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WO1998036767A1 - Modulation de l'axe hypotalamo-hypophyso-adrenalo-adipeux au moyen de ligands du recepteur de la leptine - Google Patents

Modulation de l'axe hypotalamo-hypophyso-adrenalo-adipeux au moyen de ligands du recepteur de la leptine Download PDF

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Publication number
WO1998036767A1
WO1998036767A1 PCT/US1998/002906 US9802906W WO9836767A1 WO 1998036767 A1 WO1998036767 A1 WO 1998036767A1 US 9802906 W US9802906 W US 9802906W WO 9836767 A1 WO9836767 A1 WO 9836767A1
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WO
WIPO (PCT)
Prior art keywords
replaced
gin
leptin receptor
glu
leptin
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Application number
PCT/US1998/002906
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English (en)
Inventor
Thomas W. Stephens
Mark L. Heiman
Libbey S. Craft
Jose F. Caro
Lawrence J. Slieker
Original Assignee
Eli Lilly And Company
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Filing date
Publication date
Application filed by Eli Lilly And Company filed Critical Eli Lilly And Company
Priority to AU66558/98A priority Critical patent/AU6655898A/en
Priority to EP98908553A priority patent/EP0991420A1/fr
Priority to JP53674898A priority patent/JP2001514620A/ja
Priority to CA002282341A priority patent/CA2282341A1/fr
Publication of WO1998036767A1 publication Critical patent/WO1998036767A1/fr

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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
    • A61K38/22—Hormones
    • A61K38/2264—Obesity-gene products, e.g. leptin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/38—Drugs for disorders of the endocrine system of the suprarenal hormones
    • A61P5/44—Glucocorticosteroids; Drugs increasing or potentiating the activity of glucocorticosteroids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/38—Drugs for disorders of the endocrine system of the suprarenal hormones
    • A61P5/46—Drugs for disorders of the endocrine system of the suprarenal hormones for decreasing, blocking or antagonising the activity of glucocorticosteroids

Definitions

  • This invention relates generally to a method for modulating the Hypothalamic-Pituitary-Adrenal-Adipose Axis (HPAAA) in a mammal in need of such treatment by administration of a therapeutically effective amount of a leptin receptor ligand. More specifically, this invention relates to the use of leptin receptor agonists to treat disorders of the HPAAA, including the hyperglucocorticoidemia associated with Cushing's Syndrome, fasting, or otherwise-induced stress in obese or lean mammals in need of such treatment. This invention also relates to the use of leptin receptor antagonists, or specific anti-leptin antibodies, to treat the hypoglucocorticoidemia associated with autoimmune or inflammatory conditions such as rheumatoid arthritis.
  • HPAAA Hypothalamic-Pituitary-Adrenal-Adipose Axis
  • hypothalamus, pituitary gland, and adrenal glands comprise a neuroendocrine system in mammals that is instrumental in the physiological response to stress: the hypothalamic-pituitary-adrenal axis.
  • stress including metabolic stress (e.g., hypoglycemia), neurogenic stress (e.g, anxiety, pain), and physical stress (e.g., exercise, infection, trauma) stimulate biosynthesis and release of corticotropin- releasing hormone (CRH) by paraventricular neurons in the hypothalamus.
  • CRH corticotropin- releasing hormone
  • CRH corticotropin- releasing hormone
  • glucocorticoid hormones i.e., cortisol in human and corticosterone in rat.
  • the hypothalamic-pituitary-adrenal axis responds rapidly to stress: many investigators have demonstrated increases in glucocorticoid concentration by as much as 20-fold occurring within minutes of stress. Elevated glucocorticoid levels initiate metabolic events such as rapid mobilization of amino acids and fat, accompanied by decreased glucose utilization, making substrates available for energy utilization, synthesis of other compounds (e.g., glucose) and substrates for tissues (e.g., wound healing).
  • Glucocorticoids are also very potent anti-inflammatory agents.
  • glucocorticoids ameliorate the stressful stimuli that provoke their release.
  • circulating glucocorticoids exert a direct negative feedback on hypothalamic CRH biosynthesis and release, and on anterior pituitary synthesis and release of ACTH.
  • Obesity especially that resulting from adipose tissue distributed abdominally, renders individuals more susceptible to diabetes, cardiovascular diseases, and, in women, breast and endometrial cancers.
  • Hyperinsulinemia and insulin resistance, impaired glucose tolerance, and hyperlipidemia are hormonal and metabolic abnormalities associated with such obesity and could link obesity to the disease states noted above.
  • Abdominal obesity in human and rat is associated with an increased hypothalamic-pituitary-adrenal axis activity and hypercorticism (Pasquali, R. et al. (1993) J. Clin. Endocrinol. Metab. 77:341-346; Hautanen, A. and Adlercreutz, H. (1993) J. Int. Med.
  • hypothalamic-pituitary-adrenal axis in obese mammals is regulated at an elevated level (set-point) because of lack of feedback to the hypothalamus supplied by the protein product of the obese gene (OB protein or "leptin").
  • OB protein protein product of the obese gene
  • This invention thus describes for the first time a hypothalamic-pituitary-adrenal-adipose axis (HPAAA).
  • the mouse obese gene has been cloned from obese mice (ob/ob) that suffer from a mutated ob gene that results in either no gene product or an inactive truncated protein (Zhang et al. (1994) Nature 372:425-432.).
  • the present inventors have cloned and expressed this gene in £. coli to produce native mouse and human OB proteins (mOB and hOB).
  • Ob/ob mice present not only with hyperphagia but also with very elevated corticosterone levels.
  • HPAA HPAA
  • the abnormal regulation of HPAA has been described in the obese fa/fa Zucker rat. Indeed, the mutation is associated with elevated plasma ACTH and glucocorticoid levels (Guillame-Gentil, C. et al., supra).
  • hypothalamic CRH secretion is similar to the lean wild type, suggesting at least a partial lack of negative feedback at a hypothalamic site between corticosterone and CRH feedback regulation (Plotsky, P. et al. (1992) Endocrinology 130:1931-1941).
  • the fa/fa fatty phenotype is the result of a missense mutation of they hypothalamic OB receptor (Streamson et al., (1996) Diabetes 45:1141-1143.).
  • the present inventors now also have data that demonstrate that OB inhibits hypoglycemia-mediated CRH release from the rat hypothalamus in perifusion (Fig. 2).
  • the HPAA may now be extended to include adipose tissue, and be renamed the HPAAA.
  • a major object of the present invention is to provide a method of modulating the Hypothalamic-Pituitary-Adrenal-Adipose Axis (HPAAA) in a mammal in need of such treatment by administration of a therapeutically effective amount of a leptin receptor agonist.
  • HPAAA Hypothalamic-Pituitary-Adrenal-Adipose Axis
  • this invention relates to a method of modulating the HPAAA, in particular to the use of leptin receptor agonists to treat disorders of the HPAAA, including the hypercorticoidemia associated with Cushing's Syndrome, fasting, or otherwise-induced stress in obese or lean mammals in need of such treatment by administering a leptin receptor ligand, for example, an leptin receptor agonist.
  • a leptin receptor ligand for example, an leptin receptor agonist.
  • the present invention also provides a method of treating hypoglucocorticoidemia, for example in autoimmune or inflammatory conditions such as rheumatoid arthritis, by administration of a leptin receptor antagonist, or specific anti-leptin antibodies to a mammal in need of such treatment.
  • the present invention also provides surrogate markers for early effectiveness of leptin therapy such as plasma ACTH and plasma cortisol.
  • Measurement of HPAAA hormones is a useful diagnostic for differentiating type of obesity and for determining obesity therapy.
  • Other eating disorders such as anorexia nervosa are associated with elevated CRH levels and may be a result of dysregulation of the HPAAA and oversecretion of leptin.
  • Fig. 1 Effect of various agents on secretion of leptin from isolated rat adipocytes.
  • Fig. 2 Leptin inhibition of hypoglycemia-mediated CRH secretion from rat hypothalami in perfusion. Values represent mean CRH secreted during a 30 minute period from 20 hypothalamic bisections (equivalent to 10 hypothalami). After 2.5 h perfusion with buffer containing 5.5 mM glucose, hypothalami were challenged by decreasing glucose concentration to 1.1 mM with and without addition of mouse leptin. A chamber of hypothalamic remained at 5.5 mM glucose throughout the experiment. Initial perifusion buffer was restored after 4h pe ⁇ ' fusion. Closed circles: 5.5 mM glucose; open circles: 1.1 mM glucose; closed triangles: 1.1 mM glucose + 1 nM leptin; open triangles: 1.1 mM glucose + 30 nM leptin.
  • Fig. 3 Hypoglycemia-mediated CRH secretion from rat hypothalami in perifusion. Values represent mean CRH secreted during a 30 min period from 20 hypothalamic bisections (equivalent to 10 hypothalami). At 150 min perifusion with buffer containing 5.5 mM glucose, hypothalami were challenged by decreasing glucose concentration to 2.8 mM or 1.1 mM. A chamber of hypothalami remained at 5.5 mM glucose throughought the experiment. Initial perifusion buffer was restored at 210 min. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
  • This invention arose from a desire of the inventors to provide a safe, effective treatment for disorders of the HPAAA, including the hypercorticoidemia associated with Cushing's Syndrome, fasting, or otherwise-induced stress in obese or lean mammals.
  • Base pair (bp) - refers to DNA or RNA.
  • the abbreviations A,C,G, and T correspond to the 5'-monophosphate forms of the nucleotides (deoxy)adenine, (deoxy)cytidine, (deoxy)guanine, and (deoxy)thymine, respectively, when they occur in DNA molecules.
  • the abbreviations U,C,G, and T correspond to the 5'-monophosphate forms of the nucleosides uracil, cytidine, guanine, and thymine, respectively when they occur in RNA molecules.
  • base pair may refer to a partnership of A with T or C with G.
  • base pair may refer to a partnership of T with U or C with G.
  • Chelating Peptide An amino acid sequence capable of complexing with a multivalent metal ion.
  • DNA Deoxyribonucleic acid.
  • HPAAA Hypothalamic-Pituitary-Adrenal-Adipose Axis
  • CRH released by the hypothalamus stimulates pituitary secretion of ACTH, that in turn stimulates adrenal secretion of glucocorticoids, which in turn modulates adipose tissue leptin release, that finally acts back on the hypothalamus.
  • Immunoreactive Protein(s) - a term used to collectively describe antibodies, fragments of antibodies capable of binding antigens of a similar nature as the parent antibody molecule from which they are derived, and single chain polypeptide binding molecules as described in PCT Application No. PCT/US 87/02208, International Publication No. WO 88/01649.
  • Leptin the endogenous product of expression of the ob gene; the OB protein.
  • mRNA messenger RNA.
  • MWCO an abbreviation for molecular weight cut-off.
  • Modulating - stimulating, potentiating, or inhibiting the activity of a receptor or system Plasmid - an extrachromosomal self-replicating genetic element.
  • PMSF an abbreviation for phenylmethylsulfonyl fluoride.
  • Reading frame the nucleotide sequence from which translation occurs "read” in triplets by the translational apparatus of tRNA, ribosomes and associated factors, each triplet corresponding to a particular amino acid. Because each triplet is distinct and of the same length, the coding sequence must be a multiple of three. A base pair insertion or deletion (termed a frameshift mutation) may result in two different proteins being coded for by the same DNA segment. To insure against this, the triplet codons corresponding to the desired polypeptide must be aligned in multiples of three from the initiation codon, i.e. the correct "reading frame" must be maintained.
  • Receptor agonist any compound that binds to a receptor and triggers the action of the receptor (usually an intracellular signalling event or, in the case of receptors that form transmembrane channel, the opening or closing of the channel).
  • Receptor antagonist any compound that binds to a receptor and blocks the action of the receptor (usually by out-competing the endogenous agonist for binding sites on the receptor).
  • Receptor ligand any compound that binds to a receptor.
  • Recombinant DNA Cloning Vector any autonomously replicating agent including, but not limited to, plasmids and phages, comprising a
  • DNA molecule to which one or more additional DNA segments can or have been added.
  • Recombinant DNA Expression Vector any recombinant DNA cloning vector in which a promoter has been incorporated.
  • Replicon A DNA sequence that controls and allows for autonomous replication of a plasmid or other vector.
  • RP-HPLC an abbreviation for reversed-phase high performance liquid chromatography.
  • Transcription the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence.
  • Treating - describes the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of a compound of present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder.
  • Treating obesity for example, includes the inhibition of food intake, the inhibition of weight gain, and inducing weight loss in patients in need thereof.
  • Vector - a replicon used for the transformation of cells in gene manipulation bearing polynucleotide sequences corresponding to appropriate protein molecules which, when combined with appropriate control sequences, confer specific properties on the host cell to be transformed.
  • Plasmids, viruses, and bacteriophage are suitable vectors, since they are replicons in their own right.
  • Artificial vectors are constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases. vectors include Recombinant DNA cloning vectors and Recombinant DNA expression vectors.
  • X-gal - an abbreviation for 5-bromo-4-chloro-3-idolyl beta-D- galactoside.
  • the normal HPAAA can be described as follows: metabolic, neurogenic or physical stress stimulates CRH release. This in turn stimulates ACTH release. The latter stimulates glucocorticoid release. Finally, the adrenal steroid stimulates OB release. Both glucocorticoid and OB feedback negatively to inhibit CRH release.
  • leptin receptor ligands in particular leptin receptor agonists or leptin receptor antagonists, effectively modulate the HPAAA.
  • leptin receptor agonists for use in the present invention include endogenous leptin (i.e., endogenous OB protein - the protein produced from the obesity gene following transcription and translation and deletion of introns, translation to a protein and processing to the mature protein with secretory signal peptide removed, e.g., from the N-terminal valine-proline to the C-terminal cysteine of the mature protein).
  • endogenous leptin i.e., endogenous OB protein - the protein produced from the obesity gene following transcription and translation and deletion of introns, translation to a protein and processing to the mature protein with secretory signal peptide removed, e.g., from the N-terminal valine-proline to the C-terminal cysteine of the mature protein.
  • endogenous leptin i.e., endogenous OB protein - the protein produced from the obesity gene following transcription and translation and deletion of introns, translation to a protein and processing to the mature protein with secretor
  • the rat OB protein is published in Murakami et al., Biochem. Biophys. Res. Com. 209:944-952 (1995).
  • the porcine and bovine OB genes and proteins are disclosed in EP 0 743 321 , the contents of which are incorporated by reference.
  • Various primate OB genes and proteins are disclosed in U.S. Application Serial No.08/710,483, the contents of which are incorporated by reference.
  • Also preferred for use in the present invention are leptin analogs, preferably leptin analogs having one or more amino acid substitution, more preferably less than five and most preferably less than three substitutions.
  • Particularly preferred leptin analogs for use in the present invention include proteins disclosed by Basinski et al., in WO 96/23515 and WO 96/23517 (the contents of which are incorporated by reference), of the Formula (I):
  • Xaa at position 28 is Gin or absent; said protein having at least one of the following substitutions: Gin at position 4 is replaced with Glu;
  • Gin at position 7 is replaced with Glu
  • Thr at position 27 is replaced with Ala
  • Xaa at position 28 is replaced with Glu
  • Gin at position 34 is replaced with Glu
  • Met at position 54 is replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly;
  • Gin at position 56 is replaced with Glu
  • Met at position 68 is replaced with methionine sulfoxide, Leu, He, Val, Ala, or Gly;
  • Asn at position 72 is replaced with Gin, Glu, or Asp;
  • Gin at position 75 is replaced with Glu; Ser at position 77 is replaced with Ala;
  • His at position 97 is replaced with Gin, Asn, Ala, Gly, Ser, or Pro;
  • Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val, or Leu;
  • Ala at position 101 is replaced with Ser, Asn, Gly, His, Pro, Thr, or Val;
  • Gly at position 103 is replaced with Ala; Glu at position 105 is replaced with Gin; Thr at position 106 is replaced with Lys or Ser;
  • Gly at position 111 is replaced with Asp; Gly at position 118 is replaced with Leu;
  • Gin at position 130 is replaced with Glu
  • Gin at position 134 is replaced with Glu
  • Met at position 136 is replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly; Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, lie,
  • Gin at position 139 is replaced with Glu.
  • the compounds used in the present invention are optionally substituted with a functional group.
  • Any art-recognized functional group which does not eliminate or significantly reduce the compound's ability to bind to leptin receptors are contemplated, including, but not limited to, ester, amide, acid, amine, alcohol, ether, thioether, etc.
  • Solvates e.g., hydrates of the compounds useful in the methods of the present invention, are also included within the scope of the present invention. Methods of solvation to produce such solvates are generally known in the art.
  • compositions of the leptin receptor agonists and antagonists suitable for administration by a variety of routes are known in the art and need not be described herein in detail.
  • pharmaceutically acceptable salts of the compounds and derivatives thereof according to the invention include base salts, e.g., derived from an appropriate base.
  • Pharmaceutically acceptable salts of an acid group or an amino group include, but are not limited to, saits of organic carboxylic acids such as acetic, lactic, tartaric, malic, isothionic, and lactobionic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toiylsulfonic acids, and inorganic acids such as hydrochloric, sulfuric, phosphoric and sulfamic acids.
  • Pharmaceutically-acceptable salts of a compound with a hydroxy group include, but are not limited to, the anion of the compound in combination with a suitable cation such as Na + .
  • the present invention comprises a method for modulating the HPAAA by administration of antibodies to endogenous leptin receptor agonists to a mammal in need of such treatment.
  • Such antibodies may be monoclonal or polyclonal antibodies to leptin receptor agonists, or to antigenic parts thereof. Both polyclonal and monoclonal antibodies to leptin receptor agonists are obtainable by immunization of an animal with purified leptin receptor agonists, purified recombinant leptin receptor agonists, fragments of these proteins, or purified fusion proteins of leptin receptor agonists, with another protein. In the case of monoclonal antibodies, partially purified proteins or fragments may serve as immunogens.
  • Polyclonal sera are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of purified leptin receptor agonists, or parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques.
  • Monoclonal antibodies are particularly useful because they can be produced in large quantities and with a high degree of homogeneity.
  • Hybridoma cell lines which produce monoclonal antibodies are prepared by fusing an immortal cell line with lymphocytes sensitized against the immunogenic preparation and is done by techniques which are well known to those who are skilled in the art. (See, for example, Douillard, I.Y. and Hoffman, T., "Basic Facts About Hybridomas", in Compendium of Immunology, Vol. II, L.
  • a still further part of this invention is a pharmaceutical composition of matter for modulating the HPAAA that comprises at least one of the leptin receptor agonists or antagonists described above, mixtures thereof, and/or pharmaceutical salts thereof, and a pharmaceutically-acceptable carrier therefor.
  • Such compositions are prepared in accordance with accepted pharmaceutical procedures, for example, as described in Remington's Pharmaceutical Sciences, seventeenth edition, ed. Alfonso R. Gennaro, Mack Publishing Company, Easton, PA (1985), the teachings of which are incorporated herein by reference.
  • a leptin receptor agonist or antagonist, or its salt can be conveniently administered in the form of a pharmaceutical composition containing one or more leptin receptor agonists or antagonists, or salts thereof, and a pharmaceutically acceptable carrier therefor.
  • Suitable carriers are well known in the art and vary with the desired form and mode of administration of the pharmaceutical composition. For example, they may include diluents or excipients such as fillers, binders, wetting agents, disintegrators, surface-active agents, lubricants, and the like.
  • the carrier may be a solid, liquid, or vaporizable carrier, or combinations thereof.
  • the composition is a therapeutic composition and the carrier is a pharmaceutically acceptable carrier.
  • the compounds used in the present invention, or salts thereof, may be formulated together with a carrier into any desired unit dosage form.
  • Typical unit dosage forms include tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, suppositories; injectable solutions and suspensions are particularly preferred.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients in the formulation and not injurious to the patient.
  • the carrier must be biologically acceptable and inert, i.e., it must permit the cell to conduct its metabolic reactions so that the compound of this invention may effect its inhibitory activity.
  • Formulations include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal and parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal) administration, with topical ointment formulations, and formulations appropriate for oral administration, being preferred.
  • solutions and suspensions are sterilized and are preferably isotonic to blood.
  • carriers which are commonly used in this field can also be used, for example, water, ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxyiated isostearyl alcohol, polyoxyethylene sorbitol and sorbitate esters.
  • isotonicity adjusters such as sodium chloride, glucose or glycerin can be added to make the preparations isotonic.
  • the aqueous sterile injection solutions may further contain anti-oxidants, buffers, bacteriostats, and like additions acceptable for parenteral formulations.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any method known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient with the carrier which may encompass one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.
  • Various unit dose and multidose containers e.g., sealed ampules and vials, may be used, as is well known in the art.
  • formulations of this invention may also include other agents conventional in the art for this type of pharmaceutical formulation.
  • the compounds for use in the present invention may be present in the composition in an broad proportion to the carrier.
  • the compound may be present in the amount of 0.01 to 99.9 wt%, and more preferably in about 0.1 to 99 wt%. Still more preferably, the compound may be present in an amount of about 1 to 70 wt% of the composition.
  • the dosage of the leptin receptor agonists or antagonists, pharmaceutically acceptable salts thereof, or mixtures thereof administered to a patient according to the present invention will vary depending on several factors, including, but not limited to, the age, weight, and species of the patient, the general health of the patient, the severity of the symptoms, whether the composition is being administered alone or in combination with other therapeutic agents, the incidence of side effects and the like.
  • a dose suitable for application in the modulation of the HPAAA is about 0.001 to 100 mg/kg body weight/dose, preferably about 0.01 to 60 mg/kg body weight/dose, and still more preferably about 0.1 to 40 mg/kg body weight/dose per day.
  • the desired dose may be administered as 1 to 6 or more subdoses administered at appropriate intervals throughout the day.
  • the compounds may be administered repeatedly over a period of months or years, or it may be slowly and constantly infused to the patient. Higher and lower doses may also be administered.
  • the daily dose may be adjusted taking into account, for example, the above-identified variety of parameters.
  • the present compositions may be administered in an amount of about 0.001 to 100 mg/kg body weight/day. However, other amounts may also be administered.
  • the active compounds may be administered, for instance, by intravenous injection of an approximate 0.1 to 1% solution of the active ingredient, optionally in saline, or orally administered as a bolus.
  • the active ingredient may be administered for therapy by any suitable routes, including topical, oral, rectal, nasal, vaginal and parenteral (including intraperitoneai, subcutaneous, intramuscular, intravenous, intradermal, and transdermal) routes. It will be appreciated that the preferred route will vary with the condition and age of the patient, the nature of the disorder and the chosen active ingredient including other therapeutic agents. Preferred is the oral route. Also preferred is the topical route. However, other routes may also be utilized depending on the conditions of the patient and how long-lasting the treatment is.
  • routes including topical, oral, rectal, nasal, vaginal and parenteral routes.
  • the active ingredient While it is possible for the active ingredient to be administered alone, it is preferably present as a pharmaceutical formulation.
  • the formulations of the present invention comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof and optionally other therapeutic agents.
  • the above method may be practiced by administration of the compounds by themselves or in a combination with other active ingredients, including therapeutic agents in a pharmaceutical composition.
  • Other therapeutic agents suitable for use herein are any compatible drugs that are effective by the same or other mechanisms for the intended purpose, or drugs that are complementary to those of the present agents. These include agents that are effective for the treatment of hypercorticoidemia and/or associated conditions in humans. Examples are hypothalamic serotonin antagonists such as cyproheptadine, and GABA- transaminase inhibitors such as sodium valproate, among others.
  • the compounds utilized in combination therapy may be administered simultaneously, in either separate or combined formulations, or at different times than the present compounds, e.g., sequentially, such that a combined effect is achieved.
  • the amounts and regime of administration will be adjusted by the practitioner, by preferably initially lowering their standard doses and then titrating the results obtained.
  • the therapeutic method of the invention may be used in conjunction with other therapies as determined by the practitioner.
  • Adipocytes were obtained from rat epididymal fat pads from 250- 300g male rats by coilagenase digestion. The cell suspension was filtered sequentially through 500, 250, and 100 ⁇ m mesh and washed six times with Dulbecco's modified Eagle medium / Ham's F12 (BSA/F12 3:1 , Gibco) supplemented with 20 /g/ml bovine serum albumin (BSA, RIA grade, Sigma Chemical Co., St. Louis), 20 mM Hepes, 0.1 g/l sodium selenite and 4.88 mg/l ethanolamine. Adipocytes were cultured in this medium for 24 hrs at approximately 5 x 10 5 cells/ml in either Coastar P6 trays or Corning T75 flasks.
  • RNA isolation was obtained from beneath the adipocyte layer and stored at -20 ° C until used for RIA analysis. Adipocytes were used for RNA isolation as described below. Dibutyryl cAMP (But2cAMP), isoproterenol, dexamethasone (Dex), and hydrocortisone (Hydrocort) were obtained from Sigma Chemical (St. Louis, MO). Recombinant human insulin (Ins) was from Lilly (Indianapolis, IN).
  • Recombinant murine leptin was iodinated with 125 l-Bolton Hunter reagent (Amersham Life Sciences, Arlington Heights, IL). Antibody was prepared in rabbits using recombinant murine leptin. RIA was performed in phosphate buffered saline (PBS) containing 1 mg/ml bovine serum albumin (BSA) and 0.1% Triton X-100. Using a non-equilibrium protocol to enhance sensitivity, samples were combined with anti-leptin antibody at 1 :8000 dilution and incubated at room temperature for four hours in a total volume of 400 I.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • mice Male C57BL mice, (Jackson Labs, Bar Harbor, ME), 8 weeks of age, were housed in groups of 4 under 12 h light (0600-1800) and dark (1800-0600h), and allowed free access to chow and water for 2 weeks until use.
  • the animal facility where these studies were performed is a fully accredited, institutional member of the American Association for the Accreditation of Laboratory Animal Care and provides a committee that approved the protocol used.
  • One group was injected intraperitoneally with 100 /I saline between 0700-0800h of the light cycle. The others were injected with saline, 2 ⁇ g or 4 ⁇ g mouse leptin (biosynthetically prepared in E. coli. (see ref. 9)) in 100 ⁇ saline vehicle
  • mice Male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) weighing from 250g - 300g were acclimated for at least 2 weeks in an identical environment to that described above.
  • the animal facility in which this study was performed is a fully accredited, institutional member of the American Association for the Accreditation of Laboratory Animal Care and provides a committee that approved the protocol used.
  • Five rats were housed in each cage that had water and food (Ralston-Purina, St. Louis, MO) continuously available. Rats were killed by decapitation and the brain was quickly removed.
  • Hemi-hypothalamic sections were randomly assigned to 1 of 4 wells containing 3 ml Krebs-Ringer bicarbonate buffer (KRB) with 5.5 mM glucose (KRBhiG) that was placed on ice.
  • KRB Krebs-Ringer bicarbonate buffer
  • KRBhiG 5.5 mM glucose
  • This buffer was pumped to each of 4 chambers simultaneously at 100 //l/min under an atmosphere of 95% 0 2 :5% C0 2 at 37 ° C.
  • the time- lag for buffer to reach chambers is 10 min.
  • Mouse leptin was added during this challenge period.
  • Perifusate was collected at 30 minute intervals into tubes containing 750 ⁇ 1 M trifluoroacetic acid (TFA; Aldrich, Milwaukee, Wl) and rapidly frozen.
  • [ 125 I]-CRH was purchased from DuPont NEN (Boston, MA).
  • CRH primary antisera, normal rabbit serum, and goat anti-rabbit IgG were purchased from Peninsual Laboratories (Belmont, CA) and diluted as instructed by the manufacturer.
  • Coefficient of variation calculated for a set of standards was less than 12% for both inter- and intra-assay measurements.
  • CRH release was integrated using the trapezoidal rule (SigmaPlot; Jandel Scientific, San Rafael, CA). Integrated release is presented as mean ⁇ SEM and treatment groups were compared by ANOVA followed by Scheffe's F-test (StatView, Brainpower, Inc., Calabasa, CA). Plasma leptin, ACTH and corticosterone levels were compared by ANOVA and Fisher PSLD. Significance was accepted at P ⁇ 0.05.

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Abstract

La présente invention concerne un procédé de modulation de l'axe hypotalamo-hypophyso-adrénalo-adipeux d'un mammifère justifiant d'un tel traitement, lequel procédé consiste en une administration en quantité suffisante d'un ligand du récepteur de la leptine. En l'occurrence, l'invention se rapporte à l'utilisation d'antagonistes du récepteur de la leptine pour traiter des troubles de l'axe hypotalamo-hypophyso-adrénalo-adipeux, y compris l'hyperglucocorticoïdémie associée au syndrome de Cushing, le jeûne ou de stress autrement induits chez les mammifères obèses ou maigres nécessitant un tel traitement. Cette invention concerne également l'utilisation d'antagonistes du récepteur de la leptine, ou d'anticorps spécifiquement anti-leptine, pour le traitement de l'hyperglucocorticoïdémie associée à des états auto-immunes ou inflammatoires tels que l'arthrite rhumatoïde.
PCT/US1998/002906 1997-02-25 1998-02-24 Modulation de l'axe hypotalamo-hypophyso-adrenalo-adipeux au moyen de ligands du recepteur de la leptine WO1998036767A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU66558/98A AU6655898A (en) 1997-02-25 1998-02-24 Modulation of the hypothalamic-pituitary-adrenal-adipose axis with leptin receptor ligands
EP98908553A EP0991420A1 (fr) 1997-02-25 1998-02-24 Modulation de l'axe hypotalamo-hypophyso-adrenalo-adipeux au moyen de ligands du recepteur de la leptine
JP53674898A JP2001514620A (ja) 1997-02-25 1998-02-24 レプチン受容体リガンドによる視床下部−下垂体−副腎−脂肪軸の調節
CA002282341A CA2282341A1 (fr) 1997-02-25 1998-02-24 Modulation de l'axe hypotalamo-hypophyso-adrenalo-adipeux au moyen de ligands du recepteur de la leptine

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US80466997A 1997-02-25 1997-02-25
US08/804,669 1997-02-25

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999036083A1 (fr) * 1998-01-20 1999-07-22 Beth Israel Deaconess Medical Center Methode de regulation neuro-endocrinienne permettant de traiter les troubles affectifs
WO2007080404A3 (fr) * 2006-01-12 2008-03-06 Asterion Ltd Ligands de leptines

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996023515A1 (fr) * 1995-01-31 1996-08-08 Eli Lilly And Company Proteines anti-obesite

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Publication number Priority date Publication date Assignee Title
WO1996023515A1 (fr) * 1995-01-31 1996-08-08 Eli Lilly And Company Proteines anti-obesite

Non-Patent Citations (5)

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Title
GUILLAUME-GENTIL C., ET AL.: "ABNORMAL REGULATION OF THE HYPOTHALAMO-PITUITARY-ADRENAL AXIS IN THE GENETICALLY OBESE FA/FA RAT.", ENDOCRINOLOGY, THE ENDOCRINE SOCIETY, US, vol. 126., no. 04., 1 April 1990 (1990-04-01), US, pages 1873 - 1879., XP002910862, ISSN: 0013-7227 *
KOISTINEN H A, ET AL.: "SERUM LEPTIN CONCENTRATION IS NORMAL IN CUSHING SYNDROME AND IN ACROMEGALY", DIABETOLOGIA, SPRINGER, BERLIN, DE, vol. 39, 1 August 1996 (1996-08-01), BERLIN, DE, pages A145, XP002910858, ISSN: 0012-186X *
LEAL-CERRO A, ET AL.: "SERUM IMMUNOREACTIVE-LEPTIN LEVELS ARE INCREASED IN PATIENTS WITH CUSHING'S SYNDROME", HORMONE AND METABOLIC RESEARCH, THIEME-STRATTON, STUTTGART, DE, vol. 28, no. 12, 1 January 1996 (1996-01-01), DE, pages 711 - 713, XP002910859, ISSN: 0018-5043 *
MIELL J P, ENGLARO P, BLUM W F: "DEXAMETHASONE INDUCES AN ACUTE AND SUSTAINED RISE IN CIRCULATING LEPTIN LEVELS IN NORMAL HUMAN SUBJECTS", HORMONE AND METABOLIC RESEARCH, THIEME-STRATTON, STUTTGART, DE, vol. 28, no. 12, 1 January 1996 (1996-01-01), DE, pages 704 - 707, XP002910860, ISSN: 0018-5043 *
PLOTSKI P. M., ET AL.: "HYPOTHALAMIC-PITUITARY-ADRENAL AXIS FUNCTION IN THE ZUCKER OBESE RAT.", ENDOCRINOLOGY, THE ENDOCRINE SOCIETY, US, vol. 130., no. 04., 1 April 1992 (1992-04-01), US, pages 1931 - 1941., XP002910861, ISSN: 0013-7227, DOI: 10.1210/en.130.4.1931 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999036083A1 (fr) * 1998-01-20 1999-07-22 Beth Israel Deaconess Medical Center Methode de regulation neuro-endocrinienne permettant de traiter les troubles affectifs
US6313098B1 (en) 1998-01-20 2001-11-06 Beth Israel Deaconess Medical Center, Inc. Methods of neuroendocrine regulation of affective disorders
WO2007080404A3 (fr) * 2006-01-12 2008-03-06 Asterion Ltd Ligands de leptines

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CA2282341A1 (fr) 1998-08-27
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AU6655898A (en) 1998-09-09

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