CA2243707A1 - Obesity protein compounds and formulations thereof - Google Patents
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Abstract
The present invention provides novel compounds, which comprise an obesity protein complexed with a divalent metal cation, pharmaceutical formulations thereof, and methods of using such compounds for treating obesity, and disorders associated with obesity such as diabetes, cardiovascular disease and cancer.
Description
W O 97/28824 PCTrUS97/01188 Obesity Protein Compounds and Formulations Thereof The present invention is in the field of human medicine, particularly in the treatment of obesity and disorders associated with obesity. More speci~ically, the present invention relates to compounds and formulations of an obesity protein.
Obesity, and especially upper body obesity, is a common and very serious public health problem in the United States and throughout the world. According to recent statistics, more than 25% of the United States population and 27~ of the Canadian population are overweight. Kuczmarski, ~mer. J. of Clin. Nutr. 55: 495S - 502~ (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 $150,000,000,000 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, hypertension, and insulin resistance. Many studies have demonstrated that reduction in obesity by diet and exercise reduces these risk factors dramatically. Unfortunately, these treatments are largely unsuccessful with a failure rate reaching 95%. This failure may be due to the fact that the condition is strongly associated with genetically inherited factors that contribute to increased appetite, preference for highly caloric foods, reduced physical activity, and increased lipogenic metabolism. This indicates that people inheriting these genetic traits are prone to becoming obese regardless of their efforts to combat the condition Therefore, a pharmacological agent that can correct this adiposity handicap and allow the physician to successfully _ treat obese patients in spite of their genetic inheri~ance 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 372. 425-32 (1994). This report disclosed the murine and human protein expressed in adipose tissue. Likewise, Murakami et al., in Riochemical and Bio~hvsic~l Research Communications 2Q9(3):944-52 (1995) report the cloning and expression o~ 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., Sc;ence 269: 540-543 (1995).
The present invention provides conditions under which potency of the natural obesity protein is signi~icantly enh~n~ed. Thus, effective pharmacological treatment may be achieved at lower doses that significantl~ lower the risk of toxic or other undesirable side e~~ects. In addition, because the amount of protein ~mi n; stered is less, the cost of the unit dosage form to the patient is reduced.
Accordingly, the present invention provides a novel protein-cation complex, which comprises an obesity protein complexed --with a divalent metal cation, pharmaceutical formulations thereo~, and methods of using such compounds for the treatment of obesity, and disorders associated with obesity such as diabetes, cardiovascular disease and cancer The invention provides a compound comprising an obesity protein complexed with a divalent metal cation.
The invention additionally provides parenteral pharmaceutical formulations comprising the protein-cation compounds and methods of using such compounds for treating obesity and disorders associated with obesity such as diabetes, cardiovascular disease and cancer. The invention ~urther comprises combining an obesity protein and a divalent CA 02243707 l998-07-2l W O 97/28824 PCTrUS97/01188 metal cation in an aqueous solution at a pH o~ about 4.5 to 9 .0 .
For purposes o~ the present invention, as disclosed and claimed herein, the ~ollowing terms and abbreviations are defined as ~ollows:
Base pair (bp) -- re~ers to DNA or RNA. The abbreviations A,C,G, and T correspond to the 5'-monophosphate forms o~ the nucleotides (deoxy)a~en; ne, (deoxy)cytidine, (deoxy)guanine, and (deoxy)thymine, respectively, when they occur in DNA molecules. The abbreviations U,C,G, and T
correspond to the 5'-monophosphate forms o~ the nucleosides uracil, cytidine, guanine, and thymine, respectively when they occur in RNA molecules. In double stranded DMA, base pair may re~er to a partnership of A with T or C with G. In a DN~/RNA heteroduplex, base pair may re~er to a partnership o~ T with U or C with G.
Obesity protein -- re~ers to the native m~mm~lian protein produced ~rom the native ob gene ~ollowing transcription and deletions o~ introns, translation to a protein and processing to the mature protein with secretory signal peptide removed, e.g. ~rom the N-terminal valine-proline to the C-terminal cysteine o~ the mature protein.
The human obesity protein is published in Zhang et al.
Nature 372: 425-32 (lg94). The rat obesity protein is published in Murakami et al., Biochemical ~nd Bio~hvsical Research Comm. 209(3): 944-52 (1995). Native porcine and bovine Ob proteins are disclosed in a U.S. patent application by Hansen M. Hsiung and Dennis P. Smith ~iled May 19, 1995, Serial number 08/445,305 (EPO 743 321). Other m~mm~lian Ob proteins are disclosed in U.S. patent application serial number 08/452,228, ~iled May 26, 1995 (EPO 744 408) and ~ provisional application, 60,003935, filed September 19, 1995 (EP ), herein all o~ which are incorporated by re~erence.
Obesity protein includes those proteins having a leader se~uence. A leader se~uence is one or more amino acids on the N-terminus to aid in production or puri~ication o~ the W O 97/28824 PCT~US97/01188 protein. A preferred leader sequence is Met-R1 where R1 is _ absent or any amino acid except Pro.
Plasmid -- an extrachromosomal self-replicating genetic element.
Reading frame -- the nucleotide se~uence from which translation occurs "read'~ in triplets by the translational apparatus of tRNA, ribosomes and associated factors, each triplet corresponding to a particular amino acid Because each triplet is distinct and of the same length, the coding se~uence must be a multiple of three. A base pair insertion or deletion (termed a frameshift mutation) may result in two different proteins being coded for by the same DNA segment.
To insure against this, the triplet codons corresponding to the desired polypeptide must be aligned in multiples of three from the initiation codon, i.e. the correct ~reading frame"
must be maintained. In the creation of fusion proteins containing a chelating peptide, the reading frame of the DNA
sequence encoding the structural protein must be maintained in the DNA sequence encoding the chelating peptide.
Recombinant DNA Cloning Vector -- any autonomously replicating agent including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DMA 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 se~uence of DNA is transferred to a complementary RMA se~uence.
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 for the transformation of cells in gene manipulation bearing polynucleotide se~uences corresponding to appropriate protein molecules which, when CA 02243707 l998-07-2l W O 97/2~824 PCTnUS97/01188 combined with appropriate control se~uences, confer specific properties on the host cell to be transformed. Plasmids, viruses, and bacteriophage are suitable vectors, since they are replicons in their own right. Artificial vectors are constructed by cutting and joining DMA 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 the disease, condition, or disorder and includes the administration of a compound 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 ~lm; n; stration 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 formulation to prevent the net flow of water across the cell membrane. Compounds, such as glycerin, are commonly used for such purposes at known concentrations. Other possible isotonicity agents include salts, e.g., NaCl, dextrose, and lactose.
- Physiologically tolerated buffer -- a physiologically tolerated buffer is known in the art.
Physiologically tolerated buffers include TRIS, sodium acetate, sodium phosphate, or sodium citrate. The selection and concentration of buffer is known in the art.
Pharmaceutically acceptable preservative -- a multi-use parenteral formulation must meet guidelines for preservative effectiveness to be a commercially viable product. Pharmaceutically acceptable preservatives known in the art as being acceptable in parenteral formulations include: phenol, m-cresol, benzyl alcohol, methylparaben, chlorobutanol, p-cresol, phenylmercuric nitrate, thimerosal W O 97/28824 PCT~US97/01188 and various mixtures thereof. Other preservatives may be found, e.g., in WALLHAUSE:~, K.--H., DEVELOP. BIoL. STANDARD. 24, pp. 9-28 (Basel, S. Krayer, 1974). The concentration necessary to achieve preservative effectiveness is dependent _upon the preservative used and the conditions o~ the formulation.
The nucleotide and amino acid abbreviations used herein are accepted by the United States Patent and Trademark Of~ice as set forth in 37 C.F.R. 1.822 (b)(2) (1993).
Unless otherwise indicated the amino acids are in the L
configuration.
As noted above, the invention provides a compound comprising an obesity protein complexed with a divalent metal cation. When complexed with a divalent metal cation, the ~obesity protein demonstrates significantly enhanced potency.
The presently claimed compounds comprise an obesity protein complexed with a divalent metal cation. A divalent metal cation includes, ~or example, Zn+~, Mn+~, Fe++, Co++, Cd++, Ni+~ and the like. A combination of two or more divalent metal cations is operable; however the preferred compounds comprise a single species of metal cation, most preferably Zn++. Preferably, the divalent metal cation is in excessi however, the molar ratio o~ at least one molecule of a divalent metal cation for each ten molecules of obesity protein is operable. Pre~erably, the compounds comprise from 1 to 100 divalent metal cations per molecule of obesity protein. The compounds may be amorphous or crystalline solids.
Appropriate forms of metals cations are any form of a divalent metal cation that is available to form a complex with a molecule of obesity protein of the present invention.
The metal cation may be added in solid form or it may be added as a solution. Several dif~erent cationic salts can be used in the p~esent invention. Representative examples of metal salts include the acetate, bromide, chloride, fluoride, iodide and sulfate salt forms. The skilled artisan will recognize that there are many other metal salts which also W O 97/28824 PCTnUSg7/01188 might be used in the production of the compounds of the present invention. Preferably, zinc acetate or zinc chloride is used to create the zinc-obesity protein compounds of the present invention. Most preferably, the divalent metal cationic salt is zinc chloride, Generally, the claimed compounds are prepared by techniques known in the art. For example, convenient preparation is to combine the obesity protein with the desired divalent metal cation in an aqueous solution at a pH
of about 4.5-9.0, preferably about pH 5.5-8, most preferably, about pH 6.5-7.6. The claimed compound precipitates from the solution as a crystalline or amorphous solid. Significantly, the compound is easily isolated and puri~ied by conventional separation techni~ues appreciated in the art including filtration and centrifugation. Significantly, the protein-metal cation complex is stable and may be conveniently stored as a solid or as an aqueous suspension.
The present invention further provides a pharmaceutical formulation comprising a compound o~ the present invention and water. The concentration of the obesity protein in the formulation is about 0.1 mg/mL to about 100 mg/mL; preferably about 0.5 mg/mL to about 50.0 mg/mL; most preferably, about 5.0 mg/mL.
The ~ormulation preferably comprises a pharmaceutically acceptable preservative at a 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.-30 H., DEvELoP. BIOL. STANDARD. 24, pp. 9--28(Basel, S. Krager, 1974), herein incorporated by reference.
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 ~ormulations, preferably about 1~ mg/mL glycerin.
The pH of the ~ormulation may also be buffered with a physiologically tolerated buffer. Acceptable physiologically CA 02243707 l998-07-2l W O 97/28824 PC~rUS97/01188 ~8--tolerated buffers include TRIS, sodium acetate, sodium phosphate, or sodium citrate. The selection and concentration of buffer is known in the art.
Other additives, such as a pharmaceutically acceptable excipients like Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers), BRIJ 35 (polyoxyethylene (23) lauryl ether), and PEG (polyethylene glycol) may optionally be added to the formulation to reduce aggregation.
The claimed pharmaceutical formulations are prepared in a manner known in the art, and are administered individually or in combination with other therapeutic agents.
The ~ormulations of the present invention can be prepared using conventional dissolution and mixing procedures.
Pre~erably, the claimed ~ormulations are prepared in an aqueous solution suitable for parenteral use. That is, a protein solution is prepared by mixing water for injection, buffer, and a preservative. Divalent metal cations are added to a total cation concentration of about 0.001 to 5.0 mg/mL, preferably 0.05 to 1.5 mg/mL. The pH of the solution may be adjusted to completely precipitate the obesity protein zinc complex. The compound is easily resuspended be~ore administration to the patient.
Parenteral daily doses of the compound are in the range from about 1 ng to about 10 mg per kg of body weight, although lower or higher dosages may be administered. The required dosage will be determined by the physician and will depend on the severity of the condition o~ the patient and upon such criteria as the patient's height, weight, sex, age, and medical history.
Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, if a surfactant is used, the temperature, and pH at which the ~ormulation is prepared may W O 97t28824 PCTrUS97/01188 be optimized for the concentration and means of administration used.
The pH of the formulation is generally pH 4.5 to 9.0 and preferably 5.5 to 8.0, most preferably 6.5 to 7.6;
although more acidic pH wherein a portion or all of the protein-metal cation complex is in solution is operable.
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 10 ~mi n; stration.
The obesity proteins of the present invention can be prepared by any of a variety of recognized peptide synthesis techniques including classical (solution) methods, solid phase methods, semi synthetic methods, and more recent recombinant DNA methods. Preferably, an obesity protein of the present invention is human protein of the formula:
(SEQ ID NO: 1) lo 15 Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser E~is Thr Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu 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 so 95 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys loo 105 llo His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 4 0 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 ' 45 1~5 Gly Cys CA 02243707 l998-07-2l W O 97/28824 PCT~US97/01188 - ~10 -The preparation of the human protein is known and disclosed, for example, in Halaas Jeffrey L. et al., Science 269 (1995).
The preparation of other mammalian obesity proteins are described in U.S. application serial number 08/445,305, filed May ~9, 1995 (EPO 743 221); U.S. patent application serial number 08/452,228, filed May 26, 1995 (EPO 744 408); and provisional application, 60,003935, filed September 19, 1995 (EP 96306721.0); all of which are herein incorporated by re~erence.
The obesity proteins described herein may also be produced either by recombinant DNA technology or well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution 15 : 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 in vitro or ia vivo transcription and translation of which will result in the production of the protein may be constructed by techni~ues 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 pre~erred practice of CA 02243707 l998-07-2l W O 97/28824 PCTrUS97/01188 the invention, synthesis is achieved by recombinant DNA
technology.
Methodology o~ 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. The ~NA 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 ~rom Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404). It may desirable in some applications to modi~y the coding sequence o~ the obesity protein so as to incorporate a convenient protease sensitive cleavage site, e.g., between the signal peptide and the structural protein ~acilitating the controlled excision o~ the signal peptide ~rom the fusion protein construct.
The gene encoding the obesity protein may also be created by using polymerase chain reaction (PCR). The template can be a cDNA library (commercially available ~rom CLONETECH or STRATAGENE) or mRNA isolated ~rom the desired arrival adipose tissue. Such methodologies are well known in the art Maniatis, et al. Molecular Clonin~: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989).
The constructed or isolated DNA sequences are useful ~or expressing the obesity protein either by direct expression or as ~usion protein. When the sequences are used in a fusion gene, the resulting product will require enzymatic or chemical cleavage. A variety o~ peptidases which cleave a polypeptide at speci~ic sites or digest the peptides ~rom the amino or carboxy termini (e.g.
diaminopeptidase) o~ the peptide chain are known.
Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave a polypeptide chain at speci~ic sites. The skilled artisan will appreciate the modi~ications necessary to the amino acid sequence (and synthetic or semi-synthetic coding sequence i~ recombinant means are employed) to CA 02243707 l998-07-2l W O 97/28824 PCT~US97/01188 incorporate site-specific internal cleavage sites. See U.S.
Patent No. 5,126,249; Carter P., Site Speci~ic Proteolysis o~ Fusion Protei~s, Ch. 13 in Protein Puri~icat;on: From Molecul~r Mech~n;sms to rl~r~e Scale Processes, ~merican Chemical Soc., Washington, D C. (1990).
Construction o~ suitable vectors cont~;n;ng the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or DNA ~ragments are cleaved, tailored, and religated in the ~orm desired to ~orm the plasmids required.
To e~ect the translation of the desired protein, one inserts the engineered synthetic DNA sequence in any o~ a plethora o~ appropriate recombinant DMA expression vectors through the use o~ appropriate restriction endonucleases. A
synthetic coding sequence may be designed to possess restriction endonuclease cleavage sites at either end o~ the transcript to ~acilitate isolation ~rom and integration into these expression and ampli~ication and expression plasmids.
The isolated cDNA coding sequence may be readily modi~ied by the use o~ synthetic linkers to ~acilitate the incorporation of this se~uence into the desired cloning vectors by techniques well known in the art. The particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern o~ the parent expression vector to be employed. The restriction sites are chosen so as to properly orient the coding se~uence with control sequences to achieve proper in-~rame reading and expression o~ the protein.
In general, plasmid vectors containing promoters 30 _ and control sequences which are derived ~rom 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 trans~ormed cells. For example, E. coli is typically trans~ormed using pBR322, a plasmid derived ~rom an E. coli species (Bolivar, e~ ~1-, Gene 2: 95 (1977)).
Plasmid pBR322 contains genes ~or ampicillin and tetracycline W O 97/28824 PCTrUS97/01188 resistance and thus provides easy means ~or identifying transformed cells. The pBR322 plasmid, or other microbial , plasmid must also contain or be modified to contain promoters and other control elements commonly used in recombinant DNA
technology.
The desired coding se~uence is inserted into an expression vector in the proper orientation to be transcribed from a promoter and ribosome binding site, both of which should be functional in the host cell in which the protein is to be expressed. An example of such an expression vector is a plasmid described in Belagaje et al., U.S. patent No.
5,304,493, the teachings of which are herein incorporated by reference. The gene encoding A-C-B proinsulin described in U.S. patent No. 5,304,493 can be removed from the plasmid pRB182 with restriction enzymes NdeI and BamHI. The isolated DNA se~uences can be inserted into the plasmid backbone on a NdeI/BamHI restriction fragment cassette.
In general, procaryotes are used for cloning of DNA
se~uences in constructing the vectors useful in the invention. For example, E. coli K12 strain 294 ~ATCC No.
31446) is particularly useful. other microbial strains which may be used include E. coli B and E. ÇQli X1776 (ATCC No.
31537). These examples are illustrative rather than limiting.
Procaryotes also are used for expression. The 25 aforementioned strains, as well as E. coli W3110 (prototrophic, ATCC No. 27325), bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella tv~himurium or Serratia marcescans, and various pseudomonas species may be used. Promoters suitable for use with 30 prokaryotic hosts include the ~-lactamase (vector pGX2907 [ATCC 39344] contains the replicon and ~-lactamase gene) and r lactose promoter systems (Chang et al., Nature, 275:615 (lg78); and Goeddel et ~1., Nature ~1:544 (1979)), alkaline phosphatase, the tryptophan (trp) promoter system (vector pATHl [ATCC 37695] is designed to facilitate expression of an open reading frame as a trpE fusion protein under control of the trp promoter) and hybrid promoters such as the tac CA 02243707 l998-07-2l O 97/28824 PCT~US97/0~188 promoter (isolatable from plasmid pDR540 ATCC-37282).
However, other functional bacterial promoters, whose nucleotide sequences are generally known, enable one of skill in the art to ligate them to DNA encoding the protein using linkers or adaptors to supply any re~uired restriction sites.
Promoters ~or use in bacterial systems also will contain a Shine-Dalgarno sequence operably linked to the DNA encoding protein.
The DNA molecules may also be recombinantly produced in eukaryotic expression systems. Preferred promoters controlling transcription in m~mm~l ian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. ~-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction ~ragment which also contains the SV40 viral origin of replication. Fiers, et al., Mature, 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~ (L~;m;n~, L. et al., PNAS 78:993 (1981)) and 3' (Lusky, M. L., et al., Mol. Cell Bio. 3:1108 (1983)) to the transcription unit, within an intron (sanerji, J. L. et al., Cell 33:729 (lg83)) as well as within the coding se~uence itself (Osborne, T. F., et al., ~Mol. Cell Bio. 4:1293 (1984)). Many enhancer sequences are now known from m~mm~lian genes (globin, RSV, SV40, EMC, elastase, albumin, alpha-fetoprotein and insulin).
CA 02243707 l998-07-2l W O 97/28824 PCTnUS97/~1188 Typically, however, one will use an enhancer ~rom a eukaryotic cell virus. Examples include the SV40 late enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells from other multicellular organisms) will also contain se~uences necessary ~or the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding protein The 3' untranslated regions also include transcription termination sites.
Expression vectors may contain a selection gene, also termed a selectable marker. Examples of suitable selectable markers for m~mm~l ian cells are dihydrofolate reductase (DHFR, which may be derived from the ~ n~III
restriction ~ragment of pJOD-10 [ATCC 68815]), thymidine kinase (herpes simplex virus thymidine kinase is contained on the ~mHI ~ragment of vP-5 clone [ATCC 2028]) or neomycin (G418) resistance genes (obtainable from pNN414 yeast artificial chromosome vector ~ATCC 37682]). When such selectable markers are success~ully transferred into a m~mm~l ian host cell, the trans~ected m~mm~l ian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow without a supplemented media. Two examples are: CHO D~FR- cells (ATCC
CRL-gO96) and mouse LTK- cells (L-M(TK-) ATCC CCL-2.3).
These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide - synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective CA 02243707 l998-07-2l genes, thus altering their growth requirements. Individual cells which were not trans~ormed with the DHFR or TK gene will not be capable of survival in nonsupplemented media.
The second category is dom;n~nt seiection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection.
Examples of such dominant selection use the drugs neomycin, Southern P. and Berg, P., J. Molec. A~~l. Genet. 1: 327 (1982), mycophenolic acid, Mulligan, R. C. and Berg, P.
Science ~ 1422 (1980), or hygromycin, Sugden, s. ~ al., Mol Cell. Biol. 5:410-413 (1985) . The three ~xamples given 15 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 ~rom Invitrogen Corporation, 3985 Sorrento Valle~ Blvd., San Diego, CA
92121. To confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform E. coli K12 strain DHlOs (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. 9:3Q9 (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 ampllfying 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 techni~ues o~ transforming cells with the aforementioned vectors are well known in the art and may be CA 02243707 l998-07-2l found in such general re~erences as Maniatis, et al., Molecul~r Clonin~: A Laboratorv Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, Mew York (lg89), or Current Protocols in Molecular Biolo~v (1989) and supplements.
Preferred suitable host cells for expressiny 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. et al., J. Gen Virol. 36:59-72 (1977), Viroloav 77:319-329, Virolo~v 86:10-21); baby hamster kidney cells (BHK-21(C-13), ATCC CCL-10, viroloov 16:147 (1962)); Chinese hamster ovary cells C~O-DHFR- (ATCC CRL-9096), mouse Sertoli cells (TM4, ATCC CRL-1715, Biol. Re~rod. 23:243-250 (1980)); African green monkey kidney cells (VERO 76, ATCC CRL-1587); human cervical epitheloid carcinoma cells (He~a, ATCC CC~-2); canine kidney cells (MDCK, ATCC CCL-34); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); human diploid lung cells (WI-38, ATCC CCL-75); human hepatocellular carcinoma cells (Hep G2, ATCC HB-8065);and mouse m~mm~3l~y tumor cells (~IT 060562, ATCC CCL51).
In addition to prokaryotes, unicellular eukaryotes such as yeast cult~res may also be used. Saccharomvces cerevisiae, or common baker~s yeast is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available. For expression in Saccharomyces, the 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 ~or 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 CA 02243707 l998-07-2l W O 97/28824 rCT~US97/01188 enzymes such as enolase (found on plasmid pACl ATCC 39532), glyceraldehyde-3-phosphate dehydrogenase (derived from plasmid pHcGAPCl ATCC 57090, 57091), zymomonas mobilis (United States Patent No. 5,000,000 issued March 19, 1991), 5 --hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3 -phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase other yeast promoters, which contain inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein (contained on plasmid vector pCL28XhoLHBPV
ATCC 39475, United States Patent No. 4,840,896), glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose (GALl found on plasmid pRY121 ATCC 37658) utilization. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman e~ ~l., European Patent Publication No.
73,657A. Yeast enhancers such as the UAS Gal from ~accharomvces cerevisiae (found in conjunction with the C~Cl promoter on plasmid YEpsec--hIlbeta ATCC 67024), also are advantageously used with yeast promoters.
The following examples will help describe how the invention is practiced and will illustrate the invention.
The scope of the present invention is not to be construed as merely consisting of the ~ollowing examples.
~ Pre~aration 1 Porcine 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).
W O 97/28824 PCTrUS97/01188 -19 - .
Primers were designed based on the published amino acid sequence of the human ob gene. The primers were prepared for use in polymerase chain reaction (PCR) amplification methods using a Model 380A DNA synthesizers (PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404). Primers PCROB-l (12504) (ATG CAT TGG
GGA MCC CTG TG), PCROB-2 (12505) (GG ATT CTT GTG GCT TTG GYC
CTA TCT), PCRoB-3 (12506) (TCA GCA CCC AGG GCT GAG GTC CA), and PCROB-4 (12507) (CAT GTC CTG CAG AGA CCC CTG CAG CCT GCT
CA) were prepared.
For cDNA synthesis, total RNA 1 ~L (1 ~g/~l) isolated from porcine adipose tissue and 1 ~L Perkin Elmer Random primers (50 ~M) in a total volume of 12 ~L were annealed for 10 minutes at 70~C and then cooled on ice. The following were then added to the annealed mixture: 4 ~L of BRL 5x H-reverse transcriptase (RT) reaction buffer (Gibco-BRL CAT~28025-013), 2 ~L of 0.1 M DTT, 1 ~L of 10 mM dNTPs.
This annealed mixture was then incubated at 37~C for 2 minutes before adding 1 ~L BRL M-MLV-reverse transcriptase 20 (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 wa~ cooled on ice.
For amplification of cDNA, 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 AmpliTa~ DNA polymerase (Perkin-Elmer Corporation) 10 ~1 of 10x PCR reaction buffer (Perkin-Elmer Corporation) and 50 pmol each of the sense (PCROB-l) and antisense (PCRoB-3) primers for porcine OB amplification. The condition for PCR
was 95~C for 1 minute, 57~C for 1 minute and 72-~C for 1 minute for 30 cycles using a PCR DNA Thermal Cycler (Perkin-Elmer Corporation). 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 mixture (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 W O 97/28824 PCT~US97101188 ~ragment (~0.5 mg) was puri~ied by agarose gel electrophoresis and isolated by the freeze-squeeze method.
The 500 bp fragment (_0. 2 ~g) was then ligated into SmaI
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 (100 ~g~mL). The plasmid was isolated using a Wizard Miniprep DNA
purification system (Promega) and submitted ~or DNA
se~euncing on a Applied Biosystem 370 DNA sequencer.
Preparation 2 == Bovine OB Gene and Gene Product The DNA se~uence of 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.
~
Preparation 3 Vector Construction A plasmid containing the DMA se~uence encoding the obesity protein is constructed to include MdeI and ~HI
restriction sites. The plasmid carrying the cloned PCR
product is digested with ~I and ~mHI restriction enzymes.
The small ~ 450bp fragment is gel-purified and ligated into the vector pRB182 from which the coding se~uence ~or A-C-B
proinsulin is deleted. The ligation products are transformed into E. coli DH10B (commercially available from GIBCO-B~L) and colonies growing on tryptone-yeast (DIFCO) plates supplemented with 10 mg/mL of tetracycline are analyzed.
Plasmid DNA is isolated, digested with ~I and BamHI and the resulting fragments are separated by agarose gel electrophoresis. Plasmids containing the expected ~ 450bp NdeI to ~_HI fragment are kept. E. coli R12 RV308 (available from the NRRL under deposit number B-15624) are W O 97/28824 PCT~US97/01188 - -21- =
trans~ormed with this second plasmid, resulting in a culture suitable for expressing the protein.
The techniques o~ transforming cells with the aforementioned vectors are well known in the art and may be found in such general references as Maniatis, et al. tl988) Molecular Clonino: A Laboratorv Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring ~arbor, Mew York or Current Protocols in Molecular Bioloov (1989) and supplements. The techni~ues involved in the transformation of F.. CQli cells used in the preferred practice of the invention as exemplified herein are well known in the art.
The precise conditions under which the transformed E_ cQli cells are cultured is dependent on the nature o~ the E. ç
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, E.
coli K12 RV308 cells are employed as host cells but numerous other cell lines are available such as, but not limited to, E- coli K12 L201, L687, L6g3, L507, L640, L641, L695, L814 (E. coli B). The transformed host cells are then plated on appropriate media under the selective pressure of the antibiotic corresponding to the resistance gene present on the expression plasmid. The cultures are then incubated for a time and temperature appropriate to the host cell line employed.
Proteins that are expressed in high-level bacterial expression systems characteristically aggregate in granules or inclusion bodies which contain high levels o~ the overexpressed protein. Kreuger et al., in Protein Foldino, Gierasch and King, eds., pgs 136-142 (1990), American Association for the Advancement o~ Science Publication No.
. .
:
89-18S, Washington, D.C. Such protein aggregates must be solu~ilized to provide further purl~ication and isolation of the desired protein product. I~- A variety of techniques using strongly denaturing solutions such as guanidinium-HCl 5 :~ and/or weakly denaturing solutions such as dithiothreitol ~DTT) are used to solubilize the proteins. ~radual 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 ~et-Tyr as described in U.S. Patent No. 5,126,249, herein incorporated by reference. This approach ~acilitates the e~ficient 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, af~inity chromatography, and size exclusion.
The ~ollowing examples are provided merely to further illustrate the preparation of the formulations of the invention. The scope of the invention is not construed as merely consisting of the ~ollowing examples.
~ le 1 Preparation of Protein-Zinc Formulation~
20 mg o~ human obesity protein (SEQ ID No: 1) was completely dissolved in 32 ml of an a~ueous solution containing 16 mg/ml glycerin and 2 mg/ml phenol and passed through a sterile 0.2 ~ filter. An aqueous solution containing 100 mg/ml o~ zinc in water was prepared ~rom zinc chloride and serial dilutions were made to prepare 10 mg/ml zinc and 1 mg/ml zinc solutions. Five 6-ml aliquots of the human obesity protein solution were modified as shown in Table I:
e ~
CA 02243707 l998-07-2l W O 97/28824 PCTnUS97/01188 Table I
~1 of ~l of ~l of ~l of Total mg/ml 1 mg/ml10 mg/ml100 mg/ml H2O added zinc zinc zinc zinc concentration added ad~.ed adcled Samp_e A 0 1~ C
Samp e B 17 0.0~27 Samp e C l 33 O.C54 Samp e D ~ 0 19 0 30 Samp_e E 0 92 3 1.50 Each formulation was adjusted to p~ 7.49 + 0.02 using small volumes of 2N and 5N sodium hydroxide and stored at 4~~.
Sample A was completely clear while samples B through E were cloudy suspensions.
F.~am~le 2 Analvsis of Protein-Zinc Formulations Size-exclusion chromatography was performed on the centrifuged supernatants of Samples A through E of Example 1.
For these analyses, 100 ~l of the supernatants were injected onto an analytical Superdex-75 (Pharmacia~ column equilibrated in PBS (Dulbecco's Phosphate-Buffered Saline, GibcoBRL). The column was eluted at ambient temperature at 0.5 ml/min and the human obesity protein in the eluant monitored at 214 nm. The results of this analysis are shown in Table II.
Table II
Percent of Protein Soluble in Supernatant Samp~e A 1~0 Samp e s 2'.7 Samp e C J
Samp e D
Samp_e E
F~ le 3 Bioloaical Activit~ of Protei~-Zinc Formulations 50 ~l doses of all five Samples in Example 1, each cont~;ning 30 ~g of human obesity protein, were injected subcutaneously into ob/ob mice in a single injection per day W O 97/28824 PCTrUS97/01188 for four days. In addition, a 50 ~l dose of a 16 mgtml glycerin-2 mg/ml phenol solution adjusted to pH 7.4 was administered subcutaneously as a vehicle control. The average daily food intake and cumulative change in body weight from time zero for the ob/ob mice are shown in Table III.
Table III
Food Intake, Weight Change, ~/mouse q/mouse Day Day Day Day Day Day Day Day Vehicle Control ~ .5 4, '~ 1'., ~ --~~, ' _~, ' _~ ' Samp e A . . .
Samp e B .~
Samp e C . ~ _ r _ Samp e D~.' ._ ~. .,- - . - - , - _ Samp_e E.~ . C~
Thus, the data demonstrate that the compounds of the present invention are potent and effective agents useful for treating obesity. When administered as a metal cation complex the potency of the protein was significantly enhanced. Increasing the total zinc concentration of the formulation also provides enhanced potency and improved biological effects in ob/ob mice. Accordingly, the present invention provides compounds, which comprise an obesity protein complexed with a divalent metal cation, pharmaceutical ~ormulations thereof, and methods of using such compounds for treating obesity, and disorders associated with obesity such as diabetes (particularly non-insulin dependent diabetes mellitus), cardiovascular disease and cancer.
: The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected herein, however, is not to be construed as limited to the particular forms disclosed, since they are to 30 ~be regarded as illustrative rather than restrictive.
CA 02243707 l998-07-2l W O 97/28824 PCT~US97/01188 , -2~-Variations and changes may be made by those skilled in the art without departing ~rom the spirit o~ the invention.
t
Obesity, and especially upper body obesity, is a common and very serious public health problem in the United States and throughout the world. According to recent statistics, more than 25% of the United States population and 27~ of the Canadian population are overweight. Kuczmarski, ~mer. J. of Clin. Nutr. 55: 495S - 502~ (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 $150,000,000,000 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, hypertension, and insulin resistance. Many studies have demonstrated that reduction in obesity by diet and exercise reduces these risk factors dramatically. Unfortunately, these treatments are largely unsuccessful with a failure rate reaching 95%. This failure may be due to the fact that the condition is strongly associated with genetically inherited factors that contribute to increased appetite, preference for highly caloric foods, reduced physical activity, and increased lipogenic metabolism. This indicates that people inheriting these genetic traits are prone to becoming obese regardless of their efforts to combat the condition Therefore, a pharmacological agent that can correct this adiposity handicap and allow the physician to successfully _ treat obese patients in spite of their genetic inheri~ance 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 372. 425-32 (1994). This report disclosed the murine and human protein expressed in adipose tissue. Likewise, Murakami et al., in Riochemical and Bio~hvsic~l Research Communications 2Q9(3):944-52 (1995) report the cloning and expression o~ 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., Sc;ence 269: 540-543 (1995).
The present invention provides conditions under which potency of the natural obesity protein is signi~icantly enh~n~ed. Thus, effective pharmacological treatment may be achieved at lower doses that significantl~ lower the risk of toxic or other undesirable side e~~ects. In addition, because the amount of protein ~mi n; stered is less, the cost of the unit dosage form to the patient is reduced.
Accordingly, the present invention provides a novel protein-cation complex, which comprises an obesity protein complexed --with a divalent metal cation, pharmaceutical formulations thereo~, and methods of using such compounds for the treatment of obesity, and disorders associated with obesity such as diabetes, cardiovascular disease and cancer The invention provides a compound comprising an obesity protein complexed with a divalent metal cation.
The invention additionally provides parenteral pharmaceutical formulations comprising the protein-cation compounds and methods of using such compounds for treating obesity and disorders associated with obesity such as diabetes, cardiovascular disease and cancer. The invention ~urther comprises combining an obesity protein and a divalent CA 02243707 l998-07-2l W O 97/28824 PCTrUS97/01188 metal cation in an aqueous solution at a pH o~ about 4.5 to 9 .0 .
For purposes o~ the present invention, as disclosed and claimed herein, the ~ollowing terms and abbreviations are defined as ~ollows:
Base pair (bp) -- re~ers to DNA or RNA. The abbreviations A,C,G, and T correspond to the 5'-monophosphate forms o~ the nucleotides (deoxy)a~en; ne, (deoxy)cytidine, (deoxy)guanine, and (deoxy)thymine, respectively, when they occur in DNA molecules. The abbreviations U,C,G, and T
correspond to the 5'-monophosphate forms o~ the nucleosides uracil, cytidine, guanine, and thymine, respectively when they occur in RNA molecules. In double stranded DMA, base pair may re~er to a partnership of A with T or C with G. In a DN~/RNA heteroduplex, base pair may re~er to a partnership o~ T with U or C with G.
Obesity protein -- re~ers to the native m~mm~lian protein produced ~rom the native ob gene ~ollowing transcription and deletions o~ introns, translation to a protein and processing to the mature protein with secretory signal peptide removed, e.g. ~rom the N-terminal valine-proline to the C-terminal cysteine o~ the mature protein.
The human obesity protein is published in Zhang et al.
Nature 372: 425-32 (lg94). The rat obesity protein is published in Murakami et al., Biochemical ~nd Bio~hvsical Research Comm. 209(3): 944-52 (1995). Native porcine and bovine Ob proteins are disclosed in a U.S. patent application by Hansen M. Hsiung and Dennis P. Smith ~iled May 19, 1995, Serial number 08/445,305 (EPO 743 321). Other m~mm~lian Ob proteins are disclosed in U.S. patent application serial number 08/452,228, ~iled May 26, 1995 (EPO 744 408) and ~ provisional application, 60,003935, filed September 19, 1995 (EP ), herein all o~ which are incorporated by re~erence.
Obesity protein includes those proteins having a leader se~uence. A leader se~uence is one or more amino acids on the N-terminus to aid in production or puri~ication o~ the W O 97/28824 PCT~US97/01188 protein. A preferred leader sequence is Met-R1 where R1 is _ absent or any amino acid except Pro.
Plasmid -- an extrachromosomal self-replicating genetic element.
Reading frame -- the nucleotide se~uence from which translation occurs "read'~ in triplets by the translational apparatus of tRNA, ribosomes and associated factors, each triplet corresponding to a particular amino acid Because each triplet is distinct and of the same length, the coding se~uence must be a multiple of three. A base pair insertion or deletion (termed a frameshift mutation) may result in two different proteins being coded for by the same DNA segment.
To insure against this, the triplet codons corresponding to the desired polypeptide must be aligned in multiples of three from the initiation codon, i.e. the correct ~reading frame"
must be maintained. In the creation of fusion proteins containing a chelating peptide, the reading frame of the DNA
sequence encoding the structural protein must be maintained in the DNA sequence encoding the chelating peptide.
Recombinant DNA Cloning Vector -- any autonomously replicating agent including, but not limited to, plasmids and phages, comprising a DNA molecule to which one or more additional DMA 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 se~uence of DNA is transferred to a complementary RMA se~uence.
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 for the transformation of cells in gene manipulation bearing polynucleotide se~uences corresponding to appropriate protein molecules which, when CA 02243707 l998-07-2l W O 97/2~824 PCTnUS97/01188 combined with appropriate control se~uences, confer specific properties on the host cell to be transformed. Plasmids, viruses, and bacteriophage are suitable vectors, since they are replicons in their own right. Artificial vectors are constructed by cutting and joining DMA 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 the disease, condition, or disorder and includes the administration of a compound 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 ~lm; n; stration 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 formulation to prevent the net flow of water across the cell membrane. Compounds, such as glycerin, are commonly used for such purposes at known concentrations. Other possible isotonicity agents include salts, e.g., NaCl, dextrose, and lactose.
- Physiologically tolerated buffer -- a physiologically tolerated buffer is known in the art.
Physiologically tolerated buffers include TRIS, sodium acetate, sodium phosphate, or sodium citrate. The selection and concentration of buffer is known in the art.
Pharmaceutically acceptable preservative -- a multi-use parenteral formulation must meet guidelines for preservative effectiveness to be a commercially viable product. Pharmaceutically acceptable preservatives known in the art as being acceptable in parenteral formulations include: phenol, m-cresol, benzyl alcohol, methylparaben, chlorobutanol, p-cresol, phenylmercuric nitrate, thimerosal W O 97/28824 PCT~US97/01188 and various mixtures thereof. Other preservatives may be found, e.g., in WALLHAUSE:~, K.--H., DEVELOP. BIoL. STANDARD. 24, pp. 9-28 (Basel, S. Krayer, 1974). The concentration necessary to achieve preservative effectiveness is dependent _upon the preservative used and the conditions o~ the formulation.
The nucleotide and amino acid abbreviations used herein are accepted by the United States Patent and Trademark Of~ice as set forth in 37 C.F.R. 1.822 (b)(2) (1993).
Unless otherwise indicated the amino acids are in the L
configuration.
As noted above, the invention provides a compound comprising an obesity protein complexed with a divalent metal cation. When complexed with a divalent metal cation, the ~obesity protein demonstrates significantly enhanced potency.
The presently claimed compounds comprise an obesity protein complexed with a divalent metal cation. A divalent metal cation includes, ~or example, Zn+~, Mn+~, Fe++, Co++, Cd++, Ni+~ and the like. A combination of two or more divalent metal cations is operable; however the preferred compounds comprise a single species of metal cation, most preferably Zn++. Preferably, the divalent metal cation is in excessi however, the molar ratio o~ at least one molecule of a divalent metal cation for each ten molecules of obesity protein is operable. Pre~erably, the compounds comprise from 1 to 100 divalent metal cations per molecule of obesity protein. The compounds may be amorphous or crystalline solids.
Appropriate forms of metals cations are any form of a divalent metal cation that is available to form a complex with a molecule of obesity protein of the present invention.
The metal cation may be added in solid form or it may be added as a solution. Several dif~erent cationic salts can be used in the p~esent invention. Representative examples of metal salts include the acetate, bromide, chloride, fluoride, iodide and sulfate salt forms. The skilled artisan will recognize that there are many other metal salts which also W O 97/28824 PCTnUSg7/01188 might be used in the production of the compounds of the present invention. Preferably, zinc acetate or zinc chloride is used to create the zinc-obesity protein compounds of the present invention. Most preferably, the divalent metal cationic salt is zinc chloride, Generally, the claimed compounds are prepared by techniques known in the art. For example, convenient preparation is to combine the obesity protein with the desired divalent metal cation in an aqueous solution at a pH
of about 4.5-9.0, preferably about pH 5.5-8, most preferably, about pH 6.5-7.6. The claimed compound precipitates from the solution as a crystalline or amorphous solid. Significantly, the compound is easily isolated and puri~ied by conventional separation techni~ues appreciated in the art including filtration and centrifugation. Significantly, the protein-metal cation complex is stable and may be conveniently stored as a solid or as an aqueous suspension.
The present invention further provides a pharmaceutical formulation comprising a compound o~ the present invention and water. The concentration of the obesity protein in the formulation is about 0.1 mg/mL to about 100 mg/mL; preferably about 0.5 mg/mL to about 50.0 mg/mL; most preferably, about 5.0 mg/mL.
The ~ormulation preferably comprises a pharmaceutically acceptable preservative at a 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.-30 H., DEvELoP. BIOL. STANDARD. 24, pp. 9--28(Basel, S. Krager, 1974), herein incorporated by reference.
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 ~ormulations, preferably about 1~ mg/mL glycerin.
The pH of the ~ormulation may also be buffered with a physiologically tolerated buffer. Acceptable physiologically CA 02243707 l998-07-2l W O 97/28824 PC~rUS97/01188 ~8--tolerated buffers include TRIS, sodium acetate, sodium phosphate, or sodium citrate. The selection and concentration of buffer is known in the art.
Other additives, such as a pharmaceutically acceptable excipients like Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers), BRIJ 35 (polyoxyethylene (23) lauryl ether), and PEG (polyethylene glycol) may optionally be added to the formulation to reduce aggregation.
The claimed pharmaceutical formulations are prepared in a manner known in the art, and are administered individually or in combination with other therapeutic agents.
The ~ormulations of the present invention can be prepared using conventional dissolution and mixing procedures.
Pre~erably, the claimed ~ormulations are prepared in an aqueous solution suitable for parenteral use. That is, a protein solution is prepared by mixing water for injection, buffer, and a preservative. Divalent metal cations are added to a total cation concentration of about 0.001 to 5.0 mg/mL, preferably 0.05 to 1.5 mg/mL. The pH of the solution may be adjusted to completely precipitate the obesity protein zinc complex. The compound is easily resuspended be~ore administration to the patient.
Parenteral daily doses of the compound are in the range from about 1 ng to about 10 mg per kg of body weight, although lower or higher dosages may be administered. The required dosage will be determined by the physician and will depend on the severity of the condition o~ the patient and upon such criteria as the patient's height, weight, sex, age, and medical history.
Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, if a surfactant is used, the temperature, and pH at which the ~ormulation is prepared may W O 97t28824 PCTrUS97/01188 be optimized for the concentration and means of administration used.
The pH of the formulation is generally pH 4.5 to 9.0 and preferably 5.5 to 8.0, most preferably 6.5 to 7.6;
although more acidic pH wherein a portion or all of the protein-metal cation complex is in solution is operable.
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 10 ~mi n; stration.
The obesity proteins of the present invention can be prepared by any of a variety of recognized peptide synthesis techniques including classical (solution) methods, solid phase methods, semi synthetic methods, and more recent recombinant DNA methods. Preferably, an obesity protein of the present invention is human protein of the formula:
(SEQ ID NO: 1) lo 15 Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile Ser E~is Thr Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu 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 so 95 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys loo 105 llo His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 4 0 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 ' 45 1~5 Gly Cys CA 02243707 l998-07-2l W O 97/28824 PCT~US97/01188 - ~10 -The preparation of the human protein is known and disclosed, for example, in Halaas Jeffrey L. et al., Science 269 (1995).
The preparation of other mammalian obesity proteins are described in U.S. application serial number 08/445,305, filed May ~9, 1995 (EPO 743 221); U.S. patent application serial number 08/452,228, filed May 26, 1995 (EPO 744 408); and provisional application, 60,003935, filed September 19, 1995 (EP 96306721.0); all of which are herein incorporated by re~erence.
The obesity proteins described herein may also be produced either by recombinant DNA technology or well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution 15 : 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 in vitro or ia vivo transcription and translation of which will result in the production of the protein may be constructed by techni~ues 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 pre~erred practice of CA 02243707 l998-07-2l W O 97/28824 PCTrUS97/01188 the invention, synthesis is achieved by recombinant DNA
technology.
Methodology o~ 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. The ~NA 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 ~rom Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404). It may desirable in some applications to modi~y the coding sequence o~ the obesity protein so as to incorporate a convenient protease sensitive cleavage site, e.g., between the signal peptide and the structural protein ~acilitating the controlled excision o~ the signal peptide ~rom the fusion protein construct.
The gene encoding the obesity protein may also be created by using polymerase chain reaction (PCR). The template can be a cDNA library (commercially available ~rom CLONETECH or STRATAGENE) or mRNA isolated ~rom the desired arrival adipose tissue. Such methodologies are well known in the art Maniatis, et al. Molecular Clonin~: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989).
The constructed or isolated DNA sequences are useful ~or expressing the obesity protein either by direct expression or as ~usion protein. When the sequences are used in a fusion gene, the resulting product will require enzymatic or chemical cleavage. A variety o~ peptidases which cleave a polypeptide at speci~ic sites or digest the peptides ~rom the amino or carboxy termini (e.g.
diaminopeptidase) o~ the peptide chain are known.
Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave a polypeptide chain at speci~ic sites. The skilled artisan will appreciate the modi~ications necessary to the amino acid sequence (and synthetic or semi-synthetic coding sequence i~ recombinant means are employed) to CA 02243707 l998-07-2l W O 97/28824 PCT~US97/01188 incorporate site-specific internal cleavage sites. See U.S.
Patent No. 5,126,249; Carter P., Site Speci~ic Proteolysis o~ Fusion Protei~s, Ch. 13 in Protein Puri~icat;on: From Molecul~r Mech~n;sms to rl~r~e Scale Processes, ~merican Chemical Soc., Washington, D C. (1990).
Construction o~ suitable vectors cont~;n;ng the desired coding and control sequences employ standard ligation techniques. Isolated plasmids or DNA ~ragments are cleaved, tailored, and religated in the ~orm desired to ~orm the plasmids required.
To e~ect the translation of the desired protein, one inserts the engineered synthetic DNA sequence in any o~ a plethora o~ appropriate recombinant DMA expression vectors through the use o~ appropriate restriction endonucleases. A
synthetic coding sequence may be designed to possess restriction endonuclease cleavage sites at either end o~ the transcript to ~acilitate isolation ~rom and integration into these expression and ampli~ication and expression plasmids.
The isolated cDNA coding sequence may be readily modi~ied by the use o~ synthetic linkers to ~acilitate the incorporation of this se~uence into the desired cloning vectors by techniques well known in the art. The particular endonucleases employed will be dictated by the restriction endonuclease cleavage pattern o~ the parent expression vector to be employed. The restriction sites are chosen so as to properly orient the coding se~uence with control sequences to achieve proper in-~rame reading and expression o~ the protein.
In general, plasmid vectors containing promoters 30 _ and control sequences which are derived ~rom 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 trans~ormed cells. For example, E. coli is typically trans~ormed using pBR322, a plasmid derived ~rom an E. coli species (Bolivar, e~ ~1-, Gene 2: 95 (1977)).
Plasmid pBR322 contains genes ~or ampicillin and tetracycline W O 97/28824 PCTrUS97/01188 resistance and thus provides easy means ~or identifying transformed cells. The pBR322 plasmid, or other microbial , plasmid must also contain or be modified to contain promoters and other control elements commonly used in recombinant DNA
technology.
The desired coding se~uence is inserted into an expression vector in the proper orientation to be transcribed from a promoter and ribosome binding site, both of which should be functional in the host cell in which the protein is to be expressed. An example of such an expression vector is a plasmid described in Belagaje et al., U.S. patent No.
5,304,493, the teachings of which are herein incorporated by reference. The gene encoding A-C-B proinsulin described in U.S. patent No. 5,304,493 can be removed from the plasmid pRB182 with restriction enzymes NdeI and BamHI. The isolated DNA se~uences can be inserted into the plasmid backbone on a NdeI/BamHI restriction fragment cassette.
In general, procaryotes are used for cloning of DNA
se~uences in constructing the vectors useful in the invention. For example, E. coli K12 strain 294 ~ATCC No.
31446) is particularly useful. other microbial strains which may be used include E. coli B and E. ÇQli X1776 (ATCC No.
31537). These examples are illustrative rather than limiting.
Procaryotes also are used for expression. The 25 aforementioned strains, as well as E. coli W3110 (prototrophic, ATCC No. 27325), bacilli such as Bacillus subtilis, and other enterobacteriaceae such as Salmonella tv~himurium or Serratia marcescans, and various pseudomonas species may be used. Promoters suitable for use with 30 prokaryotic hosts include the ~-lactamase (vector pGX2907 [ATCC 39344] contains the replicon and ~-lactamase gene) and r lactose promoter systems (Chang et al., Nature, 275:615 (lg78); and Goeddel et ~1., Nature ~1:544 (1979)), alkaline phosphatase, the tryptophan (trp) promoter system (vector pATHl [ATCC 37695] is designed to facilitate expression of an open reading frame as a trpE fusion protein under control of the trp promoter) and hybrid promoters such as the tac CA 02243707 l998-07-2l O 97/28824 PCT~US97/0~188 promoter (isolatable from plasmid pDR540 ATCC-37282).
However, other functional bacterial promoters, whose nucleotide sequences are generally known, enable one of skill in the art to ligate them to DNA encoding the protein using linkers or adaptors to supply any re~uired restriction sites.
Promoters ~or use in bacterial systems also will contain a Shine-Dalgarno sequence operably linked to the DNA encoding protein.
The DNA molecules may also be recombinantly produced in eukaryotic expression systems. Preferred promoters controlling transcription in m~mm~l ian host cells may be obtained from various sources, for example, the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. ~-actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction ~ragment which also contains the SV40 viral origin of replication. Fiers, et al., Mature, 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~ (L~;m;n~, L. et al., PNAS 78:993 (1981)) and 3' (Lusky, M. L., et al., Mol. Cell Bio. 3:1108 (1983)) to the transcription unit, within an intron (sanerji, J. L. et al., Cell 33:729 (lg83)) as well as within the coding se~uence itself (Osborne, T. F., et al., ~Mol. Cell Bio. 4:1293 (1984)). Many enhancer sequences are now known from m~mm~lian genes (globin, RSV, SV40, EMC, elastase, albumin, alpha-fetoprotein and insulin).
CA 02243707 l998-07-2l W O 97/28824 PCTnUS97/~1188 Typically, however, one will use an enhancer ~rom a eukaryotic cell virus. Examples include the SV40 late enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells from other multicellular organisms) will also contain se~uences necessary ~or the termination of transcription which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding protein The 3' untranslated regions also include transcription termination sites.
Expression vectors may contain a selection gene, also termed a selectable marker. Examples of suitable selectable markers for m~mm~l ian cells are dihydrofolate reductase (DHFR, which may be derived from the ~ n~III
restriction ~ragment of pJOD-10 [ATCC 68815]), thymidine kinase (herpes simplex virus thymidine kinase is contained on the ~mHI ~ragment of vP-5 clone [ATCC 2028]) or neomycin (G418) resistance genes (obtainable from pNN414 yeast artificial chromosome vector ~ATCC 37682]). When such selectable markers are success~ully transferred into a m~mm~l ian host cell, the trans~ected m~mm~l ian host cell can survive if placed under selective pressure. There are two widely used distinct categories of selective regimes. The first category is based on a cell's metabolism and the use of a mutant cell line which lacks the ability to grow without a supplemented media. Two examples are: CHO D~FR- cells (ATCC
CRL-gO96) and mouse LTK- cells (L-M(TK-) ATCC CCL-2.3).
These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide - synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media. An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective CA 02243707 l998-07-2l genes, thus altering their growth requirements. Individual cells which were not trans~ormed with the DHFR or TK gene will not be capable of survival in nonsupplemented media.
The second category is dom;n~nt seiection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells which have a novel gene would express a protein conveying drug resistance and would survive the selection.
Examples of such dominant selection use the drugs neomycin, Southern P. and Berg, P., J. Molec. A~~l. Genet. 1: 327 (1982), mycophenolic acid, Mulligan, R. C. and Berg, P.
Science ~ 1422 (1980), or hygromycin, Sugden, s. ~ al., Mol Cell. Biol. 5:410-413 (1985) . The three ~xamples given 15 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 ~rom Invitrogen Corporation, 3985 Sorrento Valle~ Blvd., San Diego, CA
92121. To confirm correct sequences in plasmids constructed, the ligation mixtures are used to transform E. coli K12 strain DHlOs (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. 9:3Q9 (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 ampllfying 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 techni~ues o~ transforming cells with the aforementioned vectors are well known in the art and may be CA 02243707 l998-07-2l found in such general re~erences as Maniatis, et al., Molecul~r Clonin~: A Laboratorv Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, Mew York (lg89), or Current Protocols in Molecular Biolo~v (1989) and supplements.
Preferred suitable host cells for expressiny 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. et al., J. Gen Virol. 36:59-72 (1977), Viroloav 77:319-329, Virolo~v 86:10-21); baby hamster kidney cells (BHK-21(C-13), ATCC CCL-10, viroloov 16:147 (1962)); Chinese hamster ovary cells C~O-DHFR- (ATCC CRL-9096), mouse Sertoli cells (TM4, ATCC CRL-1715, Biol. Re~rod. 23:243-250 (1980)); African green monkey kidney cells (VERO 76, ATCC CRL-1587); human cervical epitheloid carcinoma cells (He~a, ATCC CC~-2); canine kidney cells (MDCK, ATCC CCL-34); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); human diploid lung cells (WI-38, ATCC CCL-75); human hepatocellular carcinoma cells (Hep G2, ATCC HB-8065);and mouse m~mm~3l~y tumor cells (~IT 060562, ATCC CCL51).
In addition to prokaryotes, unicellular eukaryotes such as yeast cult~res may also be used. Saccharomvces cerevisiae, or common baker~s yeast is the most commonly used eukaryotic microorganism, although a number of other strains are commonly available. For expression in Saccharomyces, the 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 ~or 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 CA 02243707 l998-07-2l W O 97/28824 rCT~US97/01188 enzymes such as enolase (found on plasmid pACl ATCC 39532), glyceraldehyde-3-phosphate dehydrogenase (derived from plasmid pHcGAPCl ATCC 57090, 57091), zymomonas mobilis (United States Patent No. 5,000,000 issued March 19, 1991), 5 --hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3 -phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase other yeast promoters, which contain inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein (contained on plasmid vector pCL28XhoLHBPV
ATCC 39475, United States Patent No. 4,840,896), glyceraldehyde 3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose (GALl found on plasmid pRY121 ATCC 37658) utilization. Suitable vectors and promoters for use in yeast expression are further described in R. Hitzeman e~ ~l., European Patent Publication No.
73,657A. Yeast enhancers such as the UAS Gal from ~accharomvces cerevisiae (found in conjunction with the C~Cl promoter on plasmid YEpsec--hIlbeta ATCC 67024), also are advantageously used with yeast promoters.
The following examples will help describe how the invention is practiced and will illustrate the invention.
The scope of the present invention is not to be construed as merely consisting of the ~ollowing examples.
~ Pre~aration 1 Porcine 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).
W O 97/28824 PCTrUS97/01188 -19 - .
Primers were designed based on the published amino acid sequence of the human ob gene. The primers were prepared for use in polymerase chain reaction (PCR) amplification methods using a Model 380A DNA synthesizers (PE-Applied Biosystems, Inc., 850 Lincoln Center Drive, Foster City, CA 94404). Primers PCROB-l (12504) (ATG CAT TGG
GGA MCC CTG TG), PCROB-2 (12505) (GG ATT CTT GTG GCT TTG GYC
CTA TCT), PCRoB-3 (12506) (TCA GCA CCC AGG GCT GAG GTC CA), and PCROB-4 (12507) (CAT GTC CTG CAG AGA CCC CTG CAG CCT GCT
CA) were prepared.
For cDNA synthesis, total RNA 1 ~L (1 ~g/~l) isolated from porcine adipose tissue and 1 ~L Perkin Elmer Random primers (50 ~M) in a total volume of 12 ~L were annealed for 10 minutes at 70~C and then cooled on ice. The following were then added to the annealed mixture: 4 ~L of BRL 5x H-reverse transcriptase (RT) reaction buffer (Gibco-BRL CAT~28025-013), 2 ~L of 0.1 M DTT, 1 ~L of 10 mM dNTPs.
This annealed mixture was then incubated at 37~C for 2 minutes before adding 1 ~L BRL M-MLV-reverse transcriptase 20 (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 wa~ cooled on ice.
For amplification of cDNA, 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 AmpliTa~ DNA polymerase (Perkin-Elmer Corporation) 10 ~1 of 10x PCR reaction buffer (Perkin-Elmer Corporation) and 50 pmol each of the sense (PCROB-l) and antisense (PCRoB-3) primers for porcine OB amplification. The condition for PCR
was 95~C for 1 minute, 57~C for 1 minute and 72-~C for 1 minute for 30 cycles using a PCR DNA Thermal Cycler (Perkin-Elmer Corporation). 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 mixture (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 W O 97/28824 PCT~US97101188 ~ragment (~0.5 mg) was puri~ied by agarose gel electrophoresis and isolated by the freeze-squeeze method.
The 500 bp fragment (_0. 2 ~g) was then ligated into SmaI
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 (100 ~g~mL). The plasmid was isolated using a Wizard Miniprep DNA
purification system (Promega) and submitted ~or DNA
se~euncing on a Applied Biosystem 370 DNA sequencer.
Preparation 2 == Bovine OB Gene and Gene Product The DNA se~uence of 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.
~
Preparation 3 Vector Construction A plasmid containing the DMA se~uence encoding the obesity protein is constructed to include MdeI and ~HI
restriction sites. The plasmid carrying the cloned PCR
product is digested with ~I and ~mHI restriction enzymes.
The small ~ 450bp fragment is gel-purified and ligated into the vector pRB182 from which the coding se~uence ~or A-C-B
proinsulin is deleted. The ligation products are transformed into E. coli DH10B (commercially available from GIBCO-B~L) and colonies growing on tryptone-yeast (DIFCO) plates supplemented with 10 mg/mL of tetracycline are analyzed.
Plasmid DNA is isolated, digested with ~I and BamHI and the resulting fragments are separated by agarose gel electrophoresis. Plasmids containing the expected ~ 450bp NdeI to ~_HI fragment are kept. E. coli R12 RV308 (available from the NRRL under deposit number B-15624) are W O 97/28824 PCT~US97/01188 - -21- =
trans~ormed with this second plasmid, resulting in a culture suitable for expressing the protein.
The techniques o~ transforming cells with the aforementioned vectors are well known in the art and may be found in such general references as Maniatis, et al. tl988) Molecular Clonino: A Laboratorv Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring ~arbor, Mew York or Current Protocols in Molecular Bioloov (1989) and supplements. The techni~ues involved in the transformation of F.. CQli cells used in the preferred practice of the invention as exemplified herein are well known in the art.
The precise conditions under which the transformed E_ cQli cells are cultured is dependent on the nature o~ the E. ç
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, E.
coli K12 RV308 cells are employed as host cells but numerous other cell lines are available such as, but not limited to, E- coli K12 L201, L687, L6g3, L507, L640, L641, L695, L814 (E. coli B). The transformed host cells are then plated on appropriate media under the selective pressure of the antibiotic corresponding to the resistance gene present on the expression plasmid. The cultures are then incubated for a time and temperature appropriate to the host cell line employed.
Proteins that are expressed in high-level bacterial expression systems characteristically aggregate in granules or inclusion bodies which contain high levels o~ the overexpressed protein. Kreuger et al., in Protein Foldino, Gierasch and King, eds., pgs 136-142 (1990), American Association for the Advancement o~ Science Publication No.
. .
:
89-18S, Washington, D.C. Such protein aggregates must be solu~ilized to provide further purl~ication and isolation of the desired protein product. I~- A variety of techniques using strongly denaturing solutions such as guanidinium-HCl 5 :~ and/or weakly denaturing solutions such as dithiothreitol ~DTT) are used to solubilize the proteins. ~radual 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 ~et-Tyr as described in U.S. Patent No. 5,126,249, herein incorporated by reference. This approach ~acilitates the e~ficient 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, af~inity chromatography, and size exclusion.
The ~ollowing examples are provided merely to further illustrate the preparation of the formulations of the invention. The scope of the invention is not construed as merely consisting of the ~ollowing examples.
~ le 1 Preparation of Protein-Zinc Formulation~
20 mg o~ human obesity protein (SEQ ID No: 1) was completely dissolved in 32 ml of an a~ueous solution containing 16 mg/ml glycerin and 2 mg/ml phenol and passed through a sterile 0.2 ~ filter. An aqueous solution containing 100 mg/ml o~ zinc in water was prepared ~rom zinc chloride and serial dilutions were made to prepare 10 mg/ml zinc and 1 mg/ml zinc solutions. Five 6-ml aliquots of the human obesity protein solution were modified as shown in Table I:
e ~
CA 02243707 l998-07-2l W O 97/28824 PCTnUS97/01188 Table I
~1 of ~l of ~l of ~l of Total mg/ml 1 mg/ml10 mg/ml100 mg/ml H2O added zinc zinc zinc zinc concentration added ad~.ed adcled Samp_e A 0 1~ C
Samp e B 17 0.0~27 Samp e C l 33 O.C54 Samp e D ~ 0 19 0 30 Samp_e E 0 92 3 1.50 Each formulation was adjusted to p~ 7.49 + 0.02 using small volumes of 2N and 5N sodium hydroxide and stored at 4~~.
Sample A was completely clear while samples B through E were cloudy suspensions.
F.~am~le 2 Analvsis of Protein-Zinc Formulations Size-exclusion chromatography was performed on the centrifuged supernatants of Samples A through E of Example 1.
For these analyses, 100 ~l of the supernatants were injected onto an analytical Superdex-75 (Pharmacia~ column equilibrated in PBS (Dulbecco's Phosphate-Buffered Saline, GibcoBRL). The column was eluted at ambient temperature at 0.5 ml/min and the human obesity protein in the eluant monitored at 214 nm. The results of this analysis are shown in Table II.
Table II
Percent of Protein Soluble in Supernatant Samp~e A 1~0 Samp e s 2'.7 Samp e C J
Samp e D
Samp_e E
F~ le 3 Bioloaical Activit~ of Protei~-Zinc Formulations 50 ~l doses of all five Samples in Example 1, each cont~;ning 30 ~g of human obesity protein, were injected subcutaneously into ob/ob mice in a single injection per day W O 97/28824 PCTrUS97/01188 for four days. In addition, a 50 ~l dose of a 16 mgtml glycerin-2 mg/ml phenol solution adjusted to pH 7.4 was administered subcutaneously as a vehicle control. The average daily food intake and cumulative change in body weight from time zero for the ob/ob mice are shown in Table III.
Table III
Food Intake, Weight Change, ~/mouse q/mouse Day Day Day Day Day Day Day Day Vehicle Control ~ .5 4, '~ 1'., ~ --~~, ' _~, ' _~ ' Samp e A . . .
Samp e B .~
Samp e C . ~ _ r _ Samp e D~.' ._ ~. .,- - . - - , - _ Samp_e E.~ . C~
Thus, the data demonstrate that the compounds of the present invention are potent and effective agents useful for treating obesity. When administered as a metal cation complex the potency of the protein was significantly enhanced. Increasing the total zinc concentration of the formulation also provides enhanced potency and improved biological effects in ob/ob mice. Accordingly, the present invention provides compounds, which comprise an obesity protein complexed with a divalent metal cation, pharmaceutical ~ormulations thereof, and methods of using such compounds for treating obesity, and disorders associated with obesity such as diabetes (particularly non-insulin dependent diabetes mellitus), cardiovascular disease and cancer.
: The principles, preferred embodiments and modes of operation of the present invention have been described in the foregoing specification. The invention which is intended to be protected herein, however, is not to be construed as limited to the particular forms disclosed, since they are to 30 ~be regarded as illustrative rather than restrictive.
CA 02243707 l998-07-2l W O 97/28824 PCT~US97/01188 , -2~-Variations and changes may be made by those skilled in the art without departing ~rom the spirit o~ the invention.
t
Claims (19)
1. A compound comprising an obesity protein complexed with a divalent metal cation.
2. A compound of Claim 1, wherein the protein is human obesity protein, said protein optionally having a leader sequence.
3. A compound of Claim 2 wherein the protein is of the formula:
(SEQ ID NO: 1) Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr, Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys said protein having a disulfide bond between Cys at position 96 and Cys at position 146.
(SEQ ID NO: 1) Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr, Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro Gly Cys said protein having a disulfide bond between Cys at position 96 and Cys at position 146.
4. A compound of Claim 3, wherein the divalent metal cation is Zn++.
5. A parenteral pharmaceutical formulation, comprising a compound of any one of Claims 1 through 4, and water.
6. A formulation of Claim 5, which further comprises a pharmaceutically acceptable preservative.
7. A formulation of Claim 6, which further comprises an isotonicity agent.
8. A formulation of Claim 7, which further comprises a physiologically acceptable buffer.
9. A formulation of Claim 8, wherein the obesity protein is at a concentration of about 0.1 to 100 mg/mL, the preservative is phenol or m-cresol or a mixture thereof.
10. A formulation of Claim 9, wherein the total cation concentration is 0.001 to 5.0 mg/mL.
11. A formulation of Claim 12, wherein the total cation concentration is 0.05 to 1.5 mg/mL.
12. A formulation of Claim 11, wherein the pH of the formulation is about pH 4.5 to 9Ø
13. A formulation of Claim 12, wherein the pH of the formulation is about pH 5.5 to 8Ø
14. A formulation of Claim 13, wherein the pH of the formulation is pH 6.5 to 7.6.
15. A method of treating obesity, which comprises administering to a patient in need thereof an effective amount of a compound of any one of Claims 1 through 4.
16. A method of treating obesity, which comprises administering to a patient in need thereof an effective amount of a formulation of any one of Claims 5 through 14.
17. A compound of any one of Claims 1 through 4 for use in the treatment of obesity.
18. A formulation of any one of Claims 5 through 11 for use in the treatment of obesity.
19. A process of preparing a compound of any one of Claims 1 through 4, which comprises combining an obesity protein and a divalent metal cation in an aqueous solution at a pH of about 4.5 to 9Ø
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US1123896P | 1996-02-06 | 1996-02-06 | |
| US60/011,238 | 1996-02-06 | ||
| PCT/US1997/001188 WO1997028824A1 (en) | 1996-02-06 | 1997-01-24 | Obesity protein compounds and formulations thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2243707A1 true CA2243707A1 (en) | 1997-08-14 |
Family
ID=29422610
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2243707 Abandoned CA2243707A1 (en) | 1996-02-06 | 1997-01-24 | Obesity protein compounds and formulations thereof |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2243707A1 (en) |
-
1997
- 1997-01-24 CA CA 2243707 patent/CA2243707A1/en not_active Abandoned
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