CA2243512A1 - Obesity protein formulations - Google Patents

Abstract

The present invention discloses a soluble parenteral formulation, comprising an obesity protein and a preservative, said formulation having a low ionic strength. The low ionic strength allows the preparation of a commercially viable multi-use pharmaceutical product.

Description

CA 02243~l2 l998-07-l~
Wogl/26012 PCT~S97/00790 Obesity Protein Formulations This application claims the benefit o~ U.S.
Provisional Application No. 60/010,357, filed January 19, 1996.
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.
Obesity, and especially upper body obesity, is a common and very serious public health problem in the United States and throughout the world. According to recent statistics, more than 25% of the United States population and 27% of the Canadian population are overweight. Kuczmarski, 15 Amer. J. of Clin. Nutr. 55: 495S - 502S (1992); Reeder et.
al., Can. Med. ~ss. J., 23: 226-233 ~1992). Upper body obesity is the strongest risk factor known for type II
diabetes mellitus, and is a strong risk factor for cardiovascular disease and cancer as well. Recent estimates 20 ~or the medical cost of obesity are $150,000,000,~00 world wide. The problem has become serious enough that the surgeon general has begun an initiative to combat the ever increasing adiposity rampant in American society. Much of this obesity-induced pathology can be attributed to the strong association with dyslipidemia, hypertension, and insulln resistance.
Many studies have demonstrated that reduction in obesity by diet and exercise reduces these risk factors dramatically.
Un~ortunately, these treatments are largely unsuccessful with a failure rate reaching 95%. This failure may be due to the fact that the condition is strongly associated with genetically inherited factors that contribute to increased appetite, preference ~or highly caloric foods, reduced physical activity, and increased lipogenic metabolism. This indicates that people inheriting these genetic traits are prone to becoming obese regardless of their efforts to combat the condition. Therefore, a pharmacological agent that can CA 02243~l2 l998-07-l~
WO9ffl6012 PCT~S97/00790 .

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, Yiying Zhang and co-workers published the positional cloning o~ the mouse gene linked with this condition. Yiying Zhang et al. N~ture 37Z: 425-32 (1994). This report disclosed the murine and human obesity protein expressed in adipose tissue. Likewise, Murakami et al., in Biochemical and Bio~hvsical Research mmllni cations 209(3):9~4-52 (199~) report the cloning and expression of the rat obese gene. The protein, which is encoded by the o~ gene, has demonstrated an ability to effectively regulate adiposity in mice. Pelleymounter et a~., Science 269: 540-5~3 (1995).
A parenteral ~ormulation cont~; n i ng insoluble protein causes problems relating to inconsistency in the dose-response as well as unpredictability. The unpredictability is believed to be due to greater variability in the pharamacokinetics in suspension formulations. The insoluble formulations must first dissolve prior to adsorption. It is hypothesized that this step has signi~icant variability in a subcutaneous depot.
Furthermore, non-na~ive association and aggregation under physiological conditions can lead to precipitation of the protein at the site of injection, which could lead to irritation or other immune response. For these reasons, a for~ulation o~ human obesity protein that developed insoluble protein particles would be unacceptable to patients seeking its benefits and to regulatory agencies.
Unfortunately, the naturally occurring obesity proteins demonstrate a propensity to aggregate making the preparation of a soluble, pharmaceutically acceptable 35 = parenteral formulation exceedingly difficult. The molecular interactions amongst the preservative, buffer, ionic strength, pH, temperature, protein concentration, and any CA 02243~12 1998-07-1~

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additional excipients such as a surfactant, or sugar are highly unpredictable in view of the propensity for the obesity protein to aggregate and precipitate from the formulation.
The present invention provides conditions under which the obesity protein is soluble and commercially viable as a multi-use pharmaceutical product. Most unexpectedly, the present invention overcomes a specific preservative dependent interaction that is observed when the ionic strength of the formulation is greater than 10 mM. Under the specific conditions provided herein, the physical stability of the ~ormulation is greatly enhanced. That is, when formulated with select preservatives and low ionic strength, the obesity protein remains soluble at much higher concentrations and at the desired pH range. Thus a soluble, multi-use parenteral formulation may be prepared.
Accordingly, the present invention provides a soluble, parenteral ~ormulations of an obesity protein.
This invention provides a soluble parenteral formulation, comprising an obesity protein and a preservative, said ~ormulation having an ionic strength of less than about 10 mM.
Preferrably, the invention provides a soluble parenteral formulation, comprising an obesity protein and a 2~ preservative selected from the group consisting of phenol, cresol, or a combination thereof, said formulation having a ionic strength of less than 10 m~.
The invention further comprises a process for preparing a soluble parenteral formulation of Claim 1, which comprises mixing an obesity protein and a preservative, such that said ~ormulation has an ionic strength of less than about 10 mM, Additionally, the invention provides a method of treating obesity in a m~mm~ 1 in need thereof, which comprises administering to said m~mm~l a soluble parenteral formulation of Claim 1.

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For purposes o~ the present invention, as disclosed and claimed herein, the ~ollowing terms and abbreviations are de~ined as ~ollows:
Alkylparaben -- re~ers to a Cl to C4 alkyl paraben.
Pre~erably, alkylparaben is methylparaben, ethylparaben, propylparaben, or butylparaben.
Base pair (bp) -- re~ers to DMA or RNA. The abbreviations A,C,G, and T correspond to the 5'-monophosphate forms o~ 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 ~orms o~ the nucleosides uracil, cytidine, guanine, and thymine, respectively when they occur in RNA molecules. In double stranded DNA, base pair may re~er to a partnership o~ A with T or C with G. In a ~M~/RNA heteroduplex, base pair may re~er to a partnership o~ T with U or C with G.
Cresol - re~ers to meta-cresol, ortho-cresol, para-cresol, chloro-cresol, or mixtures thereo~.
Ionic Stren~th - re~ers to a measure o~ the concentration o~ ionic species in solution, weighted by the s~uare Q~ the ionic charge of each species, as described in elementary textbooks o~ physical chemistry, such as Physical C~emlstry, Gilbert w. Castellan, Addison-Wesley Publishing 25 Company, Inc., Reading, Massachusetts (1964), p. 335, or Obesity protein -- re~ers to the native mammalian obesity protein produced ~rom the native ob gene ~ollowiny transcription and deletions o~ introns, translation to a protein and processing to the mature obeisty protein with secretory signal peptide removed, e.g. ~rom the N-~erminal valine-proline to the C-terminal cvsteine o~ the mature protein. The human obesity protein is published in Zhang et al. Nature 372: 425-32 ~1994). The rat obesity protein is published in Murakami et al., Biochemical ~nd Bio~hvsical Research Comm 209(3): 944-52 (1995). Native porcine and bovine obesity proteins are disclosed in a U.S. patent application by Hansen M. Hsiung and DenniS P. Smith ~iled May -CA 02243~l2 l998-07-l~

WO97/26012 PCT~S97/0~790 19, 1995, Serial number 08/445,305 (EP0 743 321). Other m~mm~ n obesity proteins are disclosed in U.S. patent application serial number 08/452,228, filed May 26, 1995 (EP0 744 4083, U.S. provisional application, 60/003935, filed September 19, 1995 (EP ). Obesity protein includes those proteins having a leader se~uence. A leader se~uence is one or more amino acids on the M-terminus to aid in production or purification of the protein. A preferred leader sequence is Met-R1- wherein R1 is absent or any amino acid except Pro.
Plasmid -- an extrachromosomal sel~-replicating genetic el~m~n t.
Reading frame -- the nucleotide sequence from which translation occurs "read" in triplets by the translational apparatus of tRNA, ribosomes and associated factors, each triplet corresponding to a particular amino acid. Because each triplet is distinct and of the same length, the coding sequence must be a multiple o~ 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 ~e maintained. In the creation of fusion proteins containing a chelating peptide, the reading frame of the DNA
se~uence encoding the structural protein must be maintained in the DNA sequence encoding the chelating peptide.
Recombinant DNA Cloning Vector -- any autonomously replicating agent including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
Recombinant DNA Expression Vector --==any recombinant DNA cloning vector in which a promoter has been incorporated.
Replicon -- A DNA se~uence that controls and allows for autonomous replication of a plasmid or other vector.

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WO9~126012 PCT~S97/00790 Transcription -- the process whereby information contained in a nucleotide sequence o~ DNA is transferred to a complementary RNA sequence.
Translation -- the process whereby the genetic information of messenger RMA is used to specify and direct the synthesis o~ a polypeptide chain.
Vector -- a replicQn 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. Arti~icial vectors are constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases.
Vectors include Recombinant DNA cloning vectors and Recombinant DNA expression vectors.
Treating -- as used herein, describes the management and care o~ a patient for the purpose of combating 20 - the disease, condition, or disorder and includes the administration of a protein of present invention to prevent the onset of the symptoms or complications, alleviating the symptoms or complications, or eliminating the disease, condition, or disorder. Treating as used herein includes the 25 = administration of the protein for cosmetic purposes. A
cosmetic purpose seeks to control the weight of a m~mm~ 1 to improve bodily appearance.
Isotonicity agent -- isotonicity agent re~ers to an agent that is physiologically tolerated and embarks a 3~ ~ suitable tonicity to the ~ormulation to prevent the net flow o~ 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.
35 - Physiologically tolerated buffer -- a physiologically tolerated buffer is known in the art. A
physiologically tolerated buffer is preferably a phosphate CA 02243~12 1998-07-1~
WO97~26~12 PCT~S97/00790 buffer, like sodium phosphate. Other physiologically tolerated bu~fers include TRIS, sodium acetate, or sodium ~ citrate. The selection and concentration of buffer is known in the art.
The nucleotide and amino acid abbreviations are accepted by the United States Patent and Trademark O~~ice as set forth in 37 C.F.R. 1.822 (b)(2) ~1993). Unless otherwise indicated the amino acids are in the L
configuration.
As noted above, the invention provides a soluble parenteral formulation, comprising an obesity protein and a preservative, said formulation having an ionic strength of less than l0 mM. Preferably the ionic strength is less than 5 mM, and most preferably the ionic strength is less than 1 m~. Under conditions of low ionic strength, the obesity proteins rPm~in~ in solution making a soluble, parenteral formulation possible.
A parenteral formulation must meet guidelines for preservative effectiveness to be a commercially viable multi-use product. Preservatives known in the art as being acceptable in parenteral formulations include: phenol, m-cresol, benzyl alcohol, methylparaben, chlorobutanol, p-cresol, phenylmercuric nitrate, thimerosal and various mixtures thereof. See, e.g., WALLHAUSER, K.-H., DEvELoP. sIoL.
STAN~ARD. 24, pp. 9-28 (3asel, S. Krager, 1974). The alkylparabens and chlorobutanol are less sensitive to the ionic strength of the formulation, however, they are less e~fective preservative agents.
The most effective preservatives, phenol and cresol or mixtures thereo~ demonstrate a propensity to cause protein instability when formulated with the obesity protein. Under conditions described herein it is possible to formulate the protein using phenol, cresol, or a combination thereof.
Xence, the preferred preservatives of the present inventlon are selected from the group consisting of phenol, cresol, or a combination thereof.

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The concentration of obesity protein in the formulation is about 1.0 mg/mL to about 100 mg/mL; preferably about 1.5 mg/mL to about 50.0 mg/mL; most preferably, about 5.0 mg/mL to 10.0 mg/mL. The concentration of preservative required is the concentration necessary to maintain preservative ef~ectiveness. The relative amounts of preservative necessary to maintain preservative e~ectiveness varies with the preservative used. Generally, the amount necessary can be found in See, e.g., WALLHAUSER, K.-H., 10 DEVELOP. BIOL. STANDARD. 24, pp. 9-28 (sasel, S. Krager, 1974), herein incorporated by reference. The optimal concentration of the preservative depends on the preservative, its solubility, and the pH of the formulation.
An isotonicity agent, preferably glycerin, may be additionally added to the formulation. The concentration o~
the isotonicity agent is in the range known in the art ~or parenteral formulations, preferably about 1 to 20, more preferably 8 to 16 mg/mL, and still more pre~erably about 16 mg/mL. The pH of the formulation may also be buffered with a physiologically tolerated buffer, preferably a phosphate (at a concentration yielding an ionic strength o~ less than 10 mM, preferably about 1 mM to 5 mM) buffer, like sodium phosphate. Other acceptable physiologically tolerated buffers include TRIS, sodium acetate, sodium bicarbonate or sodium citrate. The selection and concentration of buffer is known in the art.
Other 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), BRIJ 35 (polyoxyethylene (23) lauryl ether), and PEG (polyethylene glycol) may optionally be added to the formulation to reduce aggregation.
35=: These additives are particularly useful if a pump or plastic container is used to administer the formulatlon. The CA 02243~12 1998-07-1~
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g presence of pharmaceutically acceptable surfactant mitigates the propensity for the protein to aggregate.
- The 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 human obesity protein in water is combined with the desired preservative in water in ~uantities sufficient to provide the protein and preservative at the desired concentration. The formulation is generally sterile ~iltered 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 surfactant used, the temperature and pH at which the formulation is prepared may be optimized for the concentration and means of administration used.
The unexpected ionic strength effect on formulation stability was demonstrated by preparing formulations of varying ionic strength and comparing the amount of protein r~m~in'ng in solution after 3 days at 4~C and 37~C. The formulations used to generate the data of Tables 1 and 2 were prepared in a manner analogous to Examples 1 and 2.

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WO9~6012 PCT~S97/00790 Table 1. Recovery o~ human o~esity protein as a ~unction o~
ionic strength and conditions at pH 7.8. Values are the least s~uare means and standard errors determined from ~itting the raw data to a factorial model o~ the second degree containing the effects o~
ionic strength, acid excursion, conditions, and ionic strength-conditions interaction (R2 = o 90, (Prob > F) - 3.63e~2~, total observations = 88).
Percentage of protein in solution is calculated using a theoretical target of 1.6 mg protein/mL.
All samples contained m cresol at 0.3%. Variation in ionic strength, ~, was achieved by addition o~
sodium chloride.
Protein in Solution (% o~ theoretical) Ionic Initial 3 days ~ 3 days strength 4~C 37~C
U (mM) 0 83.4 i 3.682.0 + 3.677.3 + 3.9 1 91.3 i 5.686.9 + 5.678.3 i 5.6 2.5 91.7 + 5.685.9 ~ 5.678.9 ~ 3.9 84.1 + 3.977.0 i 3.965.2 i 3.9 83.3 i 4.883.2 i 4.830.2 + 5.6 55.0 + 6.348.6 ~ 6.812.3 i 5.6 62.0 i 9.679.1 i 9.616.1 i 9.6 61.9 i 9.656.6 + 9.610.8 + 9.6 50.7 i 6.815.2 i 9.64.6 i 6.8 150 57.3 i 9.641.4 i 9.64.1 i 9.6 CA 02243~l2 l998-07-l~
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Table 2: The effects of ionic strength on solubility of hOB
in the presence of various preservatives. Values are the least square means and standard errors determined from fitting the raw data to a factorial model of the second degree containing the effects of preservative, acid excursion, buffer, conditions, and all two-factor interactions (R2 = 0.93, (Prob >
F) = 6.56e-47, observations = 154). Percent of protein in solution is based on a theoretical target of 1.6 mg protein/mL.
Protein in Solution (% of theoretical) Preservative Preservative low ionic high ionic Concentration strength1 strength2 (mq/mL) benzyl alcohol ~ 5.0 methylparaben + 1.6 59.5 + 3.5 42.3 + 3.5 propylparaben 0.17 benzyl alcohol 10.0 73.3 t 3.9 59.0 + 2.5 butylParaben 0.15 74.4 + 3.5 80.3 + 3.5 chlorobutanol 5.0 91.0 + 3.5 92.1 + 3.5 m-cresol 3.0 77.8 + 2.7 23.2 + 3.0 methylparaben + 1.5 72.6 i 3.5 72.6 + 3.5 pro~ylparaben 0.1g methylparaben 1.7 91.2 + 2.7 92.7 + 3.5 p-cresol 3.0 68.4 + 3.5 37.0 + 3.5 phenol 5.0 69.2 + 4.0 25.2 + 3.5 propylparaben 0.2 92.6 + 3.5 95.1 + 3.5 1 No Na2HPO~ was added.
2 65.2 mM Na1HPO4 was added, with the exception of the methylparaben studies, in which 32.5 mM Na2HPO4 was added.
The enhanced stability of the formulations prepared under low ionic strength is most unexpected and unpredictable in view of the art. The data in Table 1 demonstrate that at an ionic strength greater than 10 mM the solubility of the obesity protein sharply declines at both temperatures. The data of Ta~le 2 demonstrate the preservative effects.

CA 02243~12 1998-07-1~
WO91~26012 PC~/US97/00790 Preferably, the p~ of the present ~ormulations is about p~ 7.0 to about 8.0 and, most preferably 7.6 to 8Ø
The formulations are preferably prepared under basic conditions by mixing the obesity protein and preservative at 5 a pH greater than pH 7Ø Preferably, the pH is about 7.6 to ~-8.0, and most preferably about pH 7.8. Ideally, a preservative and water solution is mixed at pH 7.6 to 8Ø
A~ded to this solution is obesity protein in water. The pH
is adjusted, as necessary, to about pH 7.6 to 8Ø The solution is then held until the components are disso~ved, approximately 20 to 40 minutes, pre~erably about 30 minutes.
The base used to adjust the pH of the ~ormulation may be one or more pharmaceutically acceptable bases such as sodium hydroxide and potassium hydroxide. The pre~erred base ls 15= sod~um ~ydroxide.
The formulations prepared in accordance with the present invention may be used in ~ syringe, Injector, pumps or any other device recognized in the art ~or parenteral a~ministration.
Preferably, the obesity protein o~ the present invention is human obesity protein, optionally having a Met-leader se~uence, of the ~ormula:

Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lyc Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile 5~ 55 60 Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg CA 02243~l2 l998-07-l~

W O 97/26012 PCTrU~97/00790 .
- 13 ~

Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys (SEQ ID NO:l) The obesity proteins of the present invention can be prepared by any of a variety of recognized peptide synthesis techniques including classical (solution) methods, solid phase methods, semi synthetic methods, and more recent recombinant DNA methods. The preparation of the human protein is known and disclosed, for example, in Halaas Jeffrey L. et al., Science ~ (1995). The preparation of other mammalian obesity proteins is described in U.S.
application serial number 08/445,305, filed May 19, 1995 (EP0 743 321); U.S. patent application serial number 08/452,228, filed May 26, 1995 (EP0 744 408); and provisional application, 60,003935, filed September 19, 1995, (EP );~0 all of which are herein incorporated by reference.
The obesity proteins described herein may also be produced either by recombinant DNA technology or well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with~5 protein fragments coupled through conventional solution methods. Recombinant methods are preferred if a high yield is desired. The basic steps in the recombinant production of protein include:
a) construction o~ a synthetic or semi-synthetic (or isolation from natural sources) DNA
encoding the obesity protein, b) integrating the coding sequence into an expression vector in a manner suitable for the expression of the protein either alone or as a fusion protein, c) transforming an appropriate eukaryotic or prokaryotic host cell with the expression vector, and CA 02243~l2 1998-07-l~
W097/26012 PCT~S97tO0790 .

d) recovering and puri~ying the recombinantly produced protein.

Synthetic genes, the n vitro or ln Y}~Q
transcription and translation o~ which will result in the production of the protein may be constructed by techniques well known in the art. Owing ~o the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet de~inite number o~ DNA sequences may be constructed which encode the desired proteins. In the pre~erred practice o~ the invention, synthesis is achieved by recombinant DNA technology.
~ ethodology of synthetic gene construction is well known in the art. For example, see Brown, et al. (1979) Methods in En~ymology, Academic Press, N.Y., Vol. ~8, pgs.
109-151. The DNA sequence corresponding to the synthetic claimed protein gene may be generated using conventional DNA
synthesizing apparatus such as the Applied Biosystems Model 380A or 3~0B DNA synthesizers ~commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404). It may desirable in some applications to modi~y the coding sequence o~ the obesity protein so as to incorporate a convenient protease sensitive cleavage site, e.g., between the signal peptide and the structural protein 25 - facilitating the controlled excision o~ the signal peptide ~rom the ~usion protein construct.
The gene encoding the obesity protein may also be created by using polymerase chain reaction (PCR). The template can be a cDNA library (commercially available ~rom CLONETECH or STRATAGENE~ or mRNA isolated ~rom the desired arrival adipose tissue. Such methodologies are well known in the ar~ Maniatis, et al. Molecular Clonina: A L~horatorv Manua~, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989).
The constructed or isolated DNA seq=uences are use~ul ~or expressing the obesity protein either by direct expression or as ~usion protein. When the se~uences are used CA 02243~l2 l998-07-l~

in a fusion gene, the resulting product will require enzymatic or chemical cleavage. A variety of peptidases ~ which cleave a polypeptide at specific sites or digest the peptides from the amino or carboxy termini (e.g.
diaminopeptidase) of the peptide chain are known.
Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave a polypeptide chain at specific sites. The skilled artisan will appreciate the modifications necessary to the amino acid sequence (and synthetic or semi-synthetic coding sequence if recombinant means are employed) to incorporate site-speci~ic internal cleavage sites. See U.S
Patent No. 5,126,249; Carter P., ~ite Specific Proteolysis of Fusion Proteins, Ch. 13 in Protein Purification: From Molecular Mechanisms to Larae Scale Processes, American Chemical Soc., Washington, D.C. (1990).
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 may be designed to possess restriction endonuclease cleavage sites at either end of the transcript to facilitate isolation from and integration into these expression and amplification and expression plasmids.
The isolated cDNA coding sequence may be readily modified by the use of synthetic linkers to facilitate the incorporation of this se~uence into the desired cloning vectors by techniques well known in the art. The particular endonucleases employed will be dictated ~y the restriction endonuclease cleavage pattern of the parent expression vector to be employed. The restriction sites are chosen so as to properly orient the coding sequence with control sequences to CA 02243~l2 l998-07-l~
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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 origin as well as marker se~uences which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using pBR322, a plasmld derived from an 10_ E. coli species (Bolivar, et al., Gene 2: 95 (1977)).
Plasmid pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means ~or identi~ying transformed cells. The pBR322 plasmid, or other microbial plasmid must also contain or be modified to contain promoters ~5~ 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 ~unctional in the host cell in which the protein is to be expressed. An example of such an expression vector is a plasmid described in Belagaje et al., U.S. patent No.
5,304,493, the teachings o~ which are herein incorporated by reference. The gene encoding A-C-B proinsulin described in U.S. patent No. 5,304,493 can be removed from the plasmid pRB182 with restriction enzymes NdeI and BamHI. The isolated DNA se~uences can be inserted into the plasmid backbone on a NdeI/ amHI restriction ~ragment cassette.
In general, procaryotes are used for cloning o~ DNA
se~uences in constructing the vectors useful in the invention. For example, E. ~li K12 strain 294 (ATCC No.
- 31446) is particularly useful. Other microbial strains which may be used include ~. coli B and E~ ÇQll X1776 (ATCC Mo.
31537). These examples are illustrative rather than limiting.
Procaryotes also are used ~or expression. The a~orementioned strains, as well as E. coli W3110 (prototrophic, ATCC No. 27325), bacilli such as Bacillus CA 02243~l2 l998-07-l~

WO9ii26012 PCT~S97/00790 .

sllhtilis, and other enterobacteriaceae such as Salmonella tv~himurium or Serratia marcescans, and various pseudomonas species may be used. Promoters suitable for use with prokaryotic hosts include the ~-lactamase (vector pGX2907 ~ 5 [ATCC 39344] contains the replicon and ~-lactamase gene) and lactose promoter systems (Chang et al., Nature, 275: 615 (1978); and Goeddel et ~1., N~ture 281:544 (1979)), alkaline phosphatase, the tryptophan (trp) promoter system (vector pATHl [ATCC 37695] iS designed to facilitate expression of an open reading frame as a trpE fusion protein under control of the trp promoter) and hybrid promoters such as the tac promoter (isolatable from plasmid pDR540 ATCC-37282) .
However,-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 DNA encoding protein.
The DNA molecules may also be recombinantly produced in eukaryotic expression systems. Preferred promoters co~trolling transcription in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), 25 adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous m~mm~l ian 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. ~iers, et al., Nature, 273: 113 (1978) . The entire SV40 genome may be obtained from plasmid pBRSV, ATCC 45019. The immediate early promoter of the human cytomegalovirus may be obtained from plasmid pCMBb (ATCC
77177) . Of course, promoters from the host cell or related 35 species also are useful herein.
Transcription of the DNA by higher eucaryotes is increased by inserting an enhancer sequence into the vector.

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WO97/26012 PCT~S97/00790 Enhancers are cis-acting elements of DNA, usually about 10-3Q0 bp, that act on a promoter to increase its transcription.
Enhancers are relatively oriented and positioned independently and have been found 5' ~Laimins, L. e~ ~l., ~NAS 78:993 (1981)) and 3' (Lusky, M. L., et ~l., Mol. Cell Bio. 3:1108 (1~83)) to the transcription unit, within an intron (Banerji, J. L. et al., Cell 33:729 (1983)) as well as within the coding se~uence itself (Osborne, T. F., et ~l., MQ1. Cell Bio. 4:1293 (1984)). Many enhancer sequences are now known from mammalian genes (globin, RSV, SV40, EMC, elastase, albumin, alpha-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 20 := sequences necessary for the termination of transcription which may a~fect 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.
25 ~ Expression vectors may con~ain 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 BalII/~indIII
restriction fragment o~ pJOD-10 [ATCC 68815~), thymidine ~~kinase (herpes simplex virus thymidine kinase is contained on the BamHI fragment of vP-5 c~one ~ATCC 2028]) or neomycin (G418) resistance genes (obtainable from pNN414 yeast artificial chromosome vector [ATCC 37682]). When such selectable markers are successfully trans~erred into a mammalian host cell, the transfected m~mm~lian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The CA 02243~l2 l998-07-l~

W09i/26012 PCT~S97/~0790 .

first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow without a supplemented media. Two examples are: CHO DHFR- cells (ATCC
CRL-9096) and mouse LTK cells (L-M(TK-) ATCC CCL-2.3) .
These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TR 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.
~he second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection.
Examples of such dominant selection use the drugs neomycin, Southern P and Berg, P., J. Molec. A~l. Genet. 1: 327 (1982), mycophenolic acid, Mulligan, R. C. and Berg, P.
Science 209:1422 (1980), or hygromycin, Sugden, B. et al., Mol Cell. Biol. 5:410-413 (lg85). The three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively.
A preferred vector for eucaryotic expression is pRc/CMV. pRc/CMV is commercially available from Invitrogen Corporation, 3985 Sorrento Valley Blvd., San Diego, CA
92121. To confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform E. coli K12 strain DHlOB (ATCC 3144~) and successful transformants selected by antibiotic resistance where appropriate.
Plasmids from the transformants are prepared, analyzed by CA 02243~l2 l998-07-l~
WOg~/26012 PCT~S97/00790 restriction and/or sequence by the method of Messing, et al., Nucleic ~cids Res. ~:309 (19~1).
Most 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 ~or 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 ~1., ~olecular Clonina: A Laboratorv Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), or Current Protocols in Molecular Biolo~v (1989) and supplements.
Preferred suitable host cells ~or expressing the vectors encoding the claimed proteins in hiyher eucaryotes include: African green monkey kidney line cell line transformed by SV40 (COS-7, ATCC CRL-1651); trans~ormed human primary embryonal kidney cell line 2g3,(Graham, F. L. et al., J. Gen Virol. 36:59-72 (1977), Viroloov 77:319-329, Viroloov 86:10-21); baky hamster kidney cells (BHK-21~C-13), ATCC CCL-10, Virolo~v 16:147 (1962)); Chinese hamster ovary cells CHO-DHFR (ATCC CRL-9096), mouse Sertoli cells (TM4, ATCC CRL-1715, Biol. Re~rod. 23:243-2~0 (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 CCL-75); human hepatocellular carcinoma cells (Hep G2, ATCC Hs-8065);and mouse m~mm~ry tumor cells (MMT 060562, ATCC CCL51).
In addition to prokaryotes, unicellular eukaryotes such as yeast cultures may also be used. Saccharomvces 35 ~ cerevisiae, or common baker's yeast is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available. For expression in Saccharomyces, the -CA 02243~l2 l998-07-l~

WO91/26012 PCT~S97/00790 .

plasmid YRp7, for example, (ATCC-40053, Stinchcomb, et ~1-, Nature 282:39 (1979); Kingsman et al., Gene 7:141 (1979);
Tschemper et al., Gene 10:157 (1980)) is commonly used. This plasmid already contains the trp gene which provides a selection marker for a mutant strain o~ yeast lacking the ability to grow in tryptophan, for example ATCC no. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)).
Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (found on plasmid pAP12BD ATCC 53231 and described in U.S.
Patent Mo. 4,935,350, June 19, 1990) or other glycolytic enzymes such as enolase (found on plasmid pAC1 ATCC 39532), glyceraldehyde-3-phosphate dehydrogenase (derived ~rom plasmid pHcGAPC1 ATCC 57090, 57091), zymomonas mobilis (United States Patent No. 5,000,000 issued March 19, 1991), hexokinase, pyruvate decar~oxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
Other yeast promoters, which contain inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein (contained on plasmid vector pCL28XhoLHBPV
ATCC 39475, United States Patent No. 4,840,896), glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose (GAL1 found on plasmid pRY121 ATCC 37658) utilization. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman et ~l., European Patent Publication No.
73,657A. Yeast enhancers such as the UAS Gal from Saccharomvces cerevisiae (found in conjunction with the CYC1 ~ promoter on plasmid YEpsec--hIlbeta ATCC 67024), also are advantageously used with yeast promoters.
The following examples will help describe how the invention is practiced and will illustrate the invention.

CA 02243~l2 l998-07-l~
WO97/26012 PCT~S97/00790 The scope of the present invention is not ~o be construed as merely consisting of the following examples.

Pre~aration 1 Porcine OB Gene and Gene Product Total RNA was isolated ~rom porcine fat tissue obtained ~rom Pel-Freez~, Pel-Freez Inc., and the cDMA was cloned in accordance with the techni~ues described in Hsiung et al., Neuro~e~tide 25: 1-10 ~1994).
Prlmers were designed based on the published amino acid se~uence 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, 15 ~ Foster City, CA 94404). Primers PCROB-l (12504) (ATG CAT TGG
GGA MCC CTG TG), PCROB-2 (12505) (GG ATT CTT GTG GCT TTG GYC
CTA TCT), PCRoB-3 (12506) (TCA GCA CCC AGG GCT GAG GTC CA), and PCROB-4 (12507) (CAT GTC CTG CAG AGA CCC CTG CAG CCT GCT
CA) were prepared.
For cDNA synthesis, total RNA 1 ~L (1 ~g/~L) isolated from porcine adipose tissue and 1 ~L Perkin Elmer Random primers (50 ~M) in a total volume o~ 12 ~L were annealed ~or 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 (GibcQ-BRL CAT#28025-013), 2 ~L of 0.1 M DTT, 1 ~L of 10 mM dNTPs.
This annealed mixture was then incubated at 37~C ~or 2 minutes before adding 1 ~L BRL M-MLV-reverse transcriptase (200 U/~L) (cAT#28o25-ol3) 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.
For amplification of cDMA, the polymerase chain reaction (PCR) was carried out in a reaction mixture (100 ~L) contalning the above cDNA reaction mixture (1 ~L), 2.5 units 35 ~ o~ AmpliTa~ DNA polymerase (Perkin-Elmer Corporation) 10 ~L
of 10x PCR reaction bu~fer (Perkin-Elmer Corporation) and 50 CA 02243~l2 l998-07-l~

pmol each o~ the sense (PCROB-l) and antisense (PCROB-3) primers ~or porcine OB amplii~ication. The condition !~or PCR
t was 95~C :Eor 1 minute, 57~C :Eor 1 minute and 72-~C for 1 minute ~or 30 cycles using a PCR DNA Thermal Cycler (Perkin-Elmer Corporation). A~ter PCR ampli~ication, 5 ,UL BRL T4 DNA
polymerase (5 U~,UL), 2 ,UL BRL T4 polynucleotide kinase (10 U/~L), and 5 ,uL ATP (10 mM) were added to the PCR reaction mixture (100 ,Ul) directly and incubated ~or 30 minutes at 37~C. After the incubation the reaction mixture was heated at 95~C :Eor 5 minutes and then was cooled on ice. The 500 bp fragment (~0.5 mg) was puri~ied by agarose gel electrophoresis and isolated by the ~reeze-squeeze method.
The 500 bp fragment (~0.2 ~g) was then ligated into SmaI
linearized pUC18 plasmid (~1 ~lg) and the ligation mixture was used to trans~orm DH5a (BRL) competent cells. The trans~ormation 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 ,Ug/mL). The plasmid was isolated using a Wizard Miniprep DNA
puri~ication system (Promega) and submitted for DNA
seqeuncing on a Applied Biosystem 370 DNA sequencer.
Preparation 2 Bovine OB Gene and Gene Product 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 ~or bovine OB cDNA amplification.
Preparation 3 ~ctor Construction A plasmid containing the DMA sequence encoding the obesity protein is constructed to include ~I and ~I
restriction sites. The plasmid carrying the cloned PCR
product ls digested with ~I~I and ~mHI restriction enzymes.
The small ~ 450bp Eragment is gel-purii~ied and ligated into CA 02243~l2 l998-07-l~

the vector pRB182 from which the coding sequence for A-C-B
proinsulin is deleted. The ligation products are transformed into .~. coli D~lOB (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 ~I and BamHI and the resulting fragments are separated by agarose gel electrophoresi~. Plasmids cont~ining the expected ~ 450bp ~I to ~mHI fragment are kept. E. coli K12 RV308 10- (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 techniques of transforming cells with the 15~ aforementioned vectors are well known in the art and may be found in such general references as Maniatis, et al. (1988) ~olecular Clonin~. ~ Laboratorv Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York or ~urrent Protocols in Molecular Biolo~v ~1989) and supplements. The techniques involved in the transformation Of F. coli cells used in the pre~erred practice o~ the invention as exemplified herein are well known in the art.
The precise conditions under which the transformed E. coli cells are cultured is dependent on the nature of the host cell line and the expression or cloning vectors employed. For example, vectors which incorporate thermoinducible promoter-operator regions, such as the c1857 thermoinducible lambda-phage promoter-operator region, require a temperature shift ~rom about 30 to about 40 degrees C. in the culture conditions so as to induce protein synthesis.
In the preferred embodiment of the invention, E.
coli ~12 RV308 cells are employed as host cells but numerous other cell lines are available such as, but not limited to, 35 _ E. coli K12 L201, L687, L693, L507, L640, L641, L695, L814 (E. coli B). The transformed host cells are then plated on appropriate media under the selective pressure of the CA 02243~l2 l998-07-l~

WO97/26012 PCT~S97/00790 .

antibiotic corresponding to the resistance gene present on the expression plasmid. The cultures are then incubated for a time and temperature appropriate to the host cell line employed.
- 5 Proteins that are expressed in high-level bacterial expression systems characteristically aggregate in granules or inclusion bodies which contain high levels of the overexpressed protein. Kreuger et al., in Protein Foldina, Gierasch and King, eds., pgs 136-142 (1990), American Association for the Advancement of Science Publication No.
89-18S, Washington, D.C. Such protein aggregates must be solubilized to provide further purification and isolation of the desired protein product. Id. A variety of techniques using strongly denaturing solutions such as guanidinium-HCl and/or weakly denaturing solutions such as dithiothreitol (DTT) are used to solubilize the proteins. Gradual removal of the denaturing agents (often by dialysis) in a solution allows the denatured protein to assume its native conformation. The particular conditions for denaturation and folding are determined by the particular protein expression system and/or the protein in question.
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 dipeptidylpeptidases. The purification of proteins is by techniques known in the art and includes reverse phase chromatography, affinity chromatography, and size exclusion.

The following examples and preparations are provided merely to further illustrate the preparation of the formulations of the invention. The scope of the invention is not construed as merely consisting of the following examples.

CA 02243~l2 l998-07-l~
WO97/26012 P~T~S97/00790 .

Example 1 Human obesit~ Protein Formulation To generate a solution of human obesity protein (hOb) posessing a minim~l ionic strength, the lyophilized solid material was first dissolved in water to generate a stock solution (stock 1). The concentration of hOb in stock 1 was verified by W/Vis Spectrophotometry using the known extinction coefficient ~or hob at the maximum spectral absorbance (279nm or 280nm), measuring the maximum spectral absorbance (27gnm or 280nm), and utilizing the dilution ~actor. A stock preservative solution containing m-cresol, for example, was prepared by dissolving the neat liquid in water (stock 2). A solution of hOb at 1.6 mg/mL was prepared by addition of an aliquot of stock 1 to a container which held an ali~uot of stock 2 along with the re~uired quantity of water, at an alkaline pH (7.8+0.1); adjusted if absolutely necessary with trace ~L quantities of HC1 or NaOH. After an appropriate incubation period at room temperature ~30 minutes), the solution pH was examined and adjusted if absolutely necessary with trace ~L quantities o~ HC1 or NaOH, to yield an hOb solution at 1.6 mg/mL with 0.3% m-cresol pH
7.8+0.1. The solution was then hand-filtered using a glass syringe with an attached 0 22~m syringe filter into a glass vial. In order to verify the minim~l ionic strength (approaching 0 mM) of the sample, a blank solution (blank) was prepared to correspond to the hOb solution, except for the absence of stock 1. A conductivity meter, employing an epoxy-coated Pt or Au dip cell, was calibrated and then standardized with a broad series of potassium chloride (KCl) solutions. The blank solution was examined in the same fashion as the KCl standard solutions and its conductivity determined mathematically from an equation resulting from a simple linear fit of the KC1 standard data.
-CA 02243~12 1998-07-1~

WO97/26012 PCT~S97/00790 Example 2 Human Obesitv Protein Formulation - To generate a solution of Human Obesity Protein (hOb) posessing a low ionic strength, the lyophilized solid material was ~irst dissolved in water to generate a stock solution (stock l). The concentration of hOb in stock l was veri~ied by W /Vis Spectrophotometry using the known extinction coe~~icient for hOb at the maximum spectral absorbance (279nm or 280nm), measuring the maximum spectral absorbance (27gnm or 280nm), and utilizing the dilution ~actor. A stock preservative solution containing m-cresol, for example, was prepared by dissolving the neat liquid in water (stock 2). A stock solution of sodium chloride (NaCl), typically 0.l M or 0.2 M, was prepared by dissolving the solid in water (stock 3). A solution of hOb at l.6 mg/mL was prepared by addition of an aliquot of stock l to a container which held an aliquot of stock 2 along with the required quantity of water, at an alkaline pH (7.8+0.1)i adjusted if absolutely necessary with trace ~L quantities of HCl or NaOH.
After an appropriate incubation period at room temperature (30 minutes); an aliquot of stock 3 was added to provide a low, added ionic strength to the solution (approximately 2-3 mM). The pH was examined and adjusted i~ absolutely necessary with trace ~L quantities of HCl or NaOH, to yield an hOb solution at l.6 mg/mL with 0.3% m-cresol pH 7.8+0.l, and a low ionic strength; for example, 2.5 mM. The solution was then hand-filtered using a glass syringe with an attached 0.22~m syringe filter lnto a glass vial. In order to verify the low ionic strength of the sample, a blank solution (blank) was prepared to correspond to the hOb solution, except for the absence of stock l. A conductivity meter, employing an epoxy-coated Pt or Au dip cell, was calibrated and then standardized with a broad series o~ potassium chloride (KCl) solutions. The blank solution was examined in the same fashion as the KCl standard solutions and its conductivity determined mathematically from an equation resulting from a simple linear fit of the KCl standard data.

CA 02243~l2 l998-07-l~
WO9ii26012 PCT/US97/00790 .

Example 3 Human ObesitY Protein Formula~ion To generate a solution of ~uman Obesity Protein (hOb~, the solid bulk material, lyophilized from a neutral water solution, was redissolved in water to generate a stock solution (stock 1). The concentration of hOb in stock 1 was verified by UV/Vis Spectrophotometry by multiplying the maximum spectral absorbance (279nm or 280nm) by the dilution factor divided by the ~nown extinction coefficient ~or hob at the maximum spectral absorbance (279nm or 280nm). A stock preservative solution containing m-cresol, for example, was prepared by dissolving the neat liquid in water (stock 2). A
stock of an isotonicity agent, such as glycerin was prepared by dissolving the neat liquid in water ~stock 3). A so~ution of hOb at,l.6 mg/mL was prepared by addition of an aliquot of stock 1 to a container which held an aliquot of stock 2 along with an aliquot of stock 3, and the required r~uantity of water, at an alkaline pH (7.8+0.1); adjusted if absolutely necessary with trace ~L quantities of ~Cl or NaOH. After an appropriate incubation period at room temperature (30 minutes), the pH was ~mined and adjusted if absolutely necessary with trace ~L quantities of HCl or NaOH; to yield an hOb solution at 1.6 mg/mL with 0.3% m-cresol and 16 mg/mL
glycerin at pH 7.8i0.1. The solution was then hand-~iltered using a glass syringe with an attached 0.22~m syringe filter into a glass vial. In order to verify the m;nim~l ionic strength of the sample (approachin0 0 mM), a blank solution (blank) was prepared to correspond to the hOb solution, except for the absence o~ stock 1 A conductivity meter, employing an epoxy-coated Pt or Au dip cell, was r~l;hrated and then standardized with a broad series o~ potassium chloride (KCl) solutions. The blank solution was examined in the same ~ashion as the KCl standard solutions and its conductivity determined mathematically from an equation resulting ~rom a simple linear fit of the KCl standard data.

CA 02243~l2 l998-07-l~
WOg7/26012 PCT~S97/00790 .

Example 4 Human Obesitv Protein Formulation To generate a solution of Human Obesity Protein (hOb), the solid bulk material, lyophilized from a neutral water solution, was redissolved in water to generate a stock solution (stock 1). The concentration of hOb in stock 1 was verified by W /Vis Spectrophotometry by multiplying the maximum spectral absorbance (279nm or 280nm) by the dilution factor divided by the known extinction coefficient for hOb at the maximum spectral absorbance (279nm or 280nm). A stock preservative solution containing m-cresol, for example, was prepared by dissolving the neat liquid in water (stock 2). A
stock of an isotonicity agent, such as glycerin was prepared by dissolving the neat li~uid in water (stock 3). A stock solution of sodium chloride (NaCl), typically 0.1 M or 0.2 M, was prepared by dissolving the solid in water (stock 4). A
solution of hOb at 1.6 mg/mL was prepared by addition of an aliquot of stock 1 to a container which held an aliquot of stock 2 along with an aliquot of stock 3 with the required quantity of water, at an alkaline pH (7.8+0.1); adjusted if absolutely necessary with trace ~L quantities of HCl or NaOH.
After an appropriate incubation period at room temperature t30 minutes); an ali~uot of stock 4 was added to provide a low, added ionic strength to the solution. The pH was examined and adjusted if absolutely necessary with trace ~L
quantities of HCl or NaOH, to yield an hOb solution at 1.6 mg/mL with 0.3% m-cresol and 16 mg/mL glycerin at pH
7.8+0.1; with a low ionic strength, approximately 2.5 mM.
The solution was then hand-filtered using a glass syringe with an attached 0.22~m syringe filter into a glass vial. In order to verify the low ionic strength of the sample, a blank solution ~blank) was prepared to correspond to the hOb solution, except for the absence of stock 1. A conductivity meter, employing an epoxy-coated Pt or Au dip cell, was calibrated and then standardized with a broad series of potassium chloride (KCl) solutions. The blank solution was examined in the same fashion as the KCl standard solutions CA 02243~12 1998-07-l~
WO97/26012 PCT~S97/0~790 .

and its conductivity determined mathematically from an e~uation resulting from a simple linear fit of the KCl standard data.

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 ~orms disclosed, since they are to 10- 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.
.

Claims (16)

We claim:

1. A soluble parenteral formulation, comprising an obesity protein and a preservative, said formulation having an ionic strength of less than about 10 mM.

2. A formulation of Claim 1, wherein the obesity protein is the human obesity protein.

3. A formulation of Claim 1, wherein the ionic strength is less than about 5 mM.

4. A formulation of Claim 3, wherein the ionic strength is less than about 1 mM.

5. A soluble parenteral formulation, comprising an obesity protein and a preservative selected from the group consisting of phenol, cresol, or a combination thereof, said formulation having a ionic strength of less than 10 mM.

6. A formulation of Claim 5, wherein the preservative is selected from the group consisting of phenol, m- cresol, or a combination thereof.

7. A formulation of Claim 6, wherein the concentration of obesity protein is about 1.0 mg/mL to about 100 mg/mL.

8. A formulation of Claim 7, wherein the ionic strength is less than 5 mM.

9. A formulation of Claim 8, wherein the concentration of obesity protein is about 5.0 mg/mL to about 50.0 mg/mL.

10. A formulation of Claim 9, which further comprises an isotonicity agent.

11. A formulation of Claim 10, which further comprises a physiologically acceptable buffer.

12. A formulation of Claim 11, wherein the concentration of obesity protein is 5.0 to 10 mg/mL, the preservative is m-cresol, and the physiologically acceptable buffer is sodium phosphate at a concentration less than about 1.4 mM.

13. A formulation of Claim 12, wherein the obesity protein is the human obesity protein.

14. A process for preparing a soluble parenteral formulation of Claim 1, which comprises mixing an obesity protein and a preservative, such that said formulation has an ionic strength of less than about 10 mM.

15. A method of treating obesity in a mammal in need thereof, which comprises administering to said mammal a soluble parenteral formulation of Claim 1.

16. A formulation as claimed in any one of Claims 1 through 13 for use in the treatment of obesity.