AU723997B2 - Obesity protein formulations - Google Patents

Description

WO 98/31391 PCTIUS98/00939 -1- OBESITY PROTEIN FORMULATIONS Background of the Invention 1. Field of the Invention. 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.

2. Background Information. Obesity, and especially upper body obesity, is a common and very serious public health problem in the United States and throughout the world, and is expected to worsen as the population ages.

Currently, about 33 percent of Americans are overweight enough to be unhealthy, which is defined as weighing more than 26% greater than standard weight guidelines. For comparison, about 25% of Americans were overweight enough to be unhealthy in 1980, according to the United States National Center for Health Statistics. The proportion of obese adults among the well-fed populations of the world is expected to rise to more than 50% within 20 years.

Numerous studies indicate that lowering body weight dramatically reduces risk for chronic diseases, such as, diabetes, hypertension, hyperlipidemia, coronary heart disease, cancer, and muscularoskeletal diseases. Recent estimates for the medical cost of obesity are $150,000,000,000 world-wide. The problem has become serious enough that the United States Surgeon General has begun an initiative to combat the ever-increasing adiposity rampant in American society.

Obesepatients may lose weight through deliberate modification of behavior, such as changing diet and increasing exercise. Unfortunately, an estimated 33 billion dollars are spent each year on such weight-loss measures that are largely futile, with failure rates reaching SUBSTITUTE SHEET (RULE WO 98/31391 PCT/US98/00939 2 Failure may be due to genetic factors that cause increased appetite, a preference for high-fat foods, or a tendency for lipogenic metabolism. People inheriting such genetic traits are prone to becoming obese regardless of their efforts to combat the condition. Therefore, a pharmacological agent that can correct this adiposity handicap and allow the physician to successfully treat obese patients in spite of their genetic inheritance is needed.

The ob/ob mouse is a model of obesity and diabetes that is known to carry an autosomal recessive trait linked to a mutation in the sixth chromosome. Recently, Zhang and co-workers published the positional cloning of the mouse gene linked with this condition [Zhang, et al., Nature 372:425-32 (1994)]. This report disclosed the murine and human protein expressed in adipose tissue. Likewise, Murakami, 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 ob gene, has demonstrated an ability to effectively regulate adiposity in mice [Pelleymounter, et al., Science 269:540-543 (1995)].

It is well-known that 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.

Unfortunately, native obesity proteins, that is, those having sequences naturally-occurring in mammals, tend to aggregate under conditions typically preferred for protein formulations, a tendency that is aggravated by certain commonly-used, pharmaceutically-acceptable SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCT/S98/00939 3 preservatives, such as, phenol, cresol, and alkylparabens.

Because it is envisioned that a pharmaceutical product of obesity protein 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.

The molecular interactions in a formulation between obesity protein, preservative, buffer, ionic strength, pH, temperature, and excipients, such as surfactants or sugars, are complex, and the role that each factor contributes to aggregation is highly unpredictable in view of the propensity for obesity protein to aggregate and precipitate from the formulation.

As a general proposition, a slightly acidic pH is preferred for solution formulations of proteins. Among the various reasons for this preference is the concern about increasing oxidation, disulfide scrambling, and deamidation of the protein as pH increases above neutrality. For these reasons, 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.

In view of this reluctance to formulate proteins above pH 8.0, and the unpredictability of protein formulation in general, it was most unexpected to discover that, under certain conditions, as disclosed herein, the aggregation of obesity protein caused by preservatives is reduced when the pH of the formulation is greater than pH 8.0. The discovery that the physical stability of obesity protein in the presence of preservatives is greatly enhanced above pH 8 is all the more unexpected in view of the calculated titration curve for human obesity protein. The calculated titration curve for human obesity protein is given below in Table 1 as the net charge as a function of pH.

SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCTUS98/00939 4 Table 1. Net charge on human obesity protein as a function of pH.

e arge on uman bety pH Protein Predicted by Henderson- Hasselbach Equation !i net charge of human obesity protein in the pH range of preparing said soluble formulation, which comprises mixing- The data in Table 1 predict relatively little change in the net charge of human obesity protein in the pH range of to 9.0 compared with lower and higher pH levels. In particular, the predicted net charge does not change abruptly at about pH 8.0, whereas, 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.

Summary of the Invention This invention provides a soluble formulation comprising an obesity protein and a preservative, said formulation having pH greater than The invention further provides a process for preparing said soluble formulation, which comprises mixing SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCT/US98/00939 5 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 Detailed Description and Preferred Embodiments For purposes of the present invention, as disclosed and claimed herein, the following terms and abbreviations are defined as follows: 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. For the purpose of this invention, peripheral parenteral routes of administration include, without limitation, intravenous, intramuscular, subcutaneous, and intraperitoneal routes of administration.

Alkylparaben refers to a C 1 to C 4 alkyl paraben. Preferably, 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 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 monophosphate forms of the nucleosides uracil, cytidine, guanine, and thymine, respectively when they occur in RNA molecules. In double stranded DNA, base pair may refer to a partnership of A with T or C with G. In a DNA/RNA SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCT/US98/00939 6 heteroduplex, 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 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, NaC1, 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, 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, et al. Nature 372:425-32 (1994)].

Likewise, Murakami, et al., reported the cloning and expression of the rat obese gene [Biochemical and Biophysical Research Communications 209(3):944-52 (1995)].

Native porcine and bovine obesity proteins have been disclosed [Hsiung, H. et al., European Patent Publication No. 743,321, published 20 November 1996). Other mammalian obesity proteins also have been disclosed [Basinski, M. et al., European Patent Publication No.

744,408, published 27 November 1996; Basinski, M. et al., European Patent Publication No. 764,722, published March 1997]. 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-Rlwherein R1 is absent or any amino acid except Pro.

pharmaceutically acceptable buffer The pH of the formulation may be buffered with a pharmaceutically acceptable buffer, such as TRIS or a basic amino acid, such SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCT/US98/00939 7 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.

Preservative a compound added to a pharmaceutical formulation to act as an anti-microbial agent. A parenteral formulation must meet guidelines for preservative effectiveness to be a commercially viable multi-use product. Among 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. 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 SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCTIUJS98/00939 8 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).

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 SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCTIUS98/00939 9 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.

The 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 (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. To prepare 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.,

SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCTIUS98/00939 10 Develop. Biol. Standard. 24:9-28 Krager, Basel, 1974)].

The optimal concentration of the preservative depends on the preservative, its solubility, and the pH of the formulation.

Other additives, such as a pharmaceutically acceptable solubilizers like Tween 20 (polyoxyethylene sorbitan monolaurate), Tween 40 (polyoxyethylene sorbitan monopalmitate), Tween 80 (polyoxyethylene sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers), and PEG (polyethylene glycol) may optionally be added to the formulation to reduce aggregation. These additives are particularly useful if a pump or plastic container is used to administer the formulation. A pharmaceutically acceptable surfactant may reduce protein aggregation.

As noted above, the invention provides a soluble formulation comprising an obesity protein, said soluble formulation having a pH greater than pH 8.0. Preferably 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. At 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.

preferably, 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.

SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCTIUS98/00939 11 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. The 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.

Preferably, the obesity protein used in the present formulations is human obesity protein, optionally having a Met-leader sequence, of the formula: 10 Val Pro Ile Gin Lys Val Gin Asp Asp Thr Lys Thr Leu Ile Lys Thr 25 lie Val Thr Arg Ile Asn Asp Ile Ser His Thr Gin Ser Val Ser Ser 40 SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCTfUS98/00939 12 Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile 55 60 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin Ile 75 8 0 Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gin Ile Ser Asn Asp Leu 90 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 00 105 110 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 115 120 125 1 5 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 130 135 140 Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 145 (SEQ ID NO:1) Gly Cys 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 SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCTIUS98/00939 13 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.

a Gene Construction 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. et al. Methods in Enzymology, Academic Press, New York, NY, 68:109-151 (1979)]. 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 380B DNA synthesizers (Perkin Elmer, Applied Biosystems Division, Foster City, California).

It may desirable in some applications to modify the coding sequence of the protein so as to incorporate a convenient protease sensitive cleavage site, between the signal peptide and the structural protein, thereby facilitating the controlled excision of the signal peptide from the fusion protein construct.

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.

Such methodologies are well-known in the art. See, e.g., Maniatis, et al. Molecular Cloning: A Laboratory Manual, 2 nd Ed., Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989).

SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCT[US98/00939 14 b. Direct Expression or Fusion Protein 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 trypsin) which cleave a polypeptide at specific sites or digest the peptides from the amino or carboxy termini diaminopeptidase) of the peptide chain are known. Furthermore, particular chemicals cyanogen bromide) will cleave a polypeptide chain at specific sites.

The skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semisynthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See, Carter, Chapter 13 in Protein Purification: From Molecular Mechanisms to Large-Scale Processes, Ladisch,

M.,

et al. (Eds.) American Chemical Soc., Washington,

D.C.

(1990).

c. Vector Construction Construction of suitable vectors 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.

To effect the translation of the desired protein, one inserts the engineered synthetic DNA sequence in any of a plethora of appropriate recombinant DNA expression vectors through the use of appropriate restriction endonucleases.

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 SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCTIUS98/00939 15 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.

In general, 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. For example, E. coli is typically transformed using pBR322, a plasmid derived from an E. coli species [Bolivar, et al., Gene 2:95-113 (1977)]. Plasmid pBR32 2 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 NdeI and BamHI. The genes encoding the protein of the present invention can be inserted into the plasmid backbone on a NdeI/BamHI restriction fragment cassette.

d. Prokaryotic Expression In general, prokaryotes are used for cloning of DNA sequences in constructing the vectors useful in the invention. For example, E. coli K12 strain 294 (ATCC No.

31446) is particularly useful. Other microbial strains which SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCT/US98/00939 16 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 p-lactamase (vector pGX2907 [ATCC 39344] contains the replicon and p-lactamase gene) and lactose promoter systems [Chang, A. C. et al., Nature, 275:617-624 (1978); and Goeddel, D. et al., Nature 281:544-548 (1979)], alkaline phosphatase, the tryptophan (trp) promoter system (vector pATH1 [ATCC 37695] is designed to facilitate expression of an open reading frame as a trpE fusion protein under control of the trp promoter) and hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC-37282). However, other functional 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.

e. 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 adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. p-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an restriction fragment which also contains the SV40 viral origin of replication [Fiers, et al., Nature, 273:113- SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCTIUS98/00939 17 120 (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 pCMBp (ATCC 77177). Of course, promoters from the host cell or related species also are useful herein.

Transcription of a DNA encoding the protein by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that act on a promoter to increase its transcription. Enhancers are relatively orientation and position independent having been found [Laimins, L. et al., Proc. Nat'l Acad. Sci.

(USA)

78:464-468 (1981)] and 3' [Lusky, et al., Mol. Cell.

Bio. 3:1108-1122 (1983)] to the transcription unit, within an intron [Banerji, et al., Cell 33:729-740 (1983)] as well as within the coding sequence itself [Osborne, T. F., et al., Mol. Cell. Bio. 4:1293-1305 (1984)]. Many enhancer sequences are now known from mammalian genes (globin,

RSV,

EMC, elastase, albumin, a-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) 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. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR, which may be derived from the BglII/HindIII restriction fragment of pJOD-10 [ATCC 68815]), thymidine SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCTIUS98/00939 18 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). When such selectable markers are successfully transferred into a mammalian host cell, the transfected mammalian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. 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 DHFRcells (ATCC CRL-9096) and mouse LTK- cells

ATCC

CCL-2.31. 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 et al., J. Molec. Appl. Genet. 1:327-341 (1982)1, mycophenolic acid [Mulligan, R. C. et al., Science 209:1422-1427 (1980)], or hygromycin [Sugden, B. et al., Mol Cell. Biol. 5:410-413 (1985)]. The three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug, such as, G418, SUBSTITUTE SHEET (RULE 26) VO 08011 PCr.T/IUS98/00939 19 neomycin (geneticin), xgpt (mycophenolic acid), or hygromycin, respectively.

A preferred vector for eukaryotic expression is pRc/CMV. pRc/CMV is commercially available from Invitrogen Corporation, San Diego, CA. To confirm correct sequences in constructed plasmids, 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, 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, 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 (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, et al., Virology 77:319-329 (1977); Graham, F. L. et al., Virology 86:10-21 (1978)]; baby hamster kidney cells [BHK-21(C-13), ATCC MacPherson, et al, Virology 16:147-151 (1962)]; Chinese hamster ovary cells [CHO-DHFR- (ATCC CRL-9096)]; mouse Sertoli cells [TM4, ATCC CRL-1715; Mather, J. Biol.

SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCT/US98/00939 20 Reprod. 23:243-252 (1980)]; 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 human hepatocellular carcinoma cells (Hep G2, ATCC HB-8065); and mouse mammary tumor cells (MMT 060562, ATCC CCL51).

f. Yeast Expression In addition to prokaryotic and mammalian host cells, 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. For expression in Saccharomyces, the plasmid YRp7, for example, is commonly used [ATCC-40 053 Stinchcomb, D. et al., Nature 282:39-43 (1979); Kingsman, A. et al., Gene 7:141-152 (1979); Tschumper, et al., Gene 10:157-166 (1980)]. 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 ATCC 44076 or PEP4-1; Jones, E. 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 pAC1 (ATCC 39532), glyceraldehyde-3-phosphate dehydrogenase, which is derived from plasmid pHcGAPC1

(ATCC

57090, 57091), zymomonas mobilis [Ingram, et al., U.S.

Patent No. 5,000,000, issued March 19, 19911, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose- 6 phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having the additional advantage of transcription SUBSTITUTE SHEET (RULE 26) WO 98/1191 PCT/US98/00939 21 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. et al., U.S.

Patent No. 4,840,896, issued June 20, 1989], glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization, such as, the 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. 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 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. et al., in Protein Folding, Gierasch, L. M. and King, eds., American Association for the Advancement of Science Publication No. 89-18S, Washington, 136-142 (1990)].

Such protein aggregates must be dissolved to provide further purification and isolation of the desired protein product.

[Kreuger, J. et al., supra.]. A variety of techniques using strongly denaturing solutions such as guanidinium-HCl and/or weakly denaturing solutions such as urea are used to solubilize the proteins. Gradual removal of the denaturing agents (often by dialysis) in a solution allows the denatured protein to assume its native conformation. The particular conditions for denaturation and folding are SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCTUS98/00939 22 determined by the particular protein expression system and/or the protein in question.

Preferably, the proteins are expressed with a leader sequence. One of ordinary skill in the art would recognize that numerous leader sequences are operable; however, the leader sequence is preferably Met-Ri- wherein R1 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. Preferably, 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. The purification of proteins is by techniques known in the art, and includes reverse phase chromatography, affinity chromatography, and size exclusion.

Preferably, R1 is Arg, Asp, or Tyr; and most preferably, the proteins are expressed with a Met-Arg leader sequence. Interestingly, the leader sequence does not significantly affect stability or activity of the protein.

However, the leader sequence is preferably cleaved from the protein. Thus, the expressed proteins may be of the Formula: Met-Ri-(obesity protein).

The following examples will help describe how the invention is practiced and will illustrate the invention.

The scope of the present invention is not to be construed as merely consisting of the following examples.

Preparation IPorcine OB Gene Total RNA was isolated from porcine fat tissue obtained from Pel-Freez Pel-Freez Inc., and the cDNA was cloned in accordance with known techniques [Hsiung, et al., Neuropeptide 25:1-10 (1994)].

SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCT/US98/00939 23 Primers were designed based on the published amino acid sequence of the human ob gene. The primers were prepared for use in polymerase chain reaction

(PCR)

amplification methods using a Model 380A DNA synthesizers (PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404). Primers 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:4 TCA GCA CCC AGG GCT GAG GTC CA), and PCROB-4 (SEQ ID NO:5 CAT GTC CTG CAG AGA CCC CTG CAG CCT GCT CA) were prepared.

For cDNA synthesis, total RNA 1 iL (1 Ig/pL) isolated from porcine adipose tissue and 1 L Perkin Elmer Random primers (50 iM) in a total volume of 12 iL were annealed for 10 minutes at 70 0 C and then cooled on ice. The following were then added to the annealed mixture: 4 tL of BRL 5x H-reverse transcriptase (RT) reaction buffer (Gibco- BRL CAT#28025-013), 2 iL of 0.1 M DTT, 1 pL of 10 mM dNTPs.

This annealed mixture was then incubated at 37 0 C for 2 minutes before adding 1 pL BRL M-MLV-reverse transcriptase (200 U/pL) (CAT#28025-013) and incubated at 37 0 C for additional 1 hour. After incubation the mixture was heated at 95 0 C for 5 minutes and then was cooled on ice.

For amplification of cDNA, the polymerase chain reaction (PCR) was carried out in a reaction mixture (100 pL) containing the above cDNA reaction mixture (1 units of AmpliTaq.DNA polymerase (Perkin-Elmer Corporation) pL of 10x 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 0 C for 1 minute, 57 0 C for 1 minute and 72 0 C for 1 minute for 30 cycles using a PCR DNA Thermal Cycler (Perkin-Elmer Corporation). After PCR amplification, L BRL T4 DNA polymerase (5 U/lL), 2 LL BRL T4 polynucleotide kinase (10 U/IL), and 5 pL ATP (10 mM) were added to the PCR reaction mixture (100 pl) directly and incubated for 30 minutes at 37 0 C. After the incubation the reaction mixture was heated at 95 0 C for 5 minutes and then SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCTIS98/00939 24 was cooled on ice. The 500 bp fragment mg) was purified by agarose gel electrophoresis and isolated by the freeze-squeeze method. The 500 bp fragment tg) was then ligated into SmaI linearized pUC18 plasmid tg) and the ligation mixture was used to transform DH5a

(BRL)

competent cells. The transformation mixture was plated on 0.02% X-Gal TY broth plates containing ampicillin (Amp) (100 4g/mL) and was then incubated overnight at 370C. White clones were picked and were grown at 370C overnight in TY I0 broth containing Amp (100 ig/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.

Preparation 2 Bovine OB Gene The DNA sequence of the bovine OB gene was obtained by techniques analogous to Preparation i, except sense (PCROB-2) and antisense (PCROB-3) primers were used for bovine OB cDNA amplification.

Preparation 3 Vector Construction A plasmid containing the DNA sequence encoding the obesity protein is constructed to include NdeI and BamHI restriction sites. The plasmid carrying the cloned

PCR

product is digested with NdeI 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. Plasmid DNA is isolated, digested with NdeI and BamHI and the resulting fragments are separated by agarose gel electrophoresis. Plasmids containing the expected 450bp NdeI to BamHI fragment are kept. Cells of E. coli K12 SUBSTITUTE SHEET (RULE 26) WO 98/31391 PCTIUS98/00939 25 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.

The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected herein, however, is not to be construed as limited to the particular forms disclosed, since they are to be regarded as illustrative rather than restrictive. Variations and changes may be made by those skilled in the art without departing from the spirit of the invention.

SUBSTITUTE SHEET (RULE 26)

Claims (13)

1. A soluble formulation comprising an obesity protein and a preservative, said soluble formulation having pH greater-than

2. The soluble formulation of Claim 1, wherein the concentration of obesity protein is between 0.5 mg/mL and 100 mg/mL.

3. The soluble formulation of Claim 2, wherein the concentration of obesity protein is between 0.5 mg/mL and mg/mL.

4. The soluble formulation of Claim 3, wherein the pH is greater than

8.0 and less than The soluble formulation of Claim 4, wherein the pH is greater than 8.6. 6. The soluble formulation of Claim 4, wherein the pH is less than 8.6. 7. The soluble formulation of Claim 6, wherein the pH is greater than 8.3. 8. The soluble formulation of Claim 1, which further comprises a pharmaceutically acceptable buffer.

9. The soluble formulation of Claim 8, wherein the buffer is TRIS or L-arginine. The soluble formulation of Claim 9, wherein the buffer is TRIS.

11. The soluble formulation of Claim 1, wherein the preservative is selected from the group consisting of phenol, cresol, alkylparaben, benzyl alcohol and mixtures thereof. SUBSTITUTE SHEET (RULE 26) 27

12. The soluble formulation of claim 11, wherein the preservative is phenol, m-cresol, methylparaben, propylparaben, chlorocresol, benzyl alcohol, or mixtures thereof.

13. The soluble formulation of any one of claims 1 to 12, which further comprises an isotonicity agent.

14. A soluble formulation comprising an obesity protein and a preservative, said formulation being substantially as hereinbefore described with reference to any one of the examples. A process for preparing the soluble formulation of any one of claims 1 to 14, 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 a pH greater than

16. A method for the treatment or prophylaxis of obesity in a mammal requiring said treatment or prophylaxis, which method comprises administering to said mammal an effective amount S" of a formulation according to any one of claims 1 to 14.

17. A formulation according to any one of claims 1 to 14 when used in the treatment or prophylaxis of obesity. 15 18. A formulation according to any one of claims 1 to 14 for use in the treatment or prophylaxis of obesity.

19. The use of a formulation according to any one of claims 1 to 14 for the manufacture of a medicament for the treatment or prophylaxis of obesity. Dated 2 August 1999 20 ELI LILLY AND COMPANY Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON 0 C06911