AU1804002A - Methods for treatment of diabetes using peptide analogues of insulin - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 DIVISIONAL

APPLICATION

NAME OF APPLICANT(S):., a a. a a.

a Neurocrine Biosciences, Inc.

ADDRESS FOR -SERVICE: DAVIES COLLISON

GAVE

Patent Attorneys 1 Little Collins Street Melbourne, 3000.

INVENTION

TITLE:

"Methods for treatment of diabetes using peptide analogues of insulin- The following statement is a full description of this invention, including the best method of performing it known to us: Q:OPERM\TD\2507050 div-doc 22/2102 METHODS FOR TREATMENT OF DIABETES

USING

PEPTIDE ANALOGUES OF INSULIN TECHNICAL

FIELD

The present invention relates generally to peptide analogues of insulin, and more specifically to methods for treating diabetes using peptide analogues derived from residues 9-23.of human insulin B chain.

to BACKGROUND OF THE INVENTION .l Insulin dependent diabetes mellitus (IDDM) is an organ specific autoimmune disease affecting close to a million people in different age groups in the United States. The disease is characterized by extensive destruction of the insulin producing beta cells in the pancreatic islets and dysregulation of glucose metabolism S 15 leading to frank diabetes. The defining feature of IDDM is the lymphocytic infiltration of the islets. Among the invading cells, T cells appear to be one of the major mediators of autoimmune destruction.

S Type I diabetes is further characterized by increased levels of antibodies to various islet associated antigens, including insulin, GAD65. GAD67 and ICA512.

20 These antibodies can be detected much before frank disease, and an immune response to such antigens can be used as a predictor for impending diabetes in patients with susceptible genetic (HLA) haplotypes.

Currently, patients are dependent on insulin injections to maintain normoglycemia. Insulin is a polypeptide hormone consisting of two disulfide-linked chains, an A chain consisting of 21 amino acid residues and a B chain of 30 residues.

While administration of insulin provides significant benefits to patients suffering from diabetes, the short serum half-life of insulin creates difficulties for maintaining proper dosage. The use of insulin also can result in a variety of hypoglycemic side-effects and the generation of neutralizing antibodies.

In view of the problems associated with existing treatments of diabetes there is a compelling need for improved treatments that are more effective and are not associated with such disadvantages. The present invention exploits the use of peptide analogues which antagonize a T cell response to insulin to effectively treat diabetes, while further providing other related advantages.

SUMMARY OF THE INVENTION The present invention provides compounds and methods for treating and preventing diabetes. Within certain aspects, the present invention provides peptide analogues comprising residues 9 to 23 of human insulin B chain (SEQ ID NO:2) wherein the peptide analogue differs in sequence from native human insulin B chain to residues 9 to 23 due to substitutions at between I and 4 amino acid positions. Such substitutions may be made at one or more residues selected from the group consisting of residues 12, 13, 15 and 16, with or without additional substitutions at other residues.

Within certain preferred embodiments, such substitutions may occur at two or three amino acid residues within residues 9 to 23 of insulin B chain. Substitutions may also occur at residue 19. Substitutions are preferably non-conservative, and analogues wherein residue 12, 13, 15, 16 and/or 19 are altered (to, for example, alanine) are preferred. Analogues further comprising residue 24 of insulin B chain are also preferred. In certain other embodiments, the peptide analogues comprise no more than 18 residues, no more than 16 residues or no more than 15 residues of human insulin

B

20 chain.

Within further embodiments, the peptide analogues consist essentially of residues 9 to 23 or 9 to 24 of human insulin B chain (SEQ ID NO:2), wherein the peptide analogue differs in sequence from native human insulin B chain residues 9 to 23 due to substitutions at between I and 4 amino acid positions, and wherein at least one substitution occurs at a residue selected from the group consisting of residues 12, 13, 15 and 16.

Within further aspects, pharmaceutical compositions are provided, comprising a peptide analogue as described above in combination with a physiologically acceptable carrier or diluent.

The present invention further provides methods for treating and/or inhibiting the development of diabetes, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition as described above.

These and other aspects of the invention will become evident upon reference to the following detailed description and attached drawings. In addition.

various references are set forth below which describe in more detail certain procedures or compositions. These references are incorporated herein by reference in their entirety as if each were individually noted for incorporation.

to BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the amino acid sequence of residues 9-23 of insulin B chain (SEQ ID NO:2).

Figure 2 is a graph showing the proliferative response (measured in cpm) of a NOD mouse T cell clone to a native insulin B chain (9-23) peptide in the presence 1 of varying amounts of representative peptide analogues, in which different residues are substituted with alanine, as indicated.

Figure 3 is a graph showing the proliferative response (measured in cpm) S00 of a NOD mouse T cell clone to a native insulin B chain (9-23) peptide in the presence 0*.i of varying amounts of the representative peptide analogue in which amino acids at 20 positions 16 and 19 are substituted with alanine (NBI-6024; indicated by squares). For comparison the proliferative response in the presence of an unrelated control peptide derived from myelin basic protein (NBI-5096; indicated by circles) is also shown. The response is shown as mean CPM SEM of triplicate cultures.

Figures 4-6 are histograms illustrating the proliferative response (measured in cpm) of T cell lines from different diabetic patients to the native B chain (9-23) peptide or to representative peptide analogues. Peripheral blood mononuclear cells were isolated from diabetic patients and cultured in the presence of insulin B chain (9-23) peptide. After three rounds ofrestimulation with insulin B chain lxl0' T-cells and 7 x 104 irradiated autologous PBMCs were added to each well in a round bottom 96-well plate in complete medium. Cells were cultured for 5 days with NBI- 6024 (insulin B chain 9-23 with alanine substitutions at positions 16 and 19), insulin

B

chain (9-23) or medium only. On day 4. the cells were pulsed with 1H-thymidine and re-cultured for an additional 18 hours. The cultures were then harvested, counted using liquid scintillation, and the data was expressed as the mean counts per minute (cprn) of replicate samples standard error of the mean (sem).

Figure 7 is a histogram illustrating the proliferative response (measured in cpm) of a T cell line from a diabetic patient to the native B chain (9-23) peptide orto representative peptide analogues containing alanine substitutions as indicated Peripheral blood mononuclear cells were isolated from diabetic patients and cultured in to the presence of insulin B chain (9-23) peptide. After three rounds of restimulation with insulin B chain IxlO0 T-cells and 7 x 104 irradiated autologous PBMCs were added to each well in a round bottom 9 6-well plate in complete medium. Cells were cultured for 5 days with analogue, insulin B chain (9-23) or medium only (BKG), as indicated. On day 4, the cells were pulsed with H-thymidine and re-cultured for an .additional 18 hours. The cultures were then harvested, counted using liquid scintillation, and the data was expressed as the mean counts per minute (cpm) of replicate samples standard error of the mean (sem).

Figure 8 is a graph showing the percent of female NOD mice that were .i diabetic following nine weekly treatments with representative peptide analogues. Ten 20 mice each were treated subcutaneously beginning on day 24 with peptide analogues of the B chain (9-23) containing alanine substitutions at residue 12 (open triangles), 13 (squares) or 16 (solid triangles). All of the mice treated with a control peptide, neurotensin (circles), became diabetic.

Figure 9 is a graph showing the same data as in Figure 8, but contrasting only the A13 analogue-treated group with the control peptide (neurotensin)-treated group.

Figure 10 is a graph showing the percent of NOD mice that were diabetic following 13 weekly treatments with a representative peptide analogue. Ten mice were treated subcutaneously beginning on day 24 with 400 pg of neurotensin (squares),

B

chain (9-23) {diamonds} or a peptide analogue of the B chain (9-23) containing an alanine substitution at residue 13 (triangles).

Figure 11 is a graph showing the effect of representative peptide analogues on the incidence of diabetes in NOD mice. Four week old female NOD mice were treated subcutaneously with 20 mg/kg of NBI-6024 (A 6 9) at weekly intervals for 12 weeks, followed by every other week until 35 weeks of age. The control group (n=10) consisted of untreated animals. Mice with blood glucose greater than 200 mg/dL at two consecutive time points were considered to be diabetic. The data are expressed as the percent of non-diabetic over the 3 5-week study. The log-rank 10 test was used to assess whether the results of the two treatment groups were significantly different. The percent of NOD mice that were diabetic following treatments with NBI-6024

(A

6 9 is indicated at the various time points by squares, and the percent of mice that were diabetic in the control group is indicated by circles.

Figure 12 is a graph showing the effect of representative peptide analogues on the incidence of diabetes in NOD mice. Four week old female NOD mice (n=13-15) were treated subcutaneously with 20 mg/kg of NBI-6024

(A'

6 -19) or NBI- 6201 (a control peptide, neurotensin) at weekly intervals for 12 weeks, followed by every other week until 35 weeks of age. An additional control group consisted of untreated animals. Mice with blood glucose greater than 200 rg/dL at two consecutive 20 time points were considered to be diabetic. The data are expressed as the percent of non-diabetic over the 35-week study. The log-rank test was used to assess whether the results of the two treatment groups were significantly different. The percent of NOD mice that were diabetic following treatments with NBI-6024

(A

6 9 is indicated at the various time points by squares, the percent that were diabetic following treatment with the neurotensin peptide is shown by triangles, and the percent of untreated mice that were diabetic is indicated by circles.

Figures 13A-13D are graphs illustrating the immunogenicity of representative peptide analogues containing B chain residues 9-23 with an alanine substitution at residue 12 (Figure 13A), residue 13 (Figure 13B), residue 15 (Figure 13C) or residue 16 (Figure 13D). NOD mice were injected 2-4 times subcutaneously with the peptide analogue in soluble form before assaying the proliferative response of lymph node cells to varying concentrations of either the peptide analogue or native insulin B chain (9-23) peptide as indicated. Proliferative response was assessed by determining the amount of radioactive thymidine incorporated in the cells (plotted as mean counts per minute (CPM) of triplicate culture wells) by counting in a liquid scintillation counter following completion of the culture period.

Figures 14A-14F are graphs showing the immunogenicity of six different peptide analogues in the NOD mice. Peptide analogues with two alanine substitutions (A12.13; A12,15; A12,16; Al3,15; A13,16 and A15,16, as indicated) were injected in l0 NOD mice and after 10 days their lymph node cells were used in a proliferation assay using different concentrations of the immunizing peptide as stimulators. Proliferative response was assessed by determining the amount of radioactive thymidine incorporated in the cells (plotted as mean counts per minute (CPM) of triplicate culture wells) by counting in a liquid scintillation counter following completion of the culture period.

Figures 15A-15D are graphs illustrating the immunogenicity of representative double substituted peptide analogs of insulin B chain The following peptides were tested for their ability to elicit an immune response in NOD mice: A12,19 (Figure 15A); A13,19 (Figure 15B); A15,19 (Figure 15C); A16,19 (Figure 15D). Proliferative response as counts per minute of the draining lymph node 20 cells is shown in response to the immunizing analogue and also to the native insulin B chain (9-23) peptide.

Figure 16 is a graph showing the ability of a series of triple substituted peptides to evoke a T cell proliferative response in NOD mice. Mice were immunized separately with representative peptide analogues containing the following combinations of substitutions: A12,13,19; A12,15,19; A12,16,19; A13,15,19; or A13,16,19; A15.16,19. Lymph node cells were then used in a proliferation assay, and the response to each of the immunizing peptides at different concentrations is shown.

Figures 17A and 17B are graphs showing the ability of a double substituted peptide to evoke an immune response. Five female NOD mice were immunized subcutaneously with 20 mg/kg of soluble NBI-6024 on days 1. 6 and 12 On day 15, the mice were sacrificed, the inguinal lymph node cells removed and cultured in the presence of varying concentrations (0-50 pM) of either NBI-6024 (Figure 1 7A) or insulin B chain (9-23) peptide (Figure 17B). The extent of T-cell proliferation was determined using 'H-thymidine incorporation. The response is expressed as mean CPM SEM of triplicate cultures.

Figure 18 is a histogram showing a comparison of the cytokines produced by immune cells induced by (NBI-6024) peptide in the presence or absence of adjuvant. Groups of NOD mice were immunized with the NBI-6024 alone or emulsified with CFA. The cytokines IL-2 and IL-4, as indicated, were measured at 25 pM of NBI-6024 and expressed as pg/mL after subtracting the background values.

DETAILED DESCRIPTION OF THE INVENTION Prior to setting forth the present invention, it may be helpful to an understanding thereof and to provide definitions of certain terms that are used herein.

"Insulin B chain" refers to a 30 amino acid polypeptide present as one of the two disulfide-linked polypeptides that make up insulin. The sequence of human insulin B chain is provided in SEQ ID NO:I, and the sequence of residues 9-23 of human B chain is provided in Figure 1 and SEQ ID NO:2.

"Peptide analogues" of the insulin B chain comprise at least 15 amino acid residues derived from residues 9-23 of human insulin B chain (SEQ ID NO:2), with at least one difference in amino acid sequence between the analogue and the native B chain. Within a peptide analogue, at least one difference in amino acid sequence occurs at residue 12, 13, 15 and/or 16. In addition, residue 19 may be substituted, and other alterations are possible. Preferably, a peptide analogue contains between I and 4 substitutions within residues 9-23, relative to a native insulin B chain (9-23) sequence, although a greater number of substitutions 5 or 6) may be possible. Additional residues derived from insulin B chain may be included, up to the full 30 residues of native B chain, preferably up to a total of 25 residues, more preferably up to a total of 1 6 or 18 residues of the peptide analogue. Within a preferred embodiment, residue 24 of insulin B chain is also included in the peptide analogue. Sequences that are not 8 derived from insulin B chain may, but need not, be present at the amino and/or carboxy terminus of the peptide analogue. Such sequence(s) may be used, for example, to facilitate synthesis, purification or solubilization of the peptide analogue.

Unless otherwise indicated, a named amino acid refers to the L-form.

An L-amino acid residue within the native peptide sequence may be altered to any one of the 20 L-amino acids commonly found in proteins, any one of the corresponding D-amino acids, rare amino acids, such as 4 -hydroxyproline or hydroxylysine, or a nonprotein arnino acid, such as l-alanine or homoserine. Also included with the scope of the present invention are analogues comprising amino acids that have been altered by 10. chemical means such as methylation a-methylvaline); amidation of the C-terminal amino acid by an alkylamine such as ethylamine, ethanolamine or ethylene diamine; and/or acylation or methylation of an amino acid side chain function acylation of the epsilon amino group of lysine).

"Residue 12," "residue 13," "residue 15," "residue 16" and "residue 19" (also called position 12, position 13, position 15, position 16 and position 19, respectively) refer to amino acids 12, 13, 15, 16 and 19 of insulin B chain as displayed in Figure 1. More specifically, the numbering system for these residues relates to the amino acid position within the native human protein, regardless of the length of the peptide analogue or the amino acid position within the analogue. Peptide analogues 20 having an alanine substitution at residues 12, 13, 15 or 16 are referred to as the A12, A13, A15 or A16 analogues, respectively.

Peptide Analogues of Insulin B Chain As noted above, the present invention provides peptide analogues comprising at least residues 9-23 of human insulin B chain and including an alteration of the naturally occurring L-valine at position 12, L-glutamate at position 13, L-leucine at position 15 and/or L-tyrosine at position 16, to another amino acid. In one embodiment, peptide analogues contain additional alterations of one to three L-amino acids at positions 12, 13, 15, 16 and/or 19 of insulin B chain. Preferably, the peptide analogues contain two or three alterations in which one of the substituted residues is at position 19.

The portion of a peptide analogue that is derived from insulin B chain is typically 15-30 residues in length, preferably 15-18 residues in length, and more preferably 15-16 residues in length. Particularly preferred peptide analogues contain amino acids derived from insulin B chain.

As noted above, peptide analogues comprising any arnino acid alteration(s) at the positions recited above are within the scope of this invention.

Preferred peptide analogues contain non-conservative substitutions alterations to 0 amino acids having differences in charge, polarity, hydrophobicity and/or bulkiness).

Particularly preferred analogues contain alterations of one or more residues to alanine.

Peptide analogues may be synthesized by standard chemistry techniques, including automated synthesis. In general, peptide analogues may be prepared by solidphase peptide synthesis methodology which involves coupling each protected amino acid residue to a resin support, preferably a 4-methyl-benzhydrylamine resin, by activation with dicyclohexylcarbodiimide to yield a peptide with a C-terminal amide.

Alternatively, a chloromethyl resin (Merrifield resin) may be used to yield a peptide with a free carboxylic acid at the C-terminus. Side-chain functional groups may be .i protected as follows: benzyl for serine and threonine; cyclohexyl for glutamic acid and 20 aspartic acid; tosyl for histidine and arginine; 2-chlorobenzyloxycarbonyl for lysine; and 2 -bromobenzyloxycarbonyl for tyrosine. Following coupling, the tbutyloxycarbonyl protecting group on the alpha amino function of the added amino acid may be removed by treatment with trifluoroacetic acid followed by neutralization with di-isopropyl ethylamine. The next protected residue is then coupled onto the free amino group, propagating the peptide chain. After the last residue has been attached, the protected peptide-resin is treated with hydrogen fluoride to cleave the peptide from the resin and deprotect the side chain functional groups. Crude product can be further purified by gel filtration, HPLC, partition chromatography or ion-exchange chromatography, using well known procedures.

Peptide analogues within the present invention should not stimulate NOD mouse T cell clones specific to the native insulin B chain (9-23) peptide (SEQ ID NO:2). or should stimulate such clones at a level that is lower than the level stimulated by the native peptide; should not stimulate insulin B chain (9-23) specific human

T

cells from patients; should be immunogenic in the NOD mouse; should reduce the incidence of diabetes in NOD mice and may inhibit a response of T cell clones specific to the native insulin B chain (9-23) peptide (SEQ ID NO:2). Thus, candidate peptide analogues may be screened for their ability to treat diabetes by assays measuring T cell proliferation, immunogenicity in NOD mice and the effect on the 0o incidence of the disease in NOD mice. Certain representative assays for use in i evaluating candidate peptide analogues are discussed in greater detail below. Those analogs that satisfy the above criteria are useful therapeutics.

Candidate peptide analogues may initially be tested for the ability to stimulate T cells specific to the native insulin B chain (9-23) peptide (SEQ ID NO:2) (from clonal cell lines or isolated from patients). Such tests may be performed using a .i direct proliferation assay in which native B chain (9-23) reactive T cell lines or T cells isolated from patients are used as target cells. T cell lines may generally be established, using well known techniques, from lymph nodes taken from rats injected with B chain Lymph node cells may be isolated and cultured for 5 to 8 days with B chain (9- 20 23) and IL-2. Viable cells are recovered and a second round of stimulation may be performed with B chain (9-23) and irradiated splenocytes as a source of growth factors.

After 5 to 6 passages in this manner, the proliferative potential of each cell line is determined. To perform a proliferation assay, B chain 9 2 3 )-reactive T cell lines may be cultured for three days with various concentrations of peptide analogues and irradiated, autologous splenocytes. After three days, 0.5-1.0 pCi of 3 H]-thymidine is added for 12-16 hours. Cultures are then harvested and incorporated counts determined.

Mean CPM and standard error of the mean are calculated from triplicate cultures.

Peptide analogues yielding results that are less than three standard deviations of the mean response with a comparable concentration of B chain (9-23) are considered non- I I stimulatory. Peptide analogues which do not stimulate proliferation at concentrations of less than or equal to 20-50 pM are suitable for further screenings.

Candidate peptides that do not stimulate B chain (9-23) specific T cells, and preferably inhibit a response of such T cells in vitro, are further tested for their immunogenicity in the NOD mouse. Briefly, groups of NOD mice may be immunized with 100- 4 00pg of the candidate peptides subcutaneously in mannitol acetate buffer three times within a period of 10-15 days. Following the last immunization, lymph node cells and/or spleen cells may be used in a proliferation assay in which different concentrations of the Immunizing peptide are cultured with the cells for 3-4 days. The 0 last 18 hours of culture may be performed with tritiated thymidine. Cells may then be harvested and counted in a scintillation counter, and the proliferative response may be expressed as CPM SEM. Candidate peptides that induce a proliferation that is at least 2-fold higher than the background (no antigen) at 25pM of the peptide are considered to be immunogenic. Alternatively, the candidate peptide analogue is considered immunogenic if it elicits a proliferative response following immunization of S. the NOD mice in complete Freund's adjuvant. The draining lymph node cells or spleen cells, when cultured in the presence of the immunizing analogue, should induce a proliferation that is at least 2-fold higher than the background (no antigen) at 2 5pM of the peptide.

20 Candidate peptides that can inhibit proliferation by B chain (9-23) are further tested for the ability to reduce the incidence of diabetes in NOD mice. Briefly, peptides may be administered to NOD mice in soluble form or emulsified with, for example, incomplete Freund's adjuvant (IFA). Typically, weekly administration of about 400 pg of peptide is sufficient. The incidence of diabetes in the treated mice, as well as in untreated or control mice, is then evaluated by weekly monitoring of blood glucose levels. A value of 200 mg/dl or more of blood glucose on two consecutive occasions is generally considered indicative of the appearance of diabetes. Peptide analogues should result in a statistically significant decrease in the percent of NOD mice afflicted with diabetes within a monitoring period of up to about 25 weeks.

12 As noted above, peptide analogues may also inhibit the response of B chain (9-23) specific human T cells in vilro. Such inhibition may be measured by a competition assay in which candidate peptide analogues are tested for the ability to inhibit T cell proliferation induced by native B chain (9-23) (SEQ ID NO:2). In such an assay, antigen presenting cells are first irradiated and then incubated with the competing peptide analogue and the native B chain (9-23) peptide. T cells are then added to the culture. Various concentrations of candidate peptide analogues are included in cultures which may be incubated for a total of 4 days. Following the incubation period, each culture is pulsed with, for example, 1 pCi of 3 H]-thymidine for an additional 12-18 10 hours. Cultures may then be harvested on fiberglass filters and counted as above.

Mean CPM and standard error of the mean can be calculated from data determined in triplicate cultures. Peptide analogues that reduce proliferation by at least 25% at a concentration 20-50 pM are preferred.

0 1 5 Treatment and Prevention of Diabetes As noted above, the present invention provides methods for treating and preventing Type I diabetes by administering to the patient a therapeutically effective amount of a peptide analogue of insulin B chain as described herein. Diabetic patients suitable for such treatment may be identified by criteria accepted in the art for 20 establishing a diagnosis of clinically definite diabetes. Such criteria may include, but are not limited to, low (less than tenth or first percentile of controls) first phase insulin secretion following an intravenous glucose tolerance test (IVGTT) or the persistence of high titer antibodies to islet antigens such as insulin, GAD65 and/or ICA512.

Patients without clinically definite diabetes who may benefit from prophylactic treatment may generally be identified by any predictive criteria that are accepted in the art. Patients who are not frankly diabetic may be predicted to develop diabetes in the coming years (I-5yrs) based upon the following criteria: i) family history first degree relatives are automatically in the high risk group unless they have a protective HLA allele; ii) genetic make-up the presence or absence of an HLA allele that is associated with a high risk of diabetes DR3/4; DQ8); iii) presence or 13 absence of high titer autoantibodies in their blood to any or all of the antigens: insulin and/or ICA 512; and iv) intravenous glucose tolerance test (IVGTT): low firstphase insulin secretion, usually defined as below the tenth or first percentile of normal controls, typically precedes the development of type I diabetes by 1-5 years. In general, several of the above criteria may be considered. For example, the chances of developing diabetes in 5 years for a first degree relative of an individual with diabetes are estimated to be: 100% for relatives with all 3 autoantibodies listed above; 44% for relatives with 2 antibodies; 15% for relatives with one antibody; and 0.5% for relatives Swith no antibodies. Among 50 first degree relatives of patients with Type I diabetes o. to followed to the onset of diabetes, 49/50 expressed one or more of the above listed autoantibodies.

Effective treatment of diabetes may be determined in several different ways. Satisfying any of the following criteria, or other criteria accepted in the art, evidences effective treatment. Criteria may include, but are not limited to, delay in developing frank hyperglycemia, lowered frequency of hyperglycemic events and/or prolongation of normal levels of C-peptide in the blood of the patients.

Peptide analogues of the present invention may be administered either alone, or as a pharmaceutical composition. Briefly, pharmaceutical compositions of the present invention may comprise one or more of the peptide analogues described herein 20 in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like, carbohydrates such as glucose, nannose, sucrose or dextrans, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants aluminum hydroxide) and preservatives. In addition, pharmaceutical compositions of the present invention may also contain one or more additional active ingredients, such as, for example, sustained delivery systems or other immunopotentiators.

Compositions of the present invention may be formulated for the manner of administration indicated, including for example, for oral, nasal, venous, intracranial, intraperitoneal, subcutaneous, or intramuscular administration. Within other embodiments of the invention, the cornpositions described herein may be administered as part of a sustained release implant. Within yet other embodiments, compositions of the present invention may be formulated as a lyophilizate, utilizing appropriate which provide stability as a lyophilizate and/or following rehydration.

Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient. and the type and severity of the patient's disease. Within io particularly preferred embodiments of the invention, the peptide analogue may be .:...."administered at a dosage ranging from 0. 1 to 100 mg/kg, although appropriate dosages may be determined by clinical trials. Patients may be monitored for therapeutic effectiveness by delay in progression to frank diabetes and sustained use of insulin for oO~ o ,.maintaining normoglycemia as described above.

•0 15 The following examples are offered by way of illustration and not by way o f limi tation.

sees sees so: EXAMPLE 1 Preoaration of Peptides This Example illustrates the synthesis of representative peptide analogues.

Peptides were synthesized by solid phase methodology on a peptide synthesizer (Beckman model 990). Peptides with an amidated carboxyl-terminus were prepared with a p-methylbenzhydrylamine resin (MBHA resin); for peptides with a free carboxyl-terminus, a Merrifield resin coupled with the appropriately protected amino acid was used. Both resins were obtained from Bachem Fine Chemicals (Torrance, CA). Derivatized amino acids (Bachem Fine Chemicals) used in the synthesis were of Sthe L-configuration unless specified otherwise, and the N-alpha-amino function protected exclusively with the t-butyloxycarbonyl group. Side-chain functional groups were protected as follows: benzyl for serine and threonine; cyclohexyl for glutamic acid and aspartic acid; tosyl for histidine and arginine; 2 -chlorobenzyloxycarbonyl for lysine 15 and 2 -bromobenzyloxycarbonyl for tyrosine. Coupling of the carboxyl-terminal amino acid to the MBHA resin was carried out with dicyclohexylcarbodiimide and the i subsequent amino acids were coupled with dicyclohexylcarbodiimide according to Ling et al. (Proc. Nal. Acad Sci. USA 81:4302, 1984). After the last amino acid was incorporated, the t-butyloxycarbonyl protecting group was removed and the peptide- 20 resin conjugate treated with a mixture of 14 ml hydrofluoric acid 1.4 ml anisole, and 0.28 ml methylethyl sulfide per gram of resin conjugate at -20*C for 0.5 hr and at 0°C for 0.5 hr. HF was removed in vacuum at 0°C, and the resulting peptide and resin mixture was washed twice with diethyl ether and twice with chloroform and diethyl ether alternately. The peptide was extracted five times with 2 M acetic acid, and the extract lyophilized. The lyophilized product was first purified on a column of Sephadex fine (Pharmacia-LKB, Piscataway, NJ) developed in 30% acetic acid to remove the truncated fragments and inorganic salts (Ling et al., 1984). Next peptides were further purified by CM-32 carboxymethylcellulose cation-exchange chromatography (Ling et al.. 1984). Final purification was achieved by partition chromatography on Sephadex G-25 fine (Ling et al., 1984). Alternatively, the crude peptide could be 16 purified by preparative HPLC on a Biotage KP-100 gradient HPLC system. The synthetic product was characterized by amino acid analysis, mass spectrometric analysis and reversed-phase

HPLC.

EXAMPLE 2 Lone-teml Tcell lines This Example illustrates the preparation of long-term insulin-specific NOD T cell lines.

Insulin specific NOD T cell lines were established by culturing lymphocytes isolated from islet-infiltrating populations by in vitro stimulation with either porcine insulin at 25 pg/ml and irradiated NOD islet cells in the presence of irradiated NOD spleen cells as antigen presenting cells and cytokines. To obtain the infiltrating lymphocytes the following procedures were performed (see Wegmann et al., Eur. J Immunol. 24:1853, 1994): the pancreas from the NOD mouse was digested with S. 15 collagenase and individual islets were isolated manually. The infiltrating lymphocytes were then obtained by mild trypsin digestion of the islets. The insulin specific T cell lines or clones were propagated by serial stimulation in the presence of NOD spleen cells, porcine insulin and lymphokines. Clones were obtained by limiting dilution of the B chain (9-23) specific T cell lines in the presence of the antigen presenting cells 20 and porcine insulin at 25 pg/ml. Wells with a growing population of cells following limiting dilution were expanded in appropriate medium, and after one cycle of growth were tested for reactivity to the B chain (9-23) peptide of insulin by evaluating the proliferative response.

EXAMPLE 3 Effect of Peptide Analoues on Proliferation of nsulin-secifi NOD T cell Clones This Example illustrates the effect of representative peptide analogues on T cell proliferation.

Insulin B chain (9-23) (SEQ ID NO:2) specific mouse (NOD) T cell clones were isolated from infiltrated islets as described in Example 2. Peptide 17 analogues with single alanine substitutions were prepared as described in Example 1.

The effect of each analogue on T cell proliferation was then evaluated using an assay performed in 96-well flat bottom microtiter plates (see Daniel et al.. Etr. J Immurrol :1056, 1995). Briefly 25,000 T cell clones along with 1 million irradiated

NOD

spleen cells were cultured in the presence of 50 pg/ml of insulin B chain 9-23 peptide or any of the alanine substituted peptides listed below in triplicate sets. The plates were incubated for a total of 72 hours in 7% carbon dioxide atmosphere with a pulse of IpCi/well of tritiated thymidine for the last 6-8 hours of culture. Cells were harvested on a glass fiber filter and the associated radioactivity was counted in a liquid 10 scintillation counter. Results are expressed as mean counts per minute of triplicate wells.

The data obtained from five separate T cell clones showed either a lack of proliferation or significantly reduced proliferation (relative to the 9-23 native peptide of insulin B chain; SEQ ID NO:2) in the presence of the following alanine substituted I 5 analogues: A12, A13, AlS, A16, A17, and AI8. These data are presented in Tables 1 and 2, below.

S**

*o.

00 o 18 Table I Resvonse LMIL)ofQ~U92jlnSecific NOD T Cell Clones T cell clone Modified Native Position Residue Substitution PD6-4.3- PD12-240 9 S A 12861 42234 H A 12507 1409 11L A 14148 2594 12 V A 8292 671 18E A 12 19 C An67n321 20 L A 244 3868542 216 A 91 398 22 R A 6555 517322 23 G A 1404 78544 no antigen 163-682 Native 9-23 106 322721 Table2 Response (cpm) of Insulin Specific MurineO T Crrell Clones 0 0 .00.

00 0* *0 1.: Modified Native T Cell Clone Position Residue Substitution PDI2-4.4 PD 12-4.29 PD 12-4.34 9 S A 1000 18422 259 H A 823 15484 356 12 L A 4 4184 16 190 12 V 1129 15041 194 13 E A 373 891 179 14 A none L A 675 809 191 16 Y A 779 636 202 17 L A 332 1460 4360 18 V A 225 1193 721 19 C A 4295 6054 689 20 G A 1323 13736 466 21 E A 7900 4904 773 22R A 13312635 1555 23 G A3228 1842279 no antigen 357891 Naiv 92310000 14820 3614 Table 3 shows the response of four different NOD derived T cell clones to the double alanine substituted peptide analog A16, A] 9 (NBI-6024; 16Y>AII 9C>A).

NOD T cell clones were incubated in the presence of 50 jiM of either the native 3 chain (9-23) peptide or NBI-6024. The data in Table 3 represent the eno rpiaesml standard error of the mean. Within Table 3, S.1. (Stimulation Index) =proliferaion (cpm) in the presence of the peptide/proliferation (cpm) in mediurn alone. These data show a significant response when the cells were cultured with the native B chain (9-23) peptide, but little or no Proliferation over medium only (background) in the presence of NBI-6024.

Table 3 PrliertieReslonse of Insulin B Chain -11)SecfcMrieTcl Clones to 50"M of B Chain (9-23) or the Analocue A16- 9 imBJ-6024) T Cell Exp.

Clone No.

9**t

V.

V.

V.

V

V

V.

V

V Medium Only PD 12-2.35_ PD 12-2.40

PDI-.

PD 12-4.9 2 2

I

2 688£ 227 493 ±20 170 8 2152,7771 Insulin BCain (9-23)) jMean cpm~sem _S.1 120,886 7,171 175.7 100,521 i1,581 203.899 1 6,730£± 3,835 9.

176,3591 .36,306 96.16 28,593£ 66 132.99 45,541 5,222 4.99 65,624£ 4,979 9.1 NBI-6024

(A'

6 19) Mean cPml+sem

S.I.

841 *88 1.22 452*179 0.91 27 2+ 3 4 1.16 1,86-":±451 1.01 566 ±-3o 2.63 12.313£+ 1,372 1.35 6,171 *725 08 I0 EXAMPLE 4 Antap-onisrn of T cell Proliferation Assay This Example illustrates the inhibition of the response of B chain (9-23) specific mouse T cell clones to the insulin B chain (9-23) peptide by representative peptide analogues.

Peptide analogues of B chain (9-23) containing alanine substitutions at residue 12, 13., 15 or 16 or the doubly substituted peptide at positions 16 and 19 9 NBI-6024) were prepared as described ira Example 1. T cell antagonisnm was detected by evaluating the ability of the peptide analogues to inhibit T cell proliferation induced by native B chain (9-23) (SEQ ID NO:2). In this assay, antigen presenting cells were first irradiated and then incubated with the competing peptidle analogue and the native B c-ain peptide. T cells were then added to the culture. Various concentrations of candidate peptidle analogues were included in cultures which were incubated for a total of 4 days. Following this incubation period. each culture was pulsed with I pij of 3 H-]-thymidine for an additional 12-18 hours. Cultures were then harvested on fiberglass filters and counted as above. Mean CPM and standard er-ror of the mean were calculated from data determined in triplicate cultures. The results, Shown in Figure 2.

indicate that the peptide analogues containing alanine substitutions at residue 12. 1 3 or 16 are capable of attenuating the response of the pathogenic insulin B chain (9-23)

T

cells.

The ability of the doubly substituted peptide to inhibit insulin-dependent :::*proliferation by T cells is shown in Table 4 and Figure 3.Within Table 4, the control 0:::10 peptide, NBI-5096, is an unrelated peptide from myelin basic protein. The percent inhibition was calculated as: (I -experimental cpmlinsulin pepuide cpm) x 100%.

Table 4 Inhibition of Insulin B Chain (9-23)PeteRsosei Two MUrine NOD T Cell Clones by a Peptide Analogue N/A Not applicable as no inhibition was observed- The ability of NBI-6024 to block the B chain (9-23) peptide-iriduced Stimulation of NOD derived T clones suggests that the alterations at positions 16 and 19 10 of the native insulin B chain (9-23) peptidedinoalethabiyofhenlgutoe recognized by the pathogenic T cells. Moreover, these results indicate that the analogue also binds to the MI-C with sufficient affinity to allow for recognition by the insulin B chain 9 -23)D-specific T cell.

EXAMPLE Effect of Peptide Analogues on Proliferation of T cell Lines and Clones from Diabetic Patients This Example illustrates the lack of stimulation ofT cell lines and clones derived from diabetic patients by representative peptide analogues.

Peptide analogues of B chain (9-23) containing alanine substitutions at residues 13, 15, 16 or 17 or the doubly substituted alanine analog (NBI-6024) were prepared as described in Example 1. T cell lines from diabetic patients were prepared by isolating lymphocytes from the blood of the patient by subjecting the blood 1to to a density gradient separation. Isolated lymphocytes were then cultured in the presence of the insulin B chain (9-23) peptide (10pM) and recombinant human IL-2 in the presence of 5-10% of autologous serum and irradiated autologous peripheral blood lymphocytes in culture medium. Four to five days later cells were harvested and the cycle repeated for 2 or 3 times.

Proliferation of the T cell line, in response to the native B chain (9-23) peptide (SEQ ID NO:2) or to the peptide analogs, was measured by culturing 25,000 to 100,000 T cells in the presence of 50,000-200,000 irradiated autologous PBLs and ;different concentrations of the insulin B chain (9-23) peptide or a peptide analogue in triplicate cultures. Following 4-5 days of culture, including the last 18 hours with radioactive thymidine, cells were harvested and the associated radioactivity was counted in a liquid scintillation counter. Results are expressed as mean counts per minute for each of the peptide analogues tested.

The results, shown in Figures 4-7, indicate that T cell lines and clones that proliferate in response to the native insulin B chain (9-23) peptide (SEQ ID NO:2) are not stimulated by the peptide analogues. The results from these patients and others are summarized in Table 24 Table Proliferative Response of Patient PBLs to Native Insulin Peptide or the Analogue NBI-6024 Stimulation Index' Insulin B (9-23) NBI-6024 Patient Number Patient ID [50 pM] [50 pM] I 100 9.9 0.9 2 200 5.3 12 I- 3 400 7.8 4 500 2.1 0.9 5 600 5.8 1.6 6 700 3.2 7 900 2.6 0.9 8 1100 3.7 0.8 $Stimulation Index CPM with antigen/CPM with medium alone (no antigen) The results clearly demonstrate that cells from diabetic patients that are responsive to the insulin B chain (9-23) peptide do not respond to the altered peptide ligand, NBI-6024 which has substitutions at position 16 and 19. We have also determined that the APL NBI-6024 binds with similar affinity to DQ8 antigens. Thus, the absence of stimulation of the diabetic patient's T cells by NBI-6024 is not due to any incompatibility of the peptide with the presenting MHC molecules, but is more likely due to altered recognition by the B chain 9 2 3)-specific T cells.

EXAMPLE 6 Reduction of Incidence of Diabetes in NOD Mice This Example illustrates the ability of representative peptide analogs to prevent diabetes in NOD mice.

The NOD mouse spontaneously develops diabetes beginning around 3 months of age (Makino el al., in Current Topics in Clinical and Experimental Aspects

I

of Diabetes Mellitus, Sakamoto et al.. eds., p. 25-32 (Elsevier, Amsterdam, 1985)). The disease is preceded by cellular infiltration into the pancreas of T cells beginning even by one month of age. Soluble peptide analogues of B chain (9-23) containing alanine substitutions at residues 12, 13 or 16 were administered subcutaneously to NOD mice at weekly intervals. 400 p.g of each peptide were administered at each treatment to ten animals. Following 9 treatments, the percent of mice in each treatment group that had become diabetic was evaluated by measuring blood glucose levels using a glucometer at weekly intervals. A reading of more than 200mg/dl of blood glucose on two consecutive observations was considered indicative of frank diabetes.

As shown in Figure 8, treatment with each of the alanine-substituted analogues resulted in a marked reduction in the incidence of diabetes. The data for the A] 3 substituted analogue is also shown in Figure 9.

In another experiment, B chain the A13 substituted analogue or neurotensin (as a control) was administered subcutaneously to NOD mice at weekly intervals. 400 pg of each peptide were administered at each treatment to ten animals.

o* Following 13 treatments, the percent of mice in each treatment group that had become diabetic was evaluated as described above. As shown in Figure 10, the B chain (9-23) peptide reduced the incidence of diabetes. This reduction was more pronounced for the A13 substituted analogue.

To determine the ability of the double substituted peptide A 6 9

(NBI-

6024) to control the development of diabetes in the NOD mice, the peptide was administered to animals at an early age. Thus, female mice approximately 4 weeks old) were treated subcutaneously with 20mg/kg (400 pg/mouse) of NBI-6024 for twelve weeks and then every other week until Week 35. Beginning at 9-10 weeks of age, mice were then monitored weekly for hyperglycemia, measuring the blood glucose levels. As a control, a group of 10 female mice was left untreated. The results from this experiment are shown in Figure 11. As can be seen, treatment with NBI-6024 significantly reduced the incidence of diabetes by about 60-70%, compared to the untreated group, (p <0.004).

The observations were then confirmed and extended in a second experiment. Here, animals (n=13-1 5) were treated with either NBI-6024 or an unrelated peptide, neurotensin, NBI-6201 as described above. An additional group was left untreated. As shown in Figure 12, treatment with 20mg/kg of the altered peptide NBI- 6024 resulted in a reduced incidence of diabetes compared to either the neurotensin treated or untreated group.

These results demonstrate that the altered peptide ligand NBI-6024, designed around insulin B chain (9-23) peptide was capable of conferring protection to animals at risk to develop diabetes spontaneously. It is likely that T cells that recogni2e 10 other pancreas antigens are present in these animals, yet they too appear to be regulated by the insulin APL. The timing of administration was approximately at the same time that autoreactive lymphocytes begin to infiltrate the pancreas and initiate the destructive :i process. These results offer hope that early intervention with this APL may prove useful in delaying or preventing the onset of Type I diabetes in people.

EXAMPLE 7 Immunoenicity of Representative Peptide Analogues This Example illustrates the immunogenicity of representative peptide analogues in NOD mice.

Groups of 3-4 NOD mice were immunized with 100- 4 00pg of peptide Sanalogues subcutaneously in mannitol acetate buffer three times within a period of days. Following the last immunization, lymph node cells and/or spleen cells were used in a proliferation assay in which different concentrations of the immunizing peptide were cultured with the cells for 3-4 days. The last 18 hours of culture included tritiated thymidine. Cells were harvested and counted in a scintillation counter and the response is expressed as CPM SEM. These results, shown in Figure 13-16, indicate that these representative peptide analogues have the ability to bind to the mouse MHC molecules and be recognized by the corresponding T cells.

The ability of the double substituted peptide NBI-6024 1 to induce a cellular immune response in NOD mouse strain was next determined. Two female NOD mice were immunized with 10mg/kg NBI-6024 either as an aqueous suspension or, as a control, emulsified in complete Freund's adjuvant (CFA). On Day 8, three days following the last injection, the mice were sacrificed, the spleen and inguinal lymph node cells were removed and pooled, and a single-cell suspension was prepared. Cells were cultured in the presence of varying concentrations (0-25 M) of NBI-6024. The ability of these lymphoid cells to proliferate in response to NBI-6024 was measured in vitro by 3 H]-thymidine incorporation.

10 to The results are presented in Table 6. in which the response is expressed as mean CPM SEM of triplicate cultures. Lymph node cells isolated from mice immunized with the analogue in CFA showed a strong proliferative response to i challenge with the immunizing analogue in a dose-dependent manner (Table These results indicate that alterations made in the native insulin B chain (9-23) sequence at positions 16 and 19 have not affected the ability of the peptide to bind the NOD diseaseassociated MHC haplotype molecule and, more importantly, did not hinder recognition by T cells.

Table 6 Proliferative Response of Lymph Node Cells to NBI-6024 from NOD Mice Immunized with NBI-6024 in CFA NBI-6024 Proliferative Response (CPM SEM) 0 2,445 137 1 140,061 7,289 187,71 1 2,548 218.149 4,462 In addition, both the spleen and inguinal lymph nodes cells isolated from soluble administrated peptide exhibited a strong proliferative response to the APL when 28 challenged in vitro with NBI-6024 (Table 7 and Figures 1 7A and 1 7B). Even more impressive was the finding that NBI-6024-derived lymphocytes from mice immunized with the soluble peptide also responded to Insulin B chain This cross-reactive feature was not seen with CFA-emulsified peptide. This ability of the soluble peptide to induce a cross-reactive response may be desirable in controlling diabetes, as it may help to mobilize the protective NBI-6024-speciic T cells to the pathogenic target tissue.

Table 7 Proliferative Response to NBI-6024 or Native Insul in B-chain (9-23) of T cellIs from 0 NOD Mice Immunized with Soluble NBI-6024 NBI-6024 nduced T Cell Line Peptide Conc.

NuMI Mouse 1 Mouse #2 0 25 o Mouse #3 t .1 0 CPM ±L SEM CPM SEM NBI-6024 Insulin B(9-23) 219,588 226 219,588 2326 48,5119 666 16,7,262462 1260,671232369 I,482 260.07 ±1259 30,6127 3619 24,006 ±2803 24,00612803 64,239 ±9493 25,825 :L 3841- 140,836 ±11778 58.567 2737 240,278 15015 11 3,366 ±51 i

I

To determine the type of T cells produced following, soluble administration of NBI-6024, the culture supernatants from immune lymphoid cells were removed 48 hours following the initiation of culture and the levels of various cytokines measured using standard ELISA technology. Strikingly, the cytokine production profile of T cells from mice immunized with soluble NBI-6024 produced the Th2 cytokines.

interleukin-4 (Figure 18) and interieukin-5 (Table and not the Thi-derived interleukin-2. Within Table 8, values are expressed in pg/mi as mean of triplicates SEM. As a conirol, NRI-6024 emulsified with CFA did induce the expected Thi cytokine profile (IL-2) from immune T cells upon in vitro stimulation.

Table 8 Cytokirie Response of Soluble NBI-6024 Induced T cells Cultured with NBI-6024 I ~~Cytokin(pmi NBI-602411 [rMJ J IL-2 J IL-4 0 J <15 pg/mI 134 0 1,814± 332 1 15 pg/mi 684 92 9,999+ 503 5 l5 pg/mI 1.653 ±51 23 496:kAQA 9*

C

C.

C. C C C C

C.

<l5 pg/ml 2,102+±85 28,06 41 The ability of the soluble subcutaneous administration of NBI-6024 to induce Th2-like cells is a desirable feature, as such cells are associated with recovery from diabetes and other organ-specific autoimmune diseases (Sarvetnick, Exp. Med 10 i84:1597-1600, 1996; Shaw et al-, 1997; Balasa et' al., J. Exp. Med. 186:385-391, 1997). These Th2-derived cytokines have strong anti-inflammatory activities that suppress development of pro-inflammatory cytokine-secreting auto-reactive Thi cells that mediate disease.

Is From the foregoing. it will be evident that although specific embodiments of the invention have been described herein for the purpose of illustrating the invention, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the present invention is not limited except as by the appended claims.

PAOMWDOU327714 iwrisd=ojMnm 30A The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word 'comprise", and variations such as "comprises" and "cormprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

@too see* -900 006, 606.

SEQUJENCE LISTING <110> Neurocrine Biosciences, Inc.

<120> METHODS FOR TREATMENT OF DIABETES USING PEPTIDE ANALOGUES OF INSULIN <130> 690068.448PC <140> PCT <141> 1999-02-23 c160> 2 <170> Patentln Vex. *0 0 0 0 00 0 .00.

0 0 c210> 1 <211> <212> PRT c213> Homo sapiens <220> c223> Human Insulin B Chain <400> 1 Phe Val Asn Gin His 1 5 Leti Cys Gly Ser His Leu Val Glu Ala Leu Tyr 10 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 20 25 <210> 2 <211> <212> PRT <213> Homo sapiens <220> <223> Residues 9-23 <400> 2 Ser His Leu Val Glu 1 5 of human insulin B chain Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly 10 L:\690068 Neurocrine\448PC-ap.doc

Claims (24)

1. A peptide analogue comprising residues 9 to 23 of human insulin B chain, which peptide analogue is not coupled to a full length, unaltered A chain, wherein the peptide analogue differs in sequence from native human insulin B chain residues 9 to 23 due to substitutions at between 2 and 4 amino acid positions, and wherein at least one substitution occurs at a residue selected from the group consisting of residues 12, 13, 15 and 16.

2. The peptide analogue of claim 1, wherein the peptide analogue has a sequence that differs from native human insulin B chain at two amino acid residues.

3. The peptide analogue of claim 1, wherein the peptide analogue has a sequence that differs from native human insulin B chain at three amino acid residues.

4. The peptide analogue of claim 1, wherein an amino acid substitution occurs at residue 19.

5. The peptide analogue of claim I, wherein at least one amino acid substitution is non-conservative.

6. The peptide analogue of claim 1, wherein residue 12 is substituted.

7. The peptide analogue of claim 6, wherein residue 12 is an alanine residue.

8. The peptide analogue of claim 1, wherein residue 13 is substituted to an alanine residue.

9. The peptide analogue of claim 1, wherein residue 15 is substituted. The peptide analogue of claim 9, wherein residue 15 is an alanine residue. P:IOPERITDOU3Z-771 4 pifOilQdW4iMAII -32-

11. The peptide analogue of claim 1, wherein residue 16 is substituted.

12. The peptide analogue of claim 11, wherein residue 16 is an alanine residue.

13. The peptide analogue of any one of claims 6-12, wherein residue 19 is substituted.

14. The peptide analogue of claim 13, wherein residue 19 is an alanine residue.

15. The peptide analogue of claim 1, further comprising residue 24 of human insulin B chain. 0 0

16. The peptide analogue of claim 1, wherein the peptide analogue comprises no more than 18 residues of human insulin B chain.

17. The peptide analogue of claim 1, wherein the peptide analogue comprises no more than 16 residues of human insulin B chain.

18. The peptide analogue of claim 1, wherein the peptide analogue comprises no more than 15 residues of human insulin B chain.

19. A peptide analogue consisting essentially of residues 9 to 23 human insulin B hain, wherein the peptide analogue differs in sequence from native human insulin B chain residues 9 to 23 due to substitutions at between 1 and 4 amino acid positions, and wherein at least one substitution occurs at a residue selected from the group consisting of residues 12, 13, 15 and 16. A peptide analogue consisting essentially of residues 9 to 24 of human insulin B chain, wherein the peptide analogue differs in sequence from native human insulin B chain residues 9 to 23 due to substitutions at between I and 4 amino acid positions, and wherein at least one substitution occurs at a residue selected from the group consisting of residues 12, 13, 15 and 16. P'PEATmuOz274 wdii.wj -33

21. A pharmaceutical composition comprising a peptide analogue according to any one of claims 1-18 in combination with a physiologically acceptable carrier or diluent.

22. A method for inhibiting the development of diabetes, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition according to claim 21.

23. A method for treating diabetes, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition according to claim 2 1.

24. A peptide analogue comprising residues 9 to 23 of human insulin B chain, wherein the peptide analogue differs in sequence from native human insulin B chain residues 9 to 23 due to substitutions at residues 16 and 19. A pharmnaceutical composition comprising a peptide analogue according to claim 24 in combination with a physiologically acceptable carrier or diluent. .26. A method for inhibiting the development of diabetes, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition :according to claim 27?. Use of the peptide analogue of any one of claims 1-18 in the manufacture of a medicament for the inhibition of diabetes development.

28. Use of the peptide analogue of any one of claims 1-18 in the manufacture of a medicament for the treatment of diabetes. PIOP~tWOW2714 ECLFWA1IONDW~oaiwj -34-

29. Use of the peptide analogue of claim 24 in the manufacture of a medicament for the inhibition of diabetes development- DATED this 22nd day of February, 2002 Neurocrine Biosciences, Inc. by their Patent Attorneys DAVIES COLLISON CAVE