WO1998008512A1 - Methodes pour accroitre la sensibilite d'un individu a la proteine ob par accroissement de l'affinite du recepteur de la proteine ob - Google Patents

Methodes pour accroitre la sensibilite d'un individu a la proteine ob par accroissement de l'affinite du recepteur de la proteine ob Download PDF

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Publication number
WO1998008512A1
WO1998008512A1 PCT/US1997/015086 US9715086W WO9808512A1 WO 1998008512 A1 WO1998008512 A1 WO 1998008512A1 US 9715086 W US9715086 W US 9715086W WO 9808512 A1 WO9808512 A1 WO 9808512A1
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Prior art keywords
protein
analog
amino acids
amino acid
derivative
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PCT/US1997/015086
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English (en)
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Mary Ann Pelleymounter
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Amgen Inc.
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Priority to EP97940645A priority Critical patent/EP1001768A1/fr
Priority to AU42377/97A priority patent/AU4237797A/en
Priority to JP10511882A priority patent/JP2001501177A/ja
Priority to CA002263826A priority patent/CA2263826A1/fr
Publication of WO1998008512A1 publication Critical patent/WO1998008512A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/5759—Products of obesity genes, e.g. leptin, obese (OB), tub, fat
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to methods of reducing amounts of OB protein compositions administered for desired therapeutic or cosmetic effect through the use of agents which increase the sensitivity to OB protein or the affinity or availability of functional OB protein receptor .
  • OB protein protein encoded
  • OB protein protein encoded
  • the OB protein is active in vivo in both ob/ob mutant mice (mice obese due to a defect in the production of the OB gene product) as well as in normal, wild type mice.
  • the biological activity manifests itself in, among other things, weight loss.
  • OB protein As modulators for the control of weight and adiposity of animals, including mammals and humans, has been disclosed in greater detail in PCT publication WO 96/05309 (12/22/96), hereby incorporated by reference, including figures .
  • the other biological effects of OB protein are not well characterized. It is known, for instance, that in ob/ob mutant mice, administration of OB protein results in a decrease in serum insulin levels, and serum glucose levels. It is also known that administration of OB protein results in a decrease in body f t.
  • Thiazolidinediones are a class of antihyperglycemic agents with an as yet unknown mechanism of action. See Wilson et al . , J. Med. Chem., 3£: 665-668 (1996); Kallen et al., P.N.A.S., 93 . : 5793-5796 (1996); Sohda et al . , Chem. Pharm. Bull., 43.: 2168-2172 (1995); Swanson et al., Drug Dev. Res., 3_5_: 69-82 (1995); Kletzien et al . , Mol.
  • PPAR peroxisome proliferator-activated receptor
  • thiazolidinediones are thought to decrease endogenous OB protein.
  • thiazolidinediones are thought to increase insulin sensitivity, there are currently no known agents which increase an individual's sensitivity to OB protein (or leptin) .
  • Such increased sensitivity would be advantageous to leptin consumers, as increased sensitivity may contribute to lower doses required or less frequent dosing.
  • thiazolidinedione compositions provide for increased sensitivity to OB protein.
  • thiazolidinedione compositions "upregulate" OB protein receptor available for signal transduction, i.e., increase the number of OB receptor, or increase the affinity of OB receptor for its ligand (OB protein) .
  • This increase in the availability or affinity of functional OB protein receptor provides for increased signal transduction of endogenous and/or exogenous OB protein.
  • this provides means for reducing the amount and/or frequency of dosages of exogenous OB protein for therapeutic and/or cosmetic purposes.
  • the present invention provides methods of weight modulation and/or fat deposition in an individual by the administration of one or more compositions which function to increase the availability or affinity of functional OB protein receptors in an individual, such as thiazolidinedione compositions.
  • the present invention provides for methods of weight modulation and/or fat deposition in an individual by the administration of one or more compositions which function to increase the availability of functional OB protein receptors, such as a thiazolidinedione composition, in an individual, in combination with administration of an OB protein.
  • one or more compositions which function to increase the availability of functional OB protein receptors such as a thiazolidinedione composition
  • the present invention provides for methods to reduce the amount and/or frequency of dosage of exogenous an OB protein by administration of a composition which functions to increase the availability of functional OB protein receptors, such as a thiazolidinedione composition.
  • the present invention provides for methods for the treatment of co-morbidities associated with excess fat, such as diabetes, dys- or hyperlipidemias, lack of fertility, and also potentially an increase in insulin sensitivity and/or and increase in lean tissue mass.
  • Related compositions are also provided .
  • Figure 1 Recombinant urine met OB (double stranded) DNA (SEQ ID NOS: 1 and 2) and amino acid sequence (SEQ ID NO: 3).
  • Figure 2 Recombinant human met OB analog (double stranded) DNA (SEQ ID NOS: 4 and 5) and amino acid sequence (SEQ ID NO: 6).
  • composition having the ability to increase the sensitivity to OB protein or the availability of functional OB protein receptor in an individual may be selected from among various thiazolidinedione compositions, such as: 2 , 4-thiazolidinediones; opt. substituted thiazolidinediones; 5- [4- [2- (5-methyl-2- phenyl-4-oxazolyl) -2-hydroxyethoxy] enzyl] -2, 4- thiazolidinedione (AD-5070) ; clofibrate; ciglitazone; englitazone; pioglitazone; BRL 49653; troglitazone; M16209; oxazolidinediones; as well as derivatives, analogs, tautomers, enantiomer ⁇ , diastereomers , epimers, salts, solvates, esters, prodrugs and metabolites of thiazolidinediones or the compounds above.
  • the OB protein may be selected from recombinant urine set forth below (SEQ. ID No. 3 of Figure 1) , or recombinant human protein as set forth in Zhang et al . , Nature , supra , herein incorporated by reference) or those lacking a glutaminyl residue at position 28. (See Zhang et al, Nature, supra , at page 428.)
  • the murine protein is substantially homologous to the human protein, particularly as a mature protein, and, further, particularly at the N-terminus.
  • the amino acid at position 146 is cysteine
  • Rat OB protein differs from human OB protein at the following positions (using the numbering of SEQ. ID. NO. 6): 4, 32, 33, 3_5, 5 . 0 . , 68, 21, Ik, TL, 78, ££- 92- 1-2Q. 101, 102, 1_5, 106- 107, 108, 111, 118, 136, 138 and 145.
  • One may substitute with another amino acid one or more of the amino acids at these divergent positions.
  • the positions in bold print are those which in which the murine OB protein as well as the rat OB protein are divergent from the human OB protein, and thus, are particularly suitable for alteration. At one or more of these positions, one may substitute an amino acid from the corresponding rat OB protein, or another amino acid.
  • the positions from both rat and murine OB protein which diverge from the mature human OB protein are: 4, 32, 33, 35, 50, 64, 68, 71, 74, 77, 78, 89, 97, 100, 102, 105, 106, 107, 108, 111, 118, 136, 138, 142, and 145.
  • a human OB protein according to SEQ. ID. NO. 6 (with lysine at position 35 and isoleucine at position 74) having one or more of the above amino acids deleted or replaced with another amino acid, such as the amino acid found in the corresponding rat or murine sequence, may also be effective.
  • amino acids found in rhesus monkey OB protein which diverge from the mature human OB protein are (with identities noted in parentheses in one letter amino acid abbreviation): 8 (S) , 35 (R) , 48 (V) , 53(Q), 60(1), 66(1), 67 (N) , 68 ( (L) , 89 (L) , 100 (L) ,
  • a human OB protein according to SEQ. ID. NO. 6 (with lysine at position 35 and isoleucine at position 74) having one or more of the rhesus monkey divergent amino acids replaced with another amino acid, such as the amino acids in parentheses, may be effective.
  • certain rhesus divergent amino acids are also those found in the above murine species (positions 35, 68, 89, 100 and 112).
  • analogs may be prepared by deleting a part of the protein amino acid sequence.
  • the mature protein lacks a leader sequence (-22 to -1) .
  • the truncated forms may also have altered one or more of the amino acids which are divergent (in the rhesus, rat or murine OB protein) from human OB protein.
  • any alterations may be in the form of altered amino acids, such as peptidomimetics or D-amino acids.
  • the present protein (herein the term "protein” is used to include “peptide” and OB analogs, such as those recited infra, unless otherwise indicated) may also be derivatized by the attachment of one or more chemical moieties to the protein moiety.
  • the chemically modified derivatives may be further formulated for intraarterial , intraperitoneal, intramuscular, subcutaneous, intravenous, oral, nasal, pulmonary, topical or other routes of administration.
  • Chemical modification of biologically active proteins has been found to provide additional advantages under certain circumstances, such as increasing the stability and circulation time of the therapeutic protein and decreasing immunogenicity . See U.S. Patent No. 4,179,337, Davis et al . , issued December 18, 1979.
  • the chemical moieties suitable for derivatization may be selected from among various water soluble polymers .
  • the polymer selected should be water soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment.
  • the polymer will be pharmaceutically acceptable.
  • One skilled in the art will be able to select the desired polymer based on such considerations as whether the polymer/protein conjugate will be used therapeutically, and if so, the desired dosage, circulation time, resistance to proteolysis, and other considerations .
  • the effectiveness of the derivatization may be ascertained by administering the derivative, in the desired form (i.e., by osmotic pump, or, more preferably, by injection or infusion, or, further formulated for oral, pulmonary or nasal delivery, for example) , and observing biological effects as described herein .
  • the water soluble polymer may be selected from the group consisting of, for example, polyethylene glycol, copolymers of ethylene glycol/propylene glycol , carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrolidone, poly-1, 3-dioxolane, poly-1, 3 , 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random or non-random copolymers) , and dextran or poly(n-vinyl pyrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols, polystyrenemaleate and polyvinyl alcohol.
  • Polyethylene glycol propionaldenhyde may have advantages in manufacturing due to its stability in water.
  • Fusion proteins may be prepared by attaching polyaminoacids to the OB protein (or analog) moiety.
  • the polyamino acid may be a carrier protein which serves to increase the circulation half life of the protein.
  • polyamino acid should be those which have do not create neutralizing antigenic response, or other adverse response.
  • Such polyamino acid may be selected from the group consisting of serum album (such as human serum albumin) , an antibody or portion thereof (such as an antibody constant region, sometimes called "F c ”) or other polyamino acids.
  • F c antibody constant region
  • the location of attachment of the polyamino acid may be at the N- terminus of the OB protein moiety, C-terminus, or other place, and also may be connected by a chemical "linker" moiety to the OB protein.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight is between about 2 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
  • Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog) .
  • the number of polymer molecules so attached may vary, and one skilled in the art will be able to ascertain the effect on function.
  • the proportion of polymer molecules to protein (or peptide) molecules will vary, as will their concentrations in the reaction mixture. In general, the optimum ratio (in terms of efficiency of reaction in that there is no excess unreacted protein or polymer) will be determined by factors such as the desired degree of derivatization (e.g., mono, di-, tri-, etc.), the molecular weight of the polymer selected, whether the polymer is branched or unbranched, and the reaction conditions.
  • the chemical moieties should be attached to the protein with consideration of effects on functional or antigenic domains of the protein.
  • attachment methods available to those skilled in the art.
  • EP 0 401 384 herein incorporated by reference (coupling PEG to G-CSF) , see also Malik et al . , Exp. Hematol. 20.: 1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride) .
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.
  • Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residue.
  • Those having a free carboxyl group may include aspartic acid residues, glutamic acid residues, and the C-terminal amino acid residue.
  • Sulfhydrl groups may also be used as a reactive group for attaching the polyethylene glycol molecule (s).
  • Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group. Attachment at residues important for receptor binding should be avoided if receptor binding is desired.
  • One may specifically desire N-terminally chemically modified protein.
  • polyethylene glycol as an illustration of the present compositions, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
  • the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
  • Selective N-terminal chemical modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the
  • N-terminal available for derivatization in a particular protein.
  • substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
  • the water soluble polymer may be of the type described above, and should have a single reactive aldehyde for coupling to the protein.
  • Polyethylene glycol propionaldehyde, containing a single reactive aldehyde, may be used.
  • N-terminally monopegylated derivative is preferred for ease in production of a therapeutic.
  • N-terminal pegylation ensures a homogenous product as characterization of the product is simplified relative to di-, tri- or other multi pegylated products.
  • the use of the above reductive alkylation process for preparation of an N-terminal product is preferred for ease in commercial manufacturing. Complexes
  • the OB protein, analog or derivative may be administered complexed to a binding composition.
  • a binding composition may have the effect of prolonging the circulation time of the OB protein, analog or derivative.
  • Such composition may be a protein (or synonymously, peptide) .
  • An example of a binding protein is OB protein receptor or portion thereof, such as a soluble portion thereof .
  • Other binding proteins may be ascertained by examining OB protein in serum, or by empirically screening for the presence of binding. Such binding will typically not interfere with the ability of OB protein or analog or derivative to bind to endogenous OB protein receptor and/or effect signal transduction.
  • Pharmaceutical Compositions In yet another aspect of the present invention, provided are methods of using pharmaceutical compositions of the proteins, and derivatives.
  • compositions may be for administration by injection, or for oral, pulmonary, nasal, transdermal or other forms of administration.
  • pharmaceutical compositions comprising effective amounts of protein or derivative products of the invention together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and/or carriers.
  • compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite) , preservatives (e.g., Thimersol, benzyl alcohol) and bulking substances (e.g., lactose, mannitol) ; incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposome ⁇ .
  • buffer content e.g., Tris-HCl, acetate, phosphate
  • additives e.g., Tween 80, Polysorbate 80
  • anti-oxidants e.g., ascorbic acid, sodium metabisulfite
  • preservatives e.g., Thimersol,
  • Hylauronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation.
  • Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g. , Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712 which are herein incorporated by reference.
  • the compositions may be prepared in liquid form, or may be in dried powder, such as lyophilized form.
  • Implantable sustained release formulations are also contemplated, as are transdermal formulations. Contemplated for use herein are oral solid dosage forms, which are described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co.
  • Solid dosage forms include tablets, capsules, pills, troches or lozenges; cachets or pellets.
  • liposomal or proteinoid encapsulation may be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Patent No. 4,925,673).
  • Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (E.g., U.S. Patent No. 5,013,556).
  • a description of possible solid dosage forms for the therapeutic is given by Marshall, K. In: Modern Pharmaceutics Edited by G.S. Banker and CT.
  • the formulation will include the protein (or analog or derivative) , and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.
  • oral dosage forms of the above derivatized proteins Protein may be chemically modified so that oral delivery of the derivative is efficacious.
  • the chemical modification contemplated is the attachment of at least one moiety to the protein (or peptide) molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine.
  • the increase in overall stability of the protein and increase in circulation time in the body are examples of the protein (or analog or derivative) , and inert ingredients which allow for protection against the stomach environment, and release of the biologically active material in the intestine.
  • oral dosage forms of the above derivatized proteins Protein may be chemically modified so that oral delivery of the derivative is efficacious.
  • the chemical modification contemplated is the attachment of at least one moiety to the protein (or peptide) molecule itself
  • moieties include: Polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, Soluble
  • One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of the protein (or derivative) or by release of the biologically active material beyond the stomach environment, such as in the intestine.
  • a coating impermeable to at least pH 5.0 is essential .
  • examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate
  • CAT hydroxypropylmethylcellulose phthalate
  • HPMCP 50 HPMCP 55
  • PVAP polyvinyl acetate phthalate
  • Eudragit L30D Eudragit L30D
  • Aquateric cellulose acetate phthalate
  • CAP Eudragit L
  • Eudragit S and Shellac.
  • coatings may be used as mixed films.
  • a coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow.
  • Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic i.e. powder; for liquid forms, a soft gelatin shell may be used.
  • the shell material of cachets could be thick starch or other edible paper.
  • the therapeutic can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1mm.
  • the formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets .
  • the therapeutic could be prepared by compression.
  • Colorants and flavoring agents may all be included.
  • the protein (or derivative) may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.
  • these diluents could include carbohydrates, especially mannitol, a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch.
  • Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride.
  • Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.
  • Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrates include but are not limited to starch including the commercial disintegrant based on starch, Explotab.
  • Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used.
  • Another form of the disintegrants are the insoluble cationic exchange resins.
  • Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.
  • Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin.
  • MC methyl cellulose
  • EC ethyl cellulose
  • CMC carboxymethyl cellulose
  • PVP polyvinyl pyrrolidone
  • HPMC hydroxypropylmethy1 cellulose
  • Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE) , liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.
  • stearic acid including its magnesium and calcium salts
  • PTFE polytetrafluoroethylene
  • Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.
  • the glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.
  • surfactant might be added as a wetting agent.
  • Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate.
  • Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride.
  • nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.
  • Additives which potentially enhance uptake of the protein (or derivative) are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
  • Controlled release formulation may be desirable.
  • the drug could be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms i.e. gums.
  • Slowly degenerating matrices may also be incorporated into the formulation.
  • Another form of a controlled release of this therapeutic is by a method based on the Oros therapeutic system (Alza Corp.), i.e. the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some entric coatings also have a delayed release effect.
  • coatings may be used for the formulation. These include a variety of sugars which could be applied in a coating pan.
  • the therapeutic agent could also be given in a film coated tablet and the materials used in this instance are divided into 2 groups.
  • the first are the nonenteric materials and include methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose, providone and the polyethylene glycols.
  • the second group consists of the enteric materials that are commonly esters of phthalic acid. A mix of materials might be used to provide the optimum film coating. Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating.
  • pulmonary delivery of the present protein, or derivative thereof is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.
  • the protein derivative
  • the protein is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream.
  • Adjei et al . Pharmaceutical Research 7: 565-569 (1990); Adjei et al . , International Journal of Pharmaceutics £2: 135-144 (1990) (leuprolide acetate); Braquet et al . , Journal of Cardiovascular Pharmacology 13 (suppl . 5): s.143-146
  • Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.
  • Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St.
  • each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy.
  • the protein (or derivative) should most advantageously be prepared in particulate form with an average particle size of less than 10 m m (or microns) , most preferably 0.5 to 5 m m, for most effective delivery to the distal lung.
  • Carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations may include DPPC, DOPE, DSPC and DOPC . Natural or synthetic surfactants may be used. Polyethylene glycol may be used (even apart from its use in derivatizing the protein or analog) . Dextrans , such as cyclodextran, may be used. Bile salts and other related enhancers may be used. Cellulose and cellulose derivatives may be used. Amino acids may be used, such as use in a buffer formulation. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.
  • Formulations suitable for use with a nebulizer will typically comprise protein (or derivative) dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution.
  • the formulation may also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure) .
  • the nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.
  • Formulations for use with a etered-dose inhaler device will generally comprise a finely divided powder containing the protein (or derivative) suspended in a propellant with the aid of a surfactant.
  • the propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol , and 1, 1, 1, 2-tetrafluoroethane, or combinations thereof.
  • Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant .
  • Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing protein (or derivative) and may also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g. , 50 to 90% by weight of the formulation.
  • a bulking agent such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol
  • Nasal delivery of the protein (or analog or derivative) is also contemplated. Nasal delivery allows the passage of the protein to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung.
  • Formulations for nasal delivery include those with dextran or cyclodextran.
  • OB protein e.g., OB protein
  • upregulating OB protein receptor e.g., OB protein
  • the formulation of the molecule will be such that between about .10 ⁇ g/kg/day and 100 mg/kg/day will yield the desired therapeutic effect.
  • the effective dosages may be determined using diagnostic tools over time. For example, a diagnostic for measuring the amount of OB protein in the blood (or plasma or serum) may first be used to determine endogenous levels of OB protein. Such diagnostic tool may be in the form of an antibody assay, such as an antibody sandwich assay. The amount of endogenous OB protein is quantified initially, and a baseline is determined. The therapeutic dosages are determined as the quantification of endogenous and exogenous OB protein (that is, protein, analog or derivative found within the body, either self-produced or administered) is continued over the course of therapy. The dosages may therefore vary over the course of therapy, with a relatively high dosage being used initially, until therapeutic benefit is seen, and lower dosages used to maintain the therapeutic benefits.
  • the dosage in situations where solely an increase in lean body mass is desired, the dosage will be insufficient to result in weight loss.
  • dosages may be administered whereby weight loss and concomitant fat tissue decrease/lean mass increase is achieved. Once sufficient weight loss is achieved, a dosage sufficient to prevent re-gaining weight, yet sufficient to maintain desired lean mass increase (or, prevention of lean mass depletion) may be administered.
  • These dosages can be determined empirically, as the effects of OB protein are reversible. E.g. , Ca pfield et al . , Science 269 : 546-549 (1995) at 547.
  • Lean mass increase without weight loss may be achieved sufficient to decrease the amount of insulin (or, potentially, amylin or other potential diabetes treating drugs) an individual would be administered for the treatment of diabetes .
  • Lean mass increase with concomitant increase in overall strength may be achieved with doses insufficient to result in weight loss.
  • Other benefits such as an increase in red blood cells (and oxygenation in the blood) and a decrease in bone resorption or osteoporosis may also be achieved in the absence of weight loss.
  • Combinations The present methods may be used in conjunction with other medicaments, such as those useful for the treatment of diabetes (e.g., insulin, possibly thiazolidinediones, amylin or antagonists thereof) , cholesterol and blood pressure lowering medicaments
  • Such administration may be simultaneous or may be in seriatim.
  • the present methods may be used in conjunction with surgical procedures, such as cosmetic surgeries designed to alter the overall appearance of a body (e.g., liposuction or laser surgeries designed to reduce body mass, or implant surgeries designed to increase the appearance of body mass).
  • surgical procedures such as cosmetic surgeries designed to alter the overall appearance of a body (e.g., liposuction or laser surgeries designed to reduce body mass, or implant surgeries designed to increase the appearance of body mass).
  • the health benefits of cardiac surgeries such as bypass surgeries or other surgeries designed to relieve a deleterious condition caused by blockage of blood vessels by fatty deposits, such as arterial plaque, may be increased with concomitant use of the present compositions and methods .
  • Methods to eliminate gall stones, such as ultrasonic or laser methods may also be used either prior to, during or after a course of the present therapeutic methods.
  • the present methods may be used as an adjunct to surgeries or therapies for broken bones, damaged muscle, or other therapies which would be improved by an increase in lean tissue mass.
  • the present invention provides a method for increasing, in an individual, the sensitivity to OB protein or analog or derivative thereof, comprised of administering an effective amount of a composition which increases the availability, affinity, or sensitivity of functional OB protein receptor in said individual.
  • said method also involves the administration, either simultaneously or m . serriatim of an OB protein, analog or derivative thereof.
  • Said composition which increases the availability or sensitivity of functional OB protein receptor may be selected from among thiazolidinedione compositions: 2 , 4-thiazolidinediones; opt. substituted thiazolidinediones; 5- [4- [2- (5-methyl-2-phenyl-4- oxazolyl) -2-hydroxyethoxy] enzyl] -2 , 4-thiazolidinedione (AD-5070); clofibrate; ciglitazone; englitazone; pioglitazone; BRL 49653; troglitazone; M16209; oxazolidinediones; as well as derivatives, analogs, tautomers , enantiomers, diastereomers, epimers, salts, solvates, esters, prodrugs and metabolites of thiazolidinediones or the compounds above.
  • the OB protein, analog or derivative thereof may be selected from among:
  • a truncated OB protein analog selected from among: (using the numbering of SEQ. ID. NO. 6 having a lysine residue at position 35 and an isoleucine residue at position 74) :
  • amino acids 1-99 and 112-146 amino acids 1-99 and 112-146 having one or more of amino acids 100-111 sequentially placed between amino acids 99 and 112; and, (vi) the truncated OB analog of subpart
  • Example 1 is a prophetic example of human use which demonstrates that thiazolidinedione compositions increase the affinity or availability of OB protein receptors in an individual and those individuals have an increased sensitivity to said OB protein. Materials and Methods follow.
  • An obese human patient desires weight loss.
  • the patient is administered an amount of a thiazolidinedione composition effective to increase the availability of OB protein receptor for one week.
  • the patient is then dosed with an amount of OB protein, or derivative or analog thereof, sufficient to result in decrease in weight.
  • Levels of circulating OB protein or analog or derivative may be monitored using a diagnostic kit, such as an antibody assay against the OB protein (or other antigenic source if applicable) .
  • the patient loses weight, and obtains a desired body weight and/or fat mass .
  • OB protein or analog or derivative thereof optionally in combination with a thiazolidinedione composition is thereafter chronically administered for a desired period of time to maintain the desired weight and/or body fat level.
  • Sequence ID Nos. 1, 2 and 3 set forth murine recombinant OB DNA and protein (figure 1)
  • Sequence ID Nos. 4, 5 and 6 set forth an analog recombinant human OB DNA and protein (figure 2) .
  • Recombinant human OB protein as in SEQ. ID. NO. 6 having a lysine residue at position 35 and an isoleucine residue at position 74 was used in EXAMPLE 1.
  • the below murine and human analog recombinant proteins are illustrative of the OB protein which may be used in the present methods of treatment and manufacture of a medicament .
  • Other OB proteins or analogs or derivatives thereof may be used.
  • the first amino acid of the amino acid sequence for recombinant protein is referred to as +1, and is valine, and the amino acid at position -1 is methionine.
  • the C-terminal amino acid is number 146 (cysteine) .
  • the plasmid expression vector used is PCFM1656, ATCC Accession No. 69576.
  • the above DNA was ligated into the expression vector pCFM1656 linearized with Xbal and BamHI and transformed into the E. coli host ⁇ train, FM5.
  • E. coli FM5 cells were derived at Amgen Inc., Thousand Oaks, CA from E. coli K-12 strain (Bachmann, et al . , Bacteriol. Rev. 40 . : 116-167 (1976)) and contain the integrated lambda phage repressor gene, cl857 (Sus ⁇ man et al . , CR. Acad. Sci. 254 : 1517-1579
  • Fermentation Process A three-phase fermentation protocol known as a fed-batch process was used. Media compositions are set forth below.
  • a nitrogen and phosphate source were sterilized (by raising to 122 °C for 35 minutes, 18-20 psi) in the fermentation vessel (Biolafitte, 12 liter capacity) . Upon cooling, carbon, magnesium, vitamin, and trace metal sources were added aseptically. An overnight culture of the above recombinant murine protein-producing bacteria (16 hours or more) of 500 mL (grown in LB broth) was added to the fermentor.
  • Feed I Upon reaching between 4.0-6.0 OD600' cultures were fed with Feed I. The gluco ⁇ e was fed at a limiting rate in order to control the growth rate (m) . An automated system (called the Distributive Control System) was instructed to control the growth rate to 0.15 generations per hour.
  • Feed II When the OD600 had reached 30, culture temperature were slowly increased to 42°C and the feed changed to Feed II, below. The fermentation was allowed to continue for 10 hours with sampling every 2 hours. After 10 hours, the contents of the fermentor was chilled to below 20°C and harvested by centrifugation .
  • Feed II 200 g/L Bacto-tryptone
  • Vitamin Solution (Batch and Feed I) : 0.5 g Biotin, 0.4 g Folic acid, and 4.2 g riboflavin, was dissolved in 450 is H2O and 3 mis 10 N NaOH, and brought to 500 mLs in H2O. 14 g pyridoxine-HCl and 61 g niacin was dissolved 150 ml H2O and 50 ml 10 N NaOH, and brought to 250 ml in H2O. 54 g pantothenic acid was dissolved in 200 mL H2O, and brought to 250 mL. The three solutions were combined and brought to 10 liters total volume.
  • E. coli cell paste was suspended in 5 times volume of 7 mM of EDTA, pH 7.0. The cells in the EDTA were further broken by two passes through a microfluidizer . The broken cells were centrifuged at 4.2 K rpm for 1 hour in a Beckman J6-B centrifuge with a JS-4.2 rotor.
  • Inclusion body wash #1 The supernatant from above was removed, and the pellet was resuspended with 5 times volume of 7 mM EDTA, pH 7.0, and homogenized. This mixture was centrifuged as in step 1.
  • Inclusion body wash #2 The supernatant from above was removed, and the pellet was resuspended in ten times volume of 20 mM tris, pH 8.5 , 10 mM DTT, and 1% deoxycholate, and homogenized. This mixture wa ⁇ centrifuged as in step 1. 4.
  • Inclusion body wash #3 The supernatant from above was removed and the pellet was resuspended in ten times volume of distilled water, and homogenized. This mixture was centrifuged as in step 1. 5. Refolding. The pellet was refolded with
  • CM Sepharose pool of peak fractions (ascertained from ultraviolet absorbance) from the above step was made to be 0.2 M ammonium sulfate.
  • a 20 column volume reverse salt gradient was done at 5 mM NaOAC, pH 4.2, with .4 M to 0 M ammonium sulfate. This material was concentrated and diafiltered into PBS.
  • Fermentation of recombinant human OB protein analo Fermentation of the above host cells to produce recombinant human OB protein analog (SEQ. ID. NO. 6) can be- accomplished using the conditions and compositions as described above for recombinant murine material .
  • Recombinant human protein analog may be purified using methods similar to tho ⁇ e used for purification of recombinant murine protein, a ⁇ described above.
  • step 8 For preparation of recombinant human OB protein analog, step 8 should be performed by adjusting the pH of the supernatant from step 7 to pH 5.0, and loading this onto a CM Sepharose fast flow column.
  • the 20 column volume salt gradient should be performed at 20 mM NaOAC, pH 5.5, 0M to 0.5 M NaCl .
  • Step 9 should be performed by diluting the CM Sepharose pool four fold with water, and adjusting the pH to 7.5. This mixture should be made to 0.7 M ammonium sulfate.
  • CTCCCTGCCG TCCCAGAACG TTCTTCAGAT CGCTAACGAC CTCGAGAACC TTCGCGACCT 300
  • Val Leu Thr Ser Leu Pro Ser Gin Asn Val Leu Gin lie Ala Asn Asp 65 70 75 80

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Abstract

L'invention concerne des méthodes pour accroître la sensibilité du récepteur de la protéine OB fonctionnelle et ses utilisations.
PCT/US1997/015086 1996-08-30 1997-08-26 Methodes pour accroitre la sensibilite d'un individu a la proteine ob par accroissement de l'affinite du recepteur de la proteine ob WO1998008512A1 (fr)

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EP97940645A EP1001768A1 (fr) 1996-08-30 1997-08-26 Methodes pour accroitre la sensibilite d'un individu a la proteine ob par accroissement de l'affinite du recepteur de la proteine ob
AU42377/97A AU4237797A (en) 1996-08-30 1997-08-26 Methods of increasing sensitivity of an individual to OB protein by upregula ting OB protein receptor
JP10511882A JP2001501177A (ja) 1996-08-30 1997-08-26 Ｏｂタンパク質受容体をアップレギュレートすることによりｏｂタンパク質に対する個体の感受性を増加させる方法
CA002263826A CA2263826A1 (fr) 1996-08-30 1997-08-26 Methodes pour accroitre la sensibilite d'un individu a la proteine ob par accroissement de l'affinite du recepteur de la proteine ob

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Cited By (13)

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WO2002043750A2 (fr) * 2000-12-01 2002-06-06 Battelle Memorial Institute Procede de stabilisation de biomolecules dans des formulations liquides
US6420339B1 (en) 1998-10-14 2002-07-16 Amgen Inc. Site-directed dual pegylation of proteins for improved bioactivity and biocompatibility
AU2002236641B2 (en) * 2000-12-01 2006-08-03 Battelle Memorial Institute Method for the stabilizing of biomolecules (e.g. insulin) in liquid formulations
US7183254B2 (en) 2001-10-22 2007-02-27 Amgen, Inc. Use of leptin for treating human lipoatrophy and method of determining predisposition to said treatment
EP2127676A2 (fr) 2004-11-01 2009-12-02 Amylin Pharmaceuticals, Inc. Traitement de l'obésité et les maladies et troubles liés à l'obésité
US8106098B2 (en) 1999-08-09 2012-01-31 The General Hospital Corporation Protein conjugates with a water-soluble biocompatible, biodegradable polymer
US8394765B2 (en) 2004-11-01 2013-03-12 Amylin Pharmaceuticals Llc Methods of treating obesity with two different anti-obesity agents
US8501686B2 (en) 2008-06-05 2013-08-06 University Of Michigan Method of treating fatty liver diseases and conditions in non-lipodystrophic subjects
WO2014052583A1 (fr) 2012-09-27 2014-04-03 The Children's Medical Center Corporation Composés pour le traitement de l'obésité et leurs procédés d'utilisation
WO2015081093A1 (fr) 2013-11-26 2015-06-04 The Chilren's Medical Center Corporation Composés pour le traitement de l'obésité et leurs procédés d'utilisation
WO2015153933A1 (fr) 2014-04-03 2015-10-08 The Children's Medical Center Corporation Inhibiteurs hsp 90 pour le traitement de l'obésité et leurs procédés d'utilisation
WO2018049424A1 (fr) 2016-09-12 2018-03-15 Aegerion Pharmaceuticals, Inc. Procédés de détection d'anticorps neutralisants anti-leptine
US11535659B2 (en) 2010-09-28 2022-12-27 Amryt Pharmaceuticals Inc. Engineered polypeptides having enhanced duration of action

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WO1997016189A1 (fr) * 1995-11-01 1997-05-09 Merck & Co., Inc. Traitement combine du diabete et de l'obesite

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S.L. PEARSON ET AL.: "The thiazolidinedione insulin sensitiser, BRL 49653, increases the expression of PPAR-g and aP2 in adipose tissue of high-fat-fed rats.", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 229, no. 3, 1996, pages 752 - 757, XP002052407 *

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6420339B1 (en) 1998-10-14 2002-07-16 Amgen Inc. Site-directed dual pegylation of proteins for improved bioactivity and biocompatibility
US8106098B2 (en) 1999-08-09 2012-01-31 The General Hospital Corporation Protein conjugates with a water-soluble biocompatible, biodegradable polymer
WO2002043750A3 (fr) * 2000-12-01 2002-10-31 Battelle Memorial Institute Procede de stabilisation de biomolecules dans des formulations liquides
AU2002236641B2 (en) * 2000-12-01 2006-08-03 Battelle Memorial Institute Method for the stabilizing of biomolecules (e.g. insulin) in liquid formulations
WO2002043750A2 (fr) * 2000-12-01 2002-06-06 Battelle Memorial Institute Procede de stabilisation de biomolecules dans des formulations liquides
US8318666B2 (en) 2001-10-22 2012-11-27 Amgen, Inc. Use of leptin to treat metabolic abnormalities associated with lipoatrophy
EP2219031A1 (fr) 2001-10-22 2010-08-18 Amgen, Inc. Utilisation de Leptine pour traiter la Lipoatrophine humaine et procédé pour déterminer une prédisposition à ce traitement
US7183254B2 (en) 2001-10-22 2007-02-27 Amgen, Inc. Use of leptin for treating human lipoatrophy and method of determining predisposition to said treatment
EP2286837A2 (fr) 2004-11-01 2011-02-23 Amylin Pharmaceuticals, Inc. Traitement de l'obésité et de maladies liés à l'obésité
EP2286839A2 (fr) 2004-11-01 2011-02-23 Amylin Pharmaceuticals, Inc. Traitement de l'obésité et de maladies liés à l'obésité
EP2286838A2 (fr) 2004-11-01 2011-02-23 Amylin Pharmaceuticals, Inc. Traitement de l'obésité et de maladies liés à l'obésité
EP2286840A2 (fr) 2004-11-01 2011-02-23 Amylin Pharmaceuticals, Inc. Traitement de l'obésité et de maladies liés à l'obésité
EP2127676A2 (fr) 2004-11-01 2009-12-02 Amylin Pharmaceuticals, Inc. Traitement de l'obésité et les maladies et troubles liés à l'obésité
US8394765B2 (en) 2004-11-01 2013-03-12 Amylin Pharmaceuticals Llc Methods of treating obesity with two different anti-obesity agents
US8501686B2 (en) 2008-06-05 2013-08-06 University Of Michigan Method of treating fatty liver diseases and conditions in non-lipodystrophic subjects
US11535659B2 (en) 2010-09-28 2022-12-27 Amryt Pharmaceuticals Inc. Engineered polypeptides having enhanced duration of action
WO2014052583A1 (fr) 2012-09-27 2014-04-03 The Children's Medical Center Corporation Composés pour le traitement de l'obésité et leurs procédés d'utilisation
EP4082541A1 (fr) 2012-09-27 2022-11-02 The Children's Medical Center Corporation Composés pour le traitement de l'obésité et leurs procédés d'utilisation
WO2015081093A1 (fr) 2013-11-26 2015-06-04 The Chilren's Medical Center Corporation Composés pour le traitement de l'obésité et leurs procédés d'utilisation
WO2015153933A1 (fr) 2014-04-03 2015-10-08 The Children's Medical Center Corporation Inhibiteurs hsp 90 pour le traitement de l'obésité et leurs procédés d'utilisation
WO2018049424A1 (fr) 2016-09-12 2018-03-15 Aegerion Pharmaceuticals, Inc. Procédés de détection d'anticorps neutralisants anti-leptine
US10775386B2 (en) 2016-09-12 2020-09-15 Aegerion Pharmaceuticals, Inc. Methods of detecting anti-leptin neutralizing antibodies
US11709166B2 (en) 2016-09-12 2023-07-25 Amryt Pharmaceuticals Inc. Methods of detecting anti-leptin neutralizing antibodies
EP4283304A2 (fr) 2016-09-12 2023-11-29 Amryt Pharmaceuticals Inc. Procédés de détection d'anticorps neutralisants anti-leptine

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