Obesity protein analog compounds and formulations thereof

The present invention provides novel compounds, which comprise an obesity protein analog 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.

This application claims the benefit of U.S. Provisional Application No. 
60/011,055, filed Jan. 25, 1996. 
FIELD OF INVENTION 
The present invention is in the field of human medicine, particularly in 
the treatment of obesity and disorders associated with obesity. More 
specifically, the present invention relates to compounds and formulations 
of an obesity protein analog. 
BACKGROUND OF THE INVENTION 
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, 
Amer. J. of Clin. Nutr. 55: 495S-502S (1992); Reeder et. al., Can. Med. 
Ass. J., 23: 226-233 (1992). Upper body obesity is the strongest risk 
factor known for type II diabetes mellitus, and is a strong risk factor 
for cardiovascular disease and cancer as well. Recent estimates for the 
medical cost of obesity are $150,000,000,000 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 inheritance is 
needed. 
The ob/ob mouse is a model of obesity and diabetes that is known to carry 
an autosomal recessive trait linked to a mutation in the sixth chromosome. 
Recently, Yiying Zhang and co-workers published the positional cloning of 
the mouse gene linked with this condition. Yiying Zhang et al. Nature 372: 
425-32 (1994). This report disclosed the murine and human protein 
expressed in adipose tissue. Likewise, Murakami et al., in Biochemical and 
Biophysical Research Communications209(3):944-52 (1995) report the cloning 
and expression of the rat obese gene. The protein, which is encoded by the 
ob gene, has demonstrated an ability to effectively regulate adiposity in 
mice. Pelleymounter et al., Science269: 540-543 (1995). 
Obesity protein analogs have been developed and have demonstrated 
pharmacological activity. Some of these analogs demonstrate significant 
improvement in physical properties and stability making them improved 
pharmacological agents. Analogs included in the present invention are 
disclosed in Basinski et al., U.S. Ser. No. 08/383,638 and DiMarchi et 
al., U.S. provisional application Ser. Nos. 60/000,450 and 60/002,161 
(published as WO 96/23515 and 96/23517). 
The present invention provides conditions under which potency of the analog 
is significantly enhanced. Thus, effective pharmacological treatment may 
be achieved at lower doses that significantly lower the risk of toxic or 
other undesirable side effects. In addition, because the amount of protein 
administered 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 analog 
complexed with a divalent metal cation, pharmaceutical formulations 
thereof, and methods of using such compounds for the treatment of obesity, 
and disorders associated with obesity such as diabetes, cardiovascular 
disease and cancer. 
SUMMARY OF THE INVENTION 
The invention provides a compound comprising an obesity protein analog 
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 further 
provides a process of preparing such compounds, which comprises combining 
an obesity protein analog and a divalent metal cation in an aqueous 
solution at a pH of about 4.5 to 9.0. 
DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS 
For purposes of the present invention, as disclosed and claimed herein, the 
following terms and abbreviations are defined as follows: 
Base pair (bp)--refers to DNA or RNA. The abbreviations A,C,G, and T 
correspond to the 5'-monophosphate forms of the nucleotides 
(deoxy)adenine, (deoxy)cytidine, (deoxy)guanine, and (deoxy)thymine, 
respectively, when they occur in DNA molecules. The abbreviations U,C,G, 
and T correspond to the 5'-monophosphate forms of the nucleosides uracil, 
cytidine, guanine, and thymine, respectively when they occur in RNA 
molecules. In double stranded DNA, base pair may refer to a partnership of 
A with T or C with G. In a DNA/RNA heteroduplex, base pair may refer to a 
partnership of T with U or C with G. 
Obesity protein analog--refers to a protein of the Formula (I): 
##STR1## 
wherein: xaa at position 28 is Gin or absent; 
said protein having at least one of the following substitutions: 
Gln at position 4 is replaced with Glu; 
Gln at position 7 is replaced with Glu; 
Asn at position 22 is replaced with Gln or Asp; 
Thr at position 27 is replaced with Ala; 
Xaa at position 28 is replaced with Glu; 
Gln at position 34 is replaced with Glu; 
Met at position 54 is replaced with methionine sulfoxide, Leu, Ile, Val, 
Ala, or Gly; 
Gln at position 56 is replaced with Glu; 
Gln at position 62 is replaced with Glu; 
Gln at position 63 is replaced with Glu; 
Met at position 68 is replaced with methionine sulfoxide, Leu, Ile, Val, 
Ala, or Gly; 
Asn at position 72 is replaced with Gln, Glu, or Asp; 
Gln at position 75 is replaced with Glu; 
Ser at position 77 is replaced with Ala; 
Asn at position 78 is replaced with Gln or Asp; 
Asn at position 82 is replaced with Gln or Asp; 
His at position 97 is replaced with Gln, Asn, Ala, Gly, Ser, or Pro; 
Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, Ile, Phe, 
Tyr, Ser, Thr, Gly, Gln, Val or Leu; 
Ala at position 101 is replaced with Ser, Asn, Gly, His, Pro, Thr, or Val; 
Ser at position 102 is replaced with Arg; 
Gly at position 103 is replaced with Ala; 
Glu at position 105 is replaced with Gln; 
Thr at position 106 is replaced with Lys or Ser; 
Leu at position 107 is replaced with Pro; 
Asp at position 108 is replaced with Glu; 
Gly at position 111 is replaced with Asp; 
Gly at position 118 is replaced with Leu; 
Gln at position 130 is replaced with Glu; 
Gln at position 134 is replaced with Glu; 
Met at position 136 is replaced with methionine sulfoxide, Leu, Ile, Val, 
Ala, or Gly; 
Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, Ile, Phe, 
Tyr, Ser, Thr, Gly, Gln, Val or Leu; or 
Gln at position 139 is replaced with Glu; or a pharmaceutically acceptable 
salt thereof. 
Plasmid--an extrachromosomal self-replicating genetic element. 
Reading frame--the nucleotide sequence from which translation occurs "read" 
in triplets by the translational apparatus of tRNA, ribosomes and 
associated factors, each triplet corresponding to a particular amino acid. 
Because each triplet is distinct and of the same length, the coding 
sequence must be a multiple 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 DNA segments can or have been 
added. 
Recombinant DNA Expression Vector--any recombinant DNA cloning vector in 
which a promoter has been incorporated. 
Replicon--A DNA sequence that controls and allows for autonomous 
replication of a plasmid or other vector. 
Transcription--the process whereby information contained in a nucleotide 
sequence of DNA is transferred to a complementary RNA sequence. 
Translation--the process whereby the genetic information of messenger RNA 
is used to specify and direct the synthesis of a polypeptide chain. 
Vector--a replicon used for the transformation of cells in gene 
manipulation bearing polynucleotide sequences corresponding to appropriate 
protein molecules which, when combined with appropriate control sequences, 
confer specific properties on the host cell to be transformed. Plasmids, 
viruses, and bacteriophage are suitable vectors, since they are replicons 
in their own right. Artificial vectors are constructed by cutting and 
joining DNA molecules from different sources using restriction enzymes and 
ligases. Vectors include Recombinant DNA cloning vectors and Recombinant 
DNA expression vectors. 
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 protein of present invention to prevent 
the onset of the symptoms or complications, alleviating the symptoms or 
complications, or eliminating the disease, condition, or disorder. 
Treating as used herein includes the administration of the protein for 
cosmetic purposes. A cosmetic purpose seeks to control the weight of a 
mammal to improve bodily appearance. 
Isotonicity agent--isotonicity agent refers to an agent that is 
physiologically tolerated and embarks a suitable tonicity to the 
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 and various mixtures thereof. Other 
preservatives may be found, e.g., in WALLHAUSER, K.-H., DEVELOP. BIOL. 
STANDARD. 24, pp. 9-28 (Basel, S. Krager, 1974). The concentration 
necessary to achieve preservative effectiveness is dependent upon the 
preservative used and the conditions of the formulation. 
The nucleotide and amino acid abbreviations used herein are accepted by the 
United States Patent and Trademark Office as set forth in 37 C.F.R. 
.sctn.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 analog complexed with a divalent metal cation. When complexed with 
a divalent metal cation, the obesity protein analog demonstrates 
significantly enhanced potency. 
Preferred proteins of the present invention are those of Formula I, 
wherein: 
Gln at position 4 is replaced with Glu; 
Gln at position 7 is replaced with Glu; 
Asn at position 22 is replaced with Gln or Asp; 
Thr at position 27 is replaced with Ala; 
Gln at position 28 is replaced with Glu; 
Gln at position 34 is replaced with Glu; 
Met at position 54 is replaced with methionine sulfoxide, Leu, or Ala; 
Gln at position 56 is replaced with Glu; 
Gln at position 62 is replaced with Glu; 
Gln at position 63 is replaced with Glu; 
Met at position 68 is replaced with methionine sulfoxide, or Leu; 
Asn at position 72 is replaced with Glu or Asp; 
Gln at position 75 is replaced with Glu; 
Asn at position 78 is replaced with Gln or Asp; 
Asn at position 82 is replaced with Gln or Asp; 
Gln at position 130 is replaced with Glu; 
Gln at position 134 is replaced with Glu; 
Met at position 136 is replaced with methionine sulfoxide, Leu, Ile; or 
Gln at position 139 is replaced with Glu. 
Other preferred proteins are those of Formula I wherein: 
Asn at position 22 is replaced with Gln or Asp; 
Thr at position 27 is replaced with Ala; 
Met at position 54 is replaced with methionine sulfoxide, Leu, or Ala; 
Met at position 68 is replaced with methionine sulfoxide, or Leu; 
Asn at position 72 is replaced with Glu or Asp; 
Asn at position 78 is replaced with Gln or Asp; 
Asn at position 82 is replaced with Gln or Asp; or 
Met at position 136 is replaced with methionine sulfoxide, Leu, or Ile. 
Still yet additional preferred proteins are those of Formula I, wherein: 
Asn at position 22 is replaced with Gln or Asp; 
Thr at position 27 is replaced with Ala; 
Met at position 54 is replaced with Leu, or Ala; 
Met at position 68 is replaced with Leu; 
Asn at position 72 is replaced with Gln or Asp; 
Asn at position 78 is replaced with Gln or Asp; 
Asn at position 82 is replaced with Gln or Asp; or 
Met at position 136 is replaced with Leu, or Ile. 
Preferred species within Formula I include species of SEQ ID NO: 2 and SEQ 
ID NO: 3: 
##STR2## 
Most significantly, other preferred proteins of the present invention are 
specific substitutions to amino acid residues 97 to 111, and/or 138 of the 
proteins of SEQ ID NO: 1. These substitutions result in additional protein 
stability and are superior therapeutic agents. Accordingly, preferred 
embodiments are compounds comprising proteins of the Formula II: 
##STR3## 
wherein: Xaa at position 28 is Gln or absent; said protein having at least 
one substitution selected from the group consisting of: 
His at position 97 is replaced with Gln, Asn, Ala, Gly, Ser, or Pro; 
Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, Ile, Phe, 
Tyr, Ser, Thr, Gly, Gln, Val or Leu; 
Ala at position 101 is replaced with Ser, Asn, Gly, His, Pro, Thr, or Val; 
Ser at position 102 is replaced with Arg; 
Gly at position 103 is replaced with Ala; 
Glu at position 105 is replaced with Gln; 
Thr at position 106 is replaced with Lys or Ser; 
Leu at position 107 is replaced with Pro; 
Asp at position 108 is replaced with Glu; 
Gly at position 111 is replaced with Asp; or 
Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, Ile, Phe, 
Tyr, Ser, Thr, Gly, Gln, Val or Leu; or a pharmaceutically acceptable salt 
thereof. 
Preferred proteins are a protein of the Formula III: 
##STR4## 
said protein having at least one substitution selected from the group 
consisting of: 
His at position 97 is replaced with Gln, Asn, Ala, Gly, Ser, or Pro; 
Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, Ile, Phe, 
Tyr, Ser, Thr, Gly, Gln, Val or Leu; 
Ala at position 101 is replaced with Ser, Asn, Gly, His, Pro, Thr, or Val; 
Ser at position 102 is replaced with Arg; 
Gly at position 103 is replaced with Ala; 
Glu at position 105 is replaced with Gln; 
Thr at position 106 is replaced with Lys or Ser; 
Leu at position 107 is replaced with Pro; 
Asp at position 108 is replaced with Glu; 
Gly at position 111 is replaced with Asp; or 
Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, Ile, Phe, 
Tyr, Ser, Thr, Gly, Gln, Val or Leu; 
or a pharmaceutically acceptable salt thereof. 
more preferred proteins of the Formula III are those herein: 
His at position 97 is replaced with Gln, Asn, Ala, Gly, Ser or Pro; 
Trp at position 100 is replaced with Ala, Glu, Asp, Asn, Met, Ile, Phe, 
Tyr, Ser, Thr, Gly, Gln or Leu; 
Ala at position 101 is replaced with Ser, Asn, Gly, His, Pro, Thr or Val; 
Glu at position 105 is replaced with Gln; 
Thr at position 106 is replaced with Lys or Ser; 
Leu at position 107 is replaced with Pro; 
Asp at position 108 is replaced with Glu; 
Gly at position 111 is replaced with Asp; or 
Trp at position 138 is replaced with Ala, Glu, Asp, Asn, Met, Ile, Phe, 
Tyr, Ser, Thr, Gly, Gln, Val or Leu. 
Other preferred proteins of the Formula III are those wherein: 
His at position 97 is replaced with Ser or Pro; 
Trp at position 100 is replaced with Ala, Gly, Gln, Val, Ile, or Leu; 
Ala at position 101 is replaced with Thr; or 
Trp at position 138 is replaced with Ala, Ile, Gly, Gln, Val or Leu. 
Yet still additional preferred proteins of the Formula III are those 
wherein: 
His at position 97 is replaced with Ser or Pro; 
Trp at position 100 is replaced with Ala, Gln or Leu; 
Ala at position 101 is replaced with Thr; or 
Trp at position 138 is replaced with Gln. 
Most preferred species of the present invention are those proteins having a 
di-sulfide bond between Cys at position 96 and Cys at position 146. 
Examples of most preferred species include species of SEQ ID NO: 6-13: 
##STR5## 
said protein having a di-sulfide bond between Cys at position 96 and Cys 
at position 146; or a pharmaceutically acceptable salt thereof. 
##STR6## 
said protein having a di-sulfide bond between Cys at position 96 and Cys 
at position 146; or a pharmaceutically acceptable salt thereof. 
##STR7## 
said protein having a di-sulfide bond between Cys at position 96 and Cys 
at position 146; or a pharmaceutically acceptable salt thereof. 
##STR8## 
said protein having a di-sulfide bond between Cys at position 96 and Cys 
at position 146; or a pharmaceutically acceptable salt thereof. 
##STR9## 
said protein having a di-sulfide bond between Cys at position 96 and Cys 
at position 146; or a pharmaceutically acceptable salt thereof. 
##STR10## 
said protein having a di-sulfide bond between Cys at position 96 and Cys 
at position 146; or a pharmaceutically acceptable salt thereof. 
##STR11## 
said protein having a di-sulfide bond between Cys at position 96 and Cys 
at position 146; or a pharmaceutically acceptable salt thereof. 
##STR12## 
said protein having a di-sulfide bond between Cys at position 96 and Cys 
at position 146; or a pharmaceutically acceptable salt thereof. 
The presently claimed compounds comprise an obesity protein analog 
complexed with a divalent metal cation. A divalent metal cation includes, 
for 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 excess; 
however, the molar ratio of at least one molecule of a divalent metal 
cation for each ten molecules of obesity protein analog is operable. 
Preferably, the compounds comprise from 1 to 100 divalent metal cations 
per molecule of obesity protein analog. 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 
analog of the present invention. The metal cation may be added in solid 
form or it may be added as a solution. Several different cationic salts 
can be used in the present 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 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 analog 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 
analog 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, 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 purified by conventional separation techniques 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 of the present invention and water. The 
concentration of the obesity protein analog 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 formulation 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.-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 formulations, preferably about 16 mg/mL 
glycerin. The pH of the formulation may also be buffered with a 
physiologically tolerated buffer. Acceptable physiologically 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 formulations of the present invention can be 
prepared using conventional dissolution and mixing procedures. Preferably, 
the claimed formulations 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 analog-zinc complex. The 
compound is easily resuspended before 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 of 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 the 
surfactant is used, the temperature, and pH at which the formulation is 
prepared may 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 administration. 
The proteins used in the present compounds 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. 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 sequence into an expression vector in a manner 
suitable for the expression of the protein either alone or as a fusion 
protein, 
c) transforming an appropriate eukaryotic or prokaryotic host cell with the 
expression vector, and 
d) recovering and purifying the recombinantly produced protein. 
Synthetic genes, the in vitro or in vivo transcription and translation of 
which will result in the production of the protein may be constructed by 
techniques well known in the art. Owing to the natural degeneracy of the 
genetic code, the skilled artisan will recognize that a sizable yet 
definite number of DNA sequences may be constructed which encode the 
desired proteins. In the preferred practice of the invention, synthesis is 
achieved by recombinant DNA technology. 
Methodology of synthetic gene construction is well known in the art. For 
example, see Brown, et al. (1979) Methods in Enzymology, Academic Press, 
N.Y., Vol. 68, pgs. 109-151. The DNA sequence corresponding to the 
synthetic claimed protein gene may be generated using conventional DNA 
synthesizing apparatus such as the Applied Biosystems Model 380A or 380B 
DNA synthesizers (commercially available from Applied Biosystems, Inc., 
850 Lincoln Center Drive, Foster City, Calif. 94404). It may be desirable 
in some applications to modify the coding sequence of the obesity protein 
so as to incorporate a convenient protease sensitive cleavage site, e.g., 
between the signal peptide and the structural protein facilitating the 
controlled excision of the signal peptide from the 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 from CLONETECH or STRATAGENE) or mRNA isolated 
from the desired arrival adipose tissue. Such methodologies are well known 
in the art Maniatis, et al. Molecular Cloning: A Laboratory Manual, Cold 
Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, 
N.Y. (1989). 
The constructed or isolated DNA sequences are useful for expressing the 
obesity protein either by direct expression or as fusion protein. When the 
sequences are used in a fusion gene, the resulting product will require 
enzymatic or chemical cleavage. A variety of peptidases which cleave a 
polypeptide at specific sites or digest the peptides from the amino or 
carboxy termini (e.g. diaminopeptidase) of the peptide chain are known. 
Furthermore, particular chemicals (e.g. cyanogen bromide) will cleave a 
polypeptide chain at specific sites. The skilled artisan will appreciate 
the modifications necessary to the amino acid sequence (and synthetic or 
semi-synthetic coding sequence if recombinant means are employed) to 
incorporate site-specific internal cleavage sites. See U.S. Pat. No. 
5,126,249; Carter P., Site Specific Proteolysis of Fusion Proteins, Ch. 13 
in Protein Purification: From Molecular Mechanisms to Large Scale 
Processes, American Chemical Soc., Washington, D.C. (1990). 
Construction of suitable vectors containing the desired coding and control 
sequences employ standard ligation techniques. Isolated plasmids or DNA 
fragments are cleaved, tailored, and religated in the form desired to form 
the plasmids required. 
To effect the translation of the desired protein, one inserts the 
engineered synthetic DNA sequence in any of a plethora of appropriate 
recombinant DNA expression vectors through the use of appropriate 
restriction endonucleases. A synthetic coding sequence may be designed to 
possess restriction endonuclease cleavage sites at either end of the 
transcript to facilitate isolation from and integration into these 
expression and amplification and expression plasmids. The isolated cDNA 
coding sequence may be readily modified by the use of synthetic linkers to 
facilitate the incorporation of this sequence into the desired cloning 
vectors by techniques well known in the art. The particular endonucleases 
employed will be dictated by the restriction endonuclease cleavage pattern 
of the parent expression vector to be employed. The restriction sites are 
chosen so as to properly orient the coding sequence with control sequences 
to achieve proper in-frame reading and expression of the protein. 
In general, plasmid vectors containing promoters and control sequences 
which are derived from species compatible with the host cell are used with 
these hosts. The vector ordinarily carries a replication origin as well as 
marker sequences which are capable of providing phenotypic selection in 
transformed cells. For example, E. coli is typically transformed using 
pBR322, a plasmid derived from an E. coli species (Bolivar, et al., Gene2: 
95 (1977)). Plasmid pBR322 contains genes for ampicillin and tetracycline 
resistance and thus provides easy means for identifying transformed cells. 
The pBR322 plasmid, or other microbial plasmid must also contain or be 
modified to contain promoters and other control elements commonly used in 
recombinant DNA technology. 
The desired coding sequence is inserted into an expression vector in the 
proper orientation to be transcribed from a promoter and ribosome binding 
site, both of which should be functional in the host cell in which the 
protein is to be expressed. An example of such an expression vector is a 
plasmid described in Belagaje et al., U.S. Pat. No. 5,304,493, the 
teachings of which are herein incorporated by reference. The gene encoding 
A-C-B proinsulin described in U.S. Pat. No. 5,304,493 can be removed from 
the plasmid pRB182 with restriction enzymes NdeI and BamHI. The isolated 
DNA sequences can be inserted into the plasmid backbone on a NdeI/BamHI 
restriction fragment cassette. 
In general, procaryotes are used for cloning of DNA sequences in 
constructing the vectors useful in the invention. For example, E. coli K12 
strain 294 (ATCC No. 31446) is particularly useful. Other microbial 
strains which may be used include E. coli B and E. coli X1776 (ATCC No. 
31537). These examples are illustrative rather than limiting. 
Procaryotes also are used for expression. The aforementioned strains, as 
well as E. coli W3110 (prototrophic, ATCC No. 27325), bacilli such as 
Bacillus subtilis, and other enterobacteriaceae such as Salmonella 
typhimurium or Serratia marcescans, and various pseudomonas species may be 
used. Promoters suitable for use with prokaryotic hosts include the 
.beta.-lactamase (vector pGX2907 ATCC 39344! contains the replicon and 
.beta.-lactamase gene) and lactose promoter systems (Chang et al., Nature, 
275: 615 (1978); and Goeddel et al., Nature281: 544 (1979)), alkaline 
phosphatase, the tryptophan (trp) promoter system (vector pATH1 ATCC 
37695! is designed to facilitate expression of an open reading frame as a 
trpE fusion protein under control of the trp promoter) and hybrid 
promoters such as the tac promoter (isolatable from plasmid pDR540 
ATCC-37282). However, other functional bacterial promoters, whose 
nucleotide sequences are generally known, enable one of skill in the art 
to ligate them to DNA encoding the protein using linkers or adaptors to 
supply any required restriction sites. Promoters for use in bacterial 
systems also will contain a Shine-Dalgarno sequence operably linked to the 
DNA encoding protein. 
The DNA molecules may also be recombinantly produced in eukaryotic 
expression systems. Preferred promoters controlling transcription in 
mammalian host cells may be obtained from various sources, for example, 
the genomes of viruses such as: polyoma, Simian Virus 40 (SV40), 
adenovirus, retroviruses, hepatitis-B virus and most preferably 
cytomegalovirus, or from heterologous mammalian promoters, e.g. 
.beta.-actin promoter. The early and late promoters of the SV40 virus are 
conveniently obtained as an SV40 restriction fragment which also contains 
the SV40 viral origin of replication. Fiers, et al., Nature, 273: 113 
(1978). The entire SV40 genome may be obtained from plasmid pBRSV, ATCC 
45019. The immediate early promoter of the human cytomegalovirus may be 
obtained from plasmid pCMBb (ATCC 77177). Of course, promoters from the 
host cell or related species also are useful herein. 
Transcription of the DNA by higher eucaryotes is increased by inserting an 
enhancer sequence into the vector. Enhancers are cis-acting elements of 
DNA, usually about 10-300 bp, that act on a promoter to increase its 
transcription. Enhancers are relatively oriented and positioned 
independently and have been found 5' (Laimins, L. et al., PNAS78: 993 
(1981)) and 3' (Lusky, M. L., et al., Mol. Cell Bio. 3: 1108 (1983)) to 
the transcription unit, within an intron (Banerji, J. L. et al., Cell33: 
729 (1983)) as well as within the coding sequence itself (Osborne, T. F., 
et al., Mol. Cell Bio.4: 1293 (1984)). Many enhancer sequences are now 
known from mammalian genes (globin, RSV, SV40, EMC, elastase, albumin, 
alpha-fetoprotein and insulin). Typically, however, one will use an 
enhancer from a eukaryotic cell virus. Examples include the SV40 late 
enhancer, the cytomegalovirus early promoter enhancer, the polyoma 
enhancer on the late side of the replication origin, and adenovirus 
enhancers. 
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, 
plant, animal, human or nucleated cells from other multicellular 
organisms) will also contain sequences necessary for the termination of 
transcription which may affect mRNA expression. These regions are 
transcribed as polyadenylated segments in the untranslated portion of the 
mRNA encoding protein. The 3' untranslated regions also include 
transcription termination sites. 
Expression vectors may contain a selection gene, also termed a selectable 
marker. Examples of suitable selectable markers for mammalian cells are 
dihydrofolate reductase (DHFR, which may be derived from the BglII/HindIII 
restriction fragment of pJOD-10 ATCC 68815!), thymidine kinase (herpes 
simplex virus thymidine kinase is contained on the BamHI fragment 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 successfully transferred into a mammalian host 
cell, the transfected mammalian host cell can survive if placed under 
selective pressure. There are two widely used distinct categories of 
selective regimes. The first category is based on a cell's metabolism and 
the use of a mutant cell line which lacks the ability to grow without a 
supplemented media. Two examples are: CHO DHFR- cells (ATCC CRL-9096) and 
mouse LTK- cells (L-M(TK-) ATCC CCL-2.3). These cells lack the ability to 
grow without the addition of such nutrients as thymidine or hypoxanthine. 
Because these cells lack certain genes necessary for a complete nucleotide 
synthesis pathway, they cannot survive unless the missing nucleotides are 
provided in a supplemented media. An alternative to supplementing the 
media is to introduce an intact DHFR or TK gene into cells lacking the 
respective genes, thus altering their growth requirements. Individual 
cells which were not transformed with the DHFR or TK gene will not be 
capable of survival in nonsupplemented media. 
The second category is dominant selection which refers to a selection 
scheme used in any cell type and does not require the use of a mutant cell 
line. These schemes typically use a drug to arrest growth of a host cell. 
Those cells which have a novel gene would express a protein conveying drug 
resistance and would survive the selection. Examples of such dominant 
selection use the drugs neomycin, Southern P. and Berg, P., J. Molec. 
Appl. Genet.1: 327 (1982), mycophenolic acid, Mulligan, R. C. and Berg, P. 
Science209: 1422 (1980), or hygromycin, Sugden, B. et al., Mol Cell. 
Biol.5: 410-413 (1985). The three examples given above employ bacterial 
genes under eukaryotic control to convey resistance to the appropriate 
drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, 
respectively. 
A preferred vector for eucaryotic expression is pRc/CMV. pRc/CMV is 
commercially available from Invitrogen Corporation, 3985 Sorrento Valley 
Blvd., San Diego, Calif. 92121. To confirm correct sequences in plasmids 
constructed, the ligation mixtures are used to transform E. coli K12 
strain DH10B (ATCC 31446) and successful transformants selected by 
antibiotic resistance where appropriate. Plasmids from the transformants 
are prepared, analyzed by restriction and/or sequence by the method of 
Messing, et al., Nucleic Acids Res.9: 309 (1981). 
Host cells may be transformed with the expression vectors of this invention 
and cultured in conventional nutrient media modified as is appropriate for 
inducing promoters, selecting transformants or amplifying genes. The 
culture conditions, such as temperature, pH and the like, are those 
previously used with the host cell selected for expression, and will be 
apparent to the ordinarily skilled artisan. The techniques of transforming 
cells with the aforementioned vectors are well known in the art and may be 
found in such general references as Maniatis, et al., Molecular Cloning: A 
Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor 
Laboratory, Cold Spring Harbor, N.Y. (1989), or Current Protocols in 
Molecular Biology (1989) and supplements. 
Preferred suitable host cells for expressing the vectors encoding the 
claimed proteins in higher eucaryotes include: African green monkey kidney 
line cell line transformed by SV40 (COS-7, ATCC CRL-1651); transformed 
human primary embryonal kidney cell line 293,(Graham, F. L. et al., J. Gen 
Virol.36: 59-72 (1977), Virology77: 319-329, Virology86: 10-21); baby 
hamster kidney cells (BHK-21(C-13), ATCC CCL-10, Virology 16: 147 (1962)); 
Chinese hamster ovary cells CHO-DHFR- (ATCC CRL-9096), mouse Sertoli cells 
(TM4, ATCC CRL-1715, Biol. Reprod. 23: 243-250 (1980)); African green 
monkey kidney cells (VERO 76, ATCC CRL-1587); human cervical epitheloid 
carcinoma cells (HeLa, ATCC CCL-2); canine kidney cells (MDCK, ATCC 
CCL-34); buffalo rat liver cells (BRL 3A, ATCC CRL-1442); human diploid 
lung cells (WI-38, ATCC CCL-75); human hepatocellular carcinoma cells (Hep 
G2, ATCC HB-8065);and mouse mammary tumor cells (MMT 060562, ATCC CCL51). 
In addition to prokaryotes, unicellular eukaryotes such as yeast cultures 
may also be used. Saccharomyces 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., Nature282: 39 
(1979); Kingsman et al., Gene7: 141 (1979); Tschemper et al., Gene10: 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, Genetics85: 12 (1977)). 
Suitable promoting sequences for use with yeast hosts include the promoters 
for 3-phosphoglycerate kinase (found on plasmid pAP12BD ATCC 53231 and 
described in U.S. Pat. No. 4,935,350, Jun. 19, 1990) or other glycolytic 
enzymes such as enolase (found on plasmid pAC1 ATCC 39532), 
glyceraldehyde-3-phosphate dehydrogenase (derived from plasmid pHcGAPC1 
ATCC 57090, 57091), zymomonas mobilis (U.S. Pat. No. 5,000,000 issued Mar. 
19, 1991), 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, 
U.S. Pat. No. 4,840,896), glyceraldehyde 3-phosphate dehydrogenase, and 
enzymes responsible for maltose and galactose (GAL1 found on plasmid 
pRY121 ATCC 37658) utilization. Suitable vectors and promoters for use in 
yeast expression are further described in R. Hitzeman et al., European 
Patent Publication No. 73,657A. Yeast enhancers such as the UAS Gal from 
Saccharomyces cerevisiae (found in conjunction with the CYC1 promoter on 
plasmid YEpsec--hI1beta ATCC 67024), also are advantageously used with 
yeast promoters. 
Preparation 1 
The plasmid containing the DNA sequence encoding the desired protein, is 
digested with PmlI and Bsu36I. The recognition sequences for these enzymes 
lie within the coding region for the protein at nucleotide positions 275 
and 360 respectively. The cloning vector does not contain these 
recognition sequences. Consequently, only two fragments are seen following 
restriction enzyme digestion with PmlI and Bsu36I, one corresponding to 
the vector fragment, the other corresponding to the .about.85 base pair 
fragment liberated from within the protein coding sequence. This sequence 
can be replaced by any DNA sequence encoding the amino acid substitutions 
between positions 91 and 116 of the present invention. These DNA sequences 
are synthesized chemically as two oligonucleotides with complementary 
bases and ends that are compatible with the ends generated by digestion 
with PmlI and Bsu36I. The chemically synthesized oligonucleotides are 
mixed in equimolar amounts (1-10 picomoles/microliter), heated to 95 
degrees and allow to anneal by slowly decreasing the temperature to 20-25 
degrees. The annealed oligonucleotides are in a standard ligation 
reaction. Ligation products are transformed and analyzed as described in 
Example 1. Other substitutions are preferably carried out in a similar 
manner using appropriate restriction cites. 
Preparation 2 
A DNA sequence encoding SEQ ID NO: 6 with a Met Arg leader sequence was 
obtained using the plasmid and procedures described in preparation 1. The 
plasmid was digested with PmlI and Bsu36I. A synthetic DNA fragment of the 
sequence 5"-SEQ ID NO:14: 
##STR13## 
annealed with the sequence 5'-SEQ ID NO:15: 
##STR14## 
was inserted between the PmlI and the Bsu36I sites. Following ligation, 
transformation and plasmid isolation, the sequence of the synthetic 
fragment was verified by DNA sequence analysis. 
The techniques of transforming cells with the aforementioned vectors are 
well known in the art and may be found in such general references as 
Maniatis, et al. (1988) Molecular Cloning: A Laboratory Manual, Cold 
Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, 
N.Y. or Current Protocols in Molecular Biology (1989) and supplements. The 
techniques involved in the transformation of E. coli cells used in the 
preferred practice of the invention as exemplified herein are well known 
in the art. The precise conditions under which the transformed E. coli 
cells are cultured is dependent on the nature of the E. coli host cell 
line and the expression or cloning vectors employed. For example, vectors 
which incorporate thermoinducible promoter-operator regions, such as the 
c1857 thermoinducible lambda-phage promoter-operator region, require a 
temperature shift from about 30 to about 40 degrees C. in the culture 
conditions so as to induce protein synthesis. 
In the preferred embodiment of the invention 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, L693, L507, L640, L641, 
L695, L814 (E. coli B). The transformed host cells are then plated on 
appropriate media under the selective pressure of the 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 of the overexpressed protein. Kreuger et al., in Protein 
Folding, Gierasch and King, eds., pgs 136-142 (1990), American Association 
for the Advancement of Science Publication No. 89-18S, Washington, D.C. 
Such protein aggregates must be dissolved to provide further purification 
and isolation of the desired protein product. Id. A variety of techniques 
using strongly denaturing solutions such as guanidinium-HCl and/or weakly 
denaturing solutions such as urea are used to solubilize the proteins. 
Gradual removal of the denaturing agents (often by dialysis) in a solution 
allows the denatured protein to assume its native conformation. The 
particular conditions for denaturation and folding are determined by the 
particular protein expression system and/or the protein in question. 
Preparation 3 
The protein of SEQ ID NO: 6 with a Met Arg leader sequence was expressed in 
E.coli granules were isolated in 8M urea and 5 mM cysteine. The protein 
was purified by anion exchange chromatography in 8M urea, and folded by 
dilution into 8M urea (containing 5 mM cysteine) and exhaustive dialysis 
against PBS. Following final purification of the proteins by size 
exclusion chromatography the proteins were concentrated to 3-3.5 mg/mL in 
PBS. 
Preparation 4 
A DNA sequence encoding the protein of SEQ ID NO: 2 was assembled from 
chemically synthesized single stranded oligonucleotides to generate a 
double stranded DNA sequence. 
The oligonucleotides used to assemble this DNA sequence are as follows: 
##STR15## 
Oligonucleotides 16-22 were used to generate an approximately 220 
base-pair segment which extends from the NdeI site to the XbaI site at 
position 220 within the coding sequence. The oligonucleotides 23-29 were 
used to generate an approximately 240 base-pair segment which extends from 
the XbaI site to the BamHI site. 
To assemble the 220 and 240 base-pair fragments, the respective 
oligonucleotides were mixed in equimolar amounts, usually at 
concentrations of about 1-2 picomoles per microliters. Prior to assembly, 
all but the oligonucleotides at the 5"-ends of the segment were 
phosphorylated in standard kinase buffer with T4 DNA kinase using the 
conditions specified by the supplier of the reagents. The mixtures were 
heated to 95.degree. C. and allowed to cool slowly to room temperature 
over a period of 1-2 hours to ensure proper annealing of the 
oligonucleotides. The oligonucleotides were then ligated to each other and 
into a cloning vector, PUC19 was used, but others are operable using T4 
DNA ligase. The PUC19 buffers and conditions are those recommended by the 
supplier of the enzyme. The vector for the 220 base-pair fragment was 
digested with NdeI and XbaI, whereas the vector for the 240 base-pair 
fragment was digested with XbaI and BamHI prior to use. The ligation mixes 
were used to transform E. coli DH10B cells (commercially available from 
Gibco/BRL) and the transformed cells were plated on tryptone-yeast (TY) 
plates containing 100 .mu.g/mL of ampicillin, X-gal and IPTG. Colonies 
which grow up overnight were grown in liquid TY medium with 100 .mu.g/mL 
of ampicillin and were used for plasmid isolation and DNA sequence 
analysis. Plasmids with the correct sequence were kept for the assembly of 
the complete gene. This was accomplished by gel-purification of the 220 
base-pair and the 240 base-pair fragments and ligation of these two 
fragments into PUC19 linearized with NdeI and BamHI. The ligation mix was 
transformed into E. coli DH10B cells and plated as described previously. 
Plasmid DNA was isolated from the resulting transformants and digested 
with NdeI and BglII. The large vector fragment was gel-purified and 
ligated with a approximately 195 base-pair segment which was assembled as 
described previously from six chemically synthesized oligonucleotides as 
show below. 
##STR16## 
The ligation was transformed into E. coli cells as described previously. 
The DNA from the resulting transformants was isolated and the sequence was 
verified by DNA sequence analysis. The plasmid with the correct sequence 
was digested with NdeI and BamHI and the approximately 450 base-pair 
insert was recloned into an expression vector. 
The protein was expressed in E.coli, isolated and was folded either by 
dilution into PBS or by dilution into 8M urea (both containing 5 mM 
cysteine) and exhaustive dialysis against PBS. Following final 
purification of the proteins by size exclusion chromatography the proteins 
were concentrated to 3-3.5 mg/mL in PBS. Amino acid composition was 
confirmed. 
Preparation 5 
The protein of SEQ ID NO: 6 with a Met Arg leader sequence was expressed in 
E.coli, isolated and folded as described previously. The Met Arg leader 
sequence was cleaved by the addition of 6-10 milliunits dDAP per mg of 
protein. The conversion reaction was allowed to proceed for 2-8 hours at 
room temperature. The progress of the reaction was monitored by high 
performance reversed phase chromatography. The reaction was terminated by 
adjusting the pH to 8 with NaOH. The des(Met-Arg) protein was further 
purified by cation exchange chromatography in 7-8M urea and size exclusion 
chromatography in PBS. Following final purification of the proteins by 
size exclusion chromatography the proteins were concentrated to 3-3.5 
mg/mL in PBS. 
Preferably, the DNA sequences are expressed with a dipeptide leader 
sequence encoding Met-Arg or Met-Tyr as described in U.S. Pat. No. 
5,126,249, herein incorporated by reference. This approach facilitates the 
efficient expression of proteins and enables rapid conversion to the 
active protein form with Cathepsin C or other dipeptidylpeptidases. The 
purification of proteins is by techniques known in the art and includes 
reverse phase chromatography, affinity chromatography, and size exclusion. 
The following examples are provided merely to further illustrate the 
preparation of the formuations of the invention.