ANTI-OBESITY PROTEINS

PROTEINES ANTI-OBESITE

English Abstract

The present invention provides anti-obesity proteins, which when administered
to a patient regulate fat tissue. Accordingly, such agents allow patients to
overcome their obesity handicap and live normal lives with much reduced risk
for type II diabetes, cardiovascular disease and cancer.

French Abstract

La présente invention concerne des protéines anti-obésité qui, lorsqu'elles sont administrées à un patient, ont un effet régulateur sur les tissus adipeux. Ces agents permettent de combattre l'obésité et permettent aux patients souffrant de cet handicap de vivre une vie normale en diminuant considérablement les risques d'apparition du diabète sucré, de maladies cardiovasculaires et de cancer.

Claims

-35-
We claim:
1. A protein of the formula:
<IMG>
wherein:
Xaa at position 2 is Gln or Glu;
Xaa at position 17 is Asn, Asp or Gln;
Xaa at position 22 is Thr or Ala;
Xaa at position 23 is Gln, Glu or absent;
Xaa at position 29 is Gln or Glu;
Xaa at position 49 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 51 is Gln or Glu;
Xaa at position 57 is Gln or Glu;
Xaa at position 58 is Gln or Glu;
Xaa at position 63 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 67 is Asn, Asp or Gln;
Xaa at position 70 is Gln or Glu;

-36-
Xaa at position 73 is Asn, Asp or Gln;
Xaa at position 77 is Asn, Asp or Gln;
Xaa at position 95 is Trp or Gln;
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.
2. A protein of the formula:
<IMG>
wherein:
Xaa at position 17 is Asn, Asp or Gln;
Xaa at position 22 is Thr or Ala;
Xaa at position 23 is Gln, Glu or absent;
Xaa at position 29 is Gln or Glu;

-37-
Xaa at position 49 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 51 is Gln or Glu;
Xaa at position 57 is Gln or Glu;
Xaa at position 58 is Gln or Glu;
Xaa at position 63 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 67 is Asn, Asp or Gln;
Xaa at position 70 is Gln or Glu;
Xaa at position 73 is Asn, Asp or Gln;
Xaa at position 77 is Asn, Asp or Gln;
Xaa at position 95 iS Trp or Gln;
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.
3. A protein of the formula:
<IMG>

- 38 -
<IMG>
wherein:
Xaa at position 17 is Asn, Asp or Gln;
Xaa at position 22 is Thr or Ala;
Xaa at position 23 is Gln, Glu or absent
Xaa at position 29 is Gln or Glu;
Xaa at position 49 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 51 is Gln or Glu;
Xaa at position 57 is Gln or Glu;
Xaa at position 58 is Gln or Glu;
Xaa at position 63 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 67 is Asn, Asp or Gln;
Xaa at position 70 is Gln or Glu;
Xaa at position 73 is Asn, Asp or Gln;
Xaa at position 77 is Asn, Asp or Gln;
Xaa at position 95 is Trp or Gln;
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.
4. A protein of the formula:
<IMG>

-39-
<IMG>
wherein:
Xaa at position 17 is Asn, Asp or Gln;
Xaa at position 22 is Thr or Ala;
Xaa at position 23 is Gln, Glu or absent;
Xaa at position 29 is Gln or Glu;
Xaa at position 49 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 51 is Gln or Glu;
Xaa at position 57 is Gln or Glu;
Xaa at position 58 is Gln or Glu;
Xaa at position 63 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 67 is Asn, Asp or Gln;
Xaa at position 70 is Gln or Glu;
Xaa at position 73 is Asn, Asp or Gln;
Xaa at position 77 is Asn, Asp or Gln;
Xaa at position 95 is Trp or Gln;
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.

-40-
5. A protein of the formula:
<IMG>
wherein:
Xaa at position 29 is Gln or Glu;
Xaa at position 49 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 51 is Gln or Glu;
Xaa at position 57 is Gln or Glu;
Xaa at position 58 is Gln or Glu;
Xaa at position 63 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 67 is Asn, Asp or Gln;
Xaa at position 70 is Gln or Glu;
Xaa at position 73 is Asn, Asp or Gln;
Xaa at position 77 is Asn, Asp or Gln;
Xaa at position 95 is Trp or Gln;
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;

-41-
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.
6. A protein of the formula:
<IMG>
wherein:
Xaa at position 49 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 51 is Gln or Glu;
Xaa at position 57 is Gln or Glu;
Xaa at position 58 is Gln or Glu;
Xaa at position 63 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 67 is Asn, Asp or Gln;
Xaa at position 70 is Gln or Glu;
Xaa at position 73 is Asn, Asp or Gln;
Xaa at position 77 is Asn, Asp or Gln;
Xaa at position 95 is Trp or Gln;

-42-
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.
7. A protein of the formula:
<IMG>
wherein:
Xaa at position 49 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 51 is Gln or Glu;
Xaa at position 57 is Gln or Glu;
Xaa at position 58 is Gln or Glu;
Xaa at position 63 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 67 is Asn, Asp or Gln;
Xaa at position 70 is Gln or Glu;
Xaa at position 73 is Asn, Asp or Gln;
Xaa at position 77 is Asn, Asp or Gln;
Xaa at position 95 is Trp or Gln;

-43-
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.
8. A protein of the formula:
<IMG>
wherein:
Xaa at position 49 is Ile, Leu, Met, methionine
sulfoxide or absent;
Xaa at position 51 is Gln or Glu;
Xaa at position 57 is Gln or Glu;
Xaa at position 58 is Gln or Glu;
Xaa at position 63 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 67 is Asn, Asp or Gln;
Xaa at position 70 is Gln or Glu;
Xaa at position 73 is Asn, Asp or Gln;
Xaa at position 77 is Asn, Asp or Gln;
Xaa at position 95 is Trp or Gln;

-44-
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.
9. A protein of the formula:
<IMG>
wherein:
Xaa at position 57 is Gln or Glu;
Xaa at position 58 is Gln or Glu;
Xaa at position 63 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 67 is Asn, Asp or Gln;
Xaa at position 70 is Gln or Glu;
Xaa at position 73 is Asn, Asp or Gln;
Xaa at position 77 is Asn, Asp or Gln;
Xaa at position 95 is Trp or Gln;
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;

-45-
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.
10. A protein of the formula:
<IMG>
wherein:
Xaa at position 67 is Asn, Asp or Gln;
Xaa at position 70 is Gln or Glu;
Xaa at position 73 is Asn, Asp or Gln;
Xaa at position 77 is Asn, Asp or Gln;
Xaa at position 95 is Trp or Gln;
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.

-46-
11. A protein of the formula:
<IMG>
wherein:
Xaa at position 95 is Trp or Gln;
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.
12. A protein of the formula:
<IMG>
wherein:
Xaa at position 95 is Trp or Gln;
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;

-47-
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.
13. A protein of the formula:
<IMG>
wherein:
Xaa at position 70 is Gln or Glu;
Xaa at position 73 is Asn, Asp or Gln;
Xaa at position 77 is Asn, Asp or Gln;
Xaa at position 95 is Trp or Gln;
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.
14. A protein of the formula:
<IMG>

- 48 -
110 115 120
Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
125 130 135
Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser
140
Pro Gly Cys
wherein:
Xaa at position 95 is Trp or Gln;
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.
15. A protein of the formula:
100
Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser
105 110 115
Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val
120 125 130
Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa
135 140
Leu Asp Leu Ser Pro Gly Cys
wherein:
Xaa at position 95 is Trp or Gln;
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;
Xaa at position 131 is Ile, Leu, Met or methionine
sulfoxide;
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu;
or a pharmaceutically acceptable salt thereof.

- 49 -
16. A protein of any one of Claims 1 through 15,
wherein:
Xaa at position 2 is Gln;
Xaa at position 17 is Asn;
Xaa at position 22 is Thr;
Xaa at position 23 is Gln;
Xaa at position 29 is Gln;
Xaa at position 49 is Met;
xaa at position 51 is Gln;
Xaa at position 57 is Gln;
Xaa at position 58 is Gln;
Xaa at position 63 is Met;
Xaa at position 67 is Asn;
Xaa at position 70 is Gln;
Xaa at position 73 is Asn;
Xaa at position 77 is Asn;
Xaa at position 95 is Trp;
Xaa at position 125 is Gln;
Xaa at position 129 is Gln;
Xaa at position 131 is Met;
Xaa at position 133 is Trp; and
Xaa at position 134 is Gln.
17. A method of treating obesity, which comprises
administering to a mammal in need thereof a protein of any
one of Claims 1 through 15.
18. A pharmaceutical formulation, which comprises
a protein of any one of Claims 1 through 15 together with one
or more pharmaceutically acceptable diluents, carriers or
excipients therefor.

Description

CA 022ll6~6 l997-07-29
W 096123S~4 PC~U59~ 17
An~i-obesity proteins
.
The present invention is in the field of human
medicine, particularly in the treatment of obesity and
disorders associated with obesity. Most specifically the
invention relates to anti-obesity proteins that when
administered to a patient regulate fat tissue.
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 over weight. Kuczmarski,
Amer. J. of Clin. Nut. 55: 495S - 502S (1992); Reeder et.
al., Can. Med. ~s. J., ~: 226-233 (1992). upper body
obesity is the strongest risk fac~or 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
20 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
at~ributed to the strong association with dyslipidemia,
25 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
30 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
3 inheriting these genetic traits are prone to becoming obese
35 regardless of their efforts to combat the condition.

CA 022ll6~6 l997-07-29
W O96/23514 PCTrUS9Gl~17
Therefore, a new 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, ~iying 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 a gene coding for
a 167 amino acid protein with a 21 amino acid signal peptide
that is exclusively expressed in adipose tissue.
Physiologist have postulated for years that, when a
m~mm~ 1 overeats, the resulting excess fat signals to the
brain that the body is obese which, in turn, causes the body
to eat less and burn more fuel. G. R. Hervey, Nature 227:
629-631 (1969). This "feedback~ model is supported by
parabiotic experiments, which implicate a circulating hormone
controlling adiposity. Based on this model, the protein,
which is apparently encoded by the ob gene, is now
speculated to be an adiposity regulating hormone.
Pharmacological agents which are biologically active and
mimic the activity of this protein are useful to help
patients regulate their appetite and metabolism and thereby
control their adiposity. Until the present invention, such a
pharmacological agent was unknown.
The present invention provides biologically active
anti-obesity proteins. Such agents therefore allow patients
to overcome their obesity handicap and live normal lives with
a more normalized risk for type II diabetes, cardiovascular
disease and cancer.

CA 022116~6 1997-07-29
W 096~235}4 PCT/U~G~ 7
The present invention is directed to a biologically
active anti-obesity proteins of the Formula I:
Formula I: SEQ ID NO: 1
1 5 10 15
Val Xaa Asp Asp Thr Lys Thr Leu Ile Lys Thr Ile Val Thr Arg
Ile Xaa Asp Ile Ser His Xaa Xaa Ser Val Ser Ser Lys Xaa Lys
Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr
50 55 60
Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa Ile Leu
Thr Ser Xaa Pro Ser Arg Xaa Val Ile Xaa Ile Ser Xaa Asp Leu
Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
100 105
Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu
110 115 120
Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala
30125 130 135
Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu
140
Asp Leu Ser Pro Gly Cys
wherein:
Xaa at position 2 is Gln or Glu;
Xaa at position 17 iS Asn, Asp or Gln;
Xaa at position 22 is Thr or Ala;
Xaa at position 23 is Gln, Glu or absent;
Xaa at position 29 is Gln or Glu;
Xaa at position 49 is Ile, Leu, Met or methionine sul~oxide;
Xaa at position 51 is Gln or Glu;
Xaa at position 57 is Gln or Glu;
Xaa at position 58 is Gln or Glu;
Xaa at position 63 is Ile, Leu, Met or methionine sulfoxide;
Xaa at position 67 is Asn, Asp or Gln;
Xaa at position 70 is Gln or Glu;

CA 022116~6 1997-07-29
W O96/23514 PCTrUS9~G~7
Xaa at position 73 is Asn, Asp or Gln;
Xaa at position 77 is Asn, Asp or Gln;
Xaa at position 95 is Trp or Gln;
Xaa at position 125 is Gln or Glu;
Xaa at position 129 is Gln or Glu;
Xaa at position 131 is Ile, Leu, Met or methionine sulfoxide
Xaa at position 133 is Trp or Gln; and
Xaa at position 134 is Gln or Glu.
The present invention additionally includes
fragments of the proteins of Formula I. These proteins are
biologically active anti-obesity proteins and are represented
by Formulas Ia through In. For clarity purposes, the
numbering of the amino acids in Formula I is maintained in
Formulas Ia through In. Renumbering the amino acids is
unnecessary and would result in confusion. One of ordinary
skill in the art, for example, would appreciate that Formula
Ia represents amino acids 7 through 146 of SEQ ID NO: 1. In
Formulas Ia through In, the variable cites (Xaa) for each
position is the same as previously defined in Formula I
unless otherwise specified.
Formula Ia: SEQ ID NO: 2
10 15 20
Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Xaa Asp Ile Ser His
25 30 35
Xaa Xaa Ser val Ser Ser Lys Xaa Lys Val Thr Gly Leu Asp Phe
40 45 50
Ile Pro Gly Leu His Pro Ile Leu Thr Leu Ser Lys Xaa Asp Xaa
55 60 65
Thr Leu Ala val Tyr Xaa Xaa Ile Leu Thr Ser Xaa Pro Ser Arg
Xaa Val Ile Xaa Ile Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu
Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala

CA 022ll6~6 l997-07-29
W 096t23514 PCT~g~,V~3~7
100 105 110
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala
115 120 125
Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly
130 135 140
Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
10Formula Ib: SEQ ID NO: 3
Thr Ile Val Thr Arg Ile Xaa Asp Ile Ser His Xaa Xaa Ser Val
530 35 40
Ser Ser Lys Xaa Lys Val Thr Gly Leu ASp Phe Ile Pro Gly Leu
45 50 55
His Pro Ile Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val
60 65 70
Tyr Xaa Xaa Ile Leu Thr Ser Xaa Pro Ser Arg Xaa Val Ile Xaa
75 80 85
25Ile Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu
go 95 100
Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu
30105 110 115
Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser
120 125 130
Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp
135 140
Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
Formula Ic: SEQ ID NO: 4
20 25 30
Ile Xaa Asp Ile Ser His Xaa Xaa Ser Val Ser Ser Lys Xaa Lys
Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr
Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa Ile Leu
Thr Ser Xaa Pro Ser Arg Xaa Val Ile Xaa Ile Ser Xaa Asp Leu

CA 022ll6~6 l997-07-29
W O96123514 PCTAJS9~'~G~q7
Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
100 105
Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu
110 115 120
Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala
0125 130 135
Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu
140
Asp Leu Ser Pro Gly Cys
Formula Id: SEQ ID NO: 5
Xaa Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile
Leu Thr Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa
Ile Leu Thr Ser Xaa Pro Ser Arg Xaa Val Ile Xaa Ile Ser Xaa
Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser
3090 95 100
Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp
105 110 115
Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val
120 125 130
Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa
135 140
Xaa Leu Asp Leu Ser Pro Gly Cys
Formula Ie: SEQ ID NO: 6
Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile Leu Thr
Leu Ser Lys Xaa Asp Xaa Thr Leu Ala Val Tyr xaa Xaa Ile Leu
5065 70 75
Thr Ser Xaa Pro Ser Arg Xaa Val Ile Xaa Ile Ser Xaa Asp Leu

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W O96123514 PCTrUSr~'C~917
Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser
100 105
CyS HiS Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu
110 115 120
Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala
125 130 135
Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu
140
Asp Leu Ser Pro Gly Cys
Formula If: SEQ ID NO: 7
Ile Pro Gly Ieu HiS Pro Ile Leu Thr heu Ser Lys Xaa Asp Xaa
Thr Leu Ala Val Tyr Xaa Xaa Ile Leu Thr Ser Xaa Pro Ser Arg
70 75 80
Xaa Val Ile Xaa Ile Ser Xaa ASp Leu Glu Xaa Leu Arg Asp Leu
85 90 95
Leu HiS Val Leu Ala Phe Ser Lys Ser Cys HiS Leu Pro Xaa Ala
100 105 110
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala
115 120 125
35 Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly
130 135 140
Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
Formula Ig: SEQ ID NO: 8
Xaa Asp Xaa Thr Leu Ala Val Tyr Xaa Xaa Ile ~eu Thr Ser Xaa
45 65 70 75
Pro Ser Arg Xaa Val Ile Xaa Ile Ser Xaa Asp Leu Glu Xaa Leu
Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu
100 105
Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val
,, , , , _ ,

CA 022ll6~6 l997-07-29
W O96/23514 PCTrUS96/009~7
110 115 120
Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
125 130 135
Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser
140
Pro Gly Cys
wherein:
Xaa at position 49 is Ile, Leu, Met, methionine sulfoxide or
absent;
Formula Ih: SEQ ID NO: 9
Xaa Xaa Ile Leu Thr Ser Xaa Pro Ser Arg Xaa Val Ile Xaa Ile
75 80 85
Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu His Val Leu Ala
90 95 100
Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr
105 110 115
Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr
120 125 130
Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa
135 140
Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
Formula Ii: SEQ ID NO: 10
Xaa Val Ile Xaa Ile Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu
85 90 95
Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala
100 105 110
Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala
~5
115 120 125
Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly
130 135 140
Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys

CA 022ll6~6 l997-07-29
W O96/23~14 PCTrUS~CNnj~7
_g _
Formula Ij: SEQ ID NO: 11
85 90
Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro
100 105
Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu
110 115 120
Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu
125 130 135
Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro
140
Gly Cys
Formula Ik: SEQ ID NO: 12
90 95 100
Ser Lys Ser CyS HiS Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu
105 110 115
Asp Ser Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu
120 125 130
Val Val Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu
135 140
30Xaa Xaa Leu Asp Leu Ser Pro Gly Cys
Formula Il: SEQ ID NO: 13
70 75 80
35Val Ile Xaa Ile Ser Xaa Asp Leu Glu Xaa Leu Arg Asp Leu Leu
85 90 95
His Val Leu Ala Phe Ser Lys Ser Cys His Leu Pro Xaa Ala Ser
40100 105 110
Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val Leu Glu Ala Ser
115 120 125
Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu Xaa Gly Ser
130 135 140
Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser Pro Gly Cys

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W O96/23S14 PCTrUS9C~ 3~7
-10 -
Formula Im: SEQ ID NO: 14
85 90
Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu
- 100 105
Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val
110 115 120
Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
125 130 135
Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa Leu Asp Leu Ser
140
Pro Gly Cys
Formula In: SEQ ID NO: 15
20 90 95 lO0
Ser Cys His Leu Pro Xaa Ala Ser Gly Leu Glu Thr Leu Asp Ser
105 110 115
Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val
120 125 130
Ala Leu Ser Arg Leu Xaa Gly Ser Leu Xaa Asp Xaa Leu Xaa Xaa
135 140
Leu Asp Leu Ser Pro Gly Cys
The invention further provides a method of treating
obesity, which comprises administering to a m~mm~l in need
thereof a protein of any one of Formula I through In.
The invention further provides a pharmaceutical
formulation, which comprises a protein of any one of Formula
I through In together with one or more pharmaceutical
acceptable diluents, carriers or excipients therefor.
The preferred proteins of the present invention are
those wherein:
Xaa at position 2 is Gln;
Xaa at position 17 is Asn;
Xaa at position 22 is Thr;
Xaa at position 23 is Gln;

CA 022ll6~6 l997-07-29
W 096/23514 PCTrU~ 17
Xaa at position 29 is Gln,
xaa at position 49 is Met,
Xaa at position 51 is Gln;
Xaa at position 57 is Gln;
Xaa at position 58 is Gln;
Xaa at position 63 is Met;
Xaa at position 67 is Asn;
Xaa at position 70 is Gln;
Xaa at position 73 is Asn;
Xaa at position 77 is Asn;
Xaa at position 95 is Trp;
Xaa at position 125 is Gln;
xaa at position 129 is Gln;
Xaa at position 131 is Met;
Xaa at position 133 is Trp;
Xaa at position 134 is Gln.
The amino acids abbreviations are accepted by the
United States Patent and Trademark Of~ice as set forth in 37
C.F.R. 1.822 (b)(2) (1993). One skilled in the art would
recognize that certain amino acids are prone to
rearrangement. For example, Asp may rearrange to aspartimide
and isoasparigine as described in I. Schon et al., Int. J.
Pe~tide Protein Res. 14: 485-94 (1979) and references cited
therein. These rearrangement derivatives are included within
the scope of the present invention. Unless otherwise
indicated the amino acids are in the L configuration.
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

CA 022116~6 1997-07-29
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-12-
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.
Chelating Peptide -- An amino acid sequence capable
of complexing with a multivalent metal ion.
DNA -- Deoyxribonucleic acid.
EDTA -- an abbreviation for ethylenediamine
tetraacetic acid.
EDso -- an abbreviation for half-maximal value.
FAB-MS -- an abbreviation for fast atom bombardment
mass spectrometry.
Immunoreactive Protein(s) -- a term used to
collectively describe antibodies, fragments of antibodies
capable of binding antigens of a similar nature as the parent
antibody molecule from which they are derived, and single
chain polypeptide binding molecules as described in PCT
Application No. PCT/US 87/02208, International Publication
No. WO 88/01649.
mRNA -- messenger RNA.
MWCO -- an abbreviation for molecular weight cut-
off.
Plasmid -- an extrachromosomal self-replicating
genetic element.
PMSF -- an abbreviation for phenylmethylsulfonyl
fluoride.
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
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 framell

CA 022116~6 1997-07-29
W O96123514 PCTrUS~ C347
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 DMA 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 o~ a plasmid or other vector.
RNA -- ribonucleic acid.
RP-HPLC -- an abbreviation for reversed-phase high
performance liquid chromatography.
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.
Tris -- an abbreviation for tris(hydroxymethyl)-
~m; nomethane.
Treating -- 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
el;m;n~ting the disease, condition, or disorder. Treating
obesity therefor includes the inhibition of food intake, the
inhibition of weight gain, and inducing weight loss in
patients in need thereof.
- 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

CA 022ll6~6 l997-07-29
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-14-
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.
X-gal -- an abbreviation for 5-bromo-4-chloro-3-
idolyl beta-D-galactoside.
Yiying Zhang et al. in Nature 372: 425-32 (December
1994) report the cloning of the murine obese (ob) mouse gene
and present mouse DNA and the naturally occurring amino acid
sequence of the obesity protein for the mouse and human.
This protein is speculated to be a hormone that is secreted
by fat cells and controls body weight.
The present invention provides biologically active
proteins that provide effective treatment for obesity. The
proteins are also useful in the production of antibodies for
diagnostic use. Many of the claimed proteins o~er
additional advantages of stability, especially acid
stability, and improved absorption characteristics.
The claimed proteins ordinarily are prepared by
modification of the DNA encoding the claimed protein and
thereafter expressing the DNA in recombinant cell culture.
Techniques for making substitutional mutations at
predetermined sites in DNA having a known sequence are well
known, for example M13 primer mutagenesis. The mutations
that might be made in the DNA encoding the present anti-
obesity proteins must not place the sequence out of reading
frame and preferably will not create complementary regions
that could produce secondary mRNA structure. See DeBoer et
al., EP 75,444A (1983).
The compounds of the present invention may 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

CA 022ll6~6 l997-07-29
W 0961235~4 PCTrUS~'00917
protein fragments coupled through conventional solution
methods.
A. Solid Phase
The synthesis of the claimed protein may proceed by
solid phase peptide synthesis or by recombinant methods. The
principles of solid phase chemical synthesis of polypeptides
are well known in the art and may be found in general texts
in the area such as Dugas, H. and Penney, C., Biooraanic
Chemistrv Springer-Verlag, New York, pgs. 54-92 (1981). For
example, peptides may be synthesized by solid-phase
methodology utilizing an PE-Applied Biosystems 43OA peptide
synthesizer (commercially available from Applied Biosystems,
Foster City California) and synthesis cycles supplied by
Applied Biosystems. Boc amino acids and other reagents are
commercially available from PE-Applied Biosystems and other
chemical supply houses. Sequential Boc chemistry using
double couple protocols are applied to the starting p-methyl
benzhydryl amine resins for the production of C-terminal
carboxamides. For the production of C-terminal acids, the
corresponding PAM resin is used. Arginine, Asparagine,
Glutamine, Histidine and Methionine are coupled using
preformed hydroxy benzotriazole esters. The following side
chain protection may be used:
Arg, Tosyl
Asp, cyclohexyl or benzyl
Cys, 4-methylbenzyl
Glu, cyclohexyl
His, benzyloxymethyl
Lys, 2-chlorobenzyloxycarbonyl
Met, sulfoxide
Ser, Benzyl
Thr, Benzyl
Trp, formyl
Tyr, 4-bromo carbobenzoxy
Boc deprotection may be accomplished with trifluoroacetic
acid (TFA) in methylene chloride. Formyl removal from Trp is

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W O 96/23514 PCTrUS~61~0917
-16-
accomplished by treatment of the peptidyl resin with 20%
piperidine in dimethylformamide for 60 minutes at 4~C.
Met(O)
can be reduced by treatment of the peptidyl resin with
TFA/dimethylsulfide/conHCl (95/5/1) at 25~C for 60 minutes. c
Following the above pre-treatments, the peptides may be
further deprotected and cleaved from the resin with anhydrous
hydrogen fluoride containing a mixture of 10% m-cresol or m-
cresol/10% p-thiocresol or m-cresol/p-
thiocresol/dimethylsulfide. Cleavage of the side chain
protecting group(s) and of the peptide from the resin is
carried out at zero degrees Centigrade or below, pre~erably
-20~C for thirty minutes followed by thirty minutes a~ 0~C.
After removal of the HF, the peptide/resin is washed with
ether. The peptide is extracted with glacial acetic acid and
lyophilized. Purification is accomplished by reverse-phase
C18 chromatography (Vydac) column in .1% TFA with a gradient
of increasing acetonitrile concentration.
One skilled in the art recognizes that the solid
phase synthesis could also be accomplished using the FMOC
strategy and a TFA/scavenger cleavage mixture.
B. Recombinant Svnthesis
The claimed proteins may also be produced by
recombinant 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 claimed protein,
b) integrating the coding sequence into an
expression vector in a manner suitable for the
expression of the protein either alone or as a
fusion protein,
c) transforming an appropriate eukaryotic or
prokaryotic host cell with the expression
vector, and

CA 022116~6 1997-07-29
W 096S23514 PCTJU~GI'~5~7
-17-
d) recovering and purifying the recombinantly
produced protein.
2.a. Gene Construction
Synthetic genes, the n vitro or n ViVQ
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 claimed 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, CA 94404).
It may desirable in some applications to modify the
coding sequence of the claimed 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 claimed protein may also be
created by using polymerase chain reaction (PCR). The
template can be a cDNA library (commercially available from
CLONETECH or STRATAGENE) or mRNA isolated from human adipose
tissue. Such methodologies are well known in the art
- Maniatis, et ~l. Molecul~r Clonin~: A L~horatorv Manual, Cold
Spring Harbor Press, Cold Spring Harbor Laboratory, Cold
Spring Harbor, New York (1989).

CA 022116~6 1997-07-29
W O96123514 PCTrUS9G;~Sq7
.b. Direct ex~ression or Fusion Protein
The claimed protein may be made either by direct
expression or as fusion protein comprising the claimed
protein followed by enzymatic or chemical cleavage. A
variety of peptidases (e.g. trypsin) 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. ~he 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 e.g., Carter P., Site Specific
Proteolysis of Fusion Proteins, Ch. 13 in Protein
Purif;c~tion: From Molecular Mechanisms to Tarae Scale
Processes, American Chemical Soc., Washington, D.C. (1990).
~.c. Vector Construction
Construction of suitable vectors containing the
desired coding and control sequences employ standard ligation
techniques. Isolated plasmids or DNA fragments are cleaved,
tailored, and religated in the form desired to form the
plasmids required.
To effect the translation of the desired protein,
one inserts the engineered synthetic DNA sequence in any of a
plethora o~ appropriate recombinant DNA expression vectors
through the use of appropriate restriction endonucleases.
The claimed protein is a relatively large protein. A
synthetic coding sequence is designed to possess restriction
endonuclease cleavage sites at either end of the transcript
to facilitate isolation from and integration into these
expression 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

CA 022116~6 1997-07-29
W ~96123514 PCTAUS~GI'~ 7
--19--
will be dictated by the restriction endonuclease cleavage
pattern of the parent expression vector to be employed. The
choice of restriction sites are chosen so as to properly
orient the coding sequence with control se~uences to achieve
proper in-~rame reading and expression of the claimed
protein.
In general, plasmid vectors containing promoters
and control sequences which are derived from species
compatible with the host cell are used with these hosts. The
vector ordinarily carries a replication site as well as
marker sequences which are capable of providing phenotypic
selection in trans~ormed cells. For example, F.. coli is
typically transformed using pBR322, a plasmid derived from an
E. coli species (Bolivar, et al., Gene 2: 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. 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 genes
encoding the protein of the present invention can be inserted
into the plasmid backbone on a ~I/samHI restriction
fragment cassette.
2.d. Procarvotic ex~ression
In general, procaryotes are used for cloning of DNA
sequences in constructing the vectors useful in the

CA 022ll6~6 l997-07-29
W O96/Z3514 PCTrUSr~ 517
-20-
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. ~Qli X1776 (ATCC No.
31537). These examples are illustrative rather than limiting.
Prokaryotes also are used for expression. The
aforementioned strains, as well as E. coli W3110
(prototrophic, ATCC No. 27325), bacilli such as Bacillus
subtilis, and other enterobacteriaceae such as Salmonella
typh;mllrium or Serratia marcescans, and various pseudomonas
species may be used. Promoters suitable for use with
prokaryotic hosts include the ~-lactamase (vector pGX2907
[ATCC 39344] contains the replicon and ~-lactamase gene) and
lactose promoter systems (Chang ~ al., Nature, 275:615
(1978); and Goeddel et al., Natllre 281:544 (1979)), alkaline
phosphatase, the tryptophan (trp) promoter system (vector
pATHl [ATCC 37695] is designed to facilitate expression of an
open reading frame as a trpE fusion protein under control of
the trp promoter) and hybrid promoters such as the tac
promoter (isolatable from plasmid pDR540 ATCC-37282).
However, other functional bacterial promoters, whose
nucleotide sequences are generally known, enable one of skill
in the art to ligate them to 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.
2.e. F.ucarvotic ex~ression
The protein may 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 m~mm~l ian promoters,
e.g. ~-actin promoter. The early and late promoters of the
SV40 virus are conveniently obtained as an SV40 restriction

CA 022116~6 1997-07-29
W ~96123514 PCT~U~36~947
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 psRSV, ATCC
45019. The immediate early promoter of the human
cytomegalovirus may be obtained from plasmid pCMB~ (ATCC
77177). of course, promoters from the host cell or related
species also are useful herein.
Transcription of a DNA encoding the claimed protein
by higher eukaryotes is increased by inserting an enhancer
sequence into the vector. Enhancers are cis-acting elements
of DMA, usually about 10-300 bp, that act on a promoter to
increase its transcription. Enhancers are relatively
orientation and position independent having been found 5'
(Laimins, L. et al., PNAS 78:993 (1981)) and 3' (Lusky, M.
L., ~ al., Mol. Cell Bio. 3:1108 (1983)) to the
transcription unit, within an intron (Banerji, J. L. et al.,
33:729 (1983)) 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
20 m~mm~l ian genes (globin, RSV, SV40, EMC, elastase, albumin,
a-fetoprotein and insulin). Typically, however, one will use
an enhancer from a eukaryotic cell virus. Examples include
the SV40 late enhancer, the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the
replication origin, and adenovirus enhancers.
Expression vectors used in eukaryotic host cells
(yeast, fungi, insect, plant, animal, human or nucleated
cells from other multicellular organisms) will also contain
sequences necessary for the termination of transcription
which may affect mRNA expression. These regions are
transcribed as polyadenylated segments in the untranslated
portion of the mRNA encoding protein. The 3l 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 ~g1II/HindIII

CA 022116~6 1997-07-29
W 096/23S14 PCT/Us~CJ'~C917
restriction fragment of pJOD-10 [ATCC 68815]), thymidine
kinase (herpes simplex virus thymidine kinase is contained on
the E~mHI fragment of vP-5 clone [ATCC 2028]) or neomycin
tG418) resistance genes (obtainable from pNN414 yeast
artificial chromosome vector [ATCC 37682]). When such
selectable markers are successfully transferred into a
m~mm~l ian host cell, the transfected 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 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. A~l. Genet. 1: 327
(1982), mycophenolic acid, Mulligan, R. C. and Berg, P.
Science 209:1422 (1980), or hygromycin, Sugden, B. et al.,
Mol Cell. Biol. 5:410-413 (1985). The three examples given
above employ bacterial genes under eukaryotic control to
convey resistance to the appropriate drug G418 or neomycin

CA 022ll6~6 l997-07-29
W ~961235~4 PCT~US96/nO~7
-23-
(geneticin), xgpt (mycophenolic acid) or hygromycin,
respectively.
A preferred vector for eucaryotic expression is
pRc/CMV. pRc~CMV is commercially available from Invitrogen
Corporation, 3985 Sorrento Valley Blvd., San Diego, CA
92121.
To confirm correct sequences in plasmids constructed, the
ligation mixtures are used to transform ~. coli K12 strain
DH5a (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., ~ucleic Acids Res.
9:309 (1981).
Host cells may be trans~ormed 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 techni~ues 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 Clonin~: A Laboratorv Manual, Cold Spring Harbor
Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New
York (1989), or Current Protocols in Molecular Bioloov
(1989) and supplements.
Preferred suitable host cells for expressing the
vectors encoding the claimed proteins in higher eukaryotes
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), Viroloov 77:319-329, Viroloov
~ 86:10-21); baby hamster kidney cells (BHK-21(C-13), ATCC CCL-
10, Viroloov 16:147 (1362)); chinese hamster ovary cells CHO-
DHFR- (ATCC CRL-9096), mouse Sertoli cells (TM4, ATCC CRL-
1715, Biol. Re~rod. 23:243-250 (1980)); african green monkey

CA 022ll6~6 l997-07-29
W O96/23514 PCTrUS9G/00~17
-24-
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 m~mm~ry tumor cells (MMT 060562, ATCC CCL51).
2~f. Yeast ex~ression
In addition to prokaryotes, eukaryotic microbes
such as yeast cultures may also be used. Saccharomyces
cerevisiae, or common bakerls 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.,
M~t~lre 282:39 (1979); Kingsman et al., Gene 7:141 (1979);
Tschemper et al., Gene 10:157 (1980)) is commonly used. This
plasmid already contains the trp gene which provides a
selection marker for a mutant strain of yeast lacking the
ability to grow in tryptophan, for example ATCC no. 44076 or
PEP4-1 (Jones, Genetics 85:12 (1977)).
Suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase
(found on plasmid pAP12BD ATCC 53231 and described in U.S.
Patent No. 4,935,350, June 19, 1990) or other glycolytic
enzymes such as enolase (found on plasmid 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),
hexokinase, pyruvate decarboxylase, phosphofructokinase,
glucose-6-phosphate isomerase, 3-phosphoglycerate mutase,
pyruvate kinase, triosephosphate isomerase, phosphoglucose
isomerase, and glucokinase.
Other yeast promoters, which are inducible
promoters having the additional advantage of transcription
controlled by growth conditions, are the promoter regions for
alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,
degradative enzymes associated with nitrogen metabolism,

CA 022116~6 1997-07-29
w o 96/23S14 PCTrUS96/00947
metallothionein (contained on plasmid vector pCL28XhoLHBPV
ATCC 39475, United States Patent No. 4,840,896),
glyceraldehyde 3-phosphate dehydrogenase, and enzymes
responsible for ~altose 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 et al., European Patent Publlcation No.
73,657A. Yeast enhancers such as the UAS Gal from
Saccharomyces cerevisiae (found in conjunction with the CYCl
promoter on plasmid YEpsec--hIlbeta ATCC 67024), also are
advantageously used with yeast promoters.
The following examples are presented to further
illustrate the preparation of the claimed proteins. The
scope of the present invention is not to be construed as
merely Consisting of the following examples.
F.xam~le 1
A DNA sequence encoding the following protein sequence:
Met Arg - SEQ ID NO: 1.
is obtained using standard PCR methodology. A forward primer
(5'-GG GG CAT ATG AGG GTA CCT ATC CAG AAA GTC CAG GAT GAC AC)
(SEQ ID No: 16) and a reverse primer (5'-GG GG GGATC CTA TTA
GCA CCC GGG AGA CAG GTC CAG CTG CCA CAA CAT) (SEQ ID No: 17)
is used to amplify sequences from a human fat cell library
(commercially available from CLONETECH). The PCR product is
cloned into PCR-Script (available from STRATAGENE) and
sequenced.
Fxam~le 2
Vector Construction
A plasmid containing the DNA sequence encoding the
desired claimed protein is constructed to include ~I and
- 35 ~HI restriction sites. The plasmid carrying the cloned PCR
product is digested with ~I and BamHI restriction enzymes.
The small ~ 450bp fragment is gel-purified and ligated into
the vector pRB182 from which the coding sequence for A-C-B

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-26-
proinsulin is deleted. The ligation products are transformed
into F. coli l~)H10B (commercially available from GIBCO-BRL)
and colonies growing on tryptone-yeast (DIFCO) plates
supplemented with 10 ~g/mL of tetracycline are analyzed.
Plasmid DNA is isolated, digested with NdeI and ~mHI and the
resulting fragments are separated by agarose gel
electrophoresis. Plasmids containing the expected - 450bp
~I to ~mHI fragment are kept. E. coli B BL21 (DE3)
(commercially available from NOVOGEN) are transformed with
this second plasmid expression suitable for culture for
protein production.
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 Clonina: A Laboratorv Manual, Cold Spring Harbor
Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New
York or Current Protocols in Molecular Bioloov (1989) and
supplements. The techniques involved in the transformation
of F.. 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. çoli
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,
re~uire 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 F,.
coli K12 RV308 cells are employed as host cells but numerous
other cell lines are available such as, but not limited to,
. ~Ql~ 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

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-27-
a time and temperature appropriate to the host cell line
employed.
Proteins which are expressed in high-level
bacterial expression systems characteristically aggregate in
5 granules or inclusion bodies which contain high levels of the
overexpressed protein. Kreuger et al., in Protein Foldin~,
Gierasch and King, eds., pgs 136-142 (1990), American
Association ~or the Advancement of Science Publication No.
89-18S, Washington, D.C. Such protein aggregates must be
solubilized to provide further purification and isolation of
the desired protein product. Id. A variety of techniques
using strongly denaturing solutions such as guanidinium-HCl
and/or weakly denaturing solutions such as dithiothreitol
(DTT) are used to solubilize the proteins. Gradual removal
of the denaturing agents (often by dialysis) in a solution
allows the denatured protein to assume its native
con~ormation. The particular conditions for denaturation and
folding are determined by the particular protein expression
system and/or the protein in question.
Exam~le 3
A protein of the Formula:
90 95 100
Ser Cys His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser
105 110 115
Leu Gly Gly Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val
120 125 130
Ala Leu Ser Arg Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln
135 140
Leu Asp Leu Ser Pro Gly Cys
was prepared as follows.
Biosynthetic human obese gene product with an
intramolecular disulphide was prepared in E. coli, purified
and tested as described herein (hereinafter hos protein).
Human OB protein (19.2 mg) was weighed into a glass vial and
dissolved in 4.8 mL phosphate buffered saline, pH 7.4, to

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give an approximate concentration of 4.0 mg/mL. Complete
dissolution of the protein solution was achieved by briefly
adjusting the pH to 10.1 with 5N NaOH and then lowering the
pH to 7.8 with 5N HCl. The actual concentration of the
protein solution was 4.27mg/mL as calculated by W analysis.
The hOB protein solution (4.7mL) was digested with
lysyl-C endopeptidase (E:S=1:500 wt/wt) for 2 hrs. at 37~C.
The digest was removed from incubation and appeared very
turbid with a heavy precipitate at the bottom of the vial.
The digestion was terminated by acidification to pH 3.0 with
5N HCl. The digest was centrifuged at 13,600 g for 2 min and
the pellet was solubilized with 300ul glacial acetic acid.
Upon dilution of the acidified solution with 2.7mL of
distilled water some turbidity developed which was removed by
centrifugation. The clear supernatant (3mL) was transferred
to a glass vial and the remaining gelatinous pellet was
solubilized with 150~1 glacial acetic acid, diluted with an
e~ual volume of distilled water and centrifuged. This
supernatant was pooled with the above supernatant and then
directly fractionated by RP-HPLC.
The digest fragments were fractionated using a RP-HPLC
system consisting of Beckman 110A pumps and a Pharmacia
detector. Purification of the lysyl-C endopeptidase peptides
was performed on a C4-Vydac column (10 x 250 mm) with a
gradient composed of buffer A (2 parts CH3CN/98 parts 0.05M
Na2SO4, pH 2.3) and buffer B (70 parts CH3CN/30 parts 0.05M
Na2SO4, pH 2.3). The peptides were eluted at a flow rate of 2
mLJmin with a linear gradient of 20 to 80% buffer B over 180
min. The column effluent was monitored at 214 nm, R=1.0 AUFS
and collected manually. The desired peak materials eluted at
about 42.1% CH3CN and 48.2% CH3CN for the peptide fragments
54-95 and 95-146, respectively. The fractions were desalted
into 70% CH3CN/.1% TFA using Waters C2 Sep-Pak cartridges.
In order to accumulate sufficient amounts of desired
peptide sequence materials the lysyl-C endopeptidase digest
and the RP-HPLC purification process were repeated exactly as
described. The desal~ed column fractions of the desired peak

-
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-29-
materials were combined to give a total volume of 4.6-m-L for
each of the peptide sequences from the two semi-preparative
RP-HPLC purification runs. The pools were transferred into
glass vials in 400 ~l aliquots and lyophilized. Four
aliquots of 100 ~l each of the desalted pools were reserved
for amino acid and mass spectrometry analysis.
Electrospray ionization mass spectrometry of the two
desalted pools was performed on a PESciex API III mass
spectrometer equipped with a pneumatically-assisted
electrospray (Ionspray) interface. Positive ion mass spectra
were obtained by continously infusing sample into the
interface at a ~low rate o~ 5-20uL/min using a Harvard
syringe pump. Data were obtained using an inlet ori~ice
potential o~ +40V relative to the rod of~set potential.
Scans were made over a 500-2400u range in O.lu intervals for
a dwell time of lms per interval. Multiple scans (3-20) were
ac~uired per sample to provide an averaged final spectrum.
The isolated peptide fragments were hydrolyzed under
vacuum by a vapor-phase method in a PicoTag Work Station
(Waters Associates, Milford, MA) using 6N HCl at 120~C for 21
hours. The hydrolysates were dried down on the Work Station,
treated with sample buffer, and analyzed on a Model 6300
Beckman amino acid analyzer. The results of the mass
spectrometry MW observed 5489.9 (MW theoretical: 5490.2).
The results were also confirmed by amino acid analysis.
~ mnl e 4
A protein of the Formula:
50 55 60
Met Asp Gln Thr Leu Ala Val Tyr Gln Gln Ile Leu Thr Ser Met
65 70 75
Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu Glu Asn Leu
80 85 90
Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys His Leu
95 100 105
Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly Val

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-30-
110 115 120
Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg
125 130 135
Leu Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser
140
Pro Gly Cys
was prepared as follows.
hOB protein (10.Omg) was weighed into a glass vial and
dissolved in 2.Oml phosphate buffered saline, pH 7.4.
Complete dissolution of the protein solution was achieved by
briefly adjusting the pH to 10.0 with 5N NaOH and then
lowering the pH to 8.6 with 5N HCl. The actual concentration
of solution B was 5.15 mg/ml as calculated by W analysis.
The hOs protein solution B (0.78 ml) was treated with 7M
guanidine-hydrochloride (0.86 ml), diluted with phosphate
buffered saline (0. 3 6 ml) and adjusted to pH 9Ø The
digestion was carried out with lysyl-C endopeptidase
(E:S=1:1000 wt/wt) for 30 min. at 37~C. The clear solution
was removed from the incubator and acidified to pH 2.0 with
glacial acetic acid (0.15ml) to terminate the digest. The
acidified solution was directly fractionated by RP-HPLC.
The digest fragments were fractionated using a RP-HPLC
system consisting of Beckman 110A pumps and a Pharmacia
detector. Purification of the lysyl-C endopeptidase peptides
was performed on a C4-Vydac column (10 x 250mm) with a
gradient composed of buffer A (2 parts CH3CN/98 parts 0.05M
Na2SO4, pH 2.3) and buffer B (70 parts CH3CN/30 parts 0.05M
Na2SO4, pH 2. 3). The peptides were eluted at a flow rate of
2ml/min with a linear gradient of 20 to 80% buffer B over 180
min. The column effluent was monitored at 214nm, R=1.0 AUFS
and collected manually. The desired peak materials eluted at
about 42.1, 47.6, and 49.3% CH3CN for the peptide fragments
54-94, 95-146, and 54-146, respectively. The fractions were
desalted into 70% CH3CN/.l9~ TFA using Waters C2 Sep-Pak
cartridges.

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W ~961235~4 PCT/u~3GJ~17
Electrospray ionization mass spectrometry of the two
desalted pools was performed on a PESciex API III mass
spectrometer equipped with a pneumatically-assisted
electrospray (Ionspray) interface. Positive ion mass spectra
were obtained by continously infusing sample into the
interface at a flow rate of 5-20uL/min using a Harvard
syringe pump. Data were obtained using an inlet ori~ice
potential of +40V rela~ive to the rod offset potential.
Scans were made over a 500-2400u range in O.lu intervals ~or
a dwell time of lms per interval. Multiple scans (3-20) were
acquired per sample to provide an averaged final spectrum.
The results of the mass spectrometry were observed MW
10,188.4 (theoretical 10,188.7).
Preferably, the present proteins are expressed as
Met-Arg-SEQ ID NO: 1 through 15 so that the expressed
proteins may be readily converted to the claimed protein with
Cathepsin C. The purification of proteins is by techni~ues
known in the art and includes reverse phase chromatography,
affinity chromatography, and size exclusion.
The claimed proteins contain two cysteine residues.
Thus, a di-sulfide bond may be formed to stabilize the
protein. The present invention includes proteins of the
Foîmula I through In wherein the Cys at position 91 is
crosslinked to Cys at position 141 as well as those proteins
without such di-sulfide bonds.
In addition the proteins of the present invention
may exist, particularly when ~ormulated, as dimers, trimers,
tetramers, and other multimers. Such multimers are included
within the scope of the present invention.
The present invention provides a method for
treating obesity. The method comprises administering to the
organism an effective amount of anti-obesity protein in a
dose between about 1 and 1000 ~g/kg. A preferred dose is from
about 10 to 100 ~g/kg of active compound. A typical daily
dose for an adult human is from about 0.5 to 100 mg. In
practicing this method, compounds of the Formula (I) can be
administered in a single daily dose or in multiple doses per

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-32-
day. The treatment regime may require administration over
extended periods of time. The amount per administered dose
or the total amount administered will be determined by the
physician and depend on such factors as the nature and
severity of the disease, the age and general health of the
patient and the tolerance of the patient to the compound.
The instant invention further provides
pharmaceutical formulations comprising compounds of the
Formula (I through In). The proteins, preferably in the form
of a pharmaceutically acceptable salt, can be formulated for
nasal, bronchal, transdermal, or parenteral administration
for the therapeutic or prophylactic treatment of obesity.
For example, compounds of the Formula (I through In) can be
admixed with conventional pharmaceutical carriers and
excipients. The compositions comprising claimed proteins
contain from about 0.1 to 90% by weight of the active
protein, preferably in a soluble form, and more generally
from about 10 to 30%.
For intravenous (IV) use, the protein is
administered in commonly used intravenous fluid(s) and
administered by infusion. Such fluids, for example,
physiological saline, Ringer's solution or 5% dextrose
solution can be used.
For intramuscular preparations, a sterile
formulation, preferably a suitable soluble salt form of a
protein of the Formula (I through In), for example the
hydrochloride salt, can be dissolved and administered in a
pharmaceutical diluent such as pyrogen-free water
(distilled), physiological saline or 5% glucose solution. A
suitable insoluble form of the compound may be prepared and
administered as a suspension in an aqueous base or a
pharmaceutically acceptable oil base, e.g. an ester of a long
chain fatty acid such as ethyl oleate.
It may also be desirable to administer the
compounds of Formula (I through In) intranasally.
Formulations useful in the intranasal absorption of proteins
are well known in the art. Nasal formulations comprise the

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-33-
protein and carboxyvinyl polymer preferably selected from the
group comprising the acrylic acid series hydrophilic
crosslinked polymer, e.g. carbopole 934, 940, 941 (Goodrich
Co.). The polymer accelerates absorption of the protein, and
gives suitable viscosity to prevent discharge from nose.
Suitable content of the polymer is 0.05 - 2 weight ~. By
neutralisation of the polymer with basic substance,
thickening ef~ect is increased. The amount of active compound
is commonly 0.1 - 10%. The nasal preparation may be in drop
form, spraying applicator or aerosol form.
The ability of the present compounds to treat
obesity is demonstrated in vivo as follows:
~;oloaical Testina for Anti-obesitv ~roteins
Parabiotic experiments suggest that a protein is
released by peripheral adipose tissue and that the protein is
able to control body weight gain in normal, as well as obese
mice. Therefore, the most closely related biological test is
to inject the test article by any of several routes of
administration (e.g. i.v., s.c., i.p., or by minipump or
cannula) and then to monitor food and water consumption, body
weight gain, plasma chemistry or hormones (glucose, insulin,
ACTH, corticosterone, GH, T4) over various time periods.
Suitable test ~n i m~ 1 S include normal mice (ICR,
etc.) and obese mice (ob/ob, Avy/a, KK-Ay, tubby, fat). The
ob/ob mouse model of obesity and diabetes is generally
accepted in the art as being indicative of the obesity
condition. Controls for non-specific effects for these
injections are done using vehicle with or without the active
agent of similar composition in the same ~nim~l monitoring
the same parameters or the active agent itself in ~nim~l S
that are thought to lack the receptor (db/db mice, fa/fa or
cp/cp rats). Proteins demonstrating activity in these models
will demonstrate similar activity in other m~mm~l S,
particularly humans.
Since the target tissue is expected to be the
hypothalamus where food intake and lipogenic state are
regulated, a similar model is to inject the test article

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-34-
directly into the brain (e.g. i.c.v. injection via lateral or
third ventricles, or directly into specific hypothalamic
nuclei (e.g. arcuate, paraventricular, perifornical nuclei).
The same parameters as above could be measured, or the
release of neurotransmitters that are known to regulate
feeding or metabolism could be monitored (e.g. NPY, galanin,
norepinephrine, dopamine, ~-endorphin release).
Similar studies are accomplished in vitro using
isolated hypothalamic tissue in a perifusion or tissue bath
system. In this situation, the release of neurotransmitters
or electrophysiological changes is monitored.
The compounds are active in at least one of the
above biological tests and are anti-obesity agents. As such,
they are useful in treating obesity and those disorders
implicated by obesity. However, the proteins are not only
useful as therapeutic agents; one skilled in the art
recognizes that the proteins are useful in the production of
antibodies for diagnostic use and, as proteins, are useful as
feed additives for ~n; m~l S . Furthermore, the compounds are
useful for controlling weight for cosmetic purposes in
m~mm~l S . A cosmetic purpose seeks to control the weight of a
m~mm~ 1 to improve bodily appearance. The m~mm~ 1 is not
necessarily obese. Such cosmetic use forms part of the
present invention.