CA2243229A1 - Obesity protein formulations - Google Patents
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- CA2243229A1 CA2243229A1 CA 2243229 CA2243229A CA2243229A1 CA 2243229 A1 CA2243229 A1 CA 2243229A1 CA 2243229 CA2243229 CA 2243229 CA 2243229 A CA2243229 A CA 2243229A CA 2243229 A1 CA2243229 A1 CA 2243229A1
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Abstract
The present invention discloses a soluble parenteral formulation, comprising obesity protein and a preservative selected from the group consisting of alkylparaben, chlorobutanol, or a mixture thereof.
Description
CA 02243229 1998-07-1~
W O 9i~26004 PCT~US97/00864 Obesity Protein Formulations This application claims the benefit of U.S.
Provisional Application No. 60/010,229, filed January 19, t1996 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.
1~ 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 stat stics, more than 25% of the United States population and 27~ of the Canadian population are overweight. Kuczmarski, Amer. J. of Clin. Mutr. 55: 495S - 502S (1992); Reeder et al., Can. Med. ASs. 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 for the medical cost of obesity are $150,000,~00,000 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, hyper~ension, and insulin resistance. Many studies have demonstrated that reduction in obesity by diet and exercise reduces these risk factors dramatically. Unfortunately, treatments are largely unsuccessful with a failure rate reaching 95%. This failure may be due to the fact that the condi~ion is strongly associated with genetically inherited factors that contribute to increased appetite, preference for highly caloric foods, reduced physical activity, and increased lipogenic metabolism. This indicates that people ~5 inheriting these genetic traits are prone to becoming obese regardless of their effor~s to combat the condition.
CA 02243229 1998-07-1~
W O 97~26004 PCT~US97/00864 Therefore, a pharmacological agent that can correct this adiposity handicap and allow the physician to success~ully 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 of the mouse gene linked with this condition. Yiying Zhang et al. Nature ~ 425-32 ~19~4). This report disclosed the murine and human protein expressed in adipose tissue. Likewise, Murakami et a~., in Biochemical and Bio~hvsical Research Communications 209(3):944-52 (1995) report the cloning and expression of the rat obese gene. 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).
A parenteral formulation containing 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 ~ormulations. The insoluble formulations must ~irst dissolve prior to adsorption. It is hypothesized that this step has significant variability in a subcutaneous depot.
Furthermore, non-native 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 formulation 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 parente~al ~ormulation exceedingly di~icult. The molecular interactions amongst the preservative, bu~fer, ionic CA 02243229 1998-07-1~
W O 97/261)U4 PCT~S97/00864 :
strength, pH, temperature, and any additional excipients such as a surfactant, or sugar are hi~hly unpredictable in view of ~ the propensity for the obesity protein to ag~regate 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 physical stability of the ~ormulation is greatly enhanced in the presence o~ methylparaben, propylparaben, butylparaben, chlorobutanol or a mixture thereof. That is, when formulated under the conditions described herein, the obesity protein remains soluble at much higher concentrations and at a pH range acceptable for a soluble, parenteral ~ormulation. Accordingly, the present invention provides a soluble, parenteral formulations of an obesity protein.
This invention provides a soluble parenteral formulation, comprising an obesity protein and a preservative selected from the group consisting of alkylparaben, chlorobutanol, or a mixture thereof.
The invention further provides a process for preparing a soluble parenteral formulation, which comprises mixing an obesity protein and a preservative selected ~rom the group consisting of alkylparaben, chlorobutanol, or a mixture thereof.
Additionally, the invention provides a method of treating obesity in a mammal in need thereof, which comprises administering to said mammal a soluble parenteral formulation of the present invention.
For purposes of the present invention, as disclosed and -laimed herein, the following terms and abbreviations are de~ined as follows:
Alkylparaben -- refers to a Cl to C4 alkylparaben.
Preferably, alkylparaben is methylparaben, ethylparaben, ~ prop~ylparaben, or butylparaben.
Base pair (bp) -- refers to DNA or RMA. The abbreviations A,C,G, and T correspond to the 5'-monophosphate forms of the nucleotides (deoxy)adenine, (deoxy)cytidine, W O 9il26004 PCTGUS97/0086~
~deo~)guanine, and (deoxy)thymine, respectively, when they occur in DNA molecules. The abbreviations U,C,G, and T
correspond to the 5'-monophosphate forms of the nucleosides uracil, cytidine, guanine, and thymine, respectively when they occur in RNA molecules. In double stranded DNA, base pair may refer to a partnership of A with T or C with G. In a DNA/RNA heteroduplex, base pair may refer to a partnership of T with A or ~ with G.
Obesity protein -- refers to the native m~mm~l ian protein produced from the native ob gene following transcription and deletions of introns, translation to a protein and processing to the mature protein with secretory signal peptide removed, e.g. from the N-terminal valine-proline to the C-terminal cysteine of the mature protein.
The human obesity protein is published in Zhang et al.
Nature 372: 425-32 (1994). The rat obesity protein is published in Murakami et al., Biochemical and Bio~h~sical Research Comm. 209(3): 944-52 (1995). Native porcine and bovine Ob proteins are disclosed in a U.S. patent application by Hansen M. Hsiung and Dennis P. Smith filed May 19, 1995, Serial number 08/445,305 (EP0 743 321). Other mammalian Ob proteins are disclosed in U.S. patent application serial number 08f452,228, filed May 26, 1995 (EP0 744 408), U.S.
provisional application, 60/003935, filed September 19, 1995 (EP ). Obesity protein includes those proteins having a leader sequence. A leader sequence is one or more amino acids on the M-terminus to aid in production or puriflcation of the protein. A preferred leader sequence is Met-Rl-wherein Rl is absent or any amino acid except Pro.
Plasmid -- an extrachromosomal self-replicating genetic element.
Reading frame -- the nucleotide sequence from which translation occurs "readl' 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 CA 02243229 1998-07-1~
W O gil26~04 PCT~US97/00864 or deletion (termed a frameshift mutation) may result in two di~ferent 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 ~Ireading frame"
must be maintained. In the creation of fusion proteins containing a chelating peptide, the reading frame of the DNA
sequence encoding the structural protein must be maintained in the DNA sequence encoding the chelating peptide.
Recombinant DNA Cloning Vector -- any autonomously replicating agent including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
Recombinant DNA Expression Vector -- any recombinant DNA cloning vector in which a promoter has been incorporated.
Replicon -- A DNA sequence that controls and allows for autonomous replication of a plasmid or other vector.
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.
Vector -- a replicon used ~or 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, virus~s, and bacteriophage are suitable vectors, since they are replicons in their own right. Artificial vectors are constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases.
Vectors include Recombinant DNA cloning vectors and Recombinant DNA expression vectors.
Treating -- as used herein, describes the management and care of a patient for the purpose of combating CA 02243229 1998-07-1~
W O 9i/2600~ PCT~US97/00864 .
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 administration of the protein for cosmetic purposes. A
cosmetic purpose seeks to control the weight of a mammal to improve bodily appearance.
Isotonicity agent -- isotonicity agent refers to an agent that is physiologically tolerated and embarks a suitable tonicity to the ~ormulation to prevent the net flow of water across the cell membrane. Compounds, such as glycerin, are commonly used ~or such purposes at known concentrations. Other possible isotonicity agents include salts, e.g., MaCl, dextrose, mannitol, and lactose.
Physiologically tolerated buffer -- a physiologically tolerated bu~er is known in the art. A
physiologically tolerated buf~er is preferably a phosphate buffer, like sodium phosphate. Other physiologically tolerated buffers 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 Office as set forth in 37 C.F.R. ~ 1.822 (b)(2) (1993). Unless otherwise indicated the amino acids are in the L
configuration.
As noted above, the invention provides a soluble parenteral formulation, comprising human obesity protein and a preservative selected ~rom the group consisting of an alkylparaben or chlorobutanol. In the presence of these preservatives, the obesity proteins r~m~i n~ in solution making a soluble, parenteral ~ormulation possible.
A parenteral formulation must meet guidelines ~or preservative effectiveness to be a commercially viable product. Pharmaceutical preservatives known in the art as being acceptable in parenteral ~ormulations include: phenol, CA 02243229 1998-07-1~
W O 971260~4 PCTnUS97/00864 m-cresol, benzyl alcohol, methylparaben, chlorobutanol, p-cresol, phenylmercuric nitrate, thimerosal and various mixtllres thereof. See, e.g., WALLHAUSER, K.--H., DEVE:LoP. BIOL.
STANDARD. 24, pp. 9-28 (sasel~ s. Krager, 1974).
~ 5 Most unexpectedly, a select number of preservatives have been identified which provide good ~ormulation stability These select preservatives are an alkylparaben or chlorobutanol. Most preferrably, the preservative is meth~lparaben, propylparaben, or butylparaben. Most unexpectedly, the obesity protein does not aggregate in the presence of these preservatives at the conditions necessary to formulate and particularly conditions at 37~C.
The concentration of obesity protein in the formulation is about 1.0 mg/mL to about 100 mg/mL; preferably about 5.0 mg/mL to about 50.0 mg/mL; most preferably, about 10.0 mg/mL. The concentration of preservative required is the concentration necessary to maintain preservative effectiveness. The relative amounts of preservative necessary to maintain preservative effectiveness varies with the preservative used. Generally, the amount necessary can be found in WALLHAUSER, K.--H., DEVELOP. BIoL. STANDARD~ 24, pp.
9-28 (Basel, S. Krager, 1974), herein incorporated by reference. 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 of the isotonicity agent is in the range known in the art for parenteral formulations, preferably about 1 to 20 mg/mL, more preferably about 8 to 16 mg/mL, and still more preferably about 16 mg/mL. The pH of the formulation may also be ~ buffered with a physiologically tolerated buffer, preferably a phosphate buffer, like sodium phosphate. Other acceptable physiologically tolerated buffers include TRIS, sodium acetate, or sodium citrate. The selection and concentration of buffer is known in the art; however, the formulations of CA 02243229 1998-07-1~
W O 9il260Q4 PCT~US97/00864 .
the present invention are preferably prepared the mln;m~lly accpetable concentration of buffer.
Other additives, such as a pharmaceutically acceptable solubilizers like Tween 2~ (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyo ~ ethylene polyoxypropylene block copolymers), BRIJ 35 (polyoxyethylene (23) lauryl ether), 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. The presence of pharmaceutically acceptable surfactant mitigates the propensity for the protein to aggregate.
Also included as optional embodiments are agents known to be synergistic with the preservative to provide enhanced antimicrobial effect. Such agents are recognized in the art and include for example ethylene diaminetetraacetic acid (EDTA), 1,2-di(2-aminoethoxy)ethane-N,N,M'N'-tetraacetic acid (EGTA), citrate, and caprylic acid. The concentration of these agents varies with the desired preservation effect.
A preferred agent is EDTA, particularly in conjunction with an alkylparaben at a concentration of about 0.025% to 0.4%.
Notably, in preparations including EDTA or EGTA, the buffer concentration is reduced to minim; ze ionic strength.
The present formulations may optionally contain a physiologically tolerated solvent such as glycerol, propylene glycol, phenoxy ethanol, phenyl ethyl alcohol. Such solvents are generally added to enhance the solubility of the protein in the preservation effectiveness of the formulation.
The parenteral fo~mulations 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 3~ is combined with the desired preservatlve in water in quantities sufficient to provide the protein and preservative at the desired concentration. The formulation is generally CA 02243229 1998-07-1~
W O 9~/2601)4 PCTrUS97/00864 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 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 preservative e~fect on formulation stability was demonstrated by preparing formulations comprising methylparaben, propylparaben, butylparaben, chlorobutanol, cresol, phenol, benzyl alcohol, or a mixture thereof and comparing the amount o~ protein remaining in solution after 3 days at 4~C and 37~C. The data in Table 1 demonstrate that the stability and solubility of human obesity protein is enhanced in the presence o~ an alkylparaben or chlorobutanol. The formulations used to generate the data of Table 1 were prepared in a manner analogous to Examples 1 and 2.
CA 02243229 1998-07-1~
W O 9~-/26004 PCTrUS97/00864 .
Table 1. Recovery of protein as a function of preservative and conditions. Values are the calculated least square means and standard errors determined from fitting the raw data to a factorial model of the second degree containing the e~ects of preservative, acid excursion, buffer, conditions, and all two factor interactions. (R2 = 0.94, (Prob > F) = 1.96e~39, total observations = 147). Percent of protein in solution is calculated using a theoretical target o~ 1.6 mg protein/mL.
Protein in Solution (% of theoretical) PreservativePreservative 3 days ~ 3 days @
Concentration 4~C 37~C
( ) methylparaben0.17 = 0.02 ~7." = . . = .
propyl~araben0.16 0.02 9. =
butylparaben 0.~15 ,7. = ~
chlorobutanol 0.5 87., = ~-. 8r. = ~.
methylparaben ~0.18 + 0.02 76.8 = ~. 6 .
propy~paraben0.017 + 0.002 ~enzy alcohol 1.0 79.8 + 3.6 31.6 + 4.2 methy_paraben +0.16 + 0.02 62.8 + 4.3 16.7 + 4.3 propylparaben +0.02 + 0.002 benzyl :lcohol 0.~
m-creso~ 0. ~ .~- = - ~- - --' p-creso_ 0._ : = , ~. = ~.-phenol 0. J
The stabilizing ef~ect by the alkylparabens is most unexpected in view of the structural similarities to other preser~atives. The data clearly show that the alkylparabens and chlorobutanol are superior at 37~C, which is the temperature re~uired for in-use physical stability testing.
Preferably, the pH of the present formulations is about pH 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 a pH greater than pH 7Ø Preferably, the pH is about 7.6 to 8.0, and most pre~erably about pH 7.8. Ideally, a preservative and water solution is mixed at pH 7.6 to 8Ø
Added 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 dissolved, CA 02243229 l998-07-l5 W O 9~/26004 PCTnUS97/00864 approximately 20 to 40 minutes, preferably about 30 minutes.
The base used to adjust the pH of the formulation may be one or more pharmaceutically acceptable bases such as sodium hydroxide and potassium hydroxide. The preferred base is sodium hydroxide.
The formulations prepared in accordance with the present invention may be used in a syringe, injector, pumps or any other device recognized in the art for parenteral administration.
Preferably, the obesity protein employed in the formulations of the present invention is human obesity prot~in of SEQ ID NO:1.
lo 15 Val Pl~c Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr 2c 25 30 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 ~eu His Pro Ile 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 Hi~ L~u Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 3 5 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly C~s (SEQ ID NO:1) The obesity protein employed in the formulations of the present invention can be prepared by any of a variety of reco~-nized peptide synthesis techniques including classical ~solution) methods, solid phase methods, semi synthetic methods, and more recent recombinant DNA methods. The preparation of the human obesity protein is known and CA 02243229 1998-07-1~
W O 97/26004 PCT~US97/00864 .
disclosed, for example, in Halaas Jeffrey L. et al., Science 269 ~1995). The preparation of other ~mm~l ian obesity proteins are 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,~28, filed May 26, 1995 (EP0 744 408); and provisional application, 60,003935, filed September 19, 1995, (EP ); all of which are herein incorporated by reference.
The obesity proteins described herein may also be produced either ~y recombinant DNA technology or well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution methods. Recombinant methods are preferred if a high yield is desired. The basic steps in the recombinant production of protein include:
a) construction of a synthetic or semi-synthetic (or isolation from natural sources) DNA
encoding the obesity protein, b) integrating the coding se~uence into an expression vector in a manner suitable for the expression of the protein either alone or as a fusion protein, c) transforming an appropriate eukaryotic or prokaryotic host cell with the expression vector, and d) recovering and purifying the recombinantly produced protein.
Synthetic genes, the n vitro or n vivo transcription and translation o~ which will result in the production of the protein may be constructed by techniques well known in the art. Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of DNA sequences may be constructed which encode the desired proteins. In the preferred practice of the invention, synthesis is achieved by recombinant DNA
technology.
CA 02243229 l998-07-l~
w o ~7~26(~n4 PCT~US97/00864 Methodology of synthetic gene construction is well known in the art. For example, see Brown, et al. (1979) Methods in Enzymology, Academic Press, N.Y., Vol. 68, pgs 109-151. ~he 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 380B DNA synthesizers (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404). It may desirable in some applications to modify the coding se~uence of the obesity protein so as to incorporate a convenient protease sensitive cleavage site, e.g., between the signal peptide and the structural protein facilitating the controlled excision of the signal peptide from the ~usion protein construct The gene encoding the obesity protein may also be created ~y using polymerase chain reaction (PCR). The template can be a cDNA library (commercially available from CLOMETECH or STRATAGENE) or mRNA isolated from the desired arrival adipose tissue. Such methodologies are well known in the ~rt Maniatis, et al. Molecular Clonina: A Laboratorv Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold ~pring Harbor, New York (1989).
The constructed or isolated DNA sequences are usef-,ll for expressing the obesity protein either by direct expression or as fusion protein. When the sequences are used in a fusion gene, the resulting product will require enzymatic or chemical cleavage. A variety of peptidases which cleave a polypeptide at specific sites or digest the peptides from the amino or carboxy termini (e.g.
diaminopeptidase) of the peptide chain are known.
Furthermore, particular chemicals (e.g. cyanogen ~romide) - will cleave a polypeptide chain at specific sites. The skille~ artisan will appreciate the modifications necessary - to tl1e amino acid sequence (and synthetic or semi-synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See U.S.
Patent No. 5,126,249; Carter P., Site Specific Proteolysis CA 02243229 1998-07-1~
W O 97f26004 PCT~US97/00864 .
~ 14 -of Fusion Proteins, Ch. 13 in Protein Purification: From Molecular Mechanisms to Larae Scale Processes, American Chemical Soc., Washlngton, D.C. (1990).
Construction of suitable vectors containing the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or DMA 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 o~ a plethora o~ appropriate recombinant DNA expression vectors through the use of appropriate restriction erldonucleases. 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 isola~ed 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 ~y techni~ues well known in the art. The particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent expression vector to be employed. The restriction sites are chosen so as to properly orien~ 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 origin 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 ~ coli species (Bolivar, et al., Gene 2: 95 (~977)).
Plasmid pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial CA 02243229 1998-07-1~
W O 97126004 PCTrUS97/00864 plasmid must also contain or be modified to contain promoters and other control elements commonly used in recombinant DMA
technology.
The desired coding sequence is inserted into an - 5 expression vector in the proper orientation to be transcribed ~rom a promoter and ribosome binding site, both of which should be ~unctional in the host cell in which the protein is to b~ expressed. An example o~ 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 re~erence. The gene encoding A-C-s proinsulin described in U.S. patent No. 5,304,493 can be removed from the plasmid pRB182 with restriction enzymes ~I and 3amHI. The isolated DNA ~equences can be inserted into the plasmid backbone on a NdeI/BamHI restriction ~ragment cassette.
In general, procaryotes are used for cloning o~ D~A
sequences in constructing the vectors use~ul in the invention. For example, E. coli K12 strain 294 (ATCC No.
31446) is particularly use~ul. Other microbial strains which may be used include E. CQli B and E. 5~,~ X1776 (ATCC No.
31537). These examples are illustrative rather than limiting.
Procaryotes also are used for expression. The a~orementioned strains, as well as E. coli W3110 (prototrophic, ATCC No. 27325), bacilli such as Bacillus subtilis, and other enterobacteriaceae such as ~almonella tv~himurium or Serratia marcescans, and various pseudomonas species may be used. Promoters suitable for use with prokaryotic hosts include the ~-lactamase ~vector pGX2907 [ATCC 39344] contains the replicon and ~-lactamase gene) and lactose promoter systems (Chang et al., Nature, 275:615 (1978); and Goeddel et al., Nature 281:544 (1979)), alkaline phosphatase, the tryptophan (trp) promoter system (vector pATHl [ATCC 37695] is designed to ~acilitate expression o~ an open reading frame as a trpE ~usion protein under control o~
the trp promoter) and hybrid promoters such as the tac promoter (isolatable ~rom plasmid pDR54Q ATCC-37282).
However, other ~unctional bacterial promoters, whose CA 02243229 l998-07-l~
W O 97/26004 PCT~US97/00864 nucleotide sequences are ~enerally 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.
The DNA molecules may also be recom~inantly produced in eukaryotic expression systems. Preferred promoters controlling transcription in mammalian host cells may be obtained ~rom various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. ~-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication. Fiers, 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 species also are useful herein.
Transcription of the DNA by higher eucaryotes is increased by inserting an enhancer sequence into the vector.
Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that act on a promoter to increase its transcription.
Enhancers are relatively oriented and positioned independently and have been found 5' (T,~;mln~, L. et ~l., PNAS 78:993 (1981)) and 3' (Lusky/ M. L., et al., Mol. Cell Bio. 3:1108 (1983)) to the transcription unit, within an intron (Banerji, J. L. et al., Cell 33:729 (1983)) as well as within the coding sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4:1293 (1984)). Many enhancer sequences are now known from m~mm~lian gene-s (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 CA 02243229 1998-07-1~
W O 97/26~04 PCTrUS97/00864 - ~7 -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, ~ungi, insect, plant, animal, human or nucleated cells from other multicellular organisms) will also contain 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.
Expression vectors may contain a selection gene, also termed a selecta~le marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR, which may be derived from the BalII/HindIII
restriction fragment of pJOD-10 [ATCC 68815]), thymidine kinase (herpes simplex virus thymidine kinase is contained on the E~mHI fragment of vP-5 clone [ATCC 2028]) or neomycin (G418) resistance genes (obtainable ~rom pNN414 yeast artificial chromosome vector [ATCC 37682]). When such selectable markers are successfully transferred into a m~mm~l ian host cell, the transfected mammalian host cell can survive i~ placed under selective pressure. There are two widely used distinct categories of selective regimes. The 25 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 30 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 r alternative to supplementing the media is to introduce an 35 intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual CA 02243229 l998-07-l~
W O 97/26004 P~l~S97/00864 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 domin~nt 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 (1985). The three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively.
A preferred vector for eucaryotic expression is pRc/CMV. pRc/CMV is commercially available from Invitrogen Corporation, 3985 Sorrento Valley Blvd., San Diego, CA
92121. To confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform E. coli K12 strain DHlOB (ATCC 31446) and successful transformants selected by antibiotic resistance where appropriate.
Plasmids from the transformants are prepared, analyzed by restriction and/or sequence by the method of Messing, et al., Nucleic Acids Res. 9:309 (1981).
Host cells may be transformed with the expression vectors of this invention and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. The techniques of transforming cells with the aforementioned vectors are well known in the art and may be found in such general references as ManiatiS, ~t al., W O 9~/260D4 PCTrUS97/00864 Molecular Clonina: A La~oratory Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), or Cl]rrent Protocols in Molecular Bioloov (1989) and supplements.
Preferred suitable host cells for expressing the vectors encoding the claimed proteins in higher eucaryotes include: African green monkey kidney line cell line transformed by SV40 (COS-7, ATCC CRL-1651); transformed human primary embryonal kidney cell line 293,(Graham, F. L. ~ al., J. Gen Virol. 36:59-72 (1977), Viroloov 77:319-329, Virolo~Y
86:10-21); baby hamster kidney cells (BHK-21(C-13), ATCC CCL-10, Viroloov 16:147 (1962)); Chinese hamster ovary cells CHO-DHFR-- (ATCC CR~-9096), mouse Sertoli cells (TM4, ATCC CRL-1715, Biol. Re~rod. 23:243-250 (1980)); African green monkey kidLLey cells (VERO 76, ATCC CRL-1587); human cervical epitheloid carcinoma cells (HeLa, ATCC CCL-2); canine kidney cells (MDCK, ATCC CC~-34)i buffalo rat liver cells (BRL 3A, ATCC CRL-1442); human diploid lung cells (WI-38, ATCC CCL-75); human hepatocellular carcinoma cells (Hep G2, ATCC HB-8065);and mouse m~mm~ry tumor cells (MMT 060562, ATCC CCL51).
In addition to prokaryotes, unicellular eukaryotes such as yeast cultures may also be used. Saccharom~ces cere~isiae, or common baker's yeast is the most commonly used euka~yotic microorganism, although a number of other strains are commorLly available. For expression in Saccharomyces, the plasmid YRp7, for example, (ATCC-40053, Stinchcomb, et al., 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 of yeast lacking the ability to grow in tryptophan, for example ATCC no. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)).
Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (found on plasmid pAP12BD ATCC 53231 and described in U.S.
Patent No. 4,935,350, June 19, 1990) or other glycolytic enzymes such as enolase ~found on plasmid pAC1 ATCC 39532), CA 02243229 1998-07-1~
W O 97/26004 PCT~US97/00864 glyceraldehyde-3-phosphate dehydrogenase (derived from plasmid pHcGAPC1 ATCC 57090, 57091), zymomonas mobilis (United States Patent No. 5,000,000 issued March 19, 1991), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate ~inase, 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 al., 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--h~lbeta 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.
The scope of the present invention is not to be construed as merely consisting of the following examples.
Pre~aration 1 ~orcine OB Gene and Gene Product Total RNA was isolated from porcine fat tissue obtained from Pel~Freez~, Pel-Freez Inc., and the cDNA was cloned in accordance with the techniques described in Hsiung et al., Neuro~e~tide 25: 1-10 (1994).
Primers were designed based on the published amino acid sequence of the human o~ gene. The primers were prepared for use in polymerase chain reaction (PCR) CA 02243229 1998-07-1~
W O 9~260~4 PCTrUS97/00864 amplification methods using a Model 380A DNA synthesizers (PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, 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
~ 5 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 RMA 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 for 10 minutes at 70~C and then cooled on ice. The following were then added to the annealed mixture: 4 ~L o~
BRL 5x H-reverse transcriptase (RT) reaction buffer (Gibco-BRL CAT#28025-013), 2 ~L of 0.1 M DTT, 1 ~L of lQ mM dNTPs.
This annealed mixture was then incubated at 37~C for 2 minutes before adding 1 ~L BRL M-MLV-reverse transcriptase (200 U/~L) (CAT#28025-013) and incubated at 37~C for additional 1 hour. After incubation the mixture was heated at 95~C for 5 minutes and then was cooled on ice.
For amplification of cD~A, the polymerase chain reaction (PCR) was carried out in a reaction mixture (100 ~L) containing the above cDNA reaction mixture (1 ~L), 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer Corporation) 10 ~1 of 10x PCR reaction bu~er (Perkin-Elmer Corporation) and 50 pmol each of the sense (PCROB-l) and antisense (PCROB-3) primers for porcine OB amplification. The condition ~or PCR
was 9~~C ~or 1 minute, 57~C ~or 1 minute and 72-~C for 1 minute ~or 30 cycles using a PCR DNA Thermal Cycler (Perkin-Elmer Corporation). After PCR amplification, 5 ~L BRL T4 DNA
polymerase (5 U/~L), 2 ~L BRL T4 polynucleotide kinase (10 U/~L), and 5 ~L ATP (10 mM) were added to the PCR reaction mixtu:-e (100 ~1) directly and incubated for 30 minutes at 37~C. After the incubation the reaction mixture was heated at 95~C for 5 minutes and then was cooled on ice. The 500 bp ~ragment (~0.5 mg) was purified by agarose gel electrophoresis and isolated by the freeze-squeeze method.
The 500 bp fragment (_0.2 ~g) was then ligated into SmaI
CA 02243229 1998-07-1~
W O 97/26004 PCT~US97/00864 linearized pUC18 plasmid (~1 ~g) 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 ~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 (lQ0 ~g/mL). The plasmid was isolated using a Wizard Miniprep DNA
purification system (Promega) and submitted for DNA
seqeuncing on a Applied Biosystem 370 DNA sequencer.
Pre~aration 2 Bovine OB Gene and Gene Product The DNA sequence o~ the bovine OB gene was obtained by techniques analogous to Example 1, except sense (PCROB-2) and antisense (PCRoB-3) primers were used for bovine OB cDNA
amplification.
Pre~aration 3 Vector Construction A plasmid containing the DNA sequence encoding the obesity protein is constructed to include NdeI and ~HI
restriction sites. The plasmid carrying the cloned PCR
product is digested with ~I 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 F.. 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 ~mHI fragment are kept. E. coli K12 RV308 (available from the NRRL under deposit number B-15624) are transformed with this second plasmid, resulting in a culture suitable for expressing the protein.
The techniques of transforming cells with the aforementioned vectors are well known in the art and may be CA 02243229 l998-07-l~
WO 9ii260~4 PCT~US97tO0864 found in such general references as Maniatis, et al. (1988) Molecular Clonin~: A Laboratorv Manual, Cold Spring ~Iarbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York or Cllrrent Protocols in Molecular Biolo~v ~1989) and supplements. The techniques involved in the trans~ormation of E. coli cells used in the preferred practice of the invention as exemplified herein are well known in the art.
The precise conditions under wh-ich the transformed ~ coli ce~ls are cultured is dependent on the nature of the ~ coli 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 from about 30 to about 40 degrees C. in the culture conditions so as to induce protein synthesis.
In the preferred embodiment of the invention, ~.
SQ~i K12 RV308 cells are employed as host cells but numerous other cell lines are available such as, but not limited to, F 5~L~ K12 L201, L687, L693, L5~7, L640, L641, L695, L814 (E cQli B). The transformed host cells are then plated on appropriate media under the selective pressure of the an~ibiotic corresponding to the resistance gene present on the expression plasmid. The cultures are then incubated for 2S a t:ime and temperature appropriate to the host cell line emE)loyed .
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 Foldin~, 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 techni~ues us~ng strongly denaturing solutions such as guanidinium-HCl and/or weakly denaturing solutions such as dithiothreitol 5~ TE SHEET(RULE 2~) CA 02243229 1998-07-1~
W O g7/26004 PCT~US97/00864 .
(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 efficie~t 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 formuations of the invention The scope of the inve~tion is not construed as merely consisting of the following examples.
Example 1 Human Obesitv Protein Formulation To generate a solution of Human Obesity Protein (hOb), 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 Spectrophometry using the known extinction coe~ficient for hOb at the maximum spectral absorbance (279nm or 280nm), measuring the maximum spectral absorbance (279nm or 280nm), and utilizing the dilution factor. A stock preservative solution containing methylparaben was prepared by dissolving the solid 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 aliquot of stock 2 along with the required quantity of water, at an alkaline pH (7.8~0.3)i adjusted if necessary with HCl or NaOH. After an appropriate incubation period at room temperature (30 minutes), the CA 02243229 1998-07-1~
wo s7/26ao4 PCT/US97/00864 solution pH was examined and adjusted if necessary with trace ~L ~uantities of HCl or NaOH, to yield an hOb solution at 1.6 mg/mL with 0.17% methylparaben 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.
Example 2 Human Obesitv Protein Solution To generate a solution of Human Obesity Protein (hOb~, 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 Spectrophometry using the known extinction coefficient for hOb at the maximum spectral absorbance (279nm or 280nm), measuring the maximum spectral absorbance (279nm or 280nm), and utilizing the dilution factor. A stock preservative solution containing chlorobutanol, for example, was prepared by dissolving the solid in water (stock 2). A solution of hOb at 1.6 mg/mL was prepared by addition of an ali~uot of stock 1 to a container which held an aliquot of stock 2 along with the required quantity o~ water, at an alkaline pH
(7.8+0.3); adjusted if necessary with HCl or NaOH. After an appropriate incubation period at room temperature (30 minutes), the solution pH was examined and adjusted if necessary with trace ~L quantities of HCl or NaOH, to yield an hOb solution at 1.6 mg/mL with 0.50% chlorobutanol 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.
Example 3 Human Obesitv Protein Solution 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 Spectrophometry by multiplying the maximum CA 02243229 1998-07-1~
W O 97t26004 PCTrUS97/00864 spectral absorbance (279nm or 280nm) by the dilution factor divided by the known extinction coef~icient for hOb. A stock preservative solution containing methylparaben was prepared by dissolving the solid 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 stock of a physiologically-tolerated buffer, such as sodium phosphate 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 along with the required quantity of water, adjusted if necessary with HCl or NaOH to an alkaline pH (7.8+0.3). After an approp~iate incubation period at room temperature (30 minutes) an aliquot of stock 4 was added. The solution pH was then readjusted if necessary with trace ~ quantities of HCl or NaO~, to yield an hOb solution at 1.6 mg/mL with 0.17% methylparaben, 16 mg/mL glycerin, and 14mM sodium phosphate at 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.
Example 4 Hllm~n Obesitv Protein Solution 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 UV/ViS Spectrophometry by multiplying the maximum spectral absorbance (279nm or 280nm) by the dilution factor divided by the known extinction coefficient for hOb. A stock preservative solution containing chlorobutanol was prepared by dissolving the solid in water (stock 2). A stock of an isotonicity agent, such as glycerin, was prepared by dissolving the nea~ liquid in water (stock 3). A stock of a physiologically-tolerated buffer, such as sodium phosphate 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 CA 02243229 1998-07-1~
W O 97126al04 PCTAUS97/00864 aliquot of stock 1 to a container which held an aliquot of stock 2 along with an aliquot of stock 3 along with the required ~uantity of water, adjusted if necessary with HCl or NaOH to an alkaline pH (7.8+0.3). After an appropriate incubation period at room temperature (30 minutes) an aliquot of stock 4 was added. The solution pH was then readjusted if necessary with trace ~L quantities of HCl or NaOH, to yield an hOb solution,.for example, at 1.6 mg/mL with 0.5%
chlorobutanol, 16 mg/mL glycerin, and 14mM sodium phosphate (if required by the formulation) at 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.
Example 5 Pre~aration of Human Obesitv Protein Solution To generate a preserved solution of human obesity protein (hOb), the individual solution components were added in succession to a neutral water solution, allowing each component to dissolve prior to addition of the next component. To the container were added: approximately 5-6 mL of sterile water (~inal solution volume = 10.00 mL) followed by preservative solids (1.51 mg of butylparaben;
final concentration = 0.015~). The solution was mixed at medium speed with a Te~lon stir bar and heat was applied to promote dissolution o~ the solids. Care was exercised to dissolve the preservative solids, but not to overheat or boil the solution. A~ter dissolution was achieved (approximately 1-1.5 hr.), the solution was allowed to cool to room temperature while maintaining stirring. Glycerin (160.0 mg) was then added to the solution and stirred until dissolution was achieved. The final glycerin concentration was targeted to be 16 mg/mL. A 10.0 mg quantity o~ solid ethylenediamine tetra_etate (EDTA) was added to the solution and stirred to dissolve (final concentration = 0.10%). Dibasic sodium phosphate crystals (17 0 mg) were then added and allowed to dissolve by stirring. The ~inal phosphate concentration was targe~ed at 6.3 mM. Sucrose (600.0 mg) was then added and CA 02243229 1998-07-1~
W O 9~/26004 PCT~US97/00864 allowed to stir until dissolved (final concentration of 60 mg/mL). Bulk lyophilized hOb (115.4 mg) was added incrementally to the stirring solution, allowing each aliquot to dissolve prior to the addition of successive portions.
The quantity of hOb added to the solution yielded a final concentration of 10.2 mg/mL. The amount added was determined by using the "as is" weight ~, determined for the specific hOb lot used by W /Vis spectrophotometry. Following the addition of all bulk ingredients, the pH of the solution was adjusted to 8.5 using approximately 11 ~L of 10~ NaOH to promote dissolution of the solids. After stirring the solution for a 10-15 minute period, the pH was decreased to 7.8 using approximately 4-5 ~L of 10% HCl and the solution was allowed to stir for an additional 10 minutes. The solution was then transferred to a 10 mL volumetric flask, and brought to final volume with sterile water. The ~lask was then inverted 20x to thoroughly mix the contents and transferred to a glass sample vial where a recheck of the pH
yielded 7.78. A trace quantity of 10% NaOH (approximately 1 ~L) was added to yield a final pH of 7.83. The solution was then hand-filtered, using a 5 mL glass syringe with an attached 0.2~m syringe filter into a 20 mL glass vial, capped and stored refrigerated until needed.
The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. ~he invention which is intended to be protected herein, however, is not to be construed as limited to the particular forms disclosed, sinc~ 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.
W O 9i~26004 PCT~US97/00864 Obesity Protein Formulations This application claims the benefit of U.S.
Provisional Application No. 60/010,229, filed January 19, t1996 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.
1~ 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 stat stics, more than 25% of the United States population and 27~ of the Canadian population are overweight. Kuczmarski, Amer. J. of Clin. Mutr. 55: 495S - 502S (1992); Reeder et al., Can. Med. ASs. 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 for the medical cost of obesity are $150,000,~00,000 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, hyper~ension, and insulin resistance. Many studies have demonstrated that reduction in obesity by diet and exercise reduces these risk factors dramatically. Unfortunately, treatments are largely unsuccessful with a failure rate reaching 95%. This failure may be due to the fact that the condi~ion is strongly associated with genetically inherited factors that contribute to increased appetite, preference for highly caloric foods, reduced physical activity, and increased lipogenic metabolism. This indicates that people ~5 inheriting these genetic traits are prone to becoming obese regardless of their effor~s to combat the condition.
CA 02243229 1998-07-1~
W O 97~26004 PCT~US97/00864 Therefore, a pharmacological agent that can correct this adiposity handicap and allow the physician to success~ully 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 of the mouse gene linked with this condition. Yiying Zhang et al. Nature ~ 425-32 ~19~4). This report disclosed the murine and human protein expressed in adipose tissue. Likewise, Murakami et a~., in Biochemical and Bio~hvsical Research Communications 209(3):944-52 (1995) report the cloning and expression of the rat obese gene. 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).
A parenteral formulation containing 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 ~ormulations. The insoluble formulations must ~irst dissolve prior to adsorption. It is hypothesized that this step has significant variability in a subcutaneous depot.
Furthermore, non-native 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 formulation 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 parente~al ~ormulation exceedingly di~icult. The molecular interactions amongst the preservative, bu~fer, ionic CA 02243229 1998-07-1~
W O 97/261)U4 PCT~S97/00864 :
strength, pH, temperature, and any additional excipients such as a surfactant, or sugar are hi~hly unpredictable in view of ~ the propensity for the obesity protein to ag~regate 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 physical stability of the ~ormulation is greatly enhanced in the presence o~ methylparaben, propylparaben, butylparaben, chlorobutanol or a mixture thereof. That is, when formulated under the conditions described herein, the obesity protein remains soluble at much higher concentrations and at a pH range acceptable for a soluble, parenteral ~ormulation. Accordingly, the present invention provides a soluble, parenteral formulations of an obesity protein.
This invention provides a soluble parenteral formulation, comprising an obesity protein and a preservative selected from the group consisting of alkylparaben, chlorobutanol, or a mixture thereof.
The invention further provides a process for preparing a soluble parenteral formulation, which comprises mixing an obesity protein and a preservative selected ~rom the group consisting of alkylparaben, chlorobutanol, or a mixture thereof.
Additionally, the invention provides a method of treating obesity in a mammal in need thereof, which comprises administering to said mammal a soluble parenteral formulation of the present invention.
For purposes of the present invention, as disclosed and -laimed herein, the following terms and abbreviations are de~ined as follows:
Alkylparaben -- refers to a Cl to C4 alkylparaben.
Preferably, alkylparaben is methylparaben, ethylparaben, ~ prop~ylparaben, or butylparaben.
Base pair (bp) -- refers to DNA or RMA. The abbreviations A,C,G, and T correspond to the 5'-monophosphate forms of the nucleotides (deoxy)adenine, (deoxy)cytidine, W O 9il26004 PCTGUS97/0086~
~deo~)guanine, and (deoxy)thymine, respectively, when they occur in DNA molecules. The abbreviations U,C,G, and T
correspond to the 5'-monophosphate forms of the nucleosides uracil, cytidine, guanine, and thymine, respectively when they occur in RNA molecules. In double stranded DNA, base pair may refer to a partnership of A with T or C with G. In a DNA/RNA heteroduplex, base pair may refer to a partnership of T with A or ~ with G.
Obesity protein -- refers to the native m~mm~l ian protein produced from the native ob gene following transcription and deletions of introns, translation to a protein and processing to the mature protein with secretory signal peptide removed, e.g. from the N-terminal valine-proline to the C-terminal cysteine of the mature protein.
The human obesity protein is published in Zhang et al.
Nature 372: 425-32 (1994). The rat obesity protein is published in Murakami et al., Biochemical and Bio~h~sical Research Comm. 209(3): 944-52 (1995). Native porcine and bovine Ob proteins are disclosed in a U.S. patent application by Hansen M. Hsiung and Dennis P. Smith filed May 19, 1995, Serial number 08/445,305 (EP0 743 321). Other mammalian Ob proteins are disclosed in U.S. patent application serial number 08f452,228, filed May 26, 1995 (EP0 744 408), U.S.
provisional application, 60/003935, filed September 19, 1995 (EP ). Obesity protein includes those proteins having a leader sequence. A leader sequence is one or more amino acids on the M-terminus to aid in production or puriflcation of the protein. A preferred leader sequence is Met-Rl-wherein Rl is absent or any amino acid except Pro.
Plasmid -- an extrachromosomal self-replicating genetic element.
Reading frame -- the nucleotide sequence from which translation occurs "readl' 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 CA 02243229 1998-07-1~
W O gil26~04 PCT~US97/00864 or deletion (termed a frameshift mutation) may result in two di~ferent 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 ~Ireading frame"
must be maintained. In the creation of fusion proteins containing a chelating peptide, the reading frame of the DNA
sequence encoding the structural protein must be maintained in the DNA sequence encoding the chelating peptide.
Recombinant DNA Cloning Vector -- any autonomously replicating agent including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DNA segments can or have been added.
Recombinant DNA Expression Vector -- any recombinant DNA cloning vector in which a promoter has been incorporated.
Replicon -- A DNA sequence that controls and allows for autonomous replication of a plasmid or other vector.
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.
Vector -- a replicon used ~or 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, virus~s, and bacteriophage are suitable vectors, since they are replicons in their own right. Artificial vectors are constructed by cutting and joining DNA molecules from different sources using restriction enzymes and ligases.
Vectors include Recombinant DNA cloning vectors and Recombinant DNA expression vectors.
Treating -- as used herein, describes the management and care of a patient for the purpose of combating CA 02243229 1998-07-1~
W O 9i/2600~ PCT~US97/00864 .
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 administration of the protein for cosmetic purposes. A
cosmetic purpose seeks to control the weight of a mammal to improve bodily appearance.
Isotonicity agent -- isotonicity agent refers to an agent that is physiologically tolerated and embarks a suitable tonicity to the ~ormulation to prevent the net flow of water across the cell membrane. Compounds, such as glycerin, are commonly used ~or such purposes at known concentrations. Other possible isotonicity agents include salts, e.g., MaCl, dextrose, mannitol, and lactose.
Physiologically tolerated buffer -- a physiologically tolerated bu~er is known in the art. A
physiologically tolerated buf~er is preferably a phosphate buffer, like sodium phosphate. Other physiologically tolerated buffers 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 Office as set forth in 37 C.F.R. ~ 1.822 (b)(2) (1993). Unless otherwise indicated the amino acids are in the L
configuration.
As noted above, the invention provides a soluble parenteral formulation, comprising human obesity protein and a preservative selected ~rom the group consisting of an alkylparaben or chlorobutanol. In the presence of these preservatives, the obesity proteins r~m~i n~ in solution making a soluble, parenteral ~ormulation possible.
A parenteral formulation must meet guidelines ~or preservative effectiveness to be a commercially viable product. Pharmaceutical preservatives known in the art as being acceptable in parenteral ~ormulations include: phenol, CA 02243229 1998-07-1~
W O 971260~4 PCTnUS97/00864 m-cresol, benzyl alcohol, methylparaben, chlorobutanol, p-cresol, phenylmercuric nitrate, thimerosal and various mixtllres thereof. See, e.g., WALLHAUSER, K.--H., DEVE:LoP. BIOL.
STANDARD. 24, pp. 9-28 (sasel~ s. Krager, 1974).
~ 5 Most unexpectedly, a select number of preservatives have been identified which provide good ~ormulation stability These select preservatives are an alkylparaben or chlorobutanol. Most preferrably, the preservative is meth~lparaben, propylparaben, or butylparaben. Most unexpectedly, the obesity protein does not aggregate in the presence of these preservatives at the conditions necessary to formulate and particularly conditions at 37~C.
The concentration of obesity protein in the formulation is about 1.0 mg/mL to about 100 mg/mL; preferably about 5.0 mg/mL to about 50.0 mg/mL; most preferably, about 10.0 mg/mL. The concentration of preservative required is the concentration necessary to maintain preservative effectiveness. The relative amounts of preservative necessary to maintain preservative effectiveness varies with the preservative used. Generally, the amount necessary can be found in WALLHAUSER, K.--H., DEVELOP. BIoL. STANDARD~ 24, pp.
9-28 (Basel, S. Krager, 1974), herein incorporated by reference. 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 of the isotonicity agent is in the range known in the art for parenteral formulations, preferably about 1 to 20 mg/mL, more preferably about 8 to 16 mg/mL, and still more preferably about 16 mg/mL. The pH of the formulation may also be ~ buffered with a physiologically tolerated buffer, preferably a phosphate buffer, like sodium phosphate. Other acceptable physiologically tolerated buffers include TRIS, sodium acetate, or sodium citrate. The selection and concentration of buffer is known in the art; however, the formulations of CA 02243229 1998-07-1~
W O 9il260Q4 PCT~US97/00864 .
the present invention are preferably prepared the mln;m~lly accpetable concentration of buffer.
Other additives, such as a pharmaceutically acceptable solubilizers like Tween 2~ (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyo ~ ethylene polyoxypropylene block copolymers), BRIJ 35 (polyoxyethylene (23) lauryl ether), 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. The presence of pharmaceutically acceptable surfactant mitigates the propensity for the protein to aggregate.
Also included as optional embodiments are agents known to be synergistic with the preservative to provide enhanced antimicrobial effect. Such agents are recognized in the art and include for example ethylene diaminetetraacetic acid (EDTA), 1,2-di(2-aminoethoxy)ethane-N,N,M'N'-tetraacetic acid (EGTA), citrate, and caprylic acid. The concentration of these agents varies with the desired preservation effect.
A preferred agent is EDTA, particularly in conjunction with an alkylparaben at a concentration of about 0.025% to 0.4%.
Notably, in preparations including EDTA or EGTA, the buffer concentration is reduced to minim; ze ionic strength.
The present formulations may optionally contain a physiologically tolerated solvent such as glycerol, propylene glycol, phenoxy ethanol, phenyl ethyl alcohol. Such solvents are generally added to enhance the solubility of the protein in the preservation effectiveness of the formulation.
The parenteral fo~mulations 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 3~ is combined with the desired preservatlve in water in quantities sufficient to provide the protein and preservative at the desired concentration. The formulation is generally CA 02243229 1998-07-1~
W O 9~/2601)4 PCTrUS97/00864 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 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 preservative e~fect on formulation stability was demonstrated by preparing formulations comprising methylparaben, propylparaben, butylparaben, chlorobutanol, cresol, phenol, benzyl alcohol, or a mixture thereof and comparing the amount o~ protein remaining in solution after 3 days at 4~C and 37~C. The data in Table 1 demonstrate that the stability and solubility of human obesity protein is enhanced in the presence o~ an alkylparaben or chlorobutanol. The formulations used to generate the data of Table 1 were prepared in a manner analogous to Examples 1 and 2.
CA 02243229 1998-07-1~
W O 9~-/26004 PCTrUS97/00864 .
Table 1. Recovery of protein as a function of preservative and conditions. Values are the calculated least square means and standard errors determined from fitting the raw data to a factorial model of the second degree containing the e~ects of preservative, acid excursion, buffer, conditions, and all two factor interactions. (R2 = 0.94, (Prob > F) = 1.96e~39, total observations = 147). Percent of protein in solution is calculated using a theoretical target o~ 1.6 mg protein/mL.
Protein in Solution (% of theoretical) PreservativePreservative 3 days ~ 3 days @
Concentration 4~C 37~C
( ) methylparaben0.17 = 0.02 ~7." = . . = .
propyl~araben0.16 0.02 9. =
butylparaben 0.~15 ,7. = ~
chlorobutanol 0.5 87., = ~-. 8r. = ~.
methylparaben ~0.18 + 0.02 76.8 = ~. 6 .
propy~paraben0.017 + 0.002 ~enzy alcohol 1.0 79.8 + 3.6 31.6 + 4.2 methy_paraben +0.16 + 0.02 62.8 + 4.3 16.7 + 4.3 propylparaben +0.02 + 0.002 benzyl :lcohol 0.~
m-creso~ 0. ~ .~- = - ~- - --' p-creso_ 0._ : = , ~. = ~.-phenol 0. J
The stabilizing ef~ect by the alkylparabens is most unexpected in view of the structural similarities to other preser~atives. The data clearly show that the alkylparabens and chlorobutanol are superior at 37~C, which is the temperature re~uired for in-use physical stability testing.
Preferably, the pH of the present formulations is about pH 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 a pH greater than pH 7Ø Preferably, the pH is about 7.6 to 8.0, and most pre~erably about pH 7.8. Ideally, a preservative and water solution is mixed at pH 7.6 to 8Ø
Added 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 dissolved, CA 02243229 l998-07-l5 W O 9~/26004 PCTnUS97/00864 approximately 20 to 40 minutes, preferably about 30 minutes.
The base used to adjust the pH of the formulation may be one or more pharmaceutically acceptable bases such as sodium hydroxide and potassium hydroxide. The preferred base is sodium hydroxide.
The formulations prepared in accordance with the present invention may be used in a syringe, injector, pumps or any other device recognized in the art for parenteral administration.
Preferably, the obesity protein employed in the formulations of the present invention is human obesity prot~in of SEQ ID NO:1.
lo 15 Val Pl~c Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr 2c 25 30 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 ~eu His Pro Ile 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 Hi~ L~u Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 3 5 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly C~s (SEQ ID NO:1) The obesity protein employed in the formulations of the present invention can be prepared by any of a variety of reco~-nized peptide synthesis techniques including classical ~solution) methods, solid phase methods, semi synthetic methods, and more recent recombinant DNA methods. The preparation of the human obesity protein is known and CA 02243229 1998-07-1~
W O 97/26004 PCT~US97/00864 .
disclosed, for example, in Halaas Jeffrey L. et al., Science 269 ~1995). The preparation of other ~mm~l ian obesity proteins are 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,~28, filed May 26, 1995 (EP0 744 408); and provisional application, 60,003935, filed September 19, 1995, (EP ); all of which are herein incorporated by reference.
The obesity proteins described herein may also be produced either ~y recombinant DNA technology or well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution methods. Recombinant methods are preferred if a high yield is desired. The basic steps in the recombinant production of protein include:
a) construction of a synthetic or semi-synthetic (or isolation from natural sources) DNA
encoding the obesity protein, b) integrating the coding se~uence into an expression vector in a manner suitable for the expression of the protein either alone or as a fusion protein, c) transforming an appropriate eukaryotic or prokaryotic host cell with the expression vector, and d) recovering and purifying the recombinantly produced protein.
Synthetic genes, the n vitro or n vivo transcription and translation o~ which will result in the production of the protein may be constructed by techniques well known in the art. Owing to the natural degeneracy of the genetic code, the skilled artisan will recognize that a sizable yet definite number of DNA sequences may be constructed which encode the desired proteins. In the preferred practice of the invention, synthesis is achieved by recombinant DNA
technology.
CA 02243229 l998-07-l~
w o ~7~26(~n4 PCT~US97/00864 Methodology of synthetic gene construction is well known in the art. For example, see Brown, et al. (1979) Methods in Enzymology, Academic Press, N.Y., Vol. 68, pgs 109-151. ~he 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 380B DNA synthesizers (commercially available from Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404). It may desirable in some applications to modify the coding se~uence of the obesity protein so as to incorporate a convenient protease sensitive cleavage site, e.g., between the signal peptide and the structural protein facilitating the controlled excision of the signal peptide from the ~usion protein construct The gene encoding the obesity protein may also be created ~y using polymerase chain reaction (PCR). The template can be a cDNA library (commercially available from CLOMETECH or STRATAGENE) or mRNA isolated from the desired arrival adipose tissue. Such methodologies are well known in the ~rt Maniatis, et al. Molecular Clonina: A Laboratorv Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold ~pring Harbor, New York (1989).
The constructed or isolated DNA sequences are usef-,ll for expressing the obesity protein either by direct expression or as fusion protein. When the sequences are used in a fusion gene, the resulting product will require enzymatic or chemical cleavage. A variety of peptidases which cleave a polypeptide at specific sites or digest the peptides from the amino or carboxy termini (e.g.
diaminopeptidase) of the peptide chain are known.
Furthermore, particular chemicals (e.g. cyanogen ~romide) - will cleave a polypeptide chain at specific sites. The skille~ artisan will appreciate the modifications necessary - to tl1e amino acid sequence (and synthetic or semi-synthetic coding sequence if recombinant means are employed) to incorporate site-specific internal cleavage sites. See U.S.
Patent No. 5,126,249; Carter P., Site Specific Proteolysis CA 02243229 1998-07-1~
W O 97f26004 PCT~US97/00864 .
~ 14 -of Fusion Proteins, Ch. 13 in Protein Purification: From Molecular Mechanisms to Larae Scale Processes, American Chemical Soc., Washlngton, D.C. (1990).
Construction of suitable vectors containing the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or DMA 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 o~ a plethora o~ appropriate recombinant DNA expression vectors through the use of appropriate restriction erldonucleases. 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 isola~ed 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 ~y techni~ues well known in the art. The particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern of the parent expression vector to be employed. The restriction sites are chosen so as to properly orien~ 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 origin 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 ~ coli species (Bolivar, et al., Gene 2: 95 (~977)).
Plasmid pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial CA 02243229 1998-07-1~
W O 97126004 PCTrUS97/00864 plasmid must also contain or be modified to contain promoters and other control elements commonly used in recombinant DMA
technology.
The desired coding sequence is inserted into an - 5 expression vector in the proper orientation to be transcribed ~rom a promoter and ribosome binding site, both of which should be ~unctional in the host cell in which the protein is to b~ expressed. An example o~ 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 re~erence. The gene encoding A-C-s proinsulin described in U.S. patent No. 5,304,493 can be removed from the plasmid pRB182 with restriction enzymes ~I and 3amHI. The isolated DNA ~equences can be inserted into the plasmid backbone on a NdeI/BamHI restriction ~ragment cassette.
In general, procaryotes are used for cloning o~ D~A
sequences in constructing the vectors use~ul in the invention. For example, E. coli K12 strain 294 (ATCC No.
31446) is particularly use~ul. Other microbial strains which may be used include E. CQli B and E. 5~,~ X1776 (ATCC No.
31537). These examples are illustrative rather than limiting.
Procaryotes also are used for expression. The a~orementioned strains, as well as E. coli W3110 (prototrophic, ATCC No. 27325), bacilli such as Bacillus subtilis, and other enterobacteriaceae such as ~almonella tv~himurium or Serratia marcescans, and various pseudomonas species may be used. Promoters suitable for use with prokaryotic hosts include the ~-lactamase ~vector pGX2907 [ATCC 39344] contains the replicon and ~-lactamase gene) and lactose promoter systems (Chang et al., Nature, 275:615 (1978); and Goeddel et al., Nature 281:544 (1979)), alkaline phosphatase, the tryptophan (trp) promoter system (vector pATHl [ATCC 37695] is designed to ~acilitate expression o~ an open reading frame as a trpE ~usion protein under control o~
the trp promoter) and hybrid promoters such as the tac promoter (isolatable ~rom plasmid pDR54Q ATCC-37282).
However, other ~unctional bacterial promoters, whose CA 02243229 l998-07-l~
W O 97/26004 PCT~US97/00864 nucleotide sequences are ~enerally 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.
The DNA molecules may also be recom~inantly produced in eukaryotic expression systems. Preferred promoters controlling transcription in mammalian host cells may be obtained ~rom various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. ~-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication. Fiers, 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 species also are useful herein.
Transcription of the DNA by higher eucaryotes is increased by inserting an enhancer sequence into the vector.
Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that act on a promoter to increase its transcription.
Enhancers are relatively oriented and positioned independently and have been found 5' (T,~;mln~, L. et ~l., PNAS 78:993 (1981)) and 3' (Lusky/ M. L., et al., Mol. Cell Bio. 3:1108 (1983)) to the transcription unit, within an intron (Banerji, J. L. et al., Cell 33:729 (1983)) as well as within the coding sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4:1293 (1984)). Many enhancer sequences are now known from m~mm~lian gene-s (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 CA 02243229 1998-07-1~
W O 97/26~04 PCTrUS97/00864 - ~7 -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, ~ungi, insect, plant, animal, human or nucleated cells from other multicellular organisms) will also contain 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.
Expression vectors may contain a selection gene, also termed a selecta~le marker. Examples of suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR, which may be derived from the BalII/HindIII
restriction fragment of pJOD-10 [ATCC 68815]), thymidine kinase (herpes simplex virus thymidine kinase is contained on the E~mHI fragment of vP-5 clone [ATCC 2028]) or neomycin (G418) resistance genes (obtainable ~rom pNN414 yeast artificial chromosome vector [ATCC 37682]). When such selectable markers are successfully transferred into a m~mm~l ian host cell, the transfected mammalian host cell can survive i~ placed under selective pressure. There are two widely used distinct categories of selective regimes. The 25 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 30 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 r alternative to supplementing the media is to introduce an 35 intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual CA 02243229 l998-07-l~
W O 97/26004 P~l~S97/00864 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 domin~nt 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 (1985). The three examples given above employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively.
A preferred vector for eucaryotic expression is pRc/CMV. pRc/CMV is commercially available from Invitrogen Corporation, 3985 Sorrento Valley Blvd., San Diego, CA
92121. To confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform E. coli K12 strain DHlOB (ATCC 31446) and successful transformants selected by antibiotic resistance where appropriate.
Plasmids from the transformants are prepared, analyzed by restriction and/or sequence by the method of Messing, et al., Nucleic Acids Res. 9:309 (1981).
Host cells may be transformed with the expression vectors of this invention and cultured in conventional nutrient media modified as is appropriate for inducing promoters, selecting transformants or amplifying genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan. The techniques of transforming cells with the aforementioned vectors are well known in the art and may be found in such general references as ManiatiS, ~t al., W O 9~/260D4 PCTrUS97/00864 Molecular Clonina: A La~oratory Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989), or Cl]rrent Protocols in Molecular Bioloov (1989) and supplements.
Preferred suitable host cells for expressing the vectors encoding the claimed proteins in higher eucaryotes include: African green monkey kidney line cell line transformed by SV40 (COS-7, ATCC CRL-1651); transformed human primary embryonal kidney cell line 293,(Graham, F. L. ~ al., J. Gen Virol. 36:59-72 (1977), Viroloov 77:319-329, Virolo~Y
86:10-21); baby hamster kidney cells (BHK-21(C-13), ATCC CCL-10, Viroloov 16:147 (1962)); Chinese hamster ovary cells CHO-DHFR-- (ATCC CR~-9096), mouse Sertoli cells (TM4, ATCC CRL-1715, Biol. Re~rod. 23:243-250 (1980)); African green monkey kidLLey cells (VERO 76, ATCC CRL-1587); human cervical epitheloid carcinoma cells (HeLa, ATCC CCL-2); canine kidney cells (MDCK, ATCC CC~-34)i buffalo rat liver cells (BRL 3A, ATCC CRL-1442); human diploid lung cells (WI-38, ATCC CCL-75); human hepatocellular carcinoma cells (Hep G2, ATCC HB-8065);and mouse m~mm~ry tumor cells (MMT 060562, ATCC CCL51).
In addition to prokaryotes, unicellular eukaryotes such as yeast cultures may also be used. Saccharom~ces cere~isiae, or common baker's yeast is the most commonly used euka~yotic microorganism, although a number of other strains are commorLly available. For expression in Saccharomyces, the plasmid YRp7, for example, (ATCC-40053, Stinchcomb, et al., 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 of yeast lacking the ability to grow in tryptophan, for example ATCC no. 44076 or PEP4-1 (Jones, Genetics 85:12 (1977)).
Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase (found on plasmid pAP12BD ATCC 53231 and described in U.S.
Patent No. 4,935,350, June 19, 1990) or other glycolytic enzymes such as enolase ~found on plasmid pAC1 ATCC 39532), CA 02243229 1998-07-1~
W O 97/26004 PCT~US97/00864 glyceraldehyde-3-phosphate dehydrogenase (derived from plasmid pHcGAPC1 ATCC 57090, 57091), zymomonas mobilis (United States Patent No. 5,000,000 issued March 19, 1991), hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate ~inase, 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 al., 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--h~lbeta 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.
The scope of the present invention is not to be construed as merely consisting of the following examples.
Pre~aration 1 ~orcine OB Gene and Gene Product Total RNA was isolated from porcine fat tissue obtained from Pel~Freez~, Pel-Freez Inc., and the cDNA was cloned in accordance with the techniques described in Hsiung et al., Neuro~e~tide 25: 1-10 (1994).
Primers were designed based on the published amino acid sequence of the human o~ gene. The primers were prepared for use in polymerase chain reaction (PCR) CA 02243229 1998-07-1~
W O 9~260~4 PCTrUS97/00864 amplification methods using a Model 380A DNA synthesizers (PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, 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
~ 5 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 RMA 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 for 10 minutes at 70~C and then cooled on ice. The following were then added to the annealed mixture: 4 ~L o~
BRL 5x H-reverse transcriptase (RT) reaction buffer (Gibco-BRL CAT#28025-013), 2 ~L of 0.1 M DTT, 1 ~L of lQ mM dNTPs.
This annealed mixture was then incubated at 37~C for 2 minutes before adding 1 ~L BRL M-MLV-reverse transcriptase (200 U/~L) (CAT#28025-013) and incubated at 37~C for additional 1 hour. After incubation the mixture was heated at 95~C for 5 minutes and then was cooled on ice.
For amplification of cD~A, the polymerase chain reaction (PCR) was carried out in a reaction mixture (100 ~L) containing the above cDNA reaction mixture (1 ~L), 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer Corporation) 10 ~1 of 10x PCR reaction bu~er (Perkin-Elmer Corporation) and 50 pmol each of the sense (PCROB-l) and antisense (PCROB-3) primers for porcine OB amplification. The condition ~or PCR
was 9~~C ~or 1 minute, 57~C ~or 1 minute and 72-~C for 1 minute ~or 30 cycles using a PCR DNA Thermal Cycler (Perkin-Elmer Corporation). After PCR amplification, 5 ~L BRL T4 DNA
polymerase (5 U/~L), 2 ~L BRL T4 polynucleotide kinase (10 U/~L), and 5 ~L ATP (10 mM) were added to the PCR reaction mixtu:-e (100 ~1) directly and incubated for 30 minutes at 37~C. After the incubation the reaction mixture was heated at 95~C for 5 minutes and then was cooled on ice. The 500 bp ~ragment (~0.5 mg) was purified by agarose gel electrophoresis and isolated by the freeze-squeeze method.
The 500 bp fragment (_0.2 ~g) was then ligated into SmaI
CA 02243229 1998-07-1~
W O 97/26004 PCT~US97/00864 linearized pUC18 plasmid (~1 ~g) 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 ~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 (lQ0 ~g/mL). The plasmid was isolated using a Wizard Miniprep DNA
purification system (Promega) and submitted for DNA
seqeuncing on a Applied Biosystem 370 DNA sequencer.
Pre~aration 2 Bovine OB Gene and Gene Product The DNA sequence o~ the bovine OB gene was obtained by techniques analogous to Example 1, except sense (PCROB-2) and antisense (PCRoB-3) primers were used for bovine OB cDNA
amplification.
Pre~aration 3 Vector Construction A plasmid containing the DNA sequence encoding the obesity protein is constructed to include NdeI and ~HI
restriction sites. The plasmid carrying the cloned PCR
product is digested with ~I 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 F.. 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 ~mHI fragment are kept. E. coli K12 RV308 (available from the NRRL under deposit number B-15624) are transformed with this second plasmid, resulting in a culture suitable for expressing the protein.
The techniques of transforming cells with the aforementioned vectors are well known in the art and may be CA 02243229 l998-07-l~
WO 9ii260~4 PCT~US97tO0864 found in such general references as Maniatis, et al. (1988) Molecular Clonin~: A Laboratorv Manual, Cold Spring ~Iarbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York or Cllrrent Protocols in Molecular Biolo~v ~1989) and supplements. The techniques involved in the trans~ormation of E. coli cells used in the preferred practice of the invention as exemplified herein are well known in the art.
The precise conditions under wh-ich the transformed ~ coli ce~ls are cultured is dependent on the nature of the ~ coli 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 from about 30 to about 40 degrees C. in the culture conditions so as to induce protein synthesis.
In the preferred embodiment of the invention, ~.
SQ~i K12 RV308 cells are employed as host cells but numerous other cell lines are available such as, but not limited to, F 5~L~ K12 L201, L687, L693, L5~7, L640, L641, L695, L814 (E cQli B). The transformed host cells are then plated on appropriate media under the selective pressure of the an~ibiotic corresponding to the resistance gene present on the expression plasmid. The cultures are then incubated for 2S a t:ime and temperature appropriate to the host cell line emE)loyed .
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 Foldin~, 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 techni~ues us~ng strongly denaturing solutions such as guanidinium-HCl and/or weakly denaturing solutions such as dithiothreitol 5~ TE SHEET(RULE 2~) CA 02243229 1998-07-1~
W O g7/26004 PCT~US97/00864 .
(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 efficie~t 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 formuations of the invention The scope of the inve~tion is not construed as merely consisting of the following examples.
Example 1 Human Obesitv Protein Formulation To generate a solution of Human Obesity Protein (hOb), 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 Spectrophometry using the known extinction coe~ficient for hOb at the maximum spectral absorbance (279nm or 280nm), measuring the maximum spectral absorbance (279nm or 280nm), and utilizing the dilution factor. A stock preservative solution containing methylparaben was prepared by dissolving the solid 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 aliquot of stock 2 along with the required quantity of water, at an alkaline pH (7.8~0.3)i adjusted if necessary with HCl or NaOH. After an appropriate incubation period at room temperature (30 minutes), the CA 02243229 1998-07-1~
wo s7/26ao4 PCT/US97/00864 solution pH was examined and adjusted if necessary with trace ~L ~uantities of HCl or NaOH, to yield an hOb solution at 1.6 mg/mL with 0.17% methylparaben 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.
Example 2 Human Obesitv Protein Solution To generate a solution of Human Obesity Protein (hOb~, 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 Spectrophometry using the known extinction coefficient for hOb at the maximum spectral absorbance (279nm or 280nm), measuring the maximum spectral absorbance (279nm or 280nm), and utilizing the dilution factor. A stock preservative solution containing chlorobutanol, for example, was prepared by dissolving the solid in water (stock 2). A solution of hOb at 1.6 mg/mL was prepared by addition of an ali~uot of stock 1 to a container which held an aliquot of stock 2 along with the required quantity o~ water, at an alkaline pH
(7.8+0.3); adjusted if necessary with HCl or NaOH. After an appropriate incubation period at room temperature (30 minutes), the solution pH was examined and adjusted if necessary with trace ~L quantities of HCl or NaOH, to yield an hOb solution at 1.6 mg/mL with 0.50% chlorobutanol 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.
Example 3 Human Obesitv Protein Solution 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 Spectrophometry by multiplying the maximum CA 02243229 1998-07-1~
W O 97t26004 PCTrUS97/00864 spectral absorbance (279nm or 280nm) by the dilution factor divided by the known extinction coef~icient for hOb. A stock preservative solution containing methylparaben was prepared by dissolving the solid 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 stock of a physiologically-tolerated buffer, such as sodium phosphate 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 along with the required quantity of water, adjusted if necessary with HCl or NaOH to an alkaline pH (7.8+0.3). After an approp~iate incubation period at room temperature (30 minutes) an aliquot of stock 4 was added. The solution pH was then readjusted if necessary with trace ~ quantities of HCl or NaO~, to yield an hOb solution at 1.6 mg/mL with 0.17% methylparaben, 16 mg/mL glycerin, and 14mM sodium phosphate at 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.
Example 4 Hllm~n Obesitv Protein Solution 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 UV/ViS Spectrophometry by multiplying the maximum spectral absorbance (279nm or 280nm) by the dilution factor divided by the known extinction coefficient for hOb. A stock preservative solution containing chlorobutanol was prepared by dissolving the solid in water (stock 2). A stock of an isotonicity agent, such as glycerin, was prepared by dissolving the nea~ liquid in water (stock 3). A stock of a physiologically-tolerated buffer, such as sodium phosphate 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 CA 02243229 1998-07-1~
W O 97126al04 PCTAUS97/00864 aliquot of stock 1 to a container which held an aliquot of stock 2 along with an aliquot of stock 3 along with the required ~uantity of water, adjusted if necessary with HCl or NaOH to an alkaline pH (7.8+0.3). After an appropriate incubation period at room temperature (30 minutes) an aliquot of stock 4 was added. The solution pH was then readjusted if necessary with trace ~L quantities of HCl or NaOH, to yield an hOb solution,.for example, at 1.6 mg/mL with 0.5%
chlorobutanol, 16 mg/mL glycerin, and 14mM sodium phosphate (if required by the formulation) at 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.
Example 5 Pre~aration of Human Obesitv Protein Solution To generate a preserved solution of human obesity protein (hOb), the individual solution components were added in succession to a neutral water solution, allowing each component to dissolve prior to addition of the next component. To the container were added: approximately 5-6 mL of sterile water (~inal solution volume = 10.00 mL) followed by preservative solids (1.51 mg of butylparaben;
final concentration = 0.015~). The solution was mixed at medium speed with a Te~lon stir bar and heat was applied to promote dissolution o~ the solids. Care was exercised to dissolve the preservative solids, but not to overheat or boil the solution. A~ter dissolution was achieved (approximately 1-1.5 hr.), the solution was allowed to cool to room temperature while maintaining stirring. Glycerin (160.0 mg) was then added to the solution and stirred until dissolution was achieved. The final glycerin concentration was targeted to be 16 mg/mL. A 10.0 mg quantity o~ solid ethylenediamine tetra_etate (EDTA) was added to the solution and stirred to dissolve (final concentration = 0.10%). Dibasic sodium phosphate crystals (17 0 mg) were then added and allowed to dissolve by stirring. The ~inal phosphate concentration was targe~ed at 6.3 mM. Sucrose (600.0 mg) was then added and CA 02243229 1998-07-1~
W O 9~/26004 PCT~US97/00864 allowed to stir until dissolved (final concentration of 60 mg/mL). Bulk lyophilized hOb (115.4 mg) was added incrementally to the stirring solution, allowing each aliquot to dissolve prior to the addition of successive portions.
The quantity of hOb added to the solution yielded a final concentration of 10.2 mg/mL. The amount added was determined by using the "as is" weight ~, determined for the specific hOb lot used by W /Vis spectrophotometry. Following the addition of all bulk ingredients, the pH of the solution was adjusted to 8.5 using approximately 11 ~L of 10~ NaOH to promote dissolution of the solids. After stirring the solution for a 10-15 minute period, the pH was decreased to 7.8 using approximately 4-5 ~L of 10% HCl and the solution was allowed to stir for an additional 10 minutes. The solution was then transferred to a 10 mL volumetric flask, and brought to final volume with sterile water. The ~lask was then inverted 20x to thoroughly mix the contents and transferred to a glass sample vial where a recheck of the pH
yielded 7.78. A trace quantity of 10% NaOH (approximately 1 ~L) was added to yield a final pH of 7.83. The solution was then hand-filtered, using a 5 mL glass syringe with an attached 0.2~m syringe filter into a 20 mL glass vial, capped and stored refrigerated until needed.
The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. ~he invention which is intended to be protected herein, however, is not to be construed as limited to the particular forms disclosed, sinc~ 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.
Claims (11)
1. A soluble parenteral formulation, comprising an obesity protein and a preservative selected from the group consisting of alkylparaben, chlorobutanol, or a mixture thereof.
2. A formulation of Claim 1, wherein the preservative is methylparaben, ethylparaben, propylparaben, or butylparaben.
3. A formulation of Claim 2, wherein the concentration of protein is about 1.0 mg/mL to about 10 mg/mL.
4. A formulation of Claim 3, which further comprises an isotonicity agent.
5. A formulation of Claim 4, which further comprises a physiologically acceptable buffer.
6. A formulation of Claim 5, wherein the preservative is methylparaben, and the isotonicity agent is glycerin.
7. A formulation of any one of Claims 1 through 6, wherein the protein is the human obesity protein optionally having- a Met- leader sequence.
8. A formulation of any one of Claims 1 through 6, wherein the protein is the human obesity protein.
9. A process for preparing a soluble parenteral formulation of any one of Claims 1 through 8, which comprises mixing an obesity protein and a preservative selected from the group consisting of alkylparaben, chlorobutanol, or a mixture thereof.
10. A method of treating obesity in a mammal in need thereof, which comprises administering to said mammal a soluble parenteral formulation of any one of Claims 1 through 8.
11. A formulation as claimed in any one of Claims 1 through 8 for use in the treatment of obesity.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US1022996P | 1996-01-19 | 1996-01-19 | |
| US60/010,229 | 1996-01-19 | ||
| GBGB9602408.8A GB9602408D0 (en) | 1996-02-07 | 1996-02-07 | Obesity protein formulations |
| GB9602408.8 | 1996-02-07 | ||
| PCT/US1997/000864 WO1997026004A1 (en) | 1996-01-19 | 1997-01-17 | Obesity protein formulations |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2243229A1 true CA2243229A1 (en) | 1997-07-24 |
Family
ID=29424163
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2243229 Abandoned CA2243229A1 (en) | 1996-01-19 | 1997-01-17 | Obesity protein formulations |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2243229A1 (en) |
-
1997
- 1997-01-17 CA CA 2243229 patent/CA2243229A1/en not_active Abandoned
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