AU3019700A - Methods of treatment - Google Patents

Description

S&F Ref: 401767D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

r u i r i r Name and Address of Applicant Actual Inventor(s): Address for Service: Invention Title: Eli Lilly and Company Lilly Corporate Center City of Indianapolis 46285 United States of America Thomas W Stephens Spruson Ferguson St Martins Tower 31 Market Street Sydney NSW 2000 Methods of Treatment *r The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c 1 Methods of Treatment Diabetes mellitus is a metabolic disorder characterized by the failure of body tissues to store carbohydrates at the normal rate. Resistance to the action Sof insulin is the most important factor to Type II diabetes.

When this resistance exceeds the capacity of the beta cells to produce insulin, a person becomes diabetic. Durinc the last 70 years people suffering from diabetes have been grea.ly aided by receiving controlled amounts of insulin Obesity oarticularly uoper body obesitv, is of ass::L.-:c non-insulin-decendent diabetes meiius NIDDM These so called Type II diabe=ics t t are an acso ute reciremen for insulin as their be: weih e secrete insulin albeit often at di nrise-C r h Seveis. additon sucn patiens are often obese ano ma eamonstrate an inability rescond to insulin.

_s wel- known tha= a regimen o- C i t exercise leading to weight loss is the best approach for treatinc coese type Ii diabetics. Unfortunately, tese regimens are usually unsuccessful. Failure to loss weichth may be due to genetically inherited factors that contribute to increased appetite, tit a preference for high calorie foods, b. reduced physical activity, and an increased lipogenic metabolism. People inheriting such genetic predispositions are prone to obesity and often become type II diabetics, regardless of their efforts to combat the condition. The ob lob 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 (ob) 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 (hereinafter leptin) with a 21 amino acid signal peptide that is exclusively expressed in adipose tissue.

2 Circulating levels of leptin in obese individuals have been shown to vary widely. Consequently, it is now believed that a subpopulation of obese type II diabetics are particularly amenable to treatment with leptin.

Pharmacological agents which are biologically active and mimic the activity of leptin are therefore useful for treating obese type II diabetics, particularly those with abnormally high or low levels of circulating leptin.

One aspect of the present invention is a method of treating or preventing diabetes which comprises administering to an obese type II diabetic an effective amount of leptin, leptin mimetic, or a pharmaceutically acceptable salt *thereof. In a preferred embodiment, the invention includes methods for treating obese type II diabetics having low endogenous levels of leptin.

Obesity refers to a condition in which the individual has a body mass index of greater than 27 kilograms per square meter.

Leptin refers to the protein produced from the obesity gene following transcription and deletions of introns, translation to a protein and processing to the mature protein with secretory signal peptide removed, e.g., from the N-terminal valine-proline to the C-terminal cysteine of the mature protein. Mouse and human leptin protein 25 sequences are published in Zhang et al. Nature 372: 425-32 (1994). The rat leptin sequence is published in Murakami et al., Biochemical and Biohvsical Research Comm, 209(3): 944- 52 (1995). In human, murine and rat leptin the Cys associated with di-sulfide formation is positions 96 and 145.

However, particularly with murine and human leptin, a desGln(28) variant has been observed. Hence, the Cys residues associated with di-sulfide bond formation may be at positions 95 or 96 and at position 145 or 146. Leptin may also be referred to throughout this specification as obesity protein, OB, or ob gene product. Leptin therefore includes SEQ ID NOs:l-6.

3 3- Those skilled in the art will recognize that certain amino acids are prone to rearrangement. For example, Asp may rearrange to aspartimide and isoasparigine as described in I. Schbn, et al., International Journal of Peptide and Protein Research, 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.

Preferred forms of leptin useful in the presently claimed method are the native sequences. The use of human leptin is more preferred. Most preferred leptins useful in the present method include proteins of SEQ ID NOS:1-6.

Murine Leptin o 15 SEQ ID NO: 1 Val Pro Ile Gin Lys Val Gin Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 20 Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Xaa Ser Val Ser Ser 25 Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile 35 40 2 5 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin Ile 55 30 Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gln Ile Ser Asn Asp Leu 65 70 75 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 90 His Leu Pro Gin Ala Ser Gly Leu Glu Thr Leu Glu Ser Leu Gly Gly 100 105 110 Val Leu Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ser Leu Gin Asp Met Leu Gin Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145 wherein: Xaa at position 28 is Gln or absent.

-4 Val1 1 Ile Lys Leu Leu Giu 20 Pro Val Leu dly 145 Pro Val1 Gin Ser Thr As n Leu Leu Gin 130 Cys Ile Thr Arg Leu Ser Leu Pro Giu 115 Gly Trp Arg 5 Arg Ile Val Thr Ser Lys Leu Pro Arg Asp 85 Gin Ala 100 Ala Ser Ala Leu Val1 Ser Giy Met Ser 70 Leu Arg Leu Gin Gi Asj Le' As1 5! Arc Let Alz AsT 135~ Porcine Lentin SEQ ID NO: 2 n Asp Asp Thr Lys 10 p Ile Ser His Met 25 u Asp Phe Ile Pro 40 p Gin Thr Leu Ala SAsn Val Ile Gin 75 1 His Leu Leu Ala 90 Leu Giu Thr Leu 105 Ser Thr Giu Val 120 Met Leu Arg Gin Thr Gin Gly Ile Ile Ser Giu Val1 Leu 140 Leu Ser Leu Tyr Ser Ser Ser Ala 125 Asp Ile Val1 His Gin Asn Ly s Leu 110 Leu Leu Ly s Ser Pro Gin Asp Ser Gly Ser Ser Thr Ser Val1 Ile Leu Cy s Giy Arg Pro Bovine Leptin SEQ ID NO: 3 Val1 1 Ile Lys Leu Leu 65 Giu Pro Val1 Gin Ser Thr Asn Ile Thr Arg Leu Se r Leu cys Arg Val1 Ser Leu Arg Ly s 5 Ile Thr Ly s Pro Asp Val1 Asn Gly Met Ser 70 Leu Gin Asp Leu Asp 55 Arg Leu Asp Ile Asp 40 Gin Asn His Asp Ser 25 Phe Th r Val1 Leu Thr 10 His Ile Leu Val1 Leu 90 Ly s Thr Pro Ala Gin Ala Thr Xaa Gly Ile Ile Ala Leu Ser Leu Tyr Ser Ser Ile Val1 His Gin As n Ly s Ly s Ser Pro Gin Asp Ser 5 Pro Leu Pro Gin Val Arg Ala Leu Glu Ser Leu Glu Ser Leu Gly Val 100 105 110 Val Leu Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ser Leu Gin Asp Met Leu Arg Gin Leu Asp Leu Ser Pro 130 135 140 Gly Cys 145 wherein Xaa at position 28 is Gin or absent.

Human Leotin o SEQ ID NO: 4 2 Val Pro Ile Gln Lys Val Gln Asp Asp Thr Lys Thr Leu Ile Lys Thr 20 1 5 10 Ile Val Thr Arg Ile Asn Asp Ile Ser His Xaa Xaa Ser Val Ser Ser 20 25 25 Lys Gln Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile 35 40 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin Ile 55 30 Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gin Ile Ser Asn Asp Leu 65 70 75 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 5 85 90 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 100 105 110 Val Leu G l u Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 130 135 140 145 Gly Cys wherein: Xaa at position 27 is Thr or Ala; and Xaa at position 28 is Gin or absent.

Rhesus LeDtin -6 SEQ ID Val1 Thr Ser His Ile Ala Th r Th r Met Pro Ile Ser Pro Gin Ser Phe Leu Giu Leu Ile Val1 Ly s Vai Gin As n Ser Giu Vai Trp Gin Thr Gin Leu Ile Asp Ly s Ser Vali Gin Ly s Arg Arg Thr Leu Leu Ser 95 Leu 110 Ala 125 Leu 140 Val1 Ile Vali Leu Ile Glu Cy s Gly Leu Asp Gin Asn Thr Ser As n Asn His Asp Ser Leu Ser Asp G ly Gin Leu Leu Leu Val1 Arg Ser Asp Ile Leu Met Pro Arg Pro Leu Leu Pro Thr 10 Ser 25 Asp 40 Asp 55 Ser 70 Asp 85 Leu 100 Glu 115 Gin 130 Gly 145 Ly s His Phe Gin Arg Leu Ala Ala G ly :y Thr Thr Ile Thr Asn Leu Se r Se r Ser Leu Gin Pro Leu Val1 His Gly Leu Leu Ile Ser Gly Ala Ile Leu Leu Tyr Gin Ly s Val1 Leu Ile Gin Leu Giu 105 Ser 120 Asp 135 Rat Leptin SEQ ID NO:6 -5 Val 1 Ile Arg Leu Leu Giu Ser Val1 Pro Val1 Gin Ser 50 Thr As n Leu Leu Ile His Thr Arg Arg Val Leu Ser Ser Leu Leu Arg Pro Gin 100 Glu Ala Ly s 5 Ile Thr Ly s Pro Asp Thr Ser Val1 Asn Gly Met Ser 70 Leu Arg Leu Gin Asp Leu Asp 55 Gin Leu Gly Tyr Asp Ile Asp 40 Gin As n His Leu Ser Asp Ser 25 Phe Thr Val1 Leu Gin 105 Thr Thr 10 His Ile Leu Leu Leu 90 Lys Glu Ly s Thr Pro Ala Gin 75 Ala Pro Val1 Thr Gin Gly Val1 Ile Phe Giu Val1 Leu Se r Leu Ty r Ala Ser Se r Ala Ile Val1 His Gin His Ly s Leu 110 Leu Ly s Ser Pro Gin Asp Ser Asp Ser Thr Ala Ile Ile Leu Cy Gly Arg -7- 115 120 125 Leu Gin Gly Ser Leu Gin Asp Ile Leu Gin Gin Leu Asp Leu Ser Pro 130 135 140 Glu Cys 145 In addition to the above identified protein sequences, it is frequently considered expeditious to prepare such anti diabetic proteins with a one or two amino acid leader sequence, especially with a methionine containing leader. Two frequently employed leaders are Met-Arg and Met- Asp. Such proteins may be identified infra as Met-Arg-leptin 15 or Met-Asp-leptin or may be identified by Met-Arg-SEQ ID NO:X, where X is 1 to 6.

Leptin mimetics and fragments are also useful in the methods of the present invention. Leptin mimetics are generally defined by Formula (SEQ ID NO:7) as follows: Val Pro Ile Gin Lys Val Gin Asp Asp Thr Lys Thr Leu Ile Lys Thr S1 5 10 Ile Val Thr Arg Ile Xaa Asp Ile Ser His Xaa Xaa Ser Val Ser Ser 25 20 25 "o Lys Gin Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile 40 30 Leu Thr Leu Ser Lys Xaa Asp Gin Thr Leu Ala Val Tyr Gin Gin Ile 50 55 Leu Thr Ser Xaa Pro Ser Arg Xaa Val Ile Gin Ile Xaa Asn Asp Leu 70 75 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 90 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 100 105 110 Val Leu Glu Ala Ser Xaa Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 130 135 140 145 Gly Cys -8 wherein: Xaa at position 22 is Asn, Asp or Glu; Xaa at position 27 is Thr or Ala; Xaa at position 28 is Gin, Glu, or absent; Xaa at position 54 is Met or Ala; Xaa at position 68 is Met or Leu; Xaa at position 72 Asn, Asp or Glu; Xaa at position 77 is Ser or Ala; Xaa at position 118 is Gly or Leu; said protein having at least one substitution selected from the group consisting of: His at position 97 is replaced with Ser or Pro; Trp at position 100 is replaced with Gln, Ala or Leu; Ala at position 101 is replaced with Thr or Val; 15 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; or Gly at position 111 is replaced with Asp.

Preferably, leptin mimetics are those of Formula (II) (SEQ ID NO:8) as follows: Val Pro Ile Gin Lys Val Gin Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 Ile Val Thr Arg Ile Asn Asp Ile Ser His Xaa Gin Ser Val Ser Ser 20 25 Lys Gin Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile 40 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin Ile 55 Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gin Ile Xaa Asn Asp Leu 70 75 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 90 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 9 100 105 110 Val Leu Glu Ala Ser Xaa Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 130 135 140 145 Gly Cys wherein: Xaa at position 27 is Thr or Ala; Xaa at position 77 is Ser or Ala; Xaa at position 118 is Gly or Leu; said protein having at least one substitution, preferably having one to five substitutions and, most preferably, one to two substitutions selected from the group consisting of: His at position 97 is replaced with Ser; 20 Trp at position 100 is replaced with Gln; Ala at position 101 is replaced with Thr; Glu at position 105 is replaced with Gln; Thr at position 106 is replaced with Lys; Leu at position 107 is replaced with Pro; 25 Asp at position 108 is replaced with Glu; or *Gly at position 111 is replaced with Asp.

Examples of preferred proteins of the present invention include proteins of SEQ ID NO:8, wherein Xaa at position 27 is Thr; Xaa at position 77 is Ser; Xaa at position 118 is Gly; and the amino acid residues at positions 97, 100, 101, 105, 106, 107, 108, and 111 are as follows in Table I. The native human sequence is provided as a comparison to the proteins employed in the methods of the present invention.

Table I Amino Acid Position Compound 97 100 101 105 106 107 108 111 native human His Trp Ala Glu Thr Leu Asp Gly 1 Ser Trp Ala Glu Thr Leu Asp Gly 2 His Gin Ala Glu Thr Leu Asp Gly 3 His Trp Thr Glu Thr Leu Asp Gly 4 His Trp Ala Gin Thr Leu Asp Gly His Trp Ala Glu Lys Leu Asp Gly 6 His Trp Ala Glu Thr Pro Asp Gly 7 His Trp Ala Glu Thr Leu Glu Gly 8 His Trp Ala Glu Thr Leu Asp Asp 9 Ser Gin Ala Glu Thr Leu Asp Gly Ser Trp Thr Glu Thr Leu Asp Gly 11 Ser Trp Ala Gin Thr Leu Asp Gly ;12 Ser Trp Ala Glu Lys Leu Asp Gly 13 Ser Trp Ala Glu Thr Pro Asp Gly 14 Ser Trp Ala Glu Thr Leu Glu Gly 15 Ser Trp Ala Glu Thr Leu Asp Asp 16 His Gin Thr Glu Thr Leu Asp Gly 17 His Gin Ala Gin Thr Leu Asp Gly 18 His Gin Ala Glu Lys Leu Asp Gly 19 His Gin Ala Glu Thr Pro Asp Gly 20 His Gin Ala Glu Thr Leu Glu Gly 21 His Gin Ala Glu Thr Leu Asp Asp 22 His Trp Thr Gin Thr Leu Asp Gly 23 His Trp Thr Glu Lys Leu Asp Gly 24 His Trp Thr Glu Thr Pro Asp Gly His Trp Thr Glu Thr Leu Glu Gly 26 His Trp Thr Glu Thr Leu Asp Asp 27 His Trp Ala Gin Lys Leu Asp Gly 28 His Trp Ala Gin Thr Pro Asp Gly 29 His Trp Ala Gin Thr Leu Glu Gly His Trp Ala Gin Thr Leu Asp Asp 31 His Trp Ala Glu Lys Pro Asp Gly 32 His Trp Ala Glu Lys Leu Glu Gly 33 His Trp Ala Glu Lys Leu Asp Asp 34 His Trp Ala Glu Thr Pro Glu Gly His Trp Ala Glu Thr Pro Asp Asp 36 37 38 39 41 42 43 44 46 47 48 49 50 51 52 53 54 56 57 58 59 61 62 63 64 66 67 68 69 71 His Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser His His His His His His His His His His His His His His Trp Gin Gin Gin Gin Gin Gin Trp Trp Trp Trp Trp Trp T rp Trp Trp Trp Trp Trp Trp Trp Trp Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gln Gin Gin Gln Aia Thr Aia Aia Ala Aia Aia Thr Thr Thr Thr Thr Ala Al a Ala Al a Ala Ala Ala Ala Al a Ala Thr Thr Thr Thr Thr Ala Ala Ala Ala Ala Ala Ala Ala Al a Glu Glu Gin Glu Glu Glu Glu Gin Glu Glu Giu Giu Gin Gin Gin Gin Giu Giu Giu Giu Giu Glu Gin Glu Giu Glu Glu Gin Gin Gin Gin Glu Glu Glu Glu Glu Thr Thr Thr Ly s Thr Thr Thr Thr Ly s Thr Thr Thr Ly s Thr Thr Thr Ly s Ly s Ly s Thr Thr Thr Thr Lys Thr Thr Thr Ly s Thr Thr Thr Ly s Ly s Ly s Thr Thr Leu Leu Leu Leu Pro Leu Leu Leu Leu Pro Leu Leu Leu Pro Leu Leu Pro Leu Leu Pro Pro Leu Leu Leu Pro Leu Leu Leu Pro Leu Leu Pro Leu Leu Pro Pro Glu Asp Asp Asp Asp Glu Asp Asp Asp Asp Glu Asp Asp Asp Glu Asp Asp Glu Asp Glu Asp Glu Asp Asp Asp Glu Asp Asp Asp Glu Asp Asp Glu

ASP

Glu

ASP

Asp Gly G ly Gly Gly G ly Asp G ly Gly Gly G ly Asp G ly Gly Gly Asp Gly G ly Asp C ly Asp Asp G ly G ly Gly G ly Asp Gly G ly G ly Asp Gly Gly Asp C ly Asp 12 a a a a 72 73 74 76 77 78 79 81 82 83 84 85 86 87 88 89 91 92 93 94 96 97 98 99 100 101 102 103 104 105 106 107 His His His His His His His His His His His His His His His His His His His His His Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Gin Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Ala Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Thr Thr Thr Thr Thr Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Glu Gin Gin Gin Gin Glu Glu Giu Giu Giu Giu Gin Gin Gin Gin Gin Gin Glu Giu Glu Giu Gin Giu Glu Giu Glu Gin Gin Gin Gin Glu Glu Glu Giu Glu Glu Thr Ly s Thr Thr Thr Ly s Ly s Ly s Thr Thr Thr Ly s Ly s Ly s Thr Thr Thr Ly s Ly s Lys5 Thr Thr Lys5 Thr Thr Thr Ly s Thr Thr Thr Ly s Ly s Lys5 Thr Thr Thr Leu Leu Pro Leu Leu Pro Leu Leu Pro Pro Leu Pro Leu Leu Pro Pro Leu Pro Pro Leu Pro Leu Leu Pro Leu Leu Leu Pro Leu Leu Pro Leu Leu Pro Pro Leu Glu Asp Asp Glu Asp Asp Glu Asp Glu Asp Glu Asp Glu Asp Glu Asp Glu Glu Asp Glu Glu Asp Asp Asp Glu Asp Asp Asp Glu Asp Asp G lu Asp Glu Asp Glu Asp Gly Gly Gly Asp Gly Gly Asp Gly Asp Asp G ly Gly Asp G ly Asp Asp G ly Asp Asp Asp Gly Gly G ly G ly Asp G ly Gly Gly Asp G ly G ly Asp G ly Asp

ASP

0 0 13 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 S er S er S er Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser His His His His His His His His His His His His His His His His Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Ala Ala Ala Ala Ala Gin Gin Gin Gin Giu Giu Giu Glu Giu Giu Gin Gin Gin Gin Gin Gin Glu Giu Glu Glu Gin Gin Gin Gin Glu Glu Glu Glu Glu Glu Gin Gin Gin Gin Gin Gin Lys Thr Thr Thr Ly s Ly s Ly s Thr Thr Thr Ly s Ly s Ly s Thr Thr Thr Ly s Ly s Ly s Thr Ly s Thr Thr Thr Lys5 Lys5 Ly s Thr Thr Thr Ly s Ly s Lys5 Thr Thr Thr Leu Pro Leu Leu Pro Leu Leu Pro Pro Leu Pro Leu Leu Pro Pro Leu Pro Pro Leu Pro Leu Pro Leu Leu Pro Leu Leu Pro Pro Leu Pro Leu Leu Pro Pro Leu Asp Gly Asp Gly Glu Gly Asp Asp Asp Gly Glu Gly Asp Asp Giu Gly Asp Asp Giu Asp Asp Gly Giu Gly Asp Asp Glu Gly Asp Asp Glu As p Giu Gly Asp Asp Giu Asp Giu Asp Asp Gly Asp Gly Giu Gly Asp Asp Asp Gly Giu Gly Asp Asp Giu Gly Asp Asp Glu Asp ASP Gly Giu Gly Asp Asp Giu Gly ASP Asp Giu Asp Gin I Ala 14

S

0

*SS*

144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His His Gin Gin Gin Gin Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Trp Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Ala Ala Ala Ala Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Ala Ala Ala Ala Ala Ala Thr Thr Thr Thr Thr Ala Ala Ala Ala Ala Thr Thr Thr Thr Thr Thr Glu Lys Glu Lys Glu Lys Glu Thr Gin Lys Gin Lys Gin Lys Gin Thr Gin Thr Gin Thr Glu Lys Glu Lys Glu Lys Glu Thr Gin Lys Gin Lys Gin Lys Gin Thr Glu Lys Gin Lys Glu Lys Gin Thr Gin Lys Gin Lys Gin Lys Glu Lys Gin Thr Gin Lys Gin Lys Gin Lys Glu Thr Glu Lys Glu Lys Glu Lys Gin Thr Gin Thr Pro Pro Leu Pro Pro Leu Leu Pro Pro Leu Pro Pro Leu Pro Pro Pro Leu Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Leu Pro Pro Pro Leu Pro Pro Leu Pro Glu Gly Asp Asp Glu Asp Glu Asp Asp Gly Glu Gly Asp Asp Glu Gly Asp Asp Glu Asp Glu Gly Asp Asp Glu Asp Glu Asp Glu Gly Asp Asp Glu Asp Glu Asp Glu Asp Glu Asp Glu Asp Glu Asp Glu Asp Asp Asp Glu Gly Glu Asp Glu Asp Glu Asp Asp Asp Glu Gly Glu Asp Glu Asp Asp Asp Glu Gly Glu Asp Asp Asp 5* S. S a S.

55 5 4* c 55 15 180 His Gin Thr Gin Thr Pro Glu Gly 181 His Gin Thr Gin Lys Leu Asp Asp 182 His Gin Thr Gin Lys Leu Glu Gly 183 His Gin Thr Gin Lys Pro Asp Gly 184 Ser Trp Ala Glu Lys Pro Glu Asp 185 Ser Trp Ala Gin Thr Pro Glu Asp 186 Ser Trp Ala Gin Lys Leu Glu Asp 187 Ser Trp Ala Gin Lys Pro Asp Asp 188 Ser Trp Ala Gin Lys Pro Glu Gly 189 Ser Trp Thr Glu Thr Pro Glu Asp 190 Ser Trp Thr Glu Lys Leu Glu Asp 191 Ser Trp Thr Glu Lys Pro Asp Asp e* 192 Ser Trp Thr Glu Lys Pro Glu Gly S.hg 193 Ser Trp Thr Gin Thr Leu Glu Asp 194 Ser Trp Thr Gin Thr Pro Asp Asp 195 Ser Trp Thr Gin Thr Pro Glu Gly 196 Ser Trp Thr Gin Lys Leu Asp Asp 197 Ser Trp Thr Gin Lys Leu Glu Gly 198 Ser Trp Thr Gin Lys Pro Asp Gly 199 Ser Gin Ala Glu Thr Pro Glu Asp 200 Ser Gin Ala Glu Lys Leu Glu Asp 201 Ser Gin Ala Glu Lys Pro Asp Asp 202 Ser Gin Ala Glu Lys Pro Glu Gly 203 Ser Gin Ala Gin Thr Leu Glu Asp 204 Ser Gin Ala Gin Thr Pro Asp Asp 205 Ser Gin Ala Gin Thr Pro Glu Gly 206 Ser Gin Ala Gin Lys Leu Asp Asp 207 Ser Gin Ala Gin Lys Leu Glu Gly 208 Ser Gin Ala Gin Lys Pro Asp Gly 209 Ser Gin Thr Glu Thr Leu Glu Asp 210 Ser Gin Thr Glu Thr Pro Asp Asp 211 Ser Gin Thr Glu Thr Pro Glu Gly 212 Ser Gin Thr Glu Lys Leu Asp Asp 213 Ser Gin Thr Glu Lys Leu Glu Gly 214 Ser Gin Thr Glu Lys Pro Asp Gly 215 Ser Gin Thr Gin Thr Leu Asp Asp 216 217 218 219 220 22 1 222 "'23 224 225 226 227 2 28 229 230 231 232 233 234 235 23 6 237 238 239 24 0 241 242 243 244 24 5 2)4 6 247 248 249 250 251 Ser Ser Ser His His His His His His His S er Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser His Ser Ser Ser Ser Gin Gin Gin Trp Gin Gin Gin Gin Gin Gin Trp Trp Trp Trp Trp Trp Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Gin Trp Gin Gin Gin 16- Thr Thr Thr Thr Ala Thr Thr Thr Thr Thr Ala Thr Thr Thr Thr Thr Ala Ala Ala Ala Ala Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Thr Ala Thr Thr Gin Gin Gin Gin Gin Giu Gin Gin Gin Gin Gin Giu Gin Gin Gin Gin Giu Gin Gin Gin Gin Giu Giu Giu Giu Gin Gin Gin Gin Gin Gin Gin Gin Gin Giu Gin Thr Thr Ly s Ly s Ly s Ly s Thr Ly s Ly s Ly s Ly s Lys Thr Ly s Ly s Ly s Ly s Thr Ly s Ly s Ly s Thr Ly s Ly s Ly s Thr Thr Thr Ly s Ly s Ly s Ly s Ly s Ly s Ly s Thr Leu Pro Leu Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Leu Pro Pro Pro Leu Pro Pro Leu Pro Pro Leu Leu Pro Pro Pro Pro Pro Pro Giu Asp Asp Giu Giu Giu Giu Giu Asp Giu Giu Giu Giu Giu Asp Giu Giu Giu Giu Asp Giu Giu Giu Asp Giu Giu Asp Giu Asp Giu Asp Giu Giu Giu Giu Giu Gly Gly Gly Asp Asp Asp Asp Asp Asp Gly Asp Asp Asp Asp Asp Gly Asp Asp Asp Asp Gly Asp Asp Asp Gly Asp Asp Gly Asp G ly Gly Asp

ASP

Asp Asp Asp 17 252 Ser Gln Thr Gin Lys Leu Glu Asp 253 Ser Gin Thr Gin Lys Pro Asp Asp 254 Ser Gin Thr Gin Lys Pro Glu Gly 255 Ser Gln Thr Gin Lys Pro Glu Asp Other preferred proteins are those wherein Xaa at position 27 is Ala; Xaa at position 77 is Ser; Xaa at position 118 is Gly; and the amino residues at positions 97, 100, 101, 105, 106, 107, 108 and 111 are as described in Table I.

The present invention provides biologically active proteins that provide effective treatment for obese type II diabetics. Unexpectedly, the leptin proteins of Table I have improved properties due to specific substitutions to the 1 0 human obesity protein. These proteins are more stable than both the mouse and human obesity protein and, therefore, represent superior therapeutic agents.

Experiments were performed with five to six month old male, inbred normal ICR mice, inbred normal(ob/+), obese- 15 diabetic mice (ob/ob) from the Jackson Laboratories (Bar Harbor, Maine) or Harlan (England), and obese-diabetic (db/db) mice.

Both normal and diabetic mice were housed three or six per plastic cage (with bedding) and water and feed were 20 available ad libitum. The temperature of animal rooms was S. maintained at 23 2°C and lights were on from 0600 to 1800 h. Blood samples were collected from the tail vein. The most closely related biological test is, therefore, to inject the test article by any of several routes of administration 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 animals include normal mice (ICR, etc.) and obese mice (ob/ob, Avy/a, KK-Ay, tubby, fat). Controls for nonspecific effects for these injections can be done using vehicle with or without test articles of similar composition in the same 18 animal monitoring the same parameters or the test article itself in animals that are thought to lack the receptor (db/db mice, fa/fa or cp/cp rats).

Blood glucose levels were measured by a glucose oxidase method or a coupled hexokinase method. Plasma insulin was determined with radioimmunoassay kits using rat insulin as the standard. Plasma triglycerides were measured using commercial kits with glycerol as the standard.

The foregoing studies demonstrated that leptin and leptin mimetis, regulated food intake and body weight in normal ICR and genetically obese ob/ob mice. Chronic administration of leptin and leptin mimetics to ob/ob mice totally ameliorated the diabetic state of these animals showing the potential promise for these anti-diabetic 15 proteins as a treatment for obese type II diabetics. Thus, the present invention provides methods for treating obese type II diabetics, particularly those with low circulating levels of leptin.

The compounds of the present invention may be produced by well known chemical procedures, such as solution or solid-phase peptide synthesis, or semi-synthesis in solution beginning with protein fragments coupled through conventional solution methods. Such methods are well known in the art and may be found in general texts in the area such 25 as H. Dugas and C. Penney, BIOORGANIC CHEMISTRY, (1981) at pages 54-92.

Proteins useful in the presently claimed methods also may be prepared by well known recombinant DNA techniques such as those described in Maniatis, et al. (1988) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York or Current Protocols in Molecular Biolov (1989) and supplements. 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-diabetic proteins must not place the sequence out of 19 reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. e, DeBoer, et al., European Patent Publication, 075,444 A (1983) The present invention provides a method for treating obese type II diabetics. The method comprises administering an effective amount of an leptin or leptin mimetic in a dose between about 1 and 10,000 g/kg. A preferred dose is from about 20 to 10,000 g/kg. A more preferred dose is from about 200 to 600 jg/kg. In practicing this method, anti-diabetic proteins can be administered in a single daily dose or in multiple doses per day. The treatment regime may require administration over extended periods of time. The amount per administered dose or the 15 total amount administered will be determined by the physician eoee and depend on such factors as the mass of the patient, 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 present invention. The proteins, preferably in the form of a 0ooo pharmaceutically acceptable salt, can be formulated for parenteral administration. For example, compounds can be .*.admixed with conventional pharmaceutical carriers and 25 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%. Furthermore, the present proteins may be administered alone or in combination with other antiobesity agents or agents useful in treating diabetes.

For intravenous use, the protein is administered in commonly used intravenous fluids and administered by infusion. For intramuscular preparations, a sterile formulation, preferably a suitable soluble salt form of the protein, for example the hydrochloride salt, can be dissolved and administered in a pharmaceutical diluent such as pyrogenfree water or physiological saline. A suitable insoluble 20 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.

In a preferred embodiment, the present invention provides a method for treating obese type II diabetics with low leptin levels, though diabetic patients with high endogenous leptin levels may also benefit from the presently claimed methods. Methods for assaying serum and plasma leptin levels may be accomplished using standard antibodybased methodologies. Leptin assay kits are also commercially available from Linco Research, Inc. (14 Research Park Dr., St Louis, MO 63304) "Treating obese type II diabetics having leptin 15 levels between 0 and 80 ng/ml is preferred. More preferred is to treat obese type II diabetics having leptin levels between 0 and 50 ng/ml. More highly preferred is to.treat obese type II diabetics having leptin levels between 0 and ng/ml. Most preferred is to treat obese type II diabetics S 20 having leptin levels between 0 and 15 ng/ml.

By way of illustration, the following examples are provided to help describe how to make and practice the various embodiments of the invention. These example are in no way meant to limit the scope of the invention.

Example 1 A DNA sequence encoding the following protein sequence: Met-Arg-SEQ ID NO:4.

was obtained using standard PCR methodology from a human fat cell library (commercially available from CLONETECH) Briefly, degenerate primers were designed based on the published amino acid sequence of the human ob gene. The primers were prepared for use in polymerase chain reaction (PCR) amplification methods using a Model 380A DNA synthesizers (PE-Applied Biosystems, Inc., 850 Lincoln Center 21 Drive, Foster City, CA 94404). Forward primers OB.F1M GG CAT ATG AGG GTA CCT ATC CAG AAA GTC CAG GAT GAC AC)and OB.F2H (5-GG GG CAT ATG AGG GTA CCC ATC CAG AAG GTG CAG GAC GA)(and reverse primers OB.R1M (5-GG GG GGATC GAT AAT TTA GCA TCC AGG GCT AAG ATC CAA CTG CCA AAG CAT) and OB.R2H (5-GG GG GGATC CTA TTA GCA CCC GGG AGA CAG GTC CAG CTG CCA CAA CAT) were mixed together with a PCR-ready human fat cell cDNA as the template (Clontech Laboratories, Inc., 4030 Fabian Way, Palo Alto, CA 94303; Item #7128-1).

The 2 sets of PCR amplifications were performed using 2.5 units of Amplitag DNA polymerase (Perkin Elmer Cetus) or 2 units of Vent DNA polymerase (New England Biolabs) in 100 uL reactions. PCR reactions contained 1 uL of human fat cell cDNA, 10 pmol of each primer (all four were mixed). The following conditions were used for "Touchdown PCR": 2 cycles: 94 0 Cx30 sec, 60 0 Cx30 sec, 72 0 Cx45 sec 2 cycles: 94 0 Cx30 sec, 56°Cx30 sec, 72 0 Cx45 sec; 2cycles: 94 0 Cx30 sec, 52 0 Cx30 sec, 72 0 Cx45 sec; 2cycles: 94 0 Cx30 sec, .4 48 0 Cx30 sec, 72 0 Cx45 sec; 2 cycles: 94 0 Cx30 sec, 44 0 Cx30 sec, 72 0 Cx45 sec: 28 cycles 94 0 Cx30 sec, 52 0 Cx30 sec, 72 0 Cx45 sec The resultant PCR reactions products were run on a 1% agarose gel and a band of an approximate 450 bp in size was visualized by ethidium bromide staining. This band was present in both sets of PCR reactions. The bands were 25 excised and reamplified using above conditions in 30 cycles (94x30 sec, 52x30, 72x45). The PCR product obtained using Vent DNA polymerase was gel purified and cloned into a pCR- SCRIPT cloning vector (Stratagene). The vector was then used to transform E. coli cells. Plasmid DNA was isolated from white colonies of E. coli and samples from three clones were sequenced. Two such colonies, E. coli DH10B/pOJ717 and E.

coli DH10B/pOJ718 were deposited with the Northern Regional Research Laboratories (NRRL) under terms of the Budapest Treaty and are available under Accession Numbers B-21408 and B-21409 respectively.

22 Example 2 Vector Construction A plasmid containing the DNA sequence encoding a desired protein is constructed to include NdeI and BamHI restriction sites. The plasmid carrying the cloned PCR product is digested with NdeI and BamHI restriction enzymes.

The small 450bp fragment is gel-purified and ligated into the vector pRB182 from which the coding sequence for A-C-B proinsulin is deleted. The ligation products are transformed into E. coli DH1OB (commercially available) and colonies growing on tryptone-yeast plates supplemented with 10 ~g/mL of tetracycline are analyzed. Plasmid DNA is isolated, digested with NdeI and BamHI and the resulting fragments are Sseparated by agarose gel electrophoresis. Plasmids 15 containing the expected 450bp NdeI to BamHI fragment are kept. E. coli B BL21 (DE3) are transformed with this second plasmid expression suitable for culture for protein production.

The techniques of transforming cells with the S 20 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, New York (1988), or CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausabel, ed., 1989) and supplements thereof. 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°C to about 40 0 C in the culture conditions so as to induce protein synthesis.

-23 In the preferred embodiment of the invention

E.

col 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 coli 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 which are expressed in high-level bacterial expression systems characteristically aggregate in granules or inclusion bodies which contain high levels of the overexpressed protein. See, Kreuger et al. PROTEIN 15 FOLDING, (Gierasch and King, eds., 1990) at pages 136-142, American Association for the Advancement of Science Publication No. 89-18S, Washington, D.C. Such protein aggregates must be solubilized to provide further purification and isolation of the desired protein product.

Id. A variety of techniques using strongly denaturing solutions such as guanidinium-HC1 and/or weakly denaturing solutions such as dithiothreitol (DTT) are used to solubilize the proteins.

Gradual removal of the denaturing agents (often by 25 dialysis) in a solution allows the denatured protein to assume its native conformation. The particular conditions for denaturation and folding are determined by the particular protein expression system and/or the protein in question.

Preferably, the present proteins are expressed as Met-Arg-SEQ ID NO: X so that the expressed proteins may be readily converted to the claimed protein with cathepsin C (also known as diaminopeptidase). The purification of proteins is by techniques known in the art and includes reverse phase chromatography, affinity chromatography, and size exclusion chromatography.

The claimed proteins contain two cysteine residues.

Thus, a di-sulfide bond may be formed to stabilize the 24 protein. The present invention includes proteins wherein the Cys at position 96 is crosslinked to Cys at position 146 as well as those proteins without such di-sulfide bonds.

In addition the proteins useful in the present invention may exist, particularly when formulated, as dimers, trimers, tetramers, and other multimers. Such multimers are included within the scope of the present invention.

Claims (11)

1. A method for treating or preventing diabetes mellitus which comprises administering to an afflicted patient a leptin or leptin mimetic.

2. A method of claim 1 wherein said leptin or leptin mimetic is selected from the group consisting of (a) Val Pro Ile Gin 1 Ile Lys Leu Leu 65 Glu His Val Leu Gly 145 when Val Thr Arg Gin Lys Val Thr Leu Ser Thr Ser Met Asn Leu Arg Leu Pro Qln 100 Leu Glu Ala 115 Gin Gly Ser 130 Lys Val Gin Asp Asp 5 lie Asn Asp Ile Ser 25 Thr Gly Leu Asp Phe 40 Lys Met Asp Gin Thr 55 Pro Ser Arg Asn Val 70 Asp Leu Leu His Val Ala Ser Gly Leu Glu 105 Ser Gly Tyr Ser Thr 120 Leu Gin Asp Met Leu 135 Thr Lys 10 His Thr Ile Pro Leu Ala Ile Gin 75 Leu Ala 90 Thr Leu Glu Val Gin Gin Thr Leu Xaa Ser Gly Leu Val Tyr Ile Ser Phe Ser Glu Ser Val Ala 125 Leu Asp 140 Ile Val His Gin Asn Lys Leu 110 Leu Leu Lys Ser Pro Gin Asp Ser Gly Ser Ser Thr Ser Ile Ile Leu Cys Gly Arg Pro Cy~ ein: s Xaa at position 28 is Gin or absent; (SEQ ID NO: 1) (b) Val Pro Ile Trp Arg Val Gin Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 Ile Val Thr Arg Ile Ser Asp Ile Ser His Met Gin Ser Val Ser Ser 25 Lys Gin Arg Val Thr Gly Leu Asp Phe lie Pro Gly Leu His Pro Val 26 Leu Ser Leu Thr Giu Asn Pro Leu Val Leu Leu Gin 130 Giy Cys 145 Leu Ser Lys Met Asp 55 Ser Leu Pro Ser Arg 70 Leu Arg Asp Leu Leu 85 Pro Gin Ala Arg Ala 100 Giu Ala Ser Leu Tyr 115 Gly Ala Leu Gin Asp 135 (SEQ ID NO:2) (C) Val Pro Ile Cys Lys Val Gin 1 5 Ile Vai Thr Arg Ile Asn Asp Lys Gin Arg Val Thr Gly Leu Leu Ser Leu Ser Lys Met Asp 35 50 55 Leu Thr Ser Leu Pro Ser Arg 65 70 Giu Asn Leu Arg Asp Leu Leu Pro Leu Pro Gin Val Arg Ala 100 Vai Leu Giu Ala Ser Leu Tyr 115 Leu Gin Gly Ser Leu Gin Asp 130 135 Giy Cys 145 wherein Xaa at position 28 I Gir As r His Leu Ser 120 Met Asp Ile Asp 40 Gln A.sn His ,eu 'er 120 4et 1Thr Val Leu Giu 105 Thr Leu Asp Ser 25 Phe ThrI Val\ Leu 1 Glu 105 Thr C Leu A Let Ile Leu 90 Thr Giu Arg Ph r 10 H{is Ilie .eu la 1 .eu er ;iu ~rg Ala Gin 75 IAla Leu Val1 Gin Lys Thr Pro Ala Gin 75 Ala Iz Leu C Val Gin L 1i Il Il Sei Gli Val1 Leu 140 Ph r X(aa ly Ilie Ilie la ;iu al1 ~eu e Tyr 0 SSer Ser Ser Ala 125 Asp Leu Ser Leu Tyr Ser SerI Ser I 1 Aia L 125 Asp L Gli- Asn Lys Leu 110 Leu Leu Ile Val1 Mln ksn ~eu ~eu ~eu Gin Asp Ser Gly Ser Ser Ly s Ser Pro Gin Asp Ser Gly Ser Ser Ile Leu Cys Gly Arg Pro Thr Ser Leu Ile Leu :y s la 1 krg ?ro is Gin or absent; (SEQ ID NO:3) 27 (d) Val Pro Ile Gin Lys Val Gin Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 Ile Val Thr Arg Ile Asn Asp Ile Ser His Xaa Xaa Ser Val Ser Ser 25 Lys Gin Lys Vai Thr Giy Leu Asp Phe Ilie Pro Giy Leu His Pro Ile 40 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Vai Tyr Gin Gin Ile 55 Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gin Ile Ser Asn Asp Leu 65 70 75 Giu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 85 90 20 His Leu Pro Trp Ala Ser Gly Leu Giu Thr Leu Asp Ser Leu Gly Gly *100 105 110 Val Leu Giu Ala Ser Gly Tyr Ser Thr Giu VlVlAaLeuSeAg 5115 120 125 Leu Gin Giy Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 130 135 140 30 Gly Cys wherein: ooof Xaa at position 27 is Thr or Ala; and o **Xaa at position 28 is Gin or absent; *35 (SEQ ID NQ:4) (e) Val Pro Ile Gin Lys Val Gin Ser Asp Thr Lys Thr Leu Ile Lys 1 5 10 Thr Ile Val Thr Arg Ile Asn Asp Ile Ser His Thr Gin Ser Val 25 Ser Ser Lys Gin Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu 35 40 His Pro Val Leu Thr Leu Ser Gin Met Asp Gin Thr Leu Ala Ile 55 Tyr Gin Gin Ile Leu Ile Asn Leu Pro Ser Arg Asn Val Ile Gin 70 Ile Ser Asn Asp Leu Glu Asn Leu Arg Asp Leu Leu His Leu Leu 85 28 Ala Phe Ser Lys Ser Cys His Leu Pro Leu Ala Scr Gly Leu Glu 100 105 Thr Leu Glu Ser Leu Gly Asp Val Leu Glu Ala Ser Leu Tyr Ser 110 115 120 Thr Glu Val Vai Ala Leu Ser Arg Leu Gin Giy Ser Leu Gin Asp 125 130 135 Met Leu Trp Gin Leu Asp Leu Ser Pro Gly Cys 140 145 ;(SEQ ID NO: (f) Val Pro Ile His Lys Val Gin Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 Ile Val Thr Arg Ile Asn Asp Ilie Ser His Thir Gin Ser Val Ser Ala 25 Arg Gin Arg Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile 40 Leu Ser Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin Ile 55 .Leu Thr Ser Leu Pro Ser Gin Asn Vai Leu Gin Ile Aia 1-is Asp Leu 70 75 *Giu Asn Leu Arg Asp Leu Leu His Leu Leu Ala Phe Ser Lys Ser Cys 90 Ser Leu Pro Gin Thr Arg Gly Leu Gin Lys Pro Giu Ser Leu Asp Gly 100 105 110 *Val Leu Glu Ala Ser Leu Tyr Ser Thr Giu Val Val Ala Leu 5cr Arg 115 120 125 Gin Gly Ser Leu Gin Asp Ile Leu Gin Gin Leu Asp Leu Scr Pro ***130 135 140 Giu Cys 145, (SEQ ID NO: 6) (g) Val Pro Ile Gin Lys Val Gin Asp Asp Thr Lys Thr Leu Ile Lys Thr 1 5 10 Ile Val Thr Arg Ile Xaa Asp Ile Scr 1His Xaa Xaa Ser Val Ser Scr 25 Lys Gin Lys Vat Thr Gly Leu Asp Phe Ile Pro Gly Leu is Pro Ile 40 Leu Thr Leu Ser Lys Xaa Asp Gin Thr Leu Ala Val Tyr Gin Gin Ile 55 29 Leu Thr Ser Xaa Pro Ser Arg Xaa Val Ile Gin Ile Xaa Asn Asp Leu 70 75 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 90 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 100 105 110 Val Leu Glu Ala Ser Xaa Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ser Leu Gin Asp Met Leu Trp Gin Leu Asp Leu Ser Pro 130 135 140 145 Gly Cys 20 wherein: Xaa at position 22 is Asn, Asp or Glu; SXaa at position 27 is Thr or Ala; Xaa at position 28 is Gin, Glu, or absent; Xaa at position 54 is Met or Ala; 25 Xaa at position 68 is Met or Leu; Xaa at position 72 Asn, Asp or Glu; Xaa at position 77 is Ser or Ala; Xaa at position 118 is Gly or Leu; said protein having at least one substitution selected from S 30 the group consisting of: His at position 97 is replaced with Ser or Pro; Trp at position 100 is replaced with Gin, Ala or Leu; Ala at position 101 is replaced with 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 Gin; 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; or Gly at position 111 is replaced with Asp; and, (SEQ ID NO:7) (h) Val Pro Ile Gin Lys Val Gin Asp Asp Thr Lys Thr Leu Ile Lys Thr 30 6 Ile Val Thr Arg Ile Asn Asp Ile Ser His Xaa Gin Ser Val Ser Ser 25 Lys Gin Lys Val Thr Gly Leu Asp Phe Ile Pro Gly Leu His Pro Ile 40 Leu Thr Leu Ser Lys Met Asp Gin Thr Leu Ala Val Tyr Gin Gin Ile 50 55 Leu Thr Ser Met Pro Ser Arg Asn Val Ile Gin Ile Xaa Asn Asp Leu 70 75 Glu Asn Leu Arg Asp Leu Leu His Val Leu Ala Phe Ser Lys Ser Cys 90 His Leu Pro Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly Gly 2000 1 05 110 Val Leu Glu Ala Ser Xaa Tyr Ser Thr Glu Val Val Ala Leu Ser Arg 115 120 125 Leu Gin Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro 130 135 140 145 Gly Cys wherein: Xaa at position 27 is Thr or Ala; Xaa at position 77 is Ser or Ala; Xaa at position 118 is Gly or Leu; said protein having at least one substitution, preferably 35 having one to five substitutions and, most preferably, one to two substitutions selected from the group consisting of: His at position 97 is replaced with Ser; Trp at position 100 is replaced with Gln; Ala at position 101 is replaced with Thr; Glu at position 105 is replaced with Gln; Thr at position 106 is replaced with Lys; Leu at position 107 is replaced with Pro; Asp at position 108 is replaced with Glu; or Gly at position 111 is replaced with Asp, or a pharmaceutically acceptable salt or solvate thereof. (SEQ ID NO:8) S. 31

3. A method as claimed in any one of claims 1 or 2 wherein the diabetes mellitus is associated with high endogenous leptin levels.

4. A method as claimed in any one of claims 1 or 2 wherein the diabetes mellitus is associated with low endogenous leptin levels. s

5. A method as claimed in any one of claims I or 2 wherein said patient has endogenous leptin levels in the range of 0 to

6. A method as claimed in any one of claims I or 2 wherein said patient has endogenous leptin levels in the range of 0 to

7. A method as claimed in any one of claims 1 or 2 wherein said patient has endogenous leptin levels in the range of 0 to

8. A method as claimed in any one of claims I or 2 wherein said patient has endogenous leptin levels in the range of 0 to

9. A formulation for treating or preventing diabetes mellitus. comprising as an active ingredient a leptin or leptin mimetic. *r A A A A A

10. A formulation of claim 9 wherein said leptin or leptin mimetic the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2. SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8.

11. A formulation for treating or preventing diabetes mellitus. hereinbefore described with reference to any one of the Examples. is selected from SEQ ID NO: 4, substantially as Dated 28 April 2000 Eli Lilly and Company Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON A. A A *A A. 0 9 A A