covalent or non-covalent linkage of an "ERK-pathway activating compound" with a detectable molecule is useful mainly for detection, screening or diagnosis purposes.
  • a covalent or non-covalent linkage of an "ERK-pathway activating compound" with a targeting ligand is useful mainly for medical purposes.
  • detectable molecules are well known in the art.
  • a detectable molecule such as: (i) a radioactive moiety, (ii) a fluorescent molecule, (iii) a luminescent molecule, (iv) a receptor molecule specifically recognized by another ligand and (v) a metallic tag.
  • Fluorophores which may be advantageously chosen include, in particular, lanthanide complexes, metallic tags such as Europium and Terbium, fluorescein, fluorescein isothiocyanate (FITC), dichlorotriazinylamine, rhodamine, eosine, coumarin, les methyl-coumarin, pyrene, Malachite green, Cy ® or Alexa ® (Invitrogen) fluorophores such as Cy ® 3, Cy ® 5, Alexa Fluor ® 488, Lucifer Yellow, Cascade Blue, Texas Red, dansyle chloride, phycoerythrin, luciferin, GFP and its variants, bore-dipyromethene (BODIPY), and others which are known in the art such as the ones described in Haugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; The Synthegen catalog (Houston, Tex.), Lakowicz, Principles of Fluorescence
  • leptin and/or an « ERK-pathway activating compound » may be labeled with a molecule such as the ones described in Leyris et al. (2011, Anal Biochem., 408:253-262), Schaeffer et al. (2013, Proc Natl Acad Sci U S A., 110: 1512-1517) and Vauthier et al. (2013, Anal Biochem, 436: 1-9 http://dx.doi.Org/10.1016/j .ab.2012.12.013).
  • an ERK-pathway activating compound or leptin may be labelled with a d2 fluorescent probe (Cisbio), in particular labelled on lysines with a N- hydroxysuccinimiude-activated d2 dye in 100 mM P0 4 buffer at pH 8.0.
  • An ERK-pathway activating compound or leptin may be also labelled with a red fluorescent probe (Cisbio, Ref. LOO20RED).
  • the ERK-pathway activating compound may be linked covalently or non-covalently, and preferably covalently, to a cell-penetrating peptide.
  • a cell-penetrating peptide is a peptide which facilitates cellular uptake of molecular cargos.
  • Such cell-penetrating peptides are well known in the art and are used to deliver a large variety of cargoes such as proteins, DNA, antibodies, contrast agents, toxins and nanoparticular drug carriers such as liposomes.
  • a "cell-penetrating peptide”, or CPP may be an arginine-rich cell-penetrating peptide.
  • a cell-penetrating peptide may be a TAT-derived cell-penetrating peptide; the TAT peptide is an arginine-rich cell penetrating pepide derived from the transactivating protein TAT of HIV- 1 (Peitz et al., 2002, PNAS, 99:7, pp 4489-4494).
  • the said cell-penetrating peptide comprises, and preferably consists in sequence SEQ ID NO 1 :
  • G is Glycine
  • Q is Glutamine
  • K is Lysine
  • R is Arginine
  • a cell-penetrating peptide may consist of a peptide derived from maurocalcine, and preferably a peptide derived from maurocalcine as described in the PCT application n° WO 2006/051224.
  • the inventors have shown that a treatment of an individual with an ERK-pathway activating compound, such as EGF, has both the effect of accelerating weight loss and restoring leptin sensitivity in a durable manner (see examples 5 and 6 herein).
  • the ERK-pathway activating compound may be used in complement with a diet, such as a low-fat diet.
  • the invention further relates to the medical use of an ERK-pathway activating compound, either used alone or used in combination with leptin.
  • the ERK- pathway activating compound and leptin may be administered through identical or different administration modes, and preferably identical administration modes.
  • the ERK-pathway activating compound may be administered to an individual in need thereof for a long-term period.
  • a « long-term period » relates to a daily administration of more than 6 days, and preferably at least 8 weeks.
  • an ERK-pathway activating compound when administered in combination with leptin, it is preferably administered sequentially.
  • a “sequential administration” refers to the administration of two distinct compounds, wherein either (i) a first compound, such as an ERK-pathway activating compound is administered alone prior to the administration of a composition comprising the second compound, such as leptin or (ii) a first compound, such as an ERK-pathway activating compound is administered alone subsequently to the administration of a composition comprising the second compound, such as leptin.
  • a sequential administration refers to the administration of two distinct compounds, wherein a first compound, such as an ERK-pathway activating compound is administered alone prior to the administration of a composition comprising the second compound, such as leptin.
  • the ERK-pathway activating compound is administered to an individual for a long-term period, followed by administration of leptin.
  • the sequential administration of leptin after a first administration of an ERK-pathway activating compound is also referred herein as a "leptin sensitivity test".
  • an ERK-pathway activating compound such as EGF may be administered to an individual for at least a period of time of 8 weeks, then followed by a leptin sensitivity test, comprising the administration of leptin to the said individual, alone or in combination with the said ERK-pathway activating compound.
  • EGF when the ERK-pathway activating compound is EGF, EGF may be administered to an individual orally or parenterally, and is preferably administered intravenously or intraperitoneally, in an amount ranging from 0,001 to 3 mg/kg, more particularly from 0,001 to 1 mg/kg. (see Tsuda N. et al, 2008, Brain Dev: 533-543).
  • the ERK-pathway activating compound may be administered to an individual by the respiratory route, and in particular intranasally.
  • Leptin may be administered to an individual orally or parenterally, and preferably intravenously or intraperitoneally, in an amount ranging from 0.1 mg / kg to 10 mg/kg.
  • leptin may be administered to an individual by the respiratory route, and in particular intranasally.
  • the administration of ERK-pathway activating compounds and leptin is an intraperitoneal administration.
  • the invention further relates to pharmaceutical compositions comprising or consisting in an ERK-pathway activating compound, such as EGF, in combination with leptin.
  • an ERK-pathway activating compound such as EGF
  • the ERK-pathway activating compound and leptin are comprised in the same dosage unit, i.e. in the same liquid or solid dosage unit.
  • the ERK-pathway activating compound and leptin are comprised in separate dosage units in the pharmaceutical composition.
  • the pharmaceutical composition comprises, or consists of, a kit comprising:
  • a second container comprising or consisting in leptin and optionally the said ERK-pathway activating compound.
  • a « container » comprising a pharmaceutical composition according to the invention may be any pharmaceutical container known in the art, preferably a sterile container, which is either directly suitable for injection, or which may be suitable for preparing an injectable sample, alone or when mixed with a second container according to the invention.
  • examples of said containers may be selected in a group comprising pills, tablets, capsules, bottles, syringes, needles, catheters, infusion pumps, ampullas, and more generally any small sealed vial which may be used to contain and preserve a sample.
  • Said containers may be made of glass or plastic, such as polypropylene.
  • said containers are subsequently mixed in order to achieve a specific pharmaceutical composition according to the invention.
  • the amount of ERK-pathway activating compound and leptin in each container can be easily adjusted by the man skilled in the art, in order to reach a specific ratio of ERK-pathway activating compound to leptin.
  • compositions will be administered by injection.
  • pharmaceutical compositions may be formulated as sterile aqueous or non-aqueous solutions or alternatively as sterile powders for the extemporaneous preparation of sterile injectable solutions.
  • Such pharmaceutical compositions should be stable under the conditions of manufacture and storage, and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • Pharmaceutically acceptable carriers for administration by injection are solvents or dispersion media such as aqueous solutions (e.g., Hank's solution, alcoholic/aqueous solutions, or saline solutions), and non-aqueous carriers (e.g., propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate).
  • aqueous solutions e.g., Hank's solution, alcoholic/aqueous solutions, or saline solutions
  • non-aqueous carriers e.g., propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters such as ethyl oleate.
  • Injectable pharmaceutical compositions may also contain parenteral vehicles (such as sodium chloride and Ringer's dextrose), and/or intravenous vehicles (such as fluid and nutrient replenishers); as well as other conventional, pharmaceutically acceptable, nontoxic excipients and additives including salts, buffers, and preservatives such as antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thirmerosal, and the like).
  • Prolonged absorption of the injectable compositions can be brought about by adding agents that can delay absorption (e.g., aluminum monostearate and gelatin).
  • the pH and concentration of the compositions can readily be determined by those skilled in the art.
  • Sterile injectable solutions are prepared by incorporating the active compound(s) and other ingredients in the required amount of an appropriate solvent, and then by sterilizing the resulting mixture, for example, by filtration or irradiation.
  • the methods of manufacture of sterile powders for the preparation of sterile injectable solutions are well known in the art and include vacuum drying and freeze-drying techniques.
  • tanycytes may constitute the missing link in the loop that connects behaviour, hormonal changes, signal transduction, central neuronal activation and finally, behaviour again.
  • leptin resistance occurs because of deficient or insufficient leptin release by tanycytes.
  • the invention relates to an in vitro method for the screening of compounds for treating and/or preventing leptin resistance in an individual in need thereof comprising the steps of:
  • any reference value such as for instance a reference value that is measured in control cultures of tanycytes which have not been incubated with leptin and/or the said candidate compound.
  • a suitable reference value may be easily determined by the man skilled in the art on the basis of his general technical knowledge and of the content of the present specification, especially the examples herein.
  • the culture medium can be replaced by a tanycyte conditioned medium (TCM) after step b).
  • TCM tanycyte conditioned medium
  • Standard medium used to culture tanycytes may be composed of DMEM (Invitrogen, San Diego, CA) supplemented with 2% (v/v) antibiotics (penicillin/streptomycin; Invitrogen), 1% L-glutamine (Invitrogen) and 10% donor bovine serum (Invitrogen).
  • DMEM Invitrogen, San Diego, CA
  • antibiotics penicillin/streptomycin; Invitrogen
  • 1% L-glutamine Invitrogen
  • donor bovine serum Invitrogen
  • tanycytes may be cultured in a "tanycyte defined medium" or
  • TDM corresponds to the above-defined medium (without serum) applied 24h before treatment and during treatment and composed of DMEM/F12 (devoid of phenol red; Invitrogen) supplemented with 1% L-glutamine, 2% penicillin/streptomycin, 5 ⁇ g/ml insulin (Sigma, Saint Quentin Fallavier, France), and 100 ⁇ putrescine (Sigma).
  • TCM tanycyte conditioned medium
  • the fresh medium for instance a tanycyte defined medium or TDM
  • TDM tanycyte defined medium
  • the TCM has the same composition as the original medium, namely TDM.
  • the level of leptin release may be assessed using any method known in the art, such as for example western blotting, confocal imaging or ELISA methods.
  • the level of leptin release may be achieved by culturing tanycytes in a tanycyte conditioned medium (TCM), and then measuring the level of leptin which has been released by western blotting at distinct time points.
  • TCM tanycyte conditioned medium
  • the level of leptin release can then be assessed over time, when compared to a control culture.
  • leptin release may be assessed in the presence of other modulating agents such as inhibitors of ERK and/or inhibitors of leptin signaling.
  • modulating agents such as inhibitors of ERK and/or inhibitors of leptin signaling.
  • inhibitors of ERK and/or inhibitors of leptin signaling are known in the art, and described for instance in example 1.
  • the leptin is preferably a leptin labeled with a detectable molecule, such as a fluorescent leptin.
  • the level of leptin release may be assessed by measuring the decrease, over time, of the intensity of the fluorescent signal up to extinction, and by comparing it to a reference value.
  • ERK-pathway activating compounds have been proven efficient in promoting leptin release from tanycytes, those methods are also useful for providing information on the modulation of leptin uptake by a given compound, either on the same tanycyte or on a different tanycyte.
  • the invention relates to methods for the screening of compounds which further comprise a step of measuring the level of leptin uptake by the tanycytes.
  • the steps of measuring the level of (i) leptin uptake and ii) leptin release may be performed with the same cultured tanycytes or with distinct cultured tanycytes.
  • the level of leptin uptake may be measured according to any method known in the art, such as the ones described in example 2.
  • leptin uptake may be assessed by clathrin immunoprecipitation, immunohistochemistry, western blotting, confocal imaging, or ELISA methods.
  • leptin uptake is assessed by confocal-imaging
  • a time- lapse study can be performed on living cells and both leptin uptake and leptin release may be assessed on the same tanycyte (see figure 3A).
  • the invention relates to an in vitro method for the screening of compounds for treating and/or preventing leptin resistance in an individual in need thereof comprising the steps of:
  • steps c) and d) are performed with the same cultured tanycytes or with distinct cultured tanycytes.
  • the in vitro method may comprise a step of measuring the level of leptin uptake comprising the step of performing a Homogeneous Time-Resolved Fluorescence-Based Leptin Receptor binding assay, such as the one described in (Vauthier et al, 2013, Anal Biochem, 436: 1-9 http://dx.doi.Org/10.1016/j .ab.2012.12.013).
  • the invention relates to a method for the screening of compounds for treating and/or preventing leptin resistance comprising the steps of:
  • a "non-human mammal” may be a rat or a mouse, such as an adult wild-type C57B1/6 mouse, a diet-induced obese mouse, a LepR dmh or an ob/ob mutant mouse.
  • the non-human mammal may be sacrificed according to any method known in the art, such as transcardiac perfusion of a fixative solution.
  • tissue explants comprising said tanycytes
  • the said tissue explants may be the median eminence or the mediobasal hypothalamus of a non-human mammal.
  • the level of leptin release by tanycytes may be assessed on the whole tissue explant.
  • tanycytes are collected from the median eminence or the mediobasal hypothalamus of a non-human mammal, and more preferably from the median eminence.
  • the assessment of the level of ERK-phosphorylation may thus be achieved before, during or after screening.
  • the level of ERK phosphorylation, or activation, in tanycytes may be measured by immunofluorescence.
  • the invention relates to a method for the screening of compounds for treating and/or preventing leptin resistance comprising the steps of:
  • leptin may be labeled with a detectable molecule as described previously, such as a fluorescently-labeled leptin.
  • methods for the screening of compounds may comprise an additional step of quantifying the phosphorylation of a protein in a sample or a tissue explant, in particular a protein within the said tanycytes, wherein the said protein is preferably selected in the group consisting in: ERK, STAT3 and Akt, and preferably STAT3 and ERK.
  • the phosphorylation of STAT3 can be assessed to confirm the occurrence of intracellular leptin signaling.
  • the quantification of the phosphorylation of a protein can be achieved by any method known in the art, such as western blotting, immunoprecipitation and immunohistochemistry, or as shown in the examples.
  • mice were housed under a 12h/12h light-dark cycle, and provided ad libitum access to water and standard laboratory chow (Special Diet Services, Commenailles, France) or a high-fat diet containing 60% fat (Research Diets, New Brunswick, NJ, USA). ).
  • groups of diet-induced obese (DIO) mice that had been on a high- fat diet for 8 weeks were then switched to a regular diet for 4 to 10 weeks (until week 12 and 18, respectively; DIO-R).
  • Mutant LepR dmh (db/db) mice were purchased from the Jackson Laboratories and housed under the same conditions and fed with standard chow.
  • Sprague Dawley rats were purchased from Janvier (Saint Berthevin, France) and housed in a temperature-controlled room (21-23°C) with a 12h/12h light-dark cycle. Animals had ad libitum access to tap water and pelleted rat food. The protocols used here were approved by the Direction of Veterinary Departments of Nord (59-350134) and were in accordance its guidelines for the Care and Use of Laboratory Animals as well as the European Communities Council Directive of November 24th, 1986 (86/609/EEC) regarding mammalian research.
  • mice were divided into two groups, which received daily intraperitoneal (i.p.) injections of recombinant murine leptin (3 mg/kg; Peprotech) or vehicle (10 mM Tris-HCl buffer) for 3 days. Body weight was measured before and 24h after the treatment period.
  • Cell lysates were prepared in 750 ⁇ ⁇ of Tris buffer (pH 7.4; 25 mM Tris) containing 50 mM ⁇ -glycerophosphate, 1.5 mM EGTA, 0.5 mM EDTA, 1 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10 ⁇ g/mL leupeptin and pepstatin, 10 ⁇ g/mL aprotinin, and 100 ⁇ g/mL phenylmethylsulfonyl fluoride by homogenization of the fragments through 26-gauge needle. The cell lysates were cleared by centrifugation at 12,000 x g for 15 min.
  • Tris buffer pH 7.4; 25 mM Tris
  • Protein content was determined using the Bradford method (Bio- Rad, Hercules, CA). Cell lysates were incubated with gentle rocking at 4°C for 2 h with 6 ⁇ g of anti-clathrin antibody. Thereafter 50 ⁇ ⁇ of protein A-Sepharose beads in lysis buffer (1 : 1 blend) were added to each sample and incubated for 1 additional hour with gentle rocking at 4°C. The beads were pelleted by brief centrifugation and then washed three times with ice-cold lysis buffer and boiled for 5 min in 50 ⁇ ⁇ of 2X sample buffer. When necessary, the samples were stored at -80°C until use. A.6. Antibodies used for western blotting experiments
  • Rabbit polyclonal anti-phospho-STAT3 (#9131; 1 : 1000 for immunoblotting), mouse monoclonal anti-STAT3 (#9139), rabbit polyclonal anti-phospho-p44/42 ERK (#9101), rabbit polyclonal anti-p44/42 MAPK (#9102), rabbit polyclonal anti-phospho- Akt (#4060) and rabbit polyclonal anti-Akt (#4691) were obtained from Cell Signaling Technology (1 : 1000 for immunoblotting). Rabbit polyclonal anti-leptin (500-P68) was purchased from Peprotech (1 : 1000 for immunoblotting).
  • Clathrin mouse monoclonal antibody (ab2731; 1 :500 for immunoblotting) was purchased from abeam and goat polyclonal antibody anti-actin (scl616; 1 :500 for immunoblotting) from Santa Cruz Biotechnology. Secondary antibodies used for western blotting [anti-mouse IgGs (1 :2000), anti-rabbit IgGs (1 :2000) horseradish-peroxidase-conjugated antibodies] were purchased from Sigma (Saint-Quentin Fallavier, France).
  • mice were injected intraperitoneally with leptin (3 mg/kg; Peprotech) or vehicle alone (10 mM Tris-HCl buffer) and perfused 5, 15 or 45 min later with a solution of 2% paraformaldehyde. Brains were postfixed, cryoprotected overnight at 4°C in the same fixative solution containing 20% sucrose, and embedded in Tissue Tek (Miles, Elkhart, IN) before freezing.
  • mice were injected intraperitoneally with EGF (1 mg/kg; R&D Systems) or vehicle alone (10 mM acetic acid in PBS) and perfused 15 min later with a solution of 4% paraformaldehyde in pH 9.5 borate buffer. Brains were postfixed, cryoprotected overnight at 4°C in the same fixative solution containing 20% sucrose, and embedded in Tissue Tek (Miles, Elkhart, IN) before freezing.
  • Tanycytes were isolated from the median eminence of the hypothalamus of 10-day-old (P10) rats and cultured as described previously (De Seranno, S. et al, 2004, J.Neurosci. 24, 10353-1036; Prevot et a/., 2003, J. Neurosci. 23, 10622-10632).
  • the tanycyte culture medium is composed of DMEM (Invitrogen, San Diego, CA) supplemented with 2% (v/v) antibiotics (penicillin/streptomycin; Invitrogen), 1% L- glutamine (Invitrogen) and 10% donor bovine serum (Invitrogen).
  • Tanycytes of the ME express functional LepR and internalize leptin through clathrin-coated vesicles. Primary cultures of tanycytes were obtained as described previously in the material & methods.
  • the primers used for LepR mRNA detection were as follows:
  • LepRb reverse 5'-TGGATAAACCCTTGCTCTTCA-3 ' (SEQ ID N° 4);
  • LepRe forward 5 ' -TGTT AT ATCTGGTT ATTGAATGG-3 ' (SEQ ID N° 7);
  • tanycytic LepR signalling in the passage of leptin from the periphery to the hypothalamus was investigated using primary cultures of rat tanycytes, obtained from the hypothalamus or the median eminence.
  • a second sample of tanycytes from the ME is provided from Prevot et al., 2003.
  • RT-PCR and sequence analysis revealed that cultured tanycytes expressed mRNAs for LepRa, b, c and e (see figure 1).
  • leptin internalization involves a LepR-dependant but JAK2-independent mechanism.
  • leptin was assayed by clathrin immunoprecipitation (see material & methods for western blotting and suitable antibodies) in tanycytes after 15 min of leptin treatment, and by clathrin staining on tanycytes cultured on coverslips and treated for 15 min with fluorescent leptin (Vauthier et al, 2013, Anal Biochem, 436: 1-9 http://dx.doi.Org/10.1016/j .ab.2012.12.013).
  • TCM tanycyte conditioned medium
  • a leptin antagonist (LAN) known to bind LepR with the same affinity as leptin was used as a negative control for leptin internalization (1 ⁇ g/ml; Protein Laboratories Rehovot Ltd).
  • ⁇ -stack sequences (step 2 ⁇ , depth 22 ⁇ , 30 frames at 1 min intervals) were realized with a Plan Apochromatic objective (20X, 0.7 NA) using 633 nm HeNe laser excitation for the fluorescent leptin signal and a non- descanned photon multiplier (PMT) for transmission.
  • PMT photon multiplier
  • Bioactive fluorescent leptin (Cisbio Bioassays) or fluorescent leptin antagonist (LAN; Cisbio Bioassays) (Vauthier et al, 2013, Anal Biochem, 436: 1-9 http://dx.doi.Org/10.1016/j .ab.2012.12.013) were injected into the jugular vein of adult male mice (25 nmoles/animal) anesthetized with ketamine/xylazine, and animals sacrificed 3 min post-injection to assess leptin uptake by tanycytes by fluorescent microscopy. Immunofluorescent images were acquired using an Axio Imager.Zl ApoTome microscope (AxioCam MRm camera, AxioVision 4.6 software system; Zeiss).
  • leptin 3 mg/kg; Peprotech
  • EGF 1 mg/kg in vehicle; R&D systems
  • vehicle alone lOmM acetic acid in PBS
  • Variations in pERK levels were detected using extracts of individual median eminences and western blotting. The cellular localization of the pERK labeling in the brain was assessed using immunohistofluorecence (see material & methods for western blotting, suitable antibodies and pERK immunohistochemistry).
  • Fluorescent leptin was taken up by tanycytic end-feet and gradually transported towards the cell body, where it accumulated (see figure 3A and 3B), confirming that, contrary to previous assumptions (Peruzzo et al, 2004, Cell Tissue Res, 317, 147-164), clathrin- mediated transport in tanycytes proceeds in a basal-to-apical direction. The intensity of the fluorescent signal decreased over time and reached extinction (see figure 3A), suggesting that the captured leptin was eventually released.
  • leptin 1 ⁇ g/ml, 15 min
  • leptin release monitored 5 and 15 min later, the leptin content of the medium gradually increased, confirming this hypothesis.
  • EGF-mediated activation of ERK signaling in the median eminence (ME) restores leptin transport into the hypothalamus and accelerates the restoration of leptin sensitivity in obese mice once they are replaced on a normal diet.
  • EGF Epidermal growth factor
  • EGF restored peripheral leptin uptake by the MBH (see figure 4B) as well as the subsequent activation of pSTAT3 in hypothalamic neurons within 15 min by western blots (see figure 4C) and quantification with photomicrographs (data not shown).
  • EGF treatment affects glucose metabolism and restores leptin sensitivity and leptin transport. Leptin sensitivity tests were followed as shown in the material & methods.
  • mice 12 weeks old male mice were placed on HFD or standard diet for 10 weeks. Mice placed on HFD showed a rapid increase in BW. The differences in BW of HFD-fed mice became significantly different from standard chow fed mice after 4 weeks. HFD-fed mice are separated in 3 groups. DIO group did not receive any treatment. DIO-EGF8w group receive daily intraperitoneal injection of EGF (1 mg/kg) from week 2 until the glucose tolerance test at week 8. DIO-EGF6d group received daily intraperitoneal injections of EGF (lmg/kg) at week 7 during 6 days before glucose tolerance test. Glucose tolerance test (GTT)
  • BW body weight
  • EGF treatment does not impact the weight gain effect of HFD, although it may affect glucose metabolism.
  • glucose tolerance test performed after 8 weeks of HFD or standard diet revealed that long term EGF treatment in DIO mice allows a partial recovery of glucose metabolism. 6 days of EGF treatment before the test does not change the response of DIO mice to GTT (see figure 6B).
  • control mice respond to leptin by a decrease in BW. Sensitivity to leptin is maintained in DIO-EGF8w mice as their decrease in BW does not differ from control mice. After 8 weeks of HFD, DIO mice which did not receive EGF treatment lost their sensibility to leptin and did not show any change in BW after peripheral leptin administration.
  • DIO-EGF8w display an intermediary phenotype between control and DIO mice, showing that long-term EGF treatment is efficient to reduce perigonadal white adipose tissue (WAT) content in DIO mice in spite of their increased BW compared to control mice (see figure 6D).
  • WAT white adipose tissue
  • Semaphorin-7a is an ERK-pathway activating compound.
  • Tanycyte culture and pERK western blotting were performed as described previously in the material & methods, and EGF was replaced by Semaphorin-7a.
  • the antibodies used were mouse anti-Plexin CI (R&D Systems, AF5375; 1 :500), rabbit anti-Bl -Integrin (Santa Cruz, sc-8978; 1 :500), rabbit anti P- ⁇ integrin (Abeam Technology, AB5189; 1 :500), rabbit polyclonal anti-phospho-p44/42 MAPK (p- Erkl/2) (Thr202/Tyr204; Cell Signaling Technology, 9101L; 1 : 1000), rabbit polyclonal anti-p44/42 MAPK (Erkl/2) (Cell Signaling Technology, 9102L; 1 : 1000).
  • Tanycytes express the receptor to Semaphorin-7a (integrin ⁇ ), and Semaphorin-7a activates p-ERK signaling (see figure 8).
  • the phosphorylation of the ⁇ -1 integrin receptor indicates that the receptor is activated in the presence of Sema7A.
  • Semaphorin-7a is a potent ERK-pathway activating compound in tanycytes.
  • Ghrelin activates p-ERK signaling and is internalised in tanycytes in vitro.
  • Tanycyte culture, pERK immunohistochemistry and western blotting, and in vitro and in vivo ghrelin binding were performed as described previously in the material & methods, and bioactive fluorescent ghrelin was used, as described in Leyris et al (2011, Anal Biochem, 408:253-262).
  • Fluorescent bioactive ghrelin derivative (3.3 kDa) is administered intravenously to standard-chow-fed wild-type mice 3 min before sacrifice. Photomicrographs showing tanycytic processes and cell bodies were labelled by fluorescent ghrelin (25 nmoles/animal), but not fluorescent LAN (25 nmoles/animal), 3 min after intravenous injection. Sections were counterstained with Hoechst (1 : 10000; Molecular Probes, Invitrogen) and coverslipped using Mowiol® (Calbiochem, USA).
  • Bioactive fluorescent ghreline derivatives developed by Cisbio Bioassays was shown to bind arcuate neurons when injected intravenously 5 to 10 min after death (Schaeffer et al, 2013, PNAS, 110: 1512- 1517), but its initial capture by tanycytes had never been suspected so far.
  • GHSRla ligands such as ghrelin are potent ERK-pathway activating compounds in tanycytes.

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Abstract

La présente invention concerne un composé d'activation de voie ERK destiné à être utilisé pour traiter et/ou prévenir une résistance à la leptine chez un individu en ayant besoin.
PCT/IB2014/059728 2013-03-13 2014-03-13 Composé d'activation de voie erk pour prévenir ou traiter une résistance à la leptine WO2014141124A1 (fr)

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