WO1998031391A1 - Obesity protein formulations - Google Patents
Obesity protein formulations Download PDFInfo
- Publication number
- WO1998031391A1 WO1998031391A1 PCT/US1998/000939 US9800939W WO9831391A1 WO 1998031391 A1 WO1998031391 A1 WO 1998031391A1 US 9800939 W US9800939 W US 9800939W WO 9831391 A1 WO9831391 A1 WO 9831391A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- protein
- obesity
- formulation
- soluble formulation
- dna
- Prior art date
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
-
- 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.
- the ob/oh 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.
- 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 urine and human protein expressed in adipose tissue.
- 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 urine and human protein expressed in adipose tissue.
- 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 absorption. 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.
- 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 protein stability.
- the data in Table 1 predict relatively little change in the net charge of human obesity protein 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 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.
- This invention provides a soluble formulation comprising an obesity protein and a preservative, said formulation having pH greater than 8.0.
- the invention further provides a process for preparing said soluble formulation, which comprises mixing an obesity protein and a preservative to produce a soluble formulation comprising said obesity protein and said preservative, said soluble formulation having pH greater than 8.0. Additionally, 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, 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.
- Alkylparaben -- refers to a Ci to C 4 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 1 - onophosphate 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 refers to a native mammalian obesity protein produced from the native ob gene following transcription and deletions of introns, translation to a protein and processing to the mature obesity protein with secretory signal peptide removed, e . g. , from the N-terminal valine-proline to the C-terminal cysteine of the mature protein.
- the murine and human obesity proteins have been published [Zhang, Y., et al . Nature 372:425-32 (1994)].
- Murakami, T., et al . reported the cloning and expression of the rat obese gene [Biochemical and Biophysical Research Communications 209 (3) : 944-52 (1995)].
- 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-Rl- wherein Rl is absent or any amino acid except Pro.
- pH of the formulation 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, phenylmercuric 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 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 - o -
- 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
- Transcription the process whereby information contained in a nucleotide sequence of DNA is transferred to a complementary RNA sequence.
- Translation the process whereby the genetic information of messenger RNA is used to specify and direct the synthesis of a polypeptide chain.
- 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.
- 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 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 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.
- 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 [Wallhauser, 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, 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 proteins 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 proteins 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 in the body, the formulation, and the potency of the obesity protein. Where administration is intermittent, the amount per administration should also take into account the interval between doses, and the bioavailability of the obesity protein from the formulation. Administration of the formulation of the present invention could be continuous . It is within the skill of the ordinary physician to titrate the dose and rate or frequency of administration of the formulation of the present invention to achieve the desired clinical result.
- Administration of the formulations by occular, nasal, buccal, or pulmonary routes is also preferred. Administration by the pulmonary route is particularly preferred.
- Glycerin is the preferred isotonicity agent .
- concentration of the isotonicity agent is in the range known in the art for parenteral formulations, and for glycerin, is preferably about 16 mg/mL to about 25 mg/mL.
- the obesity protein used in the present formulations is human obesity protein, optionally having a Met-leader sequence, of the formula:
- Val Pro lie Gin Lys Val Gin Asp Asp Thr Lys Thr Leu lie Lys Thr
- the concentration of obesity protein in the formulation is preferably about from about 0.5 mg/mL to about 100 mg/mL. More preferably, the concentration of obesity protein in the formulation is from about 0.5 mg/mL to about 50 mg/mL. Still more preferably, the concentration of obesity protein in the formulation is from about 1 mg/mL to about 25 mg/mL. Most preferably, the concentration of obesity protein in the formulation is from about 1 mg/mL to about 10 mg/mL. Other preferred ranges of concentration of obesity protein 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 proteins 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.
- 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 mRA isolated from human adipose tissue.
- PCR polymerase chain reaction
- An obesity protein 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.
- a variety of peptidases e.g. trypsin
- 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.
- particular chemicals e.g. cyanogen bromide
- 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.
- 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.
- the gene encoding A-C-B proinsulin described in U.S. Patent No. 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 - lb -
- E. coli B and E. coli X1776 (ATCC No. 31537) . These examples are illustrative rather than limiting.
- Prokaryotes also are used for expression.
- 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 . , Nature, 275:617-624 (1978); and Goeddel, D. V., et al .
- trp tryptophan
- vector pATHl 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
- hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC-37282)
- other functional bacterial promoters whose nucleotide sequences are generally known, enable one of skill in the art to ligate them to D ⁇ A 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 D ⁇ A encoding protein.
- 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.
- 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- 12 0 (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 ⁇ ( A TCC 77177) .
- promoters from the host cell or related species also are useful herein.
- Enhancers are cis-acting elements of DNA, 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 . Nat ' l Acad . Sci . (USA) 78:464-468 (1981)] and 3' [Lusky, M. , et al., Mol . Cell . Bio. 3:1108-1122 (1983)] to the transcription unit, within an intron [Banerji, J., et al .
- enhancer sequences are now known from mammalian genes (globin, RSV, SV40, EMC, elastase, albumin, ⁇ -fetoprotein, and insulin). Typically, however, one will use 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
- yeast fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms
- Selection genes 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 Bglll/Hindlll restriction fragment of pJOD-10 [ATCC 68815] ) , thymidine - o -
- DHFR dihydrofolate reductase
- telomere kinase (herpes 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) .
- selectable markers are successfully transferred into a mammalian host cell, the transfected mammalian host cell can survive if placed under selective pressure.
- 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.
- CHO DHFR- cells ATCC CRL-9096
- 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 J., et al., J " . Molec. Appl . Genet . 1:327-341 (1982)], mycophenolic acid [Mulligan, R. C. et al . , Science 209:1422-1427 (1980)], or hygromycin [Sugden, B. et al . , Mol Cell . Biol .
- the three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug, such as, G418, neomycin (geneticin) , xgpt (mycophenolic acid) , or hygromycin, respectively.
- a preferred vector for eukaryotic expression is p R c/CMV.
- pRc/CMV is commercially available from Invitrogen C orporation, 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 Res . 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 (COS-7, ATCC CRL-1651); transformed human primary embryonal kidney cell line 293 [Graham, F. L. et al . , J. Gen Virol . 36:59-72 (1977); Harrison, T., et al . , Virology 11 -. 319- 329 (1977); Graham, F. L. et al .
- 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) . f .
- yeast 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, Stinchcomb, D. T., et al . , Nature 282:39-43 (1979); Kingsman, A. J. , et al .
- 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, but 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.
- the proteins are expressed with a leader sequence.
- the leader sequence is preferably Met-R ⁇ - , wherein R l is any amino acid except Pro or is absent, so that the expressed proteins may be readily converted to an obesity protein 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).
- Primers were designed based on the published amino acid sequence of the human oJ 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 SEQ ID NO: 2 - ATG CAT TGG GGA MCC CTG TG
- PCROB-2 SEQ ID NO: 3 - GG ATT CTT GTG GCT TTG GYC CTA TCT
- PCROB-3 SEQ ID NO: - TCA GCA CCC AGG GCT GAG GTC CA
- PCROB-4 SEQ ID NO: 5 - CAT GTC CTG CAG AGA CCC CTG CAG CCT GCT CA
- 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 .
- RT H-reverse transcriptase
- This annealed mixture was then incubated at 37°C for 2 minutes before adding 1 ⁇ L BRL M-MLV-reverse transcriptase (200 U/ ⁇ L) (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.
- BRL M-MLV-reverse transcriptase 200 U/ ⁇ L
- 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 conditions for PCR were 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 fragment ( ⁇ 0.2 ⁇ g) was then ligated into Smal linearized pUC18 plasmid Ul ⁇ 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. White clones were picked and were grown at 37°C overnight in TY broth containing Amp (100 ⁇ g/mL) .
- the plasmid was isolated using a Wizard Miniprep DNA purification system (Promega) and submitted for DNA seqeuncing on a Applied Biosystem 370 DNA sequencer .
- the DNA sequence of the bovine OB gene was obtained by techniques analogous to Preparation 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 DH10B (commercially available from GIBCO-BRL) and colonies growing on tryptone-yeast (DIFCO) plates supplemented with 10 mg/mL of tetracycline are analyzed.
- E. coli DH10B commercially available from GIBCO-BRL
- DIFCO tryptone-yeast
- Plasmid D ⁇ A 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. Cells of 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.
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Obesity (AREA)
- Endocrinology (AREA)
- Genetics & Genomics (AREA)
- Gastroenterology & Hepatology (AREA)
- Organic Chemistry (AREA)
- Dermatology (AREA)
- Immunology (AREA)
- Child & Adolescent Psychology (AREA)
- Toxicology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP98902618A EP0971742A4 (en) | 1997-01-17 | 1998-01-16 | Obesity protein formulations |
CA002276427A CA2276427A1 (en) | 1997-01-17 | 1998-01-16 | Obesity protein formulations |
AU59231/98A AU723997B2 (en) | 1997-01-17 | 1998-01-16 | Obesity protein formulations |
JP53459298A JP2001509177A (en) | 1997-01-17 | 1998-01-16 | Obesity protein preparation |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US3571497P | 1997-01-17 | 1997-01-17 | |
US60/035,714 | 1997-01-17 | ||
US5488697P | 1997-08-07 | 1997-08-07 | |
US60/054,886 | 1997-08-07 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1998031391A1 true WO1998031391A1 (en) | 1998-07-23 |
Family
ID=26712418
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1998/000939 WO1998031391A1 (en) | 1997-01-17 | 1998-01-16 | Obesity protein formulations |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0971742A4 (en) |
JP (1) | JP2001509177A (en) |
AU (1) | AU723997B2 (en) |
CA (1) | CA2276427A1 (en) |
WO (1) | WO1998031391A1 (en) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5552523A (en) * | 1995-01-31 | 1996-09-03 | Eli Lilly And Company | Anti-obesity proteins |
US5569603A (en) * | 1994-03-08 | 1996-10-29 | Heska Corporation | Dirofilaria immitis GP29 proteins, nucleic acid molecules and uses thereof |
WO1997026013A1 (en) * | 1996-01-19 | 1997-07-24 | Eli Lilly And Company | Obesity protein formulations |
EP0797999A2 (en) * | 1996-03-26 | 1997-10-01 | Eli Lilly And Company | Formulations of obesity protein |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU5539596A (en) * | 1995-04-06 | 1996-10-23 | Amylin Pharmaceuticals, Inc. | Anti-obesity agents |
AU4160597A (en) * | 1996-08-23 | 1998-03-06 | Eli Lilly And Company | Protein formulations |
-
1998
- 1998-01-16 CA CA002276427A patent/CA2276427A1/en not_active Abandoned
- 1998-01-16 EP EP98902618A patent/EP0971742A4/en not_active Withdrawn
- 1998-01-16 JP JP53459298A patent/JP2001509177A/en active Pending
- 1998-01-16 WO PCT/US1998/000939 patent/WO1998031391A1/en not_active Application Discontinuation
- 1998-01-16 AU AU59231/98A patent/AU723997B2/en not_active Ceased
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5569603A (en) * | 1994-03-08 | 1996-10-29 | Heska Corporation | Dirofilaria immitis GP29 proteins, nucleic acid molecules and uses thereof |
US5552523A (en) * | 1995-01-31 | 1996-09-03 | Eli Lilly And Company | Anti-obesity proteins |
WO1997026013A1 (en) * | 1996-01-19 | 1997-07-24 | Eli Lilly And Company | Obesity protein formulations |
EP0797999A2 (en) * | 1996-03-26 | 1997-10-01 | Eli Lilly And Company | Formulations of obesity protein |
Non-Patent Citations (1)
Title |
---|
See also references of EP0971742A4 * |
Also Published As
Publication number | Publication date |
---|---|
EP0971742A4 (en) | 2002-04-10 |
AU723997B2 (en) | 2000-09-07 |
JP2001509177A (en) | 2001-07-10 |
AU5923198A (en) | 1998-08-07 |
CA2276427A1 (en) | 1998-07-23 |
EP0971742A1 (en) | 2000-01-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5552523A (en) | Anti-obesity proteins | |
US5554727A (en) | Anti-obesity proteins | |
US5559208A (en) | Anti-obesity proteins | |
US5851995A (en) | Anti-obesity proteins | |
US5552524A (en) | Anti-obesity proteins | |
US5532336A (en) | Anti-obesity proteins | |
EP0725078A1 (en) | Anti-obesity proteins | |
US5567678A (en) | Anti-obesity proteins | |
US5569744A (en) | Anti-obesity proteins | |
US5563243A (en) | Anti-obesity proteins | |
US5574133A (en) | Anti-obesity proteins | |
US5563244A (en) | Anti-obesity proteins | |
WO1997035620A1 (en) | Formulations of ob protein | |
EP0784979A2 (en) | Obesity protein formulations | |
EP0786256A2 (en) | Obesity protein formulations | |
EP0784981A2 (en) | Obesity protein formulations | |
US5831017A (en) | Obesity protein analog compounds and formulations thereof | |
EP0784982A2 (en) | Obesity protein formulations | |
EP0849276A1 (en) | Anti-obesity proteins | |
EP0835879A2 (en) | Analogs of obesity proteins and pharmaceutical formulations comprising them | |
AU723997B2 (en) | Obesity protein formulations | |
EP0827750A2 (en) | Obesity protein formulations | |
WO1997028824A1 (en) | Obesity protein compounds and formulations thereof | |
EP0983091A1 (en) | Obesity protein formulations | |
CA2243518A1 (en) | Obesity protein 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: 2276427 Country of ref document: CA Ref country code: CA Ref document number: 2276427 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 534592 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1998902618 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 59231/98 Country of ref document: AU |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWP | Wipo information: published in national office |
Ref document number: 1998902618 Country of ref document: EP |
|
WWG | Wipo information: grant in national office |
Ref document number: 59231/98 Country of ref document: AU |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1998902618 Country of ref document: EP |