[go: up one dir, main page]

WO1998031390A1 - Composition a base de proteines de l'obesite - Google Patents

Composition a base de proteines de l'obesite Download PDF

Info

Publication number
WO1998031390A1
WO1998031390A1 PCT/US1998/000816 US9800816W WO9831390A1 WO 1998031390 A1 WO1998031390 A1 WO 1998031390A1 US 9800816 W US9800816 W US 9800816W WO 9831390 A1 WO9831390 A1 WO 9831390A1
Authority
WO
WIPO (PCT)
Prior art keywords
leu
gin
replaced
ser
asp
Prior art date
Application number
PCT/US1998/000816
Other languages
English (en)
Inventor
Joseph V. Rinella, Jr.
Original Assignee
Eli Lilly And Company
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Eli Lilly And Company filed Critical Eli Lilly And Company
Priority to CA002277282A priority Critical patent/CA2277282A1/fr
Priority to EP98902573A priority patent/EP0983091A4/fr
Priority to JP53454298A priority patent/JP2001511180A/ja
Priority to AU59199/98A priority patent/AU5919998A/en
Publication of WO1998031390A1 publication Critical patent/WO1998031390A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2264—Obesity-gene products, e.g. leptin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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

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 formulations of an obesity protein analog.
  • the ob I 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, Zhang and co-workers published the positional cloning of the mouse gene linked with this condition [Zhang, Y., et al . Nature 372:425-32 (1994)]. This report disclosed the murine and human protein expressed in adipose tissue. Likewise,
  • Murakami, T., et al. reported the cloning and expression of the rat obese gene [Biochemical and Biophysical Research Communications 209 (3) : 944-52 (1995)].
  • the protein, which is encoded by the oJ gene has demonstrated an ability to effectively regulate adiposity in mice [Pelleymounter, et al . , Science 269:540-543 (1995)].
  • parenteral formulations containing insoluble protein cause problems relating to inconsistency in the dose-response, as well as unpredictability.
  • the unpredictability is believed to be due to greater variability in the pharmacokinetics in suspension formulations.
  • Insoluble formulations must first dissolve prior to adsorption. It is hypothesized that this step has significant variability in a subcutaneous depot. Aggregation makes the preparation of a soluble, pharmaceutically-acceptable parenteral formulation exceedingly difficult.
  • native obesity proteins that is, those having sequences naturally-occurring in mammals, tend to aggregate under conditions typically preferred for protein formulations.
  • Certain analogs of native obesity proteins demonstrate activity, a reduced tendency to aggregate, and a significant improvement in physical stability in solution compared with native obesity proteins. Such analogs are disclosed in Basinski, M. B., et al . , European Patent Publication No. 725,078 (7 August 1996) and DiMarchi, R. D., et al . , European Patent Publication No. 725,079 (7 August 1996).
  • analogs of native obesity proteins retain some tendency to aggregate in solution, a tendency that is aggravated by certain commonly-used, pharmaceutically-acceptable preservatives, such as, phenol, cresol, and alkylparabens . Because it is envisioned that a pharmaceutical product of obesity protein analog for treating obesity will be a multi-use product, preservatives will be required to inhibit microbial growth. Therefore, a solution to the aggregation problem is urgently needed in order to produce a multi-use obesity protein formulation that remains free of protein aggregation.
  • a slightly acidic pH is preferred for solution formulations of proteins.
  • a slightly acidic pH is preferred for solution formulations of proteins.
  • a suggestion to formulate a protein at slightly alkaline pH, such as pH greater than 8.0, is commonly met with skepticism or concern about the chemical stability of the protein.
  • the data in Table 1 predict relatively little change in the net charge of these obesity protein analogs in the pH range of 8.0 to 9.0 compared with lower and higher pH levels .
  • the predicted net charge does not change abruptly at about pH 8.0
  • the physical stability of formulations of obesity protein analogs does change abruptly above about pH 8.0.
  • Such a lack of correspondence between the net charge and aggregation of the obesity protein molecules in the presence of phenolic preservatives is most unexpected.
  • the mechanism of such a striking increase in physical stability above pH 8.0 is unknown, but is not believed to be due to subtle changes in the conformation of the protein caused by the change in pH, such as occurs in serum albumin at acidic pH.
  • the present invention provides conditions that increase the physical stability of obesity protein analogs in the presence of preservatives, and makes possible a commercially-viable, multi-use pharmaceutical product comprising obesity protein analog to treat obesity.
  • the obesity protein analog remains soluble at much higher protein concentrations when formulated with certain preservatives, and there are less constraints on the other variables of the formulation, such as, ionic strength, temperature, etc., than at pH lower than pH 8.0.
  • This invention provides a soluble formulation comprising an obesity protein analog and a preservative, said formulation having pH greater than 8.0.
  • the invention further provides a process for preparing a soluble formulation, which comprises mixing an obesity protein analog and a preservative to produce a soluble formulation comprising said obesity protein analog and said preservative, said soluble formulation having pH greater than 8.0.
  • the invention provides a method of treating obesity in a mammal in need thereof, which comprises administering to said mammal a soluble formulation comprising an obesity protein analog, said soluble formulation having a pH higher than pH 8.0.
  • Administration -- may be via any route known to be effective by the physician of ordinary skill.
  • Parenteral administration is commonly understood in the medical literature as the injection of a dosage form into the body by a sterile syringe or some other mechanical device such as an infusion pump.
  • peripheral parenteral routes of administration include, without limitation, intravenous, intramuscular, subcutaneous, and intraperitoneal routes of administration.
  • Other routes known to the physician include occular, nasal, buccal, and pulmonary routes of administration.
  • Alkylparaben -- refers to a Ci to C alkyl paraben.
  • alkylparaben is methylparaben, ethylparaben, propylparaben, or butylparaben.
  • 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.
  • Cresol - refers to meta-cresol, ortho-cresol, para-cresol, chloro-cresol, or mixtures thereof.
  • Isotonicity agent refers to an agent that is tolerated physiologically and imparts 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, mannitol, and lactose.
  • Obesity protein analog refers to a protein having a biological activity similar to that of human obesity protein, and also having an amino acid sequence essentially the same as that of the human obesity protein, except for having one or more modifications in the amino acid sequence. Modifications in the amino acid sequence may consist of additions of amino acids, deletions of amino acids, and replacement of an amino acid or amino acids with one or more amino acids .
  • Obesity protein analog includes proteins having a leader sequence at the N-terminus the 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-Rl- wherein Rl is absent or any amino acid except Pro.
  • compositions may be buffered with a pharmaceutically acceptable buffer, such as TRIS or a basic amino acid, such as, lysine or arginine .
  • a pharmaceutically acceptable buffer such as TRIS or a basic amino acid, such as, lysine or arginine .
  • Other pharmaceutically acceptable buffers for buffering at pH above 8.0 are known in the art.
  • the selection and concentration of buffer is known in the art .
  • pH -- has its usual and well-known meaning.
  • Plasmid an extrachromosomal self-replicating genetic element .
  • a parenteral formulation must meet guidelines for preservative effectiveness to be a commercially viable multi-use product.
  • preservatives known in the art as being effective and acceptable in parenteral formulations are benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercurie nitrate, thimerosal and various mixtures thereof .
  • Reading frame the nucleotide sequence from which translation occurs is "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 poiypeptide must be aligned in multiples of three from the initiation codon, i.e., the correct "reading frame" must be maintained. In the creation of fusion proteins containing a chelating peptide, the reading frame of the DNA sequence encoding the structural protein must be maintained in the DNA sequence encoding the chelating peptide.
  • 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 .
  • Soluble refers to the relative absence of aggregated protein that is visually perceivable.
  • the degree of aggregation of formulations of proteins may be inferred by measuring the turbidity of the formulation. The greater the turbidity of the formulation, the greater the extent of aggregation of the protein in the formulation. Turbidity is commonly determined by nephelometry, and measured in Nephalometric Turbidity Units (NTU) .
  • NTU Nephalometric Turbidity Units
  • 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 formulation of the 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.
  • 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.
  • nucleotide and amino acid abbreviations 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 configuration.
  • Turbidity of the formulations was measured by nephelometry using a Hach Ratio Turbidimeter (Model 18900, or 2100N for values above 200) calibrated with Hach Formazin Turbidity Standard, 4000 NTU (Hach 2461-11, or equivalent) . Values are Nephelometric Turbidity Units (NTU) which are sample reading minus diluent blank reading .
  • NTU Nephelometric Turbidity Units
  • Formulations of the obesity protein analog of SEQ ID NO: 6 (10 mg/mL) containing m-cresol (0.3%) and TRIS buffer (5 mM) having pH ranging from 7.8 to 9.0 were prepared according to Example 2 herein. Three samples of each formulation were incubated for 14 days at each of three temperatures, 5, 25, and 37°C. Turbidity of the formulations was measured by nephelometry using a Hach Ratio Turbidimeter (Model 18900) calibrated with Hach Formazin Turbidity Standard, 4000 NTU (Hach 2461-11, or equivalent) . Results are presented in Table 3, below.
  • each formulation contained 15 mg/ml protein and either 10 mM phosphate buffer (pH 7.8) or 5 mM arginine buffer (pH 8.5).
  • Turbidity of the formulations was measured by nephelometry using a Hach Ratio Turbidimeter (Model 18900, or 2100N for values above 200) calibrated with Hach Formazin Turbidity Standard, 4000 NTU (Hach 2461-11, or equivalent) . Values are Nephelometric Turbidity Units (NTU) which are sample reading minus diluent blank reading.
  • NTU Nephelometric Turbidity Units
  • Parenteral formulations of the present invention can be prepared using conventional dissolution and mixing procedures.
  • a suitable formulation for example, a measured amount of obesity protein analog in water is combined with the desired preservative in water in quantities sufficient to provide the protein and preservative at the desired concentration.
  • the pH of the formulation may be adjusted to greater than 8.0 either before or after combining the obesity protein analog and the preservative.
  • the formulation is generally sterile filtered prior to administration. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, the order in which pH is adjusted, the surfactant used, if any, the temperature and ionic strength at which the formulation is prepared, may be optimized for the concentration and means of administration used.
  • the most effective preservatives phenol and cresol, or mixtures thereof, cause protein aggregation when formulated with obesity protein analog.
  • the concentration of preservative is that required to maintain preservative effectiveness.
  • the relative amounts of preservative necessary to maintain preservative effectiveness varies with the preservative used. Generally, the amount of preservative necessary is known in the art [ allhauser, K. , Develop . Biol . Standard. 24:9-28 (S . Krager, Basel, 1974)].
  • the optimal concentration of the preservative depends on the preservative, its solubility, and the pH of the formulation.
  • additives such as a pharmaceutically acceptable solubilizers 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) , and PEG (polyethylene glycol) may optionally be added to the formulation to reduce aggregation.
  • a pharmaceutically acceptable solubilizers 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) , and PEG (polyethylene glycol) may optionally be added to the formulation to reduce aggregati
  • the invention provides a soluble formulation comprising an obesity protein analog, said soluble formulation having a pH greater than pH 8.0.
  • the pH is between about 8.0 and about 8.6, and more preferably between about 8.3 and about 8.6.
  • Other preferred pH ranges for the formulations of the present invention are between about pH 8.2 and about pH 9.0, between about pH 8.2 and about pH 8.8, between about pH 8.2 and about pH 8.6, between about pH 8.3 and about pH 9.0, between about pH 8.3 and about pH 8.8, between about pH 8.4 and about pH 9.0, between about pH 8.4 and about pH 8.8, between about pH 8.4 and about pH 8.6, between about pH 8.5 and about pH 9.0, between about pH 8.5 and about pH 8.8, and between about pH 8.6 and about pH 9.0.
  • pH above 8.0 obesity protein analogs remain in solution in the presence of certain preservatives, making possible a multi-use parenteral formulation containing those preservatives that is relatively free of protein aggregation.
  • the solubility of the obesity protein analogs in the present formulations is such that the turbidity of the formulation is lower than 50 NTU. More preferably, the turbidity is lower than 20 NTU. Most preferably, the turbidity is lower than 10 NTU.
  • Peripheral, parenteral administration is preferred.
  • the formulations prepared in accordance with the present invention may be administered using a syringe, injector, pump, or any other device recognized in the art for parenteral administration.
  • the amount of a formulation of the present invention that would be administered to treat obesity will depend on a number of factors, among which are included, without limitation, the patient's sex, weight and age, the underlying causes of obesity, the route of administration and bioavailability, the persistence of administered obesity protein analog in the body, the formulation, and the potency of the obesity protein analog. Where administration is intermittent, the amount per administration should also take into account the interval between doses, and the bioavailability of the obesity protein analog from the formulation. Administration of the formulation of the present invention could be continuous.
  • Preferred obesity protein analogs employed in the formulations of the present invention are those of the Formula (I) :
  • Val Pro lie Gin Lys Val Gin Asp Asp Thr Lys Thr Leu lie Lys Thr 20 25 30 lie Val Thr Arg lie Asn Asp lie Ser His Thr Xaa Ser Val Ser Ser Ser
  • Xaa at position 28 is Gin or absent; and said protein having at least one of the following substitutions : Gin at position 4 is replaced with Glu;
  • Gin at position 7 is replaced with Glu
  • Thr at position 27 is replaced with Ala
  • Gin at position 63 is replaced with Glu;
  • Met at position 68 is replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly;
  • Asn at position 72 is replaced with Gin, Glu, or Asp;
  • Gin at position 75 is replaced with Glu; Ser at position 77 is replaced with Ala; Asn at position 78 is replaced with Gin or Asp; Asn at position 82 is replaced with Gin or Asp; His at position 97 is replaced with Gin, Asn, Ala,
  • Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu; Ala at position 101 is replaced with Ser, Asn,
  • Gin at position 134 is replaced with Glu; Met at position 136 is replaced with methionine sulfoxide, Leu, lie, Val, Ala, or Gly;
  • Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu; or Gin at position 139 is replaced with Glu; or a pharmaceutically acceptable salt thereof .
  • a further group of preferred embodiments are formulations of proteins of Formula I, wherein: Gin at position 4 is replaced with Glu;
  • Gin at position 7 is replaced with Glu
  • Thr at position 27 is replaced with Ala
  • Met at position 54 is replaced with methionine sulfoxide, Leu, or Ala;
  • Gin at position 56 is replaced with Glu
  • Met at position 68 is replaced with methionine sulfoxide, or Leu;
  • Gin at position 75 is replaced with Glu; Asn at position 78 is replaced with Gin or Asp;
  • Trp at position 100 is replaced with Ala or Asp;
  • Gin at position 130 is replaced with Glu
  • Gin at position 134 is replaced with Glu; Met at position 136 is replaced with methionine sulfoxide, Leu, lie; or
  • Gin at position 139 is replaced with Glu.
  • Thr at position 27 is replaced with Ala
  • Met at position 54 is replaced with methionine sulfoxide, Leu, or Ala; Met at position 68 is replaced with methionine sulfoxide, or Leu;
  • Thr at position 27 is replaced with Ala; Met at position 54 is replaced with Leu, or Ala;
  • Asn at position 82 is replaced with Gin or Asp; or Met at position 136 is replaced with Leu, or lie.
  • Preferred species employed in the formulations of the present invention are those of SEQ ID NO: 2 and SEQ ID NO: 3:
  • preferred embodiments are formulations comprising obesity protein analogs of the Formula I, wherein:
  • Xaa at position 28 is Gin or absent; and said protein having at least one substitution selected from the group consisting of :
  • His at position 97 is replaced with Gin, Asn, Ala, Gly, Ser, or Pro;
  • Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, lie, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu;
  • Ala at position 101 is replaced with Ser, Asn, Gly, His, Pro, Thr, or Val;
  • Ser at position 102 is replaced with Arg; Gly at position 103 is replaced with Ala;
  • Glu at position 105 is replaced with Gin
  • Thr at position 106 is replaced with Lys or Ser;
  • Leu at position 107 is replaced with Pro; Asp at position 108 is replaced with Glu;
  • Gly at position 111 is replaced with Asp;
  • Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, He, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu; or a pharmaceutically acceptable salt thereof .
  • compositions comprising obesity protein analogs of the Formula I, wherein Xaa at position 28 is Gin, and said protein has at least one substitution selected from the group consisting of:
  • His at position 97 is replaced with Gin, Asn, Ala, Gly, Ser, or Pro;
  • Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, He, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu;
  • Ala at position 101 is replaced with Ser, Asn, Gly, His, Pro, Thr, or Val;
  • Gly at position 103 is replaced with Ala; Glu at position 105 is replaced with Gin;
  • Thr at position 106 is replaced with Lys or Ser;
  • Gly at position 111 is replaced with Asp; or Trp at position 138 is replaced with Ala, Glu,
  • More preferred embodiments are formulations comprising obesity protein analogs of the Formula I, wherein:
  • Xaa at position 28 is Gin
  • Trp at position 100 is replaced with Ala, Glu,
  • Glu at position 105 is replaced with Gin
  • Thr at position 106 is replaced with Lys or Ser; Leu at position 107 is replaced with Pro;
  • Gly at position 111 is replaced with Asp;
  • Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, He, Phe, Tyr, Ser, Thr, Gly, Gin, Val or Leu.
  • compositions comprising obesity protein analogs of the Formula I, wherein:
  • Xaa at position 28 is Gin; and His at position 97 is replaced with Ser or Pro;
  • Trp at position 100 is replaced with Ala, Gly, Gin, Val, He, or Leu;
  • Ala at position 101 is replaced with Thr;
  • Trp at position 138 is replaced with Ala, He, Gly, Gin, Val or Leu.
  • Additional preferred embodiments are formulations comprising obesity protein analogs of the Formula I, wherein:
  • Xaa at position 28 is Gin; and His at position 97 is replaced with Ser or Pro;
  • Trp at position 100 is replaced with Ala, Gin or Leu;
  • Ala at position 101 is replaced with Thr;
  • Trp at position 138 is replaced with Gin.
  • Another group of preferred proteins for the formulations of the present invention is described by Formula II:
  • Xaa at position 28 is Gin or absent ;
  • Xaa at position 73 is Val or Met ; said protein having at least one of the following substitutions:
  • Trp at position 100 is replaced with Glu, Asp, His, Lys, or Arg;
  • Trp at position 138 is replaced with Glu, Asp, His, Lys, or Arg; or a pharmaceutically acceptable salt thereof.
  • Xaa at position 22 is Asn
  • Xaa at position 28 is Gin
  • Xaa at position 72 in Asn or Asp
  • Xaa at position 73 is Val.
  • Trp at position 100 is replaced with Glu or Asp; or Trp at position 138 is replaced with Glu or Asp.
  • Particularly preferred are proteins of Formula II wherein Xaa at position 72 is Asp.
  • Additional preferred proteins are those of Formula II wherein Trp at position 100 is replaced with His, Lys, or Arg.
  • Other preferred proteins of the Formula II for the present formulations are those wherein Trp at position 100 is replaced with Lys or Arg; or Trp at position 138 is replaced with Lys or Arg.
  • Another group of preferred embodiments consists of formulations comprising an obesity protein analog of the Formula (III) :
  • Xaa at position 1 is Val or absent ;
  • Xaa at position 2 is Pro or absent ;
  • Xaa at position 22 is Asn or Ser ;
  • Xaa at position 28 is Gin or absent ;
  • Xaa at position 72 is Asn, Gin, Glu or Asp ;
  • Xaa at position 73 is Val or Met ;
  • Xaa at position 100 is Trp , Gin , Glu , Asp , Ser ,
  • Xaa at position 138 is Trp , Gin , Glu , Asp , Ser , Thr , Lys , His , or Arg ; said protein having at least one of the following substitutions :
  • Xaa at position 1 is replaced with Glu, Asp, Ser, Thr, Lys, His, or Arg;
  • Xaa at position 2 is replaced with Glu, Asp, Ser,
  • He at position 3 is replaced with Glu, Asp, Arg, Lys, or His;
  • Val at position 30 is replaced with Glu, Asp, Arg, Lys, or His;
  • Val at position 36 is replaced with Glu, Asp, Arg, Lys, or His;
  • Phe at position 41 is replaced with Glu, Asp, Arg, Lys, or His; He at position 42 is replaced with Glu, Asp, Arg,
  • Pro at position 43 is replaced with Glu, Asp, Arg, Lys, or His;
  • Leu at position 45 is replaced with Glu, Asp, Arg, Lys, or His;
  • His at position 46 is replaced with Glu, Asp, Arg, or Lys;
  • Pro at position 47 is replaced with Glu, Asp, Arg, Lys, or His; He at position 48 is replaced with Glu, Asp, Arg,
  • Leu at position 49 is replaced with Glu, Asp, Arg, Lys, or His;
  • Thr at position 50 is replaced with Glu, Asp, Arg, Lys, or His;
  • He at position 74 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser;
  • Val at position 89 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; Phe at position 92 is replaced with Gin, Glu, Asp,
  • Pro at position 99 is replaced with Gin, Glu, Asp, Arg, Lys, His, Thr or Ser; or
  • Leu at position 142 is replaced with Glu, Asp, Arg, Lys, or His; or a pharmaceutically acceptable salt thereof .
  • Preferred embodiments of the formulations of the present invention comprise an obesity protein of SEQ ID NO: 5, wherein:
  • Phe at position 92 is replaced with Asp, Glu, Lys, Arg, or His, or pharmaceutically acceptable salts thereof.
  • Preferred embodiments of the formulations of the present invention comprise an obesity protein of SEQ ID NO: 5, wherein:
  • Phe at position 92 is replaced with Asp or Glu, or pharmaceutically acceptable salts thereof.
  • compositions of the present invention comprise an obesity protein of SEQ ID NO: 5, wherein:
  • Xaa at position 22 is Asn
  • Xaa at position 28 is Gin or absent
  • Xaa at position 72 is Asn
  • Xaa at position 73 is Val; Xaa at position 100 is Trp, Glu, Asp, Ser, Thr,
  • Xaa at position 138 is Trp, Glu, Asp, Ser, Thr, Lys, His, or Arg; said protein having at least one of the following substitutions:
  • Leu at position 45 is replaced with Glu, Asp, Arg, Lys, or His;
  • His at position 46 is replaced with Glu, Asp, Arg, or Lys; Pro at position 47 is replaced with Glu, Asp, Arg,
  • Lys, or His He at position 48 is replaced with Glu, Asp, Arg, Lys, or His; or
  • Leu at position 142 is replaced with Glu, Asp, Arg, Lys, or His; or pharmaceutically acceptable salts thereof .
  • Xaa at position 1 is Val
  • Xaa at position 2 is Pro; Xaa at position 28 is Gin or absent;
  • Xaa at position 72 is Asn
  • Xaa at position 100 is Trp, Glu, or Asp;
  • Xaa at position 138 is Trp, Glu or Asp; said protein having at least one of the following substitutions:
  • His at position 46 is replaced with Glu, Asp, Arg, or Lys;
  • He at position 48 is replaced with Glu, Asp, Arg, Lys, or His; or Phe at position 92 is replaced with Glu, Asp, Arg,
  • Xaa at position 2 is Pro
  • Xaa at position 28 is Gin or absent
  • Xaa at position 72 is Asn
  • Xaa at position 100 is Trp, Glu, or Asp
  • Xaa at position 138 is Trp, Glu or Asp
  • said protein having at least one of the following substitutions :
  • His at position 46 is replaced with Glu, Asp, Arg, or Lys ; or He at position 48 is replaced with Glu, Asp, Arg,
  • Xaa at position 1 is Val
  • Xaa at position 2 is Pro; Xaa at position 28 is Gin or absent;
  • Xaa at position 72 is Asn
  • Xaa at position 100 is Trp or Asp
  • Xaa at position 138 is Trp; said protein having at least one of the following substitutions:
  • His at position 46 is replaced with Glu, Asp, Arg, or Lys;
  • He at position 48 is replaced with Asp, Arg, Lys, or His; or Phe at position 92 is replaced with Asp, Glu, Arg,
  • proteins of SEQ ID NO: 5 wherein: Xaa at position 1 is Val;
  • Xaa at position 2 is Pro
  • Xaa at position 28 is Gin or absent
  • Xaa at position 72 is Asn
  • Xaa at position 100 is Trp or Asp; Xaa at position 138 is Trp; said protein having at least one of the following substitutions :
  • His at position 46 is replaced with Glu, Asp, Arg, or Lys ; or He at position 48 is replaced with Asp, Arg, Lys, or His; or pharmaceutically acceptable salts thereof .
  • Xaa at position 1 is Val or absent;
  • Xaa at position 2 is Pro or absent
  • Xaa at position 28 is Gin or absent;
  • Xaa at position 72 is Asn, Glu or Asp;
  • Xaa at position 100 is Trp, Glu, Asp, Ser, Thr, Lys, His, or Arg;
  • Xaa at position 138 is Trp, Glu, Asp, Ser, Thr, Lys, His, or Arg; said protein having at least one of the following substitutions :
  • Xaa at position 1 is replaced with Glu, Asp, Ser, Thr, Lys, His, Arg, or absent;
  • Xaa at position 2 is replaced with Glu, Asp, Ser,
  • Val at position 89 is replaced with Glu, Asp, Arg, Lys, or His; or
  • Phe at position 92 is replaced with Glu, Asp, Arg, Lys, or His; or a pharmaceutically acceptable salt thereof .
  • Xaa at position 28 is Gin or absent
  • Xaa at position 72 is Asn, Glu or Asp;
  • Xaa at position 100 is Glu, Asp, Lys, His, or Arg;
  • Xaa at position 138 is Trp, Glu, Asp, Ser, Thr, Lys, His, or Arg; said protein having at least one of the following substitutions :
  • Phe at position 92 is replaced with Glu, Asp, Arg, Lys, or His; Pro at position 99 is replaced with Glu, Asp, Arg,
  • the most preferred embodiments are formulations comprising obesity protein analogs having a di-sulfide bond between Cys at position 96 and Cys at position 146.
  • Examples of the most preferred embodiments include formulations comprising obesity protein analogs of SEQ ID NO: 2 , or one of the proteins described by SEQ ID NO: 6-13, said obesity protein analogs having intramolecular disulfide bonds between Cys at position 96 and Cys at position 14 6, or pharmaceutically acceptable salts thereof.
  • the concentration of obesity protein analog in the formulation is preferably about from about 0.5 mg/mL to about 100 mg/mL. More preferably, the concentration of obesity protein analog in the formulation is from about 0.5 mg/mL to about 50 mg/mL. Still more preferably, the concentration of obesity protein analog in the formulation is from about 1 mg/mL to about 25 mg/mL. Most preferably, the concentration of obesity protein analog in the formulation is from about 1 mg/mL to about 15 mg/mL. Other preferred ranges of concentration of obesity protein analog in the formulation are from about 0.5 mg/mL to about 20 mg/mL, from 0.5 mg/mL to about 5 mg/mL, and from about 2 mg/mL to about 20 mg/mL.
  • the obesity protein analogs used in the formulations of the present invention are preferably bio- synthesized in a host cell transformed with recombinant DNA.
  • the basic steps biosynthesis of a heterologous protein using the methods of recombinant technology include : a) construction of a synthetic or semi-synthetic (or isolation from natural sources) DNA encoding the 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 biosynthesized 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 proteins . Methodologies of synthetic gene construction are well-known in the art [Brown, E. L., et al . Methods in Enzymology, Academic Press, New York, NY, 68:109-151 (1 9 7 9 )]. The DNA sequence corresponding to the synthetic protein gene may be generated using conventional DNA synthesizing apparatus such as the Applied Biosystems Model 380A or 38OB DNA synthesizers (Perkin Elmer, Applied Biosystems Division, Foster City, California) .
  • a convenient protease sensitive cleavage site e . g.
  • the gene encoding the 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 human adipose tissue.
  • An obesity protein analog may be made either by direct expression, or as fusion protein comprising the protein, in which case expression may be followed by enzymatic or chemical cleavage to form the obesity protein analog.
  • a variety of peptidases e.g. trypsin which cleave a poiypeptide at specific sites or digest the peptides from the amino or carboxy termini (e.g. diaminopeptidase) of the peptide chain are known.
  • peptidases e.g. trypsin
  • 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 is designed to possess restriction endonuclease cleavage sites at either end of the transcript to facilitate isolation from and integration into these expression, 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 endonuclease cleavage pattern of the parent expression vector to be employed. 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 site 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, F., et al., Gene 2:95-113 (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, R. M., et al . , U.S. patent No. 5,304,473, issued, April 19, 1994, the teachings of which are herein incorporated by reference.
  • 5,304,473 can be removed from the plasmid pRB182 with restriction enzymes Ndel and BamHI .
  • the genes encoding the protein of the present invention can be inserted into the plasmid backbone on a Ndel/BamHI restriction fragment cassette . d. Prokaryotic Expression
  • prokaryotes are used for cloning of DNA sequences in constructing the vectors useful in the invention.
  • E. coli K12 strain 294 ATCC No. 31446
  • Other microbial strains which may be used include E. coli B and E. coli X1776 (ATCC No. 31537) . These examples are illustrative rather than limiting.
  • Prokaryotes also are used for expression.
  • the aforementioned strains, as well as E. coli W3110 (prototrophic, ATCC No. 27325) , bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella typhimurium or Serratia marcescans, and various pseudomonas species may be used.
  • 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, A. C. Y., et al .
  • trp tryptophan promoter system
  • vector pATHl ATCC 37695
  • tac promoter isolated from plasmid pDR540 ATCC-37282
  • bacterial promoters whose nucleotide sequences are generally known, enable one of skill in the art to ligate them to DNA encoding the protein using linkers or adaptors to supply any required restriction sites. Promoters for use in bacterial systems also will contain a Shine-Dalgarno sequence operably linked to the DNA encoding protein.
  • Eukaryotic Expression The protein may 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.
  • 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, W., et al., Nature, 273:113- 120 (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 pCMB ⁇ (ATCC 77177) .
  • promoters from the host cell or related species also are useful herein. Transcription of a D ⁇ A encoding the protein by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of D ⁇ A, usually about 10-300 bp, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent having been found 5' [Laimins, L. A., et al . , Proc.
  • enhancer sequences are now known from mammalian genes (globin, RSV, S V 4 0, EMC, elastase, albumin, ⁇ -fetoprotein, and insulin) .
  • 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.
  • DHFR dihydrofolate reductase
  • thymidine kinase pes simplex virus thymidine kinase is contained on the BamHI fragment of vP-5 clone (ATCC 2028) or neomycin (G418) resistance genes, which are obtainable from pNN414 yeast artificial chromosome vector (ATCC 37682) .
  • 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 LTKX 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 J. , et al., J. Molec. Appl . Genet . 1:327-341* (1982)], mycophenolic acid [Mulligan, R. C. et al . , Science 209:1422-1427 (1980)], or hygro ycin [Sugden, B. et al . , Mol Cell .
  • a preferred vector for eukaryotic expression is pRc/CMV.
  • pRc/CMV is commercially available from Invitrogen Corporation, San Diego, CA.
  • the ligation mixtures are used to transform E. coli K12 strain DH5a (ATCC 31446) and successful transformants are selected by antibiotic resistance where appropriate. Plasmids from the transformants are prepared, analyzed by restriction and/or sequenced by the method of Messing, J., et al . , Nucleic Acids J.es. 9:309-321 (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, T., et al. Molecular Cloning: A Laboratory Manual , 2 nd Ed., 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 proteins in higher eukaryotes include: African green monkey kidney cell line transformed by SV40
  • African green monkey kidney cells (VERO 76, ATCC CRL-1587) ; human cervical epitheloid carcinoma cells (HeLa, ATCC CCL-2) ; canine kidney cells (MDCK, ATCC CCL-34) ; buffalo rat liver cells (BRL 3A, ATCC CRL-1442) ; human diploid lung cells (WI-38, ATCC CCL-75) ; human hepatocellular carcinoma cells (Hep G2 , ATCC HB-8065) ; and mouse mammary tumor cells (MMT 060562, ATCC CCL51) .
  • Yeast Expression (VERO 76, ATCC CRL-1587) ; human cervical epitheloid carcinoma cells (HeLa, ATCC CCL-2) ; canine kidney cells (MDCK, ATCC CCL-34) ; buffalo rat liver cells (BRL 3A, ATCC CRL-1442) ; human diploid lung cells (WI-38, ATCC CCL-75) ; human hepatocellular carcinoma cells (Hep G2 , ATCC
  • eukaryotic microorganisms such as yeast may also be used as host cells.
  • Saccharomyces cerevisiae common baker's yeast, is the most commonly-used eukaryotic microorganism for expressing heterologous proteins, although a number of other strains are commonly available .
  • the plasmid YRp7 for example, is commonly used [ATCC-40053, Stinchco b, D. T., et al . , Nature 282:39-43 (1979); Kingsman, A. J. , et al . , Gene
  • 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 [e.g., ATCC 44076 or PEP4-1; Jones, E. W., Genetics 85:23-33 (1977)].
  • Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase, which is found on plasmid pAP12BD ATCC 53231 [Patel, A. C, et al . , U.S. Patent No. 4,935,350, issued June 19, 1990] or other glycolytic enzymes such as enolase, which is found on plasmid pACl (ATCC 39532) , glyceraldehyde-3-phosphate dehydrogenase, which is derived from plasmid pHcGAPCl (ATCC 57090, 57091), zymomonas mobilis [Ingram, L.O., et al . , U.S. Patent No.
  • yeast promoters which are 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, which is contained on plasmid vector pCL28XhoLHBPV [ATCC 39475; Reddy, V. B., et al . , U.S. Patent No.
  • GAL1 promoter which may be found on plasmid pRY121 (ATCC 37658) .
  • Suitable vectors and promoters for use in yeast expression are further described in Hitzeman, R. A., et al., European Patent Publication No. 73,657A1, published March 9, 1983.
  • Yeast enhancers such as the UAS Gal from Saccharomyces cerevisiae, which is found in conjunction with the CYC1 promoter on plasmid YEpsec--hIlbeta (ATCC 67024) , also are advantageously used with yeast promoters .
  • the preferred host cell line for biosynthesizing the proteins used in the present formulations is E. coli K12 RV308, but numerous other cell lines are available, such as, b ut not limited to, E. coli K12 L201, L687, L693, L507, L640, L641, L695, L814 (E. coli B) .
  • 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, J. K. , et al . , in Protein Folding, Gierasch, L. M. and King, J., eds . , American Association for the Advancement of Science Publication No. 89-18S, Washington, D.C., 136-142 (1990)].
  • Such protein aggregates must be dissolved to provide further purification and isolation of the desired protein product. [Kreuger, J. K. , et al . , supra . ] .
  • the proteins are expressed with a leader sequence.
  • the leader sequence is preferably Met-R ⁇ - , wherein Ri is any amino acid except Pro or is absent, so that the expressed proteins may be readily converted to an obesity protein analog not having a leader sequence with Cathepsin C, or other suitable aminopeptidases .
  • 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 dipeptidylaminopeptidases .
  • Ri is Arg, Asp, or Tyr; and most preferably, the proteins are expressed with a Met-Arg leader sequence.
  • the leader sequence does not significantly affect stability or activity of the protein.
  • the leader sequence is preferably cleaved from the protein.
  • the expressed proteins may be of the Formula: Met-R ⁇ - (obesity protein analog).
  • Preparation 1 The plasmid containing the DNA sequence encoding the desired protein, is digested with Pmll and Bsu36I.
  • the recognition sequences for these enzymes lie within the coding region for the protein at nucleotide positions 275 and 360 respectively.
  • the cloning vector does not contain these recognition sequences. Consequently, only two fragments are seen following restriction enzyme digestion with Pmll and Bsu36I, one corresponding to the vector fragment, the other corresponding to the -85 base pair fragment liberated from within the protein coding sequence.
  • This sequence can be replaced by any DNA sequence encoding the amino acid substitutions between positions 91 and 116 of the obesity protein.
  • DNA sequences are synthesized chemically as two oligonucieotides with complementary bases and ends that are compatible with the ends generated by digestion with Pmll and Bsu36I.
  • the chemically synthesized oligonucieotides are mixed in equimolar amounts (1-10 picomoles/microliter) , heated to 95°C and allowed to anneal by slowly decreasing the temperature to 20-25°C.
  • the annealed oligonucieotides are used in a standard ligation reaction. Ligation products are transformed and analyzed using standard techniques. Other substitutions are preferably carried out in a similar manner using appropriate restriction sites.
  • Preparation 2 A DNA sequence encoding SEQ ID NO: 6 with a Met-Arg leader sequence was obtained using the plasmid and procedures described in Preparation 1. The plasmid was digested with Pmll and Bsu36I. A synthetic DNA fragment of the sequence 5 " -SEQ ID NO : 14 : (SEQ ID NO: 14)
  • Preparation 3 A DNA sequence encoding human obesity protein was assembled from chemically- synthesized, single-stranded oligonucieotides to generate a double- stranded DNA sequence.
  • the oligonucieotides used to assemble this DNA sequence are as follows : (SEQ ID NO:16)
  • Oligonucieotides having SEQ ID NO: 16 through 22 were used to generate an approximately 220 base-pair segment which extends from the Ndel site to the Xbal site at position 220 within the coding sequence.
  • the oligonucieotides having SEQ ID NO: 23 through 29 were used to generate an approximately 240 base-pair segment which extends from the Xbal site to the BamHI site.
  • the respective oligonucieotides were mixed in equimolar amounts, usually at concentrations of about 1-2 picomoles per microliters.
  • oligonucieotides at the 5' -ends of the segment were phosphorylated in standard kinase buffer with T4 DNA kinase using the conditions specified by the supplier of the reagents.
  • the mixtures were heated to 95°C and allowed to cool slowly to room temperature over a period of 1-2 hours to ensure proper annealing of the oligonucieotides .
  • the oligonucieotides were then ligated to each other and into a cloning vector, PUC19 was used, but others are operable using T4 DNA ligase.
  • the PUC19 buffers and conditions are those recommended by the supplier of the enzyme.
  • the vector for the 220 base-pair fragment was digested with Ndel and Xbal, whereas the vector for the 240 base-pair fragment was digested with Xbal and BamHI prior to use.
  • the ligation mixes were used to transform E. coli DH10B cells (commercially available from Gibco/BRL) and the transformed cells were plated on tryptone-yeast (TY) plates containing 100 ⁇ g/ml of ampicillin, X-gal and IPTG. Colonies which grow up overnight were grown in liquid TY medium with 100 ⁇ g/ml of ampicillin and were used for plasmid isolation and DNA sequence analysis . Plasmids with the correct sequence were kept for the assembly of the complete gene.
  • Preparation 4 Protein of SEQ ID NO: 6 with a Met-Arg leader sequence was expressed in E. coli . Inclusion bodies were isolated in 8 M urea and 5 mM cysteine. The protein was purified by anion exchange chromatography in 8 M urea, and folded by dilution into 8 M urea (containing 5 mM cysteine) and exhaustive dialysis against phosphate-buffered saline (PBS) . Little to no aggregation of protein was seen in either of these procedures. Following final purification of the proteins by size-exclusion chromatography, the protein was concentrated to 3-3.5 mg/mL in PBS. Amino acid composition was confirmed.
  • the Met-Arg leader sequence was cleaved by the addition of 6-10 milliunits of a diaminopeptidase which was isolated from Dicteolstelium discoidium per mg of protein [Atkinson, P. R. , et al . , U.S. Patent No. 5,565,330, issued October 15, 1996, incorporated herein expressly by reference] .
  • the conversion reaction was allowed to proceed for 2-8 hours at room temperature. The progress of the reaction was monitored by high performance reversed phase chromatography. The reaction was terminated by adjusting the pH to 8 with NaOH.
  • the des (Met-Arg) protein was further purified by cation exchange chromatography in 7-8 M urea, and then by size exclusion chromatography in PBS. Following final purification of the proteins by size exclusion chromatography the proteins were concentrated to 3-3.5 mg/mL in PBS.
  • Protein NO: 6 Lyophilized, obesity protein analog of SEQ ID NO: 6 (hereinafter Protein NO: 6) was first dissolved in arginine buffer (15.2 mM, pH 8.5) to form a Protein NO: 6 stock solution, having a concentration of Protein NO: 6 of 30.3 mg/mL.
  • arginine buffer 15.2 mM, pH 8.5
  • a stock solution containing preservative at 2 times the desired final preservative concentration was prepared by dissolving the preservative in water.
  • To prepare formulations comprising the Protein NO: 6 at pH greater than 8.0 5 volumes of the appropriate preservative stock and 3.3 volumes of the Protein NO: 6 stock solution were combined. Water was added to bring the combined volume to 10 volumes. The formulations were placed at 25°C.
  • Protein NO: 6 the stock solution concentration of Protein
  • Protein NO: 2 Lyophilized, obesity protein analog of SEQ ID NO: 2 (Protein NO: 2) was dissolved in Arginine buffer (28.6 mM, pH 8.5) to form a Protein NO: 2 stock solution having a concentration of Protein NO: 2 of 85.7 mg/mL.
  • the Protein NO: 2 stock solution was sterile-filtered using a 0.2 micron filter. Separate stock solutions of m-cresol, phenol, and chlorocresol at concentrations of 0.60%, 1.0%, and 0.38%, (w/v) , respectively, were prepared by dissolving the appropriate preservative in water.
  • Control formulations in phosphate buffer were prepared as described above, except that the Protein NO: 2 stock solution was prepared by dissolving lyophilized, obesity protein analog of SEQ ID NO: 2 (Protein NO: 2) with sodium phosphate buffer (62.5 mM, pH 7.8) to form a Protein NO: 2 stock solution having a concentration of Protein NO: 2 of 93.8 mg/mL.
  • the pH of the formulation was adjusted, as needed, to pH 7.8 after mixing 1.6 parts of the Protein NO: 2 stock solution with 5 parts of the appropriate preservative stock solution.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Obesity (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Endocrinology (AREA)
  • Zoology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Child & Adolescent Psychology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Diabetes (AREA)
  • Hematology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)

Abstract

La présente invention concerne une composition stable lorsqu'elle est stockée et soluble, qui comporte un analogue de protéines de l'obésité et un conservateur. Ladite composition a un pH supérieur à 8,0 et est utilisée en tant que produit pharmaceutique à usages multiples permettant de traiter l'obésité.
PCT/US1998/000816 1997-01-17 1998-01-16 Composition a base de proteines de l'obesite WO1998031390A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002277282A CA2277282A1 (fr) 1997-01-17 1998-01-16 Composition a base de proteines de l'obesite
EP98902573A EP0983091A4 (fr) 1997-01-17 1998-01-16 Composition a base de proteines de l'obesite
JP53454298A JP2001511180A (ja) 1997-01-17 1998-01-16 肥満タンパク質製剤
AU59199/98A AU5919998A (en) 1997-01-17 1998-01-16 Obesity protein formulations

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
US3571597P 1997-01-17 1997-01-17
US60/035,715 1997-01-17
US5496997P 1997-08-07 1997-08-07
US60/054,969 1997-08-07
US6535297P 1997-11-12 1997-11-12
US60/065,352 1997-11-12

Publications (1)

Publication Number Publication Date
WO1998031390A1 true WO1998031390A1 (fr) 1998-07-23

Family

ID=27364892

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/000816 WO1998031390A1 (fr) 1997-01-17 1998-01-16 Composition a base de proteines de l'obesite

Country Status (5)

Country Link
EP (1) EP0983091A4 (fr)
JP (1) JP2001511180A (fr)
AU (1) AU5919998A (fr)
CA (1) CA2277282A1 (fr)
WO (1) WO1998031390A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996023515A1 (fr) * 1995-01-31 1996-08-08 Eli Lilly And Company Proteines anti-obesite
WO1996027661A1 (fr) * 1995-03-07 1996-09-12 Dade International Inc. Solutions pour stabiliser des proteines et des peptides
WO1996034885A2 (fr) * 1995-05-05 1996-11-07 Smithkline Beecham Plc Fragments peptidiques biologiquement actifs de proteines ob

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU5539596A (en) * 1995-04-06 1996-10-23 Amylin Pharmaceuticals, Inc. Anti-obesity agents
AU2548097A (en) * 1996-03-26 1997-10-17 Eli Lilly And Company Formulations of ob protein
WO1998007446A1 (fr) * 1996-08-23 1998-02-26 Eli Lilly And Company Formulations de proteines

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996023515A1 (fr) * 1995-01-31 1996-08-08 Eli Lilly And Company Proteines anti-obesite
WO1996027661A1 (fr) * 1995-03-07 1996-09-12 Dade International Inc. Solutions pour stabiliser des proteines et des peptides
WO1996034885A2 (fr) * 1995-05-05 1996-11-07 Smithkline Beecham Plc Fragments peptidiques biologiquement actifs de proteines ob

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
"PARENTERAL FORMULATIONS OF PROTEINS AND PEPTIDES: STABILITY AND STABILIZERS.", JOURNAL OF PARENTERAL SCIENCE AND TECHNOLOGY., PARENTAL DRUF ASSOCIATION, US, vol. 04., no. 02S., 1 January 1988 (1988-01-01), US, pages S01 - S26., XP002912221, ISSN: 0279-7976 *
AVIS K. E.: "PARENTERAL PREPARATIONS.", REMINGTON'S PHARMACEUTICAL SCIENCES, MACK PUBLISHING CO., US, 1 January 1990 (1990-01-01), US, pages 1545 - 1569., XP002912218 *
GEIGERT J.: "OVERVIEW OF THE STABILITY AND HANDLING OF RECOMBINANT PROTEIN DRUGS", JOURNAL OF PARENTERAL SCIENCE AND TECHNOLOGY., PARENTAL DRUF ASSOCIATION, US, vol. 43., no. 05., 1 September 1989 (1989-09-01), US, pages 220 - 224., XP002912220, ISSN: 0279-7976 *
MANNING M. C., ET AL.: "STABILITY OF PROTEIN PHARMACEUTICALS.", PHARMACEUTICAL RESEARCH, SPRINGER NEW YORK LLC, US, vol. 06., no. 11., 1 January 1989 (1989-01-01), US, pages 903 - 918., XP002912219, ISSN: 0724-8741, DOI: 10.1023/A:1015929109894 *
See also references of EP0983091A4 *
TAMI J., EVENS R. P.,: "EVALUATION OF BIOTECHNOLOGY PRODUCTS.", PHARMACOTHERAPY : THE JOURNAL OF HUMAN PHARMACOLOGY AND DRUG THERAPY, WILEY-BLACKWELL, US, vol. 16., no. 04., 1 January 1996 (1996-01-01), US, pages 527 - 536., XP002912217, ISSN: 0277-0008 *
WANG Y.-C. J., ET AL.: "REVIEW OF EXCIPIENTS AND PH'S FOR PARENTERAL PRODUCTS USED IN THE UNITED STATES.", JOURNAL OF THE PARENTERAL DRUG ASSOCIATION., PARENTERAL DRUG ASSOCIATION, PHILADELPHIA, PA., US, vol. 34., no. 06., 1 November 1980 (1980-11-01), US, pages 452 - 462., XP002912222, ISSN: 0161-1933 *
ZHANG Y., ET AL.: "POSITIONAL CLONING OF THE MOUSE OBESE GENE AND ITS HUMAN HOMOLOGUE.", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 372., 1 December 1994 (1994-12-01), United Kingdom, pages 425 - 433., XP002912223, ISSN: 0028-0836, DOI: 10.1038/372425a0 *

Also Published As

Publication number Publication date
EP0983091A4 (fr) 2002-04-10
EP0983091A1 (fr) 2000-03-08
JP2001511180A (ja) 2001-08-07
AU5919998A (en) 1998-08-07
CA2277282A1 (fr) 1998-07-23

Similar Documents

Publication Publication Date Title
CA2211664A1 (fr) Proteines anti-obesite
WO1997035620A1 (fr) Formulations de proteine ob
EP0784979A2 (fr) Formulations contenant une protéine de l'obésité
US5831017A (en) Obesity protein analog compounds and formulations thereof
EP0786256A2 (fr) Préparation des protéines contre l'obésité
EP0784981A2 (fr) Préparation des protéines contre l'obésité
EP0784982A2 (fr) Préparation des protéines contre l'obésité
EP0849276A1 (fr) Proteines contre l'obésité
WO1998016545A1 (fr) Proteines therapeutiques
AU723997B2 (en) Obesity protein formulations
WO1998031390A1 (fr) Composition a base de proteines de l'obesite
EP0827750A2 (fr) Préparations des protéines contre l'obésité
WO1997028824A1 (fr) Compose contenant la proteine de l'obesite et leurs formulations
EP1007648A1 (fr) Proteines anti-obesite
HK1009391A (en) Obesity protein formulations
CA2243518A1 (fr) Formulations de proteine de l'obesite
CA2243825A1 (fr) Composes d'analogues de proteines de l'obesite et preparations a base de ceux-ci
CA2243229A1 (fr) Preparations a base de proteine d'obesite
HK1014336A (en) Anti-obesity proteins
CA2243512A1 (fr) Formulations d'une proteine de l'obesite
CA2243707A1 (fr) Compose contenant la proteine de l'obesite et leurs formulations

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH GM GW HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref document number: 2277282

Country of ref document: CA

Ref country code: CA

Ref document number: 2277282

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase

Ref country code: JP

Ref document number: 1998 534542

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 1998902573

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1998902573

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1998902573

Country of ref document: EP