EP0889737A1 - Compose contenant la proteine de l'obesite et leurs formulations - Google Patents

Compose contenant la proteine de l'obesite et leurs formulations

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Publication number
EP0889737A1
EP0889737A1 EP97903961A EP97903961A EP0889737A1 EP 0889737 A1 EP0889737 A1 EP 0889737A1 EP 97903961 A EP97903961 A EP 97903961A EP 97903961 A EP97903961 A EP 97903961A EP 0889737 A1 EP0889737 A1 EP 0889737A1
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EP
European Patent Office
Prior art keywords
leu
ser
formulation
protein
obesity
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Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP97903961A
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German (de)
English (en)
Inventor
James A. Hoffmann
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Eli Lilly and Co
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Eli Lilly and Co
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Publication of EP0889737A1 publication Critical patent/EP0889737A1/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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is in the field of human medicine, particularly in the treatment of obesity and disorders associated with obesity. More specifically, the present invention relates to compounds and formulations of an obesity protein.
  • the ob/ob mouse is a model of obesity and diabetes that is known to carry an autosomal recessive trait linked to a mutation in the sixth chromosome. Recently, Yiying Zhang and co-workers published the positional cloning of the mouse gene linked with this condition. Yiying Zhang et al. Nature 372: 425-32 (1994). This report disclosed the murine and human protein expressed in adipose tissue. Likewise, Murakami et al. , in Biochemical and Biophysical Research
  • the present invention provides conditions under which potency of the natural obesity protein is significantly enhanced.
  • effective pharmacological treatment may be achieved at lower doses that significantly lower the risk of toxic or other undesirable side effects.
  • the cost of the unit dosage form to the patient is reduced.
  • the present invention provides a novel protein- cation complex, which comprises an obesity protein complexed with a divalent metal cation, pharmaceutical formulations thereof, and methods of using such compounds for the treatment of obesity, and disorders associated with obesity such as diabetes, cardiovascular disease and cancer.
  • the invention provides a compound comprising an obesity protein complexed with a divalent metal cation.
  • the invention additionally provides parenteral pharmaceutical formulations comprising the protein-cation compounds and methods of using such compounds for treating obesity and disorders associated with obesity such as diabetes, cardiovascular disease and cancer.
  • the invention further comprises combining an obesity protein and a divalent metal cation in an aqueous solution at a pH of about 4.5 to 9.0.
  • Base pair (bp) -- refers to DNA or RNA.
  • the abbreviations A,C,G, and T correspond to the 5 ' -monophosphate forms of the nucleotides (deoxy)adenine, (deoxy) cytidine, (deoxy)guanine, and (deoxy)thymine, respectively, when they occur in DNA molecules.
  • the abbreviations U,C,G, and T correspond to the 5 ' -monophosphate forms of the nucleosides uracil, cytidine, guanine, and thymine, respectively when they occur in RNA molecules.
  • base pair may refer to a partnership of A with T or C with G.
  • base pair may refer to a partnership of T with U or C with G.
  • Obesity protein refers to the native mammalian protein produced from the native ob gene following transcription and deletions of introns, translation to a protein and processing to the mature protein with secretory signal peptide removed, e.g. from the N-terminal valine- proline to the C-terminal cysteine of the mature protein.
  • the human obesity protein is published in Zhang et al . Nature 372: 425-32 (1994).
  • the rat obesity protein is published in Murakami et al. , Biochemical and Biophysical Research Comm. 209(3) : 944-52 (1995).
  • Native porcine and bovine Ob proteins are disclosed in a U.S. patent application by Hansen M. Hsiung and Dennis P.
  • Obesity protein includes those proteins having a leader sequence.
  • a leader sequence is one or more amino acids on the N-terminus to aid in production or purification of the protein.
  • a preferred leader sequence is Met-Ri where Ri is absent or any amino acid except Pro.
  • Reading frame the nucleotide sequence from which translation occurs "read” in triplets by the translational apparatus of tRNA, ribosomes and associated factors, each triplet corresponding to a particular amino acid. Because each triplet is distinct and of the same length, the coding sequence must be a multiple of three. A base pair insertion or deletion (termed a frameshift mutation) may result in two different proteins being coded for by the same DNA segment. To insure against this, the triplet codons corresponding to the desired polypeptide must be aligned in multiples of three from the initiation codon, i.e. the correct "reading frame" must be maintained.
  • Recombinant DNA Cloning Vector any autonomously replicating agent including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
  • Recombinant DNA Expression Vector any recombinant DNA cloning vector in which a promoter has been incorporated.
  • Replicon A DNA sequence that controls and allows for autonomous replication of a plasmid or other vector.
  • Transcription the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence.
  • Vector a replicon used for the transformation of cells in gene manipulation bearing polynucleotide sequences corresponding to appropriate protein molecules which, when combined with appropriate control sequences, confer specific properties on the host cell to be transformed. Plasmids, viruses, and bacteriophage are suitable vectors, since they are replicons in their own right. Artificial vectors are constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases. Vectors include Recombinant DNA cloning vectors and Recombinant DNA expression vectors.
  • Treating -- as used herein describes the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of a compound of present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. Treating as used herein includes the administration of the protein for cosmetic purposes.
  • a cosmetic purpose seeks to control the weight of a mammal to improve bodily appearance.
  • Isotonicity agent refers to an agent that is physiologically tolerated and embarks a suitable tonicity to the formulation to prevent the net flow of water across the cell membrane.
  • Compounds, such as glycerin, are commonly used for such purposes at known concentrations.
  • Other possible isotonicity agents include salts, e.g., NaCl, dextrose, and lactose.
  • Physiologically tolerated buffer a physiologically tolerated buffer is known in the art.
  • Physiologically tolerated buffers include TRIS, sodium acetate, sodium phosphate, or sodium citrate. The selection and concentration of buffer is known in the art.
  • compositions must meet guidelines for preservative effectiveness to be a commercially viable product.
  • Pharmaceutically acceptable preservatives known in the art as being acceptable in parenteral formulations include: phenol, m-cresol, benzyl alcohol, methylparaben, chlorobutanol, p-cresol, phenylmercuric nitrate, thimerosal and various mixtures thereof.
  • Other preservatives may be found, e.g., in WALLHAUSER, K.-H. , DEVELOP, BIOL. STANDARD. 24, pp. 9-28 (Basel, S. Krager, 1974) .
  • the concentration necessary to achieve preservative effectiveness is dependent upon the preservative used and the conditions of the formulation.
  • nucleotide and amino acid abbreviations used herein are accepted by the United States Patent and Trademark Office as set forth in 37 C.F.R. ⁇ 1.822 (b) (2) (1993) . Unless otherwise indicated the amino acids are in the L configura ion.
  • the invention provides a compound comprising an obesity protein complexed with a divalent metal cation.
  • the obesity protein demonstrates significantly enhanced potency.
  • the presently claimed compounds comprise an obesity protein complexed with a divalent metal cation.
  • a divalent metal cation includes, for example, Zn ++ , Mn ++ , Fe ++ , Co ++ , Cd ++ , Ni ++ and the like.
  • a combination of two or more divalent metal cations is operable; however the preferred compounds comprise a single species of metal cation, most preferably Zn ++ .
  • the divalent metal cation is in excess; however, the molar ratio of at least one molecule of a divalent metal cation for each ten molecules of obesity protein is operable.
  • the compounds comprise from 1 to 100 divalent metal cations per molecule of obesity protein.
  • the compounds may be amorphous or crystalline solids.
  • metals cations are any form of a divalent metal cation that is available to form a complex with a molecule of obesity protein of the present invention.
  • the metal cation may be added in solid form or it may be added as a solution.
  • Several different cationic salts can be used in the present invention. Representative examples of metal salts include the acetate, bromide, chloride, fluoride, iodide and sulfate salt forms. The skilled artisan will recognize that there are many other metal salts which also might be used in the production of the compounds of the present invention.
  • zinc acetate or zinc chloride is used to create the zinc-obesity protein compounds of the present invention.
  • the divalent metal cationic salt is zinc chloride,
  • the claimed compounds are prepared by techniques known in the art. For example, convenient preparation is to combine the obesity protein with the desired divalent metal cation in an aqueous solution at a pH of about 4.5-9.0, preferably about pH 5.5-8, most preferably, about pH 6.5-7.6.
  • the claimed compound precipitates from the solution as a crystalline or amorphous solid.
  • the compound is easily isolated and purified by conventional separation techniques appreciated in the art including filtration and centrifugation.
  • the protein- metal cation complex is stable and may be conveniently stored as a solid or as an aqueous suspension.
  • the present invention further provides a pharmaceutical formulation comprising a compound of the present invention and water.
  • concentration of the obesity protein in the formulation is about 0.1 mg/mL to about 100 mg/mL; preferably about 0.5 mg/mL to about 50.0 mg/mL; most preferably, about 5.0 mg/mL.
  • the formulation preferably comprises a pharmaceutically acceptable preservative at a concentration necessary to maintain preservative effectiveness.
  • the relative amounts of preservative necessary to maintain preservative effectiveness varies with the preservative used. Generally, the amount necessary can be found in WALLHAUSER, K.- H., DEVELOP. BIOL. STANDARD. 24, pp. 9-28 (Basel, S. Krager, 1974), herein incorporated by reference.
  • An isotonicity agent preferably glycerin
  • the concentration of the isotonicity agent is in the range known in the art for parenteral formulations, preferably about 16 mg/mL glycerin.
  • the pH of the formulation may also be buffered with a physiologically tolerated buffer. Acceptable physiologically tolerated buffers include TRIS, sodium acetate, sodium phosphate, or sodium citrate. The selection and concentration of buffer is known in the art.
  • additives such as a pharmaceutically acceptable excipients like Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate) , Tween 80 (polyoxyethylene (20) sorbitan monooleate) , Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers) , BRIJ 35 (polyoxyethylene (23) lauryl ether), and PEG (polyethylene glycol) may optionally be added to the formulation to reduce aggregation.
  • a pharmaceutically acceptable excipients like Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate) , Tween 80 (polyoxyethylene (20) sorbitan monooleate) , Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers) , BRIJ 35 (polyoxyethylene (23) la
  • the claimed pharmaceutical formulations are prepared in a manner known in the art, and are administered individually or in combination with other therapeutic agents.
  • the formulations of the present invention can be prepared using conventional dissolution and mixing procedures.
  • the claimed formulations are prepared in an aqueous solution suitable for parenteral use. That is, a protein solution is prepared by mixing water for injection, buffer, and a preservative. Divalent metal cations are added to a total cation concentration of about 0.001 to 5.0 mg/mL, preferably 0.05 to 1.5 mg/mL.
  • the pH of the solution may be adjusted to completely precipitate the obesity protein zinc complex. The compound is easily resuspended before administration to the patient.
  • Parenteral daily doses of the compound are in the range from about 1 ng to about 10 mg per kg of body weight, although lower or higher dosages may be administered.
  • the required dosage will be determined by the physician and will depend on the severity of the condition of the patient and upon such criteria as the patient's height, weight, sex, age, and medical history.
  • the order the components are added, if a surfactant is used, the temperature, and pH at which the formulation is prepared may be optimized for the concentration and means of administration used.
  • the pH of the formulation is generally pH 4.5 to 9.0 and preferably 5.5 to 8.0, most preferably 6.5 to 7.6; although more acidic pH wherein a portion or all of the protein-metal cation complex is in solution is operable.
  • formulations prepared in accordance with the present invention may be used in a syringe, injector, pumps or any other device recognized in the art for parenteral administration.
  • the obesity proteins of the present invention can be prepared by any of a variety of recognized peptide synthesis techniques including classical (solution) methods, solid phase methods, semi synthetic methods, and more recent recombinant DNA methods.
  • an obesity protein of the present invention is human protein of the formula:
  • Val Pro lie Gin Lys Val Gin Asp Asp Thr Lys Thr Leu lie Lys Thr
  • the obesity proteins described herein may also be produced either by recombinant DNA technology or well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution methods. Recombinant methods are preferred if a high yield is desired.
  • the basic steps in the recombinant production of protein include: a) construction of a synthetic or semi-synthetic (or isolation from natural sources) DNA encoding the obesity protein, b) integrating the coding sequence into an expression vector in a manner suitable for the expression of the protein either alone or as a fusion protein, c) transforming an appropriate eukaryotic or prokaryotic host cell with the expression vector, and d) recovering and purifying the recombinantly produced protein.
  • Synthetic genes the in vitro or in vivo transcription and translation of which will result in the production of the protein may be constructed by techniques well known in the art. Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of DNA sequences may be constructed which encode the desired proteins. In the preferred practice of -li ⁇ the invention, synthesis i ⁇ achieved by recombinant DNA technology.
  • the gene encoding the obesity protein may also be created by using polymerase chain reaction (PCR) .
  • the template can be a cDNA library (commercially available from CLONETECH or STRATAGENE) or mRNA isolated from the desired arrival adipose tissue.
  • PCR polymerase chain reaction
  • Such methodologies are well known in the art Maniatis, ei al- Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989) .
  • the constructed or isolated DNA sequences are useful for expressing the obesity protein either by direct expression or as fusion protein. When the sequences are used in a fusion gene, the resulting product will require enzymatic or chemical cleavage.
  • a variety of peptidases which cleave a polypeptide at specific sites or digest the peptides from the amino or carboxy termini (e.g. diaminopeptidase) of the peptide chain are known. Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave a polypeptide chain at specific sites. The skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi-synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See U.S. Patent No. 5,126,249; Carter P., Site Specific Proteolysis of Fusion Proteins, Ch. 13 in Protein Purification: From Molecular Mechanisms to Lar ⁇ e Scale Processes. American Chemical Soc, Washington, D.C. (1990) .
  • Plasmids containing the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or DNA fragments are cleaved, tailored, and religated in the form desired to form the plasmids required.
  • a synthetic coding sequence may be designed to possess restriction endonuciease cleavage sites at either end of the transcript to facilitate isolation from and integration into these expression and amplification and expression plasmids.
  • the isolated cDNA coding sequence may be readily modified by the use of synthetic linkers to facilitate the incorporation of this sequence into the desired cloning vectors by techniques well known in the art.
  • the particular endonucleases employed will be dictated by the restriction endonuciease cleavage pattern of the parent expression vector to be employed.
  • the restriction sites are chosen so as to properly orient the coding sequence with control sequences to achieve proper in-frame reading and expression of the protein.
  • plasmid vectors containing promoters and control sequences which are derived from species compatible with the host cell are used with these hosts.
  • the vector ordinarily carries a replication origin as well as marker sequences which are capable of providing phenotypic selection in transformed cells.
  • E__. coli is typically transformed using pBR322, a plasmid derived from an E. coli species (Bolivar, e_£. al. , Gene 2 : 95 (1977)).
  • Plasmid pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells.
  • the pBR322 plasmid, or other microbial plasmid must also contain or be modified to contain promoters and other control elements commonly used in recombinant DNA technology.
  • the desired coding sequence is inserted into an expression vector in the proper orientation to be transcribed from a promoter and ribosome binding site, both of which should be functional in the host cell in which the protein is to be expressed.
  • An example of such an expression vector is a plasmid described in Belagaje et al., U.S. patent No. 5,304,493, the teachings of which are herein incorporated by reference.
  • the gene encoding A-C-B proinsulin described in U.S. patent No. 5,304,493 can be removed from the plasmid pRBl82 with restriction enzymes Ndel and BamHl. The isolated
  • DNA sequences can be inserted into the plasmid backbone on a Ndel/BamHI restriction fragment cassette.
  • procaryotes are used for cloning of DNA sequences in constructing the vectors useful in the invention.
  • E_ ⁇ coli K12 strain 294 ATCC No.
  • Promoters suitable for use with prokaryotic hosts include the ⁇ -lactamase (vector pGX2907
  • ATCC 39344 contains the replicon and ⁇ -lactamase gene) and lactose promoter systems (Chang e_L al.. , Nature, 275 : 615 (1978); and Goeddel ££ al. * Nature 281:544 (1979)), alkaline phosphatase, the tryptophan (trp) promoter system (vector pATHl [ATCC 37695] is designed to facilitate expression of an open reading frame as a trpE fusion protein under control of the trp promoter) and hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC-37282) .
  • the DNA molecules may also be recombinantly produced in eukaryotic expression systems.
  • Preferred promoters controlling transcription in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. ⁇ -actin promoter.
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication. Fiers, e_£. al- • Nature. 273 : 113 (1978) .
  • the entire SV40 genome may be obtained from plasmid pBRSV, ATCC 45019.
  • the immediate early promoter of the human cytomegalovirus may be obtained from plasmid pCMBb (ATCC 77177) .
  • promoters from the host cell or related species also are useful herein. Transcription of the DNA by higher eucaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10- 300 bp, that act on a promoter to increase its transcription. Enhancers are relatively oriented and positioned independently and have been found 5' (Laimins, L. eJ al..
  • an enhancer from a eukaryotic cell virus examples include the SV40 late enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • Expression vectors used in eukaryotic host cells will also contain sequences necessary for the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding protein. The 3' untranslated regions also include transcription termination sites.
  • Expression vectors may contain a selection gene, also termed a selectable marker.
  • selectable markers for mammalian cells are dihydrofolate reductase (DHFR, which may be derived from the B ⁇ lll/Hindlll restriction fragment of pJOD-10 [ATCC 68815] ) , thymidine kinase (herpes simplex virus thymidine kinase is contained on the BamHl fragment of vP-5 clone [ATCC 2028] ) or neomycin (G418) resistance genes (obtainable from pNN414 yeast artificial chromosome vector [ATCC 37682]) .
  • DHFR dihydrofolate reductase
  • thymidine kinase herepes simplex virus thymidine kinase is contained on the BamHl fragment of vP-5 clone [ATCC 2028]
  • neomycin (G418) resistance genes obtainable from
  • the transfected mammalian host cell can survive if placed under selective pressure.
  • selectable markers are successfully transferred into a mammalian host cell
  • the first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow without a supplemented media.
  • Two examples are: CHO DHFR " cells (ATCC CRL-9096) and mouse LTK ⁇ cells (L-M(TK-) ATCC CCL-2.3) .
  • These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media.
  • An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells which were not transformed with the DHFR or TK gene will not be capable of survival in nonsupplemented media.
  • the second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell.
  • Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection.
  • Examples of such dominant selection use the drugs neomycin. Southern P. and Berg, P., J. Molec. APPI. Genet. : 327 (1982), mycophenolic acid, Mulligan, R. C. and Berg, P. Science 2IL£:1422 (1980), or hygromycin, Sugden, B. e_£. al. , Mol Cell. Biol. 5:410-413 (1985) .
  • the three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin) , xgpt (mycophenolic acid) or hygromycin, respectively.
  • a preferred vector for eucaryotic expression is pRc/CMV.
  • pRc/CMV is commercially available from Invitrogen Corporation, 3985 Sorrento Valley Blvd., San Diego, CA 92121.
  • the ligation mixtures are used to transform £. coli K12 strain DH10B (ATCC 31446) and successful transformants selected by antibiotic resistance where appropriate.
  • Plasmids from the transformants are prepared, analyzed by restriction and/or sequence by the method of Messing, e_t al. , Nucleic Acids Res. 1:309 (1981) .
  • Host cells may be transformed with the expression vectors of this invention and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the techniques of transforming cells with the aforementioned vectors are well known in the art and may be found in such general references as Maniatis, e_t al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), or Current Protocols in Molecular Biology (1989) and supplements.
  • Preferred suitable host cells for expressing the vectors encoding the claimed proteins in higher eucaryotes include: African green monkey kidney line cell line transformed by SV40 (COS-7, ATCC CRL-1651); transformed human primary embryonal kidney cell line 293, (Graham, F. L. e£ al. , J. Gen Virol. 36:59-72 (1977), Virology 77:319-329. Virology ££ • •10-21); baby hamster kidney cells (BHK-21 (C-13 ) , ATCC CCL- 10, Virology 1£:147 (1962)); Chinese hamster ovary cells CHO-
  • DHFR human cervical epitheloid carcinoma cells
  • canine kidney cells MDCK, ATCC CCL-34
  • buffalo rat liver cells BBL 3A, ATCC CRL-1442
  • human diploid lung cells WI-38, ATCC CCL- 75
  • human hepatocellular carcinoma cells Hep G2, ATCC HB-
  • yeast cultures may also be used. Saccharomyces cerevisiae. or common baker's yeast is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available.
  • Saccharomyces cerevisiae. or common baker's yeast is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available.
  • the plasmid YRp7 for example, (ATCC-40053, Stinchcomb, et al. , Nature 282:39 (1979); Kingsman e£ al- , Gene 7:141 (1979); Tschemper e_t aL. , Gene 1£:157 (1980)) is commonly used.
  • This plasmid already contains the trp gene which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC no. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)).
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (found on plasmid pAPl2BD ATCC 53231 and described in U.S. Patent No. 4,935,350, June 19, 1990) or other glycolytic enzymes such as enolase (found on plasmid pACl ATCC 39532), glyceraldehyde-3-phosphate dehydrogenase (derived from plasmid pHcGAPCl ATCC 57090, 57091), zymomonas mobilis (United States Patent No.
  • yeast promoters which contain inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein (contained on plasmid vector pCL28XhoLHBPV ATCC 39475, United States Patent No. 4,840,896) , glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose (GALl found on plasmid PRY121 ATCC 37658) utilization. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman e_t al.
  • yeast enhancers such as the UAS Gal from Saccharomyces cerevisiae (found in conjunction with the CYCl promoter on plasmid YEpsec--hIlbeta ATCC 67024), also are advantageously used with yeast promoters.
  • UAS Gal from Saccharomyces cerevisiae
  • YEpsec--hIlbeta ATCC 67024 also are advantageously used with yeast promoters.
  • RNA Porcine OB Gene and Gene Product Total RNA was isolated from porcine fat tissue obtained from Pel-Freez®, Pel-Freez Inc., and the cDNA was cloned in accordance with the techniques described in Hsiung et al., Neurooe tide 25: 1-10 (1994). Primers were designed based on the published amino acid sequence of the human ob gene. The primers were prepared for use in polymerase chain reaction (PCR) amplification methods using a Model 380A DNA synthesizers (PE-Applied Biosystems, Inc., 850 Lincoln Center Drive,
  • PCROB-1 (ATG CAT TGG GGA MCC CTG TG)
  • PCROB-2 (12505)
  • GG ATT CTT GTG GCT TTG GYC CTA TCT GG ATT CTT GTG GCT TTG GYC CTA TCT
  • PCROB-3 (12506)
  • TCA GCA CCC AGG GCT GAG GTC CA PCROB-4
  • CAT GTC CTG CAG AGA CCC CTG CAG CCT GCT CA were prepared.
  • RNA 1 ⁇ L (1 ⁇ g/ ⁇ l) isolated from porcine adipose tissue and 1 ⁇ L Perkin Elmer Random primers (50 ⁇ M) in a total volume of 12 ⁇ L were annealed for 10 minutes at 70°C and then cooled on ice. The following were then added to the annealed mixture: 4 ⁇ L of
  • BRL 5x H-reverse transcriptase (RT) reaction buffer (Gibco- BRL CAT#28025-013) , 2 ⁇ L of 0.1 M DTT, 1 ⁇ L of 10 mM dNTPs.
  • This annealed mixture was then incubated at 37°C for 2 minutes before adding 1 ⁇ L BRL M-MLV-reverse transcriptase (200 U/ ⁇ D (CAT#28025-013) and incubated at 37°C for additional 1 hour. After incubation the mixture was heated at 95°C for 5 minutes and then was cooled on ice.
  • PCR polymerase chain reaction
  • a reaction mixture 100 ⁇ L containing the above cDNA reaction mixture (1 ⁇ L) , 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer Corporation) 10 ⁇ l of lOx PCR reaction buffer (Perkin-Elmer Corporation) and 50 pmol each of the sense (PCROB-1) and antisense (PCROB-3) primers for porcine OB amplification.
  • the condition for PCR was 95°C for 1 minute, 57°C for 1 minute and 72 _ °C for 1 minute for 30 cycles using a PCR DNA Thermal Cycler (Perkin- Elmer Corporation) .
  • the 500 bp frag ent ( ⁇ 0.5 mg) was purified by agarose gel electrophoresis and isolated by the freeze-squeeze method.
  • the 500 bp fragment (_0.2 ⁇ g) was then ligated into Smal linearized pUCl ⁇ plasmid ( ⁇ 1 ⁇ g) and the ligation mixture was used to transform DH5 ⁇ (BRL) competent cells.
  • the transformation mixture was plated on 0.02% X-Gal TY broth plates containing ampicillin (Amp) (100 ⁇ g/mL) and was then incubated overnight at 37°C.
  • the DNA sequence of the bovine OB gene was obtained by techniques analogous to Example 1, except sense (PCROB-2) and antisense (PCROB-3) primers were used for bovine OB cDNA amplification.
  • a plasmid containing the DNA sequence encoding the obesity protein is constructed to include Ndel and BamHI restriction sites.
  • the plasmid carrying the cloned PCR product is digested with Ndel and BamHI restriction enzymes.
  • the small ⁇ 450bp fragment is gel-purified and ligated into the vector pRB182 from which the coding sequence for A-C-B proinsulin is deleted.
  • the ligation products are transformed into E. coli DHlOB (commercially available from GIBCO-BRL) and colonies growing on tryptone-yeast (DIFCO) plates supplemented with 10 mg/mL of tetracycline are analyzed. Plasmid DNA is isolated, digested with Ndel and BamHI and the resulting fragments are separated by agarose gel electrophoresis. Plasmids containing the expected ⁇ 450bp Ndel to BamHI fragment are kept. E. coli K12 RV308 (available from the NRRL under deposit number B-15624) are transformed with this second plasmid, resulting in a culture suitable for expressing the protein.
  • E,. coli K12 RV308 cells are employed as host cells but numerous other cell lines are available such as, but not limited to, £. coli K12 L201, L687 , L693 , L507 , L640, L641, L695, L814 (E. coli B) .
  • the transformed host cells are then plated on appropriate media under the selective pressure of the antibiotic corresponding to the resistance gene present on the expression plasmid.
  • the cultures are then incubated for a time and temperature appropriate to the host cell line employed.
  • Proteins that are expressed in high-level bacterial expression systems characteristically aggregate in granules or inclusion bodies which contain high levels of the overexpressed protein. Kreuger et al. , in Protein Folding. Gierasch and King, eds., pgs 136-142 (1990), American Association for the Advancement of Science Publication No. 89-18S, Washington, D.C. Such protein aggregates must be solubilized to provide further purification and isolation of the desired protein product. JLd,. A variety of techniques using strongly denaturing solutions such as guanidinium-HCl and/or weakly denaturing solutions such as dithiothreitol (DTT) are used to solubilize the proteins. Gradual removal of the denaturing agents (often by dialysis) in a solution allows the denatured protein to assume its native conformation. The particular conditions for denaturation and folding are determined by the particular protein expression system and/or the protein in question.
  • the DNA sequences are expressed with a dipeptide leader sequence encoding Met-Arg or Met-Tyr as described in U.S. Patent No. 5,126,249, herein incorporated by reference.
  • This approach facilitates the efficient expression of proteins and enables rapid conversion to the active protein form with Cathepsin C or other dipeptidylpeptidases.
  • the purification of proteins is by techniques known in the art and includes reverse phase chromatography, affinity chromatography, and size exclusion.
  • the following examples are provided merely to further illustrate the preparation of the formulations of the invention. The scope of the invention is not construed as merely consisting of the following examples.
  • human obesity protein SEQ ID No: 1
  • 20 mg of human obesity protein SEQ ID No: 1
  • An aqueous solution containing 100 mg/ml of zinc in water was prepared from zinc chloride and serial dilutions were made to prepare 10 mg/ml zinc and 1 mg/ml zinc solutions.
  • Five 6-ml aliquots of the human obesity protein solution were modified as shown in Table I: Table J ⁇ l of ⁇ l of ⁇ l of ⁇ l of Total mg/ml
  • Example 2 50 ⁇ l doses of all five Samples in Example 1, each containing 30 ⁇ g of human obesity protein, were injected subcutaneously into ob/ob mice in a single injection per day for four days.
  • a 50 ⁇ l dose of a 16 mg/ml glycerin-2 mg/ml phenol solution adjusted to pH 7.4 was administered subcutaneously as a vehicle control.
  • the average daily food intake and cumulative change in body weight from time zero for the ob/ob mice are shown in Table III.
  • the compounds of the present invention are potent and effective agents useful for treating obesity.
  • the potency of the protein was significantly enhanced.
  • Increasing the total zinc concentration of the formulation also provides enhanced potency and improved biological effects in ob/ob mice.
  • the present invention provides compounds, which comprise an obesity protein complexed with a divalent metal cation, pharmaceutical formulations thereof, and methods of using such compounds for treating obesity, and disorders associated with obesity such as diabetes (particularly non-insulin dependent diabetes mellitus) , cardiovascular disease and cancer.

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Abstract

La présente invention concerne de nouveaux composés comprenant une protéine de l'obésité sous forme de complexe avec un cation métallique divalent, et des compositions pharmaceutiques contenant ces composés. L'invention concerne également des procédés faisant appel à ces composés pour traiter l'obésité et des maladies associées à l'obésité telles que le diabète, les maladies cardiovasculaires et le cancer.
EP97903961A 1996-02-06 1997-01-24 Compose contenant la proteine de l'obesite et leurs formulations Withdrawn EP0889737A1 (fr)

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US1123896P 1996-02-06 1996-02-06
US11238P 1996-06-26
PCT/US1997/001188 WO1997028824A1 (fr) 1996-02-06 1997-01-24 Compose contenant la proteine de l'obesite et leurs formulations

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US6001968A (en) * 1994-08-17 1999-12-14 The Rockefeller University OB polypeptides, modified forms and compositions
US6656508B2 (en) 1997-04-17 2003-12-02 Amgen Inc. Sustained-release alginate gels

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US5569603A (en) * 1994-03-08 1996-10-29 Heska Corporation Dirofilaria immitis GP29 proteins, nucleic acid molecules and uses thereof
US5827734A (en) * 1995-01-20 1998-10-27 University Of Washington Materials and methods for determining ob protein in a biological sample
EP0725079A1 (fr) * 1995-01-31 1996-08-07 Eli Lilly And Company Protéine contre obesité

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