WO1997038014A1 - Compositions pharmaceutiques renfermant la fibuline et procedes connexes - Google Patents

Compositions pharmaceutiques renfermant la fibuline et procedes connexes Download PDF

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Publication number
WO1997038014A1
WO1997038014A1 PCT/US1997/006280 US9706280W WO9738014A1 WO 1997038014 A1 WO1997038014 A1 WO 1997038014A1 US 9706280 W US9706280 W US 9706280W WO 9738014 A1 WO9738014 A1 WO 9738014A1
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Prior art keywords
protein
amino acids
fibulin
cys
ser
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PCT/US1997/006280
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English (en)
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Larry G. Bennett
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Amgen Inc.
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Priority to AU26708/97A priority Critical patent/AU2670897A/en
Publication of WO1997038014A1 publication Critical patent/WO1997038014A1/fr

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    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to fibulin protein, fibulin/OB protein complex and pharmaceutical compositions thereof.
  • the present invention also relates to methods of making and using the above proteins and compositions.
  • OB gene OB gene
  • protein protein encoded
  • Zhang et al. Nature 372 : 425-432 (1994); see also, the Correction at Nature 374: 479 (1995) .
  • 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. See generally, Barinaga, "Obese” Protein Slims Mice, Science 269: 475-476 (1995) .
  • OB protein in blood has not been accomplished to any significant degree. For example, it is not known whether OB protein circulates complexed to other substances in blood, and, if so, what effects these other substances may have. Such binding complexes or proteins may, for example, act as antagonist, or may have a protective effect, and contribute to the stability of either exogenously administered or naturally occurring OB protein present in the body.
  • fibulins are known to be plasma proteins. S_ee Tran et al. , J. Biol. Chem. 270: 19458-19464 (1990) . There are two known types of fibulins, type 1 and type 2. Agraves et al . , J. Cell Biol. Ill: 3155-3164 (1990) . Of fibulin type 1, there are 4 variants or isoforms, denoted in the literature as "A", "B” , "C", and "D” . Id. at Figure 6. Generally, fibulin type 1 "A" is a 537 amino acid protein. Argraves, et al.
  • Variants “B” , “C” and “D” have conserved amino acids 1-537, but have altered C-termini: “B” has amino acids 538-572, “C” has amino acids 538- 654, and “D” has amino acids 538-674.
  • the polypeptides predicted from the nucleotide sequences of the CDNA's have molecular masses of 58,670, 62,561, 71,551 and 77,274 for fibulin type 1 variants A-D respectively. See Argraves et al. , supra. These forms are also rich in cysteine, approximately 11 mole %. .Id. at 3160.
  • Fibulin type 1 is known to co-purify with fibrinogen, and to bind to various extracellular matrix proteins such as fibronectin, laminin, and nidogen. See Tran et al. , supra. Fibulin type 1 has been observed in the blood in concentrations of 30-40 ⁇ g/ml, id., and is thought to be, among other things, a structural protein that contributes to the elastic properties of connective tissue, or is involved in the process of fibrogenesis . Roark et al. , J. Histochem. Cytochem. 4_3: 401-411 (1995) . Fibulin protein, the DNA encoding it, as well as antibodies reactive with fibulin, have also been characterized by Argraves et al . in the PCT published application No. WO 91/02755.
  • fibulin type 2 As for fibulin type 2, it is larger than fibulin type 1 and possesses a 408 amino acid N-terminal domain not found in fibulin type 1. See Zhang et al . , Genomics 2 : 425-430 (1994) . Fibulin type 2 has been observed in the skin, including the epidermal layer, the dermis and adipose tissue, as well as the keratinocyte layer of the hair, the epithelial layer of the cornea, the aortic intima, and around small vessels in the kidney and liver. Id. at 428. In addition, serum levels of fibulin type 2 have been noted to be 1000 fold lower than fibulin type 1. if], at 1269. It is desirable to have a pharmaceutical composition which enhances the effectiveness of either exogenously administered or endogenous OB protein. Such compositions would be useful for example, to reduce or eliminate the need for exogenous OB protein administration. SUMMARY OF THE INVENTION
  • fibulin type 1 binds to OB protein. Such binding is thought to have a potentiating effect of OB protein. While not wishing to be bound by theory, such potentiating effect is thought to be related to fibulin' s protection of OB protein in circulation. Such protection may increase the stability of OB protein in the blood, and therefore permit biological activity enhanced relative to OB protein alone. Accordingly, fibulin can be used as a pharmaceutical composition to enhance the effectiveness of either exogenously administered or endogenous OB protein. Therefore, one aspect of the present invention is a pharmaceutical composition containing fibulin type 1 protein.
  • This composition can contain either fibulin type 1 variant "A” (amino acids 1-537) , fibulin type 1 variant “B” (amino acids 1-572) , fibulin type 1 variant “C” (amino acids 1-654), fibulin type 1 variant "D"
  • the pharmaceutical composition can also contain the methionyl form of any of the above fibulin type 1 variants having a methionyl residue at the N-terminus (at the -1 position) .
  • Other analogs, amino acid substitutions, derivatives, truncated versions of fibulin type 1 variants and combinations thereof are also within the scope of this invention. This would include fibulin type 2 as well since type 2 is the same as type 1 with an additional 408 amino acid N-terminal domain added.
  • the fibulin can also be in a dimer form.
  • the pharmaceutical composition includes a pharmaceutically acceptable carrier, diluent, adjuvant, solubilizer and/or stabilizer.
  • a second aspect of the present invention includes OB protein complexed to the fibulin type 1 protein whether variant A, B, C, D or a combination thereof is used.
  • the OB protein can be chemically, covalently, or ionically attached or complexed to fibulin type 1, as well as, any other means of association. In addition, such binding can occur in the presence of divalent cations, especially calcium, (Ca ++ ) .
  • divalent cations especially calcium, (Ca ++ )
  • the N-terminal methionyl form of fibulin type 1 can also be used.
  • various forms of the OB protein, analogs or derivations thereof can be used as well as various forms of fibulin type 1, analogs, derivatives or combinations thereof .
  • a third aspect of the present invention provides a pharmaceutical composition containing the OB protein/fibulin type 1 complex as described above in a pharmaceutically acceptable carrier, diluent, adjuvant solubilizer and/or stabilizer.
  • a fourth aspect of the present invention includes a method of treating obesity, hyperlypedemia, Type II diabetes and other obesity related disorders by administering an effective amount of one or more of the pharmaceutical compositions containing fibulin type 1 or fibulin type 1/OB protein complex.
  • Such treatment includes use of variants, analogs, derivations or combinations of fibulin type 1 and/or OB protein.
  • Treatment using the above includes, but is not limited to, the reduction of serum lipid levels, cholesterol and triglyceride levels, reduction or prevention of arterial plaque, and the reduction of hypertension and related gall stone formation.
  • a fifth aspect of the present invention is a method of preparing the pharmaceutical compositions discussed above by admixing purified and isolated fibulin type 1 or OB protein/fibulin type 1 complex, with a pharmaceutically acceptable carrier, diluent, adjuvant solubilizer and/or stabilizer.
  • This method includes use of any variants, N-terminal methionyl versions, analogs, derivatives or combinations of fibulin type 1 or OB protein/fibulin type 1 complex.
  • Another aspect of the present invention includes a method for detecting the presence of OB protein in a biological sample.
  • Such a method comprises the steps of incubating the sample with fibulin type 1, or variants, analogs or derivatives as described above, under conditions that allow binding of the fibulin type 1 to OB protein in a sample. The amount of OB protein bound to the fibulin type 1 in the complex is then determined.
  • Figure 1 represents the presence of fibulin type 1 binding to OB protein. Lane 1 contains the wash from the control resin. Lane 2, contains the wash from the OB-linked resin. Lanes 3-10 contain various wash fractions and controls. Fibulin is located in Lane 2 at band ⁇ 85 kD. Figure 2 represents the amino acid and DNA sequences for recombinant murine met-OB protein (SEQ. ID. NOs. 1, 2 and 3) .
  • Figure 3 represents the amino acid and DNA sequences for recombinant human met-OB analog (SEQ. ID. NOs. 4, 5 and 6) .
  • Figure 4 represents the amino acid sequence for human fibulin type 1 isoform or variant A (SEQ. ID. NO. 7) .
  • Figure 5 represents the amino acid sequence for human fibulin type 1 isoform or variant B (SEQ. ID. NO. 8) .
  • Figure 6 represents the amino acid sequence for human fibulin type 1 isoform or variant C (SEQ. ID. NO. 9) .
  • Figure 7 represents the amino acid sequence for human fibulin type 1 isoform or variant D (SEQ. ID. NO. 10) .
  • Fibulin may be selected from fibulin type 1 isoforms or variants A, B, C or D.
  • fibulin type 1 may be selected from amino acids 1-537 (variant A) of SEQ. ID. NO. 7 ( Figure 4) ; amino acids 1-572 (variant B) of SEQ. ID. NO. 8 ( Figure 5); amino acids 1-654 (variant C) of SEQ. ID. NO. 9 ( Figure 6); or amino acids 1-674 (variant D) of SEQ. ID. NO. 10 ( Figure 7) .
  • the amino acid sequences above can contain a methionyl residue at the -1 position, however, as with any of the present fibulin proteins and analogs, the methionyl residue may be absent.
  • any fibulin protein used can be isolated and purified from naturally occurring sources or recombinantly produced.
  • Such fibulins may also include, incident to expression or otherwise, the leader sequence at position -29 to -1.
  • Such fibulins can also include a dimer form of fibulin.
  • what is needed is that portion of the fibulin type 1 protein sufficient to complex to the type of OB protein used. This is readily determined by preparing deletion or substitution analogs (for example, using the methods of Alton et al . PCT published application No.
  • WO 83/04053 or other methods available to those skilled in the art) , contacting such prepared portion of fibulin type 1 with the desired OB protein under suitable conditions (e.g., physiological conditions) , including but not limited to the presence of divalent cations such as Calcium (Ca ++ ) and ascertaining the presence or amount of fibulin/OB protein complex formed.
  • suitable conditions e.g., physiological conditions
  • divalent cations such as Calcium (Ca ++ )
  • one or more cysteine residues may be replaced by an alanine or serine residue and one or more tyrosine residues replaced by a phenylalanine residue.
  • chemical moieties can include water soluble polymers, such as polyethylene glycol or polyamino acid.
  • the chemical moiety can be attached at solely the N-terminus of the fibulin protein.
  • Hybrid molecules based on the fibulin type 1 variants A, B, C or D can also be prepared.
  • the fibulin type 1 variants have conserved sequences 1-537 but differ from amino acids 538-674. One may substitute another amino acid or add to the existing amino acids one or more of the amino acids from these alternative isoforms to obtain a hybrid molecule.
  • fibulin type 2 which is the same as fibulin type 1 with an additional 408 amino acid N-terminal domain added.
  • consensus molecules include but are not limited to the following fibulin protein molecules (using the numbering of SEQ. ID. NOS. 7, 8, 9 and 10) :
  • the OB protein may be selected from recombinant murine set forth below (SEQ. ID. NOs. 1, 2 or 3 ) , 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, 22., 33, 3_5, 5.0, 68, 71, 74, 77, 78, .81, 9J . , 100, 101, 102, 105. 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 identitites 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), 108(E), 112 (D) , and 118 (L) . Since the recombinant human OB protein is active in cynomolgus monkeys, a human OB protein according to SEQ. ID. NO.
  • rhesus monkey divergent amino acids may be effective.
  • certain rhesus divergent amino acids are also those found in the above murine species (positions 35, 68, 89, 100 and 112) .
  • a murine/rhesus/human consensus molecule having (using the numbering of SEQ.ID. NO.
  • amino acids 1-99 and (connected to) 112-146 amino acids 1-99 and (connected to) 112-146 having one or more of amino acids 100-111 placed between amino acids 99 and 112.
  • 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 fibulin and/or OB protein may also be derivatized by the attachment of one or more chemical moieties to the protein moiety. If the present pharmaceutical compositions contain as the active ingredient a complex of fibulin and OB protein, one or both of such proteins may be derivatized.
  • the chemically modified derivatives may be further formulated for intraarterial, intraperitoneal, intramuscular, subcutaneous, intravenous, oral, nasal, pulmonary, topical or other routes of administration.
  • 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 fibulin or OB protein (or analog) moiety.
  • the polyamino acid may be a carrier protein which serves to increase the circulation half life of the protein.
  • such 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 "Fc”) or other polyamino acids.
  • the location of attachment of the polyamino acid may be at the N-terminus of the fibulin or OB protein moiety, or other place, and also may be connected by a chemical "linker" moiety to the fibulin or 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.
  • polymer molecules so attached may vary, and one skilled in the art will be able to ascertain the effect on function.
  • One may mono-derivatize, or may provide for a di-, tri-, tetra- or some combination of derivatization, with the same or different chemical moieties (e.g., polymers, such as different weights of polyethylene glycols) .
  • the proportion of polymer molecules to protein (or peptide) molecules will vary, as will their concentrations in the reaction mixture.
  • the optimum ratio in terms of efficiency of reaction in that there is no excess unreacted protein or polymer
  • 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. 2J): 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.
  • 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) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules.
  • 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. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
  • attachment of a water soluble polymer to a protein is controlled: the conjugation with the polymer takes place predominantly at the N-terminus of the protein and no significant modification of other reactive groups, such as the lysine side chain amino groups, occurs.
  • 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 pro ionaldehyde containing a single reactive aldehyde, may be used.
  • An 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.
  • compositions in yet another aspect of the present invention, methods of using pharmaceutical compositions of the proteins, and derivatives are provided.
  • Such pharmaceutical 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-HCI, 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 liposomes.
  • buffer content e.g., Tris-HCI, acetate, phosphate
  • additives e.g., Tween 80, Polysorbate 80
  • anti-oxidants e.g., ascorbic acid, sodium metabisulfite
  • preservatives e.g., Thimersol, benzy
  • 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 n 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.
  • 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 (e.g. , 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) .
  • 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.
  • 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 examples include: polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline.
  • Abuchowski and Davis Soluble polymer-Enzyme Adducts: "Enzymes as Drugs", Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY,
  • the location of release may be the stomach, the small intestine (the duodenum, the jejunem, or the ileum) , or the large intestine.
  • 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.
  • cellulose acetate trimellitate cellulose acetate trimellitate
  • HPMCP 50 hydroxypropylmethylcellulose phthalate
  • HPMCP 55 polyvinyl acetate phthalate
  • PVAP polyvinyl acetate phthalate
  • Eudragit L30D Eudragit L30D
  • Aquateric cellulose acetate phthalate
  • CAP Eudragit L
  • Eudragit S and Shellac.
  • 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.
  • moist massing techniques can be used.
  • 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, ⁇ -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 hydroxypropylmethyl 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 si1icoaluminate.
  • 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.
  • 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.
  • Other 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.
  • Film coating may be carried out in a pan coater or in a fluidized bed or by compression coating.
  • pulmonary delivery of the present proteins, 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 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_L 565-569 (1990); Adjei et al. , International Journal of Pharmaceutics 6_3: 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.
  • Ultravent nebulizer manufactured by Mallinckrodt, Inc., St. Louis, Missouri
  • Acorn II nebulizer manufactured by Marquest Medical Products, Englewood, Colorado
  • the Ventolin metered dose inhaler manufactured by Glaxo Inc., Research Triangle Park, North Carolina
  • the Spinhaler powder inhaler manufactured by Fisons Corp., Bedford, Massachusetts.
  • 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 proteins (or derivative) should most advantageously be prepared in particulate form with an average particle size of less than 10 ⁇ m (or microns) , most preferably 0.5 to 5 ⁇ 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.
  • liposomes are contemplated.
  • microcapsules or microspheres inclusion complexes, or other types of carriers.
  • 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 metered-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 in amounts which facilitate dispersal of the powder from the device, e.g. 50 to 90% by weight of the formulation.
  • Nasal delivery of the protein 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. Delivery via transport across other mucus membranes is also contemplated.
  • One skilled in the art will be able to ascertain effective dosages by administration and observing the desired therapeutic effect.
  • the formulation of the molecule or complex in a pharmaceutical composition will be such that between about .10 ⁇ g/kg/day and 10 ⁇ g/kg/day will yield the desired therapeutic effect.
  • the effective dosages may be determined using diagnostic tools over time.
  • a diagnostic for measuring the amount of OB or fibulin protein in the blood (or plasma or serum) may first be used to determine endogenous levels of OB or fibulin protein.
  • Such diagnostic tools may be in the form of an antibody assay, such as an antibody sandwich assay.
  • the amount of OB protein can also be determined by means of the methods disclosed as part of the invention herein. These include incubating biological samples with fibulin protein and determining the amount of OB protein bound in the OB/fibulin complex. The amount of endogenous OB or fibulin protein is quantified initially, and a baseline is determined.
  • the therapeutic dosages are determined as the quantification of endogenous and exogenous OB or fibulin 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.
  • Methods of Use Therapeutic benefits include the reduction of excess weight and/or excess fat. When excess weight and/or fat is reduced or eliminated, other therapeutic benefits may concomitantly occur, such as a treatment of Type II diabetes. There may also be a reduction in blood lipid levels and related cardio-vacular problems, such as a reduction in LDL or VLDL, cholesterol, a reduction in artherial plaque or the rate of formation thereof, treatment of hyper-tension, and a reduction in the rate of gall stone formation. Additional therapeutic benefits include the relative or absolute increase in lean mass along with loss in fat. Increase in lean tissue mass may also result in an increase in sensitivity to insulin, and improvement in overall strength, a decrease in bone resorption, and an increase in red blood cells or oxygenation of tissue.
  • the present compositions may have solely cosmetic benefits in weight reduction or fat reduction.
  • the present methods may be used in conjunction with other medicaments, such as those useful for the treatment of diabetes (e.g. , insulin, and possibly amylin) , cholesterol and blood pressure lowering medicaments (such as those which reduce blood lipid levels or other cardiovascular medicaments) , and activity increasing medicaments (e.g. , amphetamines) .
  • Appetite suppressants may also be used.
  • 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. Therefore, the present invention provides a method for treating disorders selected from among excess weight, high blood levels, high cholesterol, high triglycerides, Type II diabetes, or other related disorders, comprised of administering an effective amount of fibulin type I protein selected from among the following:
  • fibulin protein analog sufficient to bind an OB protein, analog or derivative thereof;
  • a hybrid molecule of fibulin protein selected from among the following:
  • amino acids 1-537 and (connected to) amino acids 573-654 having one or more of amino acids 538-572 placed between amino acids 537 and 573;
  • amino acids 1-537 and (connected to) amino acids 655-674 having one or more of amino acids 538-572 and/or 573-654, placed between amino acids 537 and 655;
  • amino acids 1-572 and (connected to) amino acids 655-674 having one or more of amino acids 573-654 placed between amino acids 572 and 655;
  • the present invention also provides a method for treating disorders selected from among excess weight, high blood levels, high cholesterol, high triglycerides, Type II diabetes, or other related disorders comprised of administering a therapeutically effective amount of a complex consisting of fibulin type I, or analogs or derivatives thereof as discussed above, complexed to an OB protein, analog or derivative thereof selected from among the following: (a) the amino acid sequence 1-146 as set forth in SEQ. ID. NO. 3 (below) or SEQ ID. NO. 6 (below) ,
  • 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) : (i) amino acids 98-146;
  • amino acids 1-32 amino acids 40-116; 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;
  • Fibulin may also be used as an affinity reagent to purify or detect the presence of OB protein in a biological sample.
  • the fibulin protein is immobilized on CnBr-activated Sepharose and a column of protein-Sepharose conjugate is used to remove OB protein from liquid samples.
  • Fibulin protein may also be immobilized on other support materials which are well known in the art.
  • Fibulin can also be used as a diagnostic reagent to detect and quantitate OB protein in biological samples.
  • Such methods utilize fibulin protein as a capture agent under conditions that allow OB protein in a biological sample to bind to fibulin protein forming a fibulin/OB complex. The amount of OB protein or the presence of OB protein in a sample can then be determined through known methods.
  • Example 1 demonstrates that fibulin binds to OB protein.
  • Example 2 is a prophetic example illustrating the use of the present fibulin pharmaceutical compositions for the treatment of obesity.
  • Example 3 is a prophetic example illustrating the use of the present fibulin/OB protein complex pharmaceutical composition for the treatment of obesity. Materials and Methods follow.
  • the resin capsule was removed from the serum, and washed until the eluant was relatively protein-free, having an OD below .01 at A280-
  • the first was buffer contained 50 mM Tris, pH 7.5, 150 mM NaCl, 2 mM CaCl2 and 2 mM MgCl2 • One milliliter fractions were collected.
  • SDS-PAGE The SDS-PAGE (non-reducting conditions) results are shown in FIGURE 1.
  • Lane 4 contains molecular weight standards ranging from 200 kD to 6kD.
  • the presence of fibulin binding to OB protein is demonstrated by comparing lane 1 containing the EDTA elution from the control resin (first lane on the left) and the lane 2, containing the EDTA elution from the OB- linked resin (second lane from the left) , As can be seen, there is a band at approximately 85 kD seen in lane 2.
  • Fibulin dimer binding to OB protein is demonstrated by comparing lane 1 containing the EDTA elutions from the control resin and lane 2, containing the EDTA elution from the OB-linked resin. As can be seen, there is a band at approxiamtely 200 kD in lane 2.
  • Sequencing Confirmation Sequencing confirmed the identity of fibulin in the 85 kD band.
  • the N-terminal 13 amino acids were identical to the N-terminal amino acids of the mature fibulin protein (GenBank accession No. P37888) .
  • This Example demonstrates the use of fibulin pharmaceutical compositions for the treatment of obesity.
  • An obese human, with a BMI greater than 30, is treated with an effective dose of fibulin in a pharmaceutically acceptable diluent, adjuvant or carrier.
  • the fibulin is selected from amino acids
  • variant B 1-572 of SEQ. ID. NO. 8; amino acids (variant C) 1-654 of SEQ. ID. NO. 9; amino acids
  • fibulin levels in the serum are monitored using an anti-fibulin selective binding molecule, such as a monoclonal antibody.
  • This Example demonstrates the use of fibulin/OB protein complex pharmaceutical compositions for the treatment of obesity.
  • An obese human, with a BMI greater than 30, is treated with an effective dose of fibulin/OB protein complex, in a pharmaceutically acceptable diluent, adjuvant or carrier.
  • the fibulin and OB protein are selected from the amino acids, analogs and derivates described herein.
  • the fibulin can be monitored using an anti-fibulin selective binding molecule, such as a monoclonal antibody.
  • fibulin type 1 can be produced using standard methods well known in the art.
  • fibulin type 1 can also be isolated and purified from naturally occurring sources such as human placenta or human serum or plasma using standard methods well known in the art. Such methods have previously been described in Balbona et al. , J. Biol. Chem. 267: 20120- 20125 (1992) and Argraves et al. , J. Cell Biol. Ill: 3155- 3164 (1990) .
  • standard methods for producing methionyl analogs and other derivatives can also be used in the preparation of fibulin type 1 analogs or derivatives.
  • 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 strain, 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, CI857 (Sussman et al., CR. Acad. Sci. 254.: 1517-1579 (1962)) .
  • Fermentation Process A three-phase fermentation protocol known as a fed-batch process was used. Media compositions are set forth below. Batch: 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 glucose was fed at a limiting rate in order to control the growth rate ( ⁇ ) .
  • the Distributive Control System 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 I 50 g/L Bacto-tryptone
  • Feed II 200 g/L Bacto-tryptone
  • 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 was centrifuged as in step 1.
  • 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 the above host cells to produce recombinant human OB protein analog 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 those used for purification of recombinant murine protein.
  • 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.
  • Recombinant human OB protein of SEQ.ID.NO.6 having lysine 35 and isoleucine 74 can be formulated in a buffer containing 10 mM histidine, 4.3% arginine, at pH 6.0. Methods of producing fibulin Type 1/OB Protein Complex.
  • Fibulin type 1/OB protein complex can be prepared by admixing purified and isolated Fibulin type 1 with purified and isolated OB protein. The fibulin type 1/OB protein complex will form due to the ability of fibulin type 1 to associate with OB protein.
  • CTCCCTGCCG TCCCAGAACG TTCTTCAGAT CGCTAACGAC CTCGAGAACC TTCGCGACCT 300
  • MOLECULE TYPE protein
  • Val Leu Thr Ser Leu Pro Ser Gin Asn Val Leu Gin lie Ala Asn Asp 65 70 75 80
  • Thr lie Val Thr Arg lie Asn Asp lie Ser His Thr Gin Ser Val Ser 20 25 30 Ser Lys Gin Arg Val Thr Gly Leu Asp Phe lie Pro Gly Leu His Pro 35 40 45 lie Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin 50 55 60 lie Leu Thr Ser Met Pro Ser Arg Asn Val Leu Gin lie Ser Asn Asp 65 70 75 80

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Abstract

La présente invention se rapporte à la protéine fibuline, au complexe de protéine OB/fibuline et à des compositions pharmaceutiques contenant celles-ci. La présente invention se rapporte également à des procédés de fabrication et d'utilisation des protéines et compositions précitées.
PCT/US1997/006280 1996-04-04 1997-04-03 Compositions pharmaceutiques renfermant la fibuline et procedes connexes WO1997038014A1 (fr)

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US5935810A (en) * 1994-08-17 1999-08-10 The Rockefeller University Mammalian ob polypeptides capable of modulating body weight, corresponding nucleic acids, and diagnostic and therapeutic uses thereof
WO2000009165A1 (fr) * 1998-08-10 2000-02-24 Amgen Inc. Conjugues dextrane-leptine, compositions pharmaceutiques et methodes connexes
WO2001089548A2 (fr) * 2000-05-24 2001-11-29 Schering Aktiengesellschaft Utilisation pharmaceutique de fibuline-1
US6420339B1 (en) 1998-10-14 2002-07-16 Amgen Inc. Site-directed dual pegylation of proteins for improved bioactivity and biocompatibility
WO2002072138A1 (fr) * 2001-03-08 2002-09-19 Hyseq, Inc. Methodes et materiels se rapportant a des polypeptides et a des polynucleotides apparentes a la fibuline
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
WO2009055864A1 (fr) * 2007-10-31 2009-05-07 Crc For Asthma And Airways Ltd Procédés et compositions pour réguler un remodelage du tissu des voies aériennes
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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Cited By (29)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5935810A (en) * 1994-08-17 1999-08-10 The Rockefeller University Mammalian ob polypeptides capable of modulating body weight, corresponding nucleic acids, and diagnostic and therapeutic uses thereof
US7521258B2 (en) 1994-08-17 2009-04-21 The Rockefeller University Methods of detecting, measuring, and evaluating modulators of body weight in biological samples, and diagnostic, monitoring, and therapeutic uses thereof
WO2000009165A1 (fr) * 1998-08-10 2000-02-24 Amgen Inc. Conjugues dextrane-leptine, compositions pharmaceutiques et methodes connexes
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
WO2001089548A2 (fr) * 2000-05-24 2001-11-29 Schering Aktiengesellschaft Utilisation pharmaceutique de fibuline-1
WO2001089548A3 (fr) * 2000-05-24 2003-01-23 Schering Ag Utilisation pharmaceutique de fibuline-1
WO2002072138A1 (fr) * 2001-03-08 2002-09-19 Hyseq, Inc. Methodes et materiels se rapportant a des polypeptides et a des polynucleotides apparentes a la fibuline
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
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
EP2286840A2 (fr) 2004-11-01 2011-02-23 Amylin Pharmaceuticals, Inc. Traitement de l'obésité et de maladies liés à l'obésité
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
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
EP2286838A2 (fr) 2004-11-01 2011-02-23 Amylin Pharmaceuticals, Inc. Traitement de l'obésité et de maladies liés à l'obésité
WO2009055864A1 (fr) * 2007-10-31 2009-05-07 Crc For Asthma And Airways Ltd Procédés et compositions pour réguler un remodelage du tissu des voies aériennes
AU2008318288B2 (en) * 2007-10-31 2013-11-14 The University Of Sydney Methods and compositions for regulating airway tissue remodelling
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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