MXPA00008268A - Methods for treatment of diabetes using peptide analogues of insulin - Google Patents

Abstract

The present invention is directed toward peptide analogues of insulin (B) chain that are generally derived from peptides comprising residues (9 to 23) of the native (B) chain sequence. The analogues are altered from the native sequence at position (12, 13, 15) and/or (16), and may be additionally altered at position (19) and/or other positions. Pharmaceutical compositions containing these peptide analogues are provided. The peptide analogues are useful for treating and inhibiting the development of diabetes.

Description

METHODS FOR THE TREATMENT OF DIABETES USING INSULIN PEPTIDE ANALOGS TECHNICAL FIELD The present invention relates generally to insulin peptide analogues and more specifically to methods for the treatment of diabetes using peptide analogues derived from residues 9-23 of the B chain of human insulin.

BACKGROUND OF THE INVENTION Insulin dependent diabetes mellitus (IDDM) is an organ-specific autoimmune disease that affects nearly one million people in different age groups in the United States. The disease is characterized by the extensive destruction of beta cells that produce insulin in pancreatic islets and dysregulation of glucose metabolism that leads to natural diabetes. The defined characteristic of the IDDM is the lymphocytic infiltration of the islets. Among the invading cells, T cells seem to be one of the main mediators of autoimmune destruction. Type I diabetes is also characterized by increased levels of antibodies to several islets associated with antigens, including insulin, GAD65, GAD67 and ICA512. These antibodies can be detected well before the natural disease, and an immune response for such antigens can be used as a predictor for impending diabetes in patients with susceptible genetic haplotypes (HLA). Currently, patients depend on insulin injections to maintain normoglycemia. Insulin is a polypeptide hormone consisting of two linked disulfide chains, an A chain consisting of 21 amino acid residues and a 30 residue B chain. Although the administration of insulin provides significant benefits for patients suffering from diabetes, the short half-life of insulin serum creates difficulties in maintaining the appropriate dose. The use of insulin can also result in a variety of hypoglycemic side effects and the generation of neutralizing antibodies. In view of the problems associated with existing diabetes treatments, there is an urgent need for improved treatments that are more effective and are not associated with such disadvantages. The present invention exploits the use of peptide analogs that antagonize a T cell response to insulin to effectively treat diabetes, while also providing other related advantages.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides compounds and methods for treating and preventing diabetes. Within certain aspects, the present invention provides peptide analogs comprising residues 9 to 23 of the B chain of human insulin (SEQ ID NO: 2), wherein the peptide analogue differs in sequence from residues 9 to 23 of the B chain of native human insulin due to substitutions at amino acid positions 1 and 4. Such substitutions can be made in one or more residues selected from the group consisting of residues 12, 13, 15 and 16, with or without substitutions additional waste. Within certain preferred embodiments, such substitutions can occur in two or three amino acid residues within residues 9 to 23 of the B chain of insulin. Substitutions may also occur at residue 19. Substitutions are preferably non-conservative, and analogs are preferred wherein residues 12, 13, 15, 16 and / or 19 are altered (eg, alanine). Analogs which further comprise residue 24 of the insulin B chain are also preferred. In certain other embodiments, the peptide analogs comprise no more than 18 residues, no more than 16 residues, or no more than 15 residues of the B chain of human insulin. Within further embodiments, the peptide analogs consist essentially of residues 9 to 23 or 9 to 24 of the B chain of human insulin (SEQ ID NO: 2), wherein in peptide analogue it differs in sequence from residues 9 to 23 of the B chain of human insulin due to substitutions in amino acid positions 1 and 4, and wherein at least one substitution occurs in a residue selected from the group consisting of residues 12, 13, 15 and 16. Within the additional aspects, pharmaceutical compositions are provided, comprising a peptide analog as described above in combination with a physiologically acceptable diluent or carrier. The present invention provides other 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 be apparent from the reference to the following detailed description and accompanying drawings. In addition, several references are set forth below, which disclose in more detail certain procedures or compositions. These references are incorporated for reference in their entirety as if each will be considered individually for incorporation BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 represents the amino acid sequence of residues 9 to 23 of the 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 peptide (9-23) of the native insulin B chain in the presence of varying amounts of peptide analogues representative, in which different residues are substituted with alanine, as indicated. Figure 3 is a graph showing the proliferative response (measured in cpm) of a NOD mouse T cell clone to a peptide (9-23) of the native insulin B chain in the presence of varying amounts of the peptide analogue representative, in which amino acids at positions 16 and 19 are substituted with alanine (NBI-6024, indicated by squares). For comparison, the proliferative response is also shown in the presence of an unlinked control peptide of the myelin basic protein (NBI-5096, indicated by circles). The answer is shown as CPM + SEM average of truplicated crops. Figures 4-6 are histograms illustrating the proliferative response (measured in cpm) of the T cell lines of different diabetic patients to the peptide (9-23) of the native B chain or to representative peptide analogs. The peripheral blood mononuclear cells were isolated from diabetic patients and cultured in the presence of the peptide (9-23) of the insulin B chain. After three rounds of restimulation with the B chain of insulin (9-23), 1 x 10 5 T cells and 7x10 4 irradiated derived PBMCs were added to each well in a 96-well round-bottomed plate in complete medium. The cells were cultured for 5 days with NBI-6024 (9-23 of the B chain of insulin with alanine substitutions at positions 16 and 19), of the B chain of insulin (9-23), or only medium. On day 4, the cells were pulsed with 3H-thymidine and re-cultured for a further 18 hours. The cultures were collected, counted using a liquid scintillation, and the data were expressed as the average counts per minute (cpm) of the replicated samples + standard error of the average (sem).

Figure 7 is a histogram illustrating the proliferative response (measured in cpm) of a T-cell line from a diabetic patient to the peptide (9-23) of the native B chain or to representative peptide analogs containing alanine substitutions as indicated. Peripheral blood mononuclear cells were isolated from the diabetic patients and cultured in the presence of the peptide (9-23) of the insulin B chain. After three rounds of restimulation with (9-23) of the insulin B chain, 1 x 150 T cells and 7x10 4 irradiated derived PBMCs were added to each well in a 96-well round bottom plate in complete medium. . The cells were cultured for 5 days with (9-23) of the insulin B chain, analogously or only with medium (BKG), as indicated. On day 4, the cells were pulsed with 3H-thymidine and re-cultured for a further 18 hours. The cultures were collected, counted using liquid scintillation, and the data were expressed as the average counts per minute (cpm) of the replicated samples + standard error of the average (sem). Figure 8 is a graph showing the percent of female NOD mice that are diabetic that nine weekly treatments with representative peptide analogues. Ten mice were treated subcutaneously beginning on day 24 with the peptide analogues (9-23) of the B chain containing alanine substitutions at residue 12 (open triangles), 13 (squares) or 16 (solid triangles). All 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 in the group treated with analog A13 with the group treated with the control peptide (neurotensin). Figure 10 is a graph showing the percent of NOD mice that are diabetic following 13 weekly treatments with a representative peptide analogue. Ten mice were treated subcutaneously started on day 24 with 400 μg of neurotensin (squares), (9-23) of chain B (diamonds) or a peptide analogue of (9-23) of chain B that contains a substitution of alanine at residue 13 (triangles). Figure 11 is a graph showing the effect of representative peptide analogs on the incidence of diabetes in NOD mice. Female four-week NOD mice (n = 9) were treated subcutaneously with 20 mg / kg of NBI-6024 (A16 19) at weekly intervals for 12 weeks, followed by every other week until 35 weeks of age. The control group (n = 10) consists of untreated animals. Mice with blood glucose greater than 200 mg / dL at two consecutive time points are considered diabetic. The data are expressed as the percent of non-diabetics during the 35-week study. The log-order test was used to evaluate whether the results of the two treatment groups were significantly different. The percent of NOD mice that are diabetic following treatments with NBI-6024 (A16 19) is indicated at various points of time by squares, and the percent of mice that are diabetic in the control group is indicated by circles. Figure 12 is a graph showing the effect of representative peptide analogs on the incidence of diabetes in NOD mice. Female four-week NOD mice (n = 1 3-15) were treated subcutaneously with 20 mg / kg of NBI-6024 (A16 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 (n = 8) consists of untreated animals. Mice with blood glucose greater than 200 mg / dL at two consecutive time points are considered diabetic. The data are expressed as the percent of non-diabetics during the 35-week study. The log-order test was used to evaluate whether the results of the two treatment groups were significantly different. The percent of NOD mice that are diabetics following treatments with NBI-6024 (A16 19) is indicated at various points of time by squares, the percent who are diabetics following treatment with the neurotensin peptide is shown by triangles , and the percent of untreated mice that are diabetic is indicated by circles. Figures 13A-13D are graphs illustrating the immunogenicity of representative peptide analogs containing residues (9-23) of the B chain with a substitution of alanine at residue 1 2 (Figure 13A), residue 1 3 (Figure 13B), residue 15 (Figure 1 3C) or residue 16 (Figure 13D). NDO mice were injected 2-4 times subcutaneously with the peptide analogue in soluble form before evaluating the proliferative response of lymph node cells at varying concentrations of either the peptide analogue or the peptide (9-23) of the B chain of insulin, as indicated. The proliferative response was evaluated by determining the amount of radioactive thymidine incorporated in the cells (plotted as average counts per minute (CPM) of triplicated cell cavities) by counting in a liquid scintillation counter after the termination of the culture period. Figures 14A-14F are graphs showing the immunogenicity of six different peptide analogs in NOD mice. Peptide analogs with two alanine substitutions (A12.1 3; A12.15; A12.16; A13.1 5; A13.16 and A15.16, as indicated) were injected into 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. The proliferative response was assessed by determining the amount of radioactive thymidine incorporated in the cells (plotted as average counts per minute (CPM) of triplicate culture cavities) by counting in a liquid scintillation counter after the termination of the culture period. Figures 15A-15D are graphs illustrating the immunogenicity of double substituted peptide analogs representative of the B chain of insulin (9-23). The following peptides were tested for their ability to elicit an immune response in NOD: A12.19 mice (Figure 15A); A13.19 (Figure 1 5B); A15.19 (Figure 15C); A16.19 (Figure 15D). The proliferative response as counts per minute of draining lymph node cells is shown in response to the immunizing analogue and also to the peptide (9-23) of the insulin B chain. Figure 16 is a graph showing the ability of a series of triple substituted peptides to evoke a proliferative response of the T cell in NOD mice. Mice were separately immunized 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. The lymph node cells were then used in a proliferation assay, and the response to each of the immunizing peptides in different concentrations is shown. Figures 17A and 17B are graphs showing the ability of a double substituted peptide (A16, 19) 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 were removed and cultured in the presence of varying concentrations (0-50 μM) of either NBI-6024 (Figure 17A) or peptide (9-23) of the B chain of insulin (Figure 17B). The degree of proliferation of the T cell was determined using an incorporation of 3 H-thymidine. The response is expressed as CPM + _ SEM average of triplicate cultures.

Figure 18 is a histogram showing a comparison of the cytokines produced by immune cells induced by peptide A16, 19 (NBI-6024) in the presence or absence of adjuvant. Groups of NOD mice are immunized with NBI-6024 alone or emulsified with CFA. The cytokines IL-2 and IL-4, as indicated, are measured in 25 μM of NBI-6024 and expressed as pg / mL after subtracting background values.

DETAILED DESCRIPTION OF THE INVENTION Before establishing the present invention, it may be useful for an understanding thereof and for providing definitions of certain terms used therein. "Insulin chain B" refers to a 30 amino acid polypeptide present as one of the two bisulfide-defined polypeptides that produce insulin. The sequence of the human insulin B chain is provided in SEQ ID NO: 1, and the sequence of residues 9-23 of the human B chain is provided in Figure 1 and SEQ ID NO: 2. "Peptide Analogs" of the insulin B chain comprise at least 15 amino acid residues derived from residues 9-23 of the B chain of human insulin (SEQ ID NO: 2), with at least one difference in the amino acid sequence between the analog and the chain Native B Within a peptide analogue, at least one difference in amino acid sequence occurs at residues 12, 13, 15 and / or 16. In addition, residue 19 can be substituted, and other alterations are possible. Preferably, a peptide analogue contains between 1 and 4 substitutions with residues 9-23, relative to the sequence (9-23) of the B chain of native insulin, although a greater number of substitutions may be possible (e.g. 5 or 6). Additional residues derived from the insulin B chain may be included, up to the total of 30 residues of the native B chain, preferably up to a total of 25 residues, more preferably up to a total of 16 or 18 residues of the peptide analogue. Within a preferred embodiment, residue 24 of the insulin B chain is also included in the peptide analogue. Sequences that are not derived from the insulin B chain may, but need not, be present at the amino and / or carboxy terminus of the peptide analogue. Such sequence (s) can be used, for example, to facilitate the synthesis, purification or solubilization of the peptide analogue. Unless otherwise indicated, a named amino acid refers to the form L. An L-amino acid residue within the native peptide sequence can be altered to any of the L-20 amino acids commonly found in proteins, any of which Corresponding D-amino acids, rare amino acids, such as 4-hydroxyproline or hydroxylysine, or a protein amino acid, such as β-alanine or homoserin. Also included with scope of the present invention are analogs comprising amino acids that have been altered by chemical means such as methylation (e.g., α-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 (eg, 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 and position 19, respectively), refer to amino acids 12, 13, 15, 16 and 19 of the insulin B chain as shown in Figure 1. More specifically, the number system for these residues refers to the position of the amino acid within the native human protein, without considering the length of the peptide analogue or the position of the amino acid within the analogue. Peptide analogs having a substitution of alanine at residues 12, 13, 15 or 16 are referred to as analogues A12, A13, A15 or A16, respectively.

Peptide Analogues of Insulin Chain B As noted above, the present invention provides peptide analogs comprising at least residues 9-23 of the human insulin B chain and including an alteration of L-valine that occurs naturally in position 12. L-glutamate at position 13, L-leucine in position 15 and / or L-tyrosine in position 16, to another amino acid. In one embodiment, the peptide analogs contain additional alterations of one to three L-amino acids at positions 12, 13, 1, 5, 16 and / or 1 9 of the B chain of insulin. Preferably, the peptide analogs contain two to three alterations in which one of the substituted residues is in the 1-position. 9. The portion of an analogous peptide that is derived from the B-chain of insulin is typically 5-30 residues. in length, preferably 15-18 residues in length and more preferably 15-16 residues in length. Particularly preferred peptide analogs contain 15 amino acids derived from the B chain of insulin. As noted above, peptide analogs comprising any amino acid alteration (s) in the above-mentioned positions are within the scope of this invention. Preferred peptide analogs contain non-conservative substitutions (ie, alterations to amino acids that differ in charge, polarity, hydrophobicity and / or volume). Particularly preferred peptide analogs contain alterations of one or more residues to alanine. Peptide analogs can be synthesized by standard chemical techniques, including automated synthesis. In general, peptide analogs can be prepared by the solid phase peptide synthesis methodology which includes coupling each protected amino acid residue to a resin support, preferably a 4-methyl-benchidrylamine resin, by activation with dicyclohexylcarbodiimide to produce a peptide with a C-terminal amide. Alternatively, a chloromethyl resin (Merrifield resin) can be used to produce a peptide with a free carboxylic acid in the C-terminus. The side chain functional groups can be protected as follows: tosyl for histidine and arginine; 2-chlorobenzyloxycarbonyl for lysine; and 2-bromobenzyloxycarbonyl for tyrosine. After coupling, the t-butyloxycarbonyl protecting group on the alpha amino function of the added amino acid can be removed by treatment with trifluoroacetic acid followed by neutralization with di-isopropyl ethylamine. The next protected residue is then coupled onto the amino-free group, which propagates the peptide chain. After the last residue has been attached, the protected peptide-resin is treated with hydrogen fluoride to remove the resin from the resin and deprotect the side chain functional groups. The crude product can be further purified by gel filtration, HPLC, partition chromatography or ion exchange chromatography using well known procedures. The peptide analogs within the present invention (a) should not stimulate NOD mouse T cell clones specific for the peptide (9-23) of the native insulin B chain (SEQ ID NO; 2), or stimulating such clones at a level that is lower than the level stimulated by the native peptides; (b) they must not stimulate the T cells specific for the insulin B chain (9-23) of the patients; (c) they must be immunogenic in the NOD mouse; (d) they must reduce the incidence of diabetes in NOD mice, and (e) they can inhibit a response of cell clones specific for the peptide (9-23) of the native insulin B chain (SEQ ID NO: 2) . In this manner, candidate peptide analogs can be selected for their ability to treat diabetes by assays that measure T cell proliferation, immunogenicity in NOD mice and the effect on disease incidence in NOD mice. Certain representative assays for use in evaluating candidate peptide analogs are discussed in more detail below. Those analogous that satisfy the above criterion are useful therapeutics. Candidate peptide analogs can be tested initially for the ability to stimulate T cells specific for the peptide (9-23) of the native insulin B chain (SEQ ID NO: 2) (from clonal cell lines or isolated from patients). Such tests can be carried out using direct proliferation analysis, in which the reactive T cell lines of the native B chain (9-23) or T cells isolated from patients are used as target cells. The T-cell lines can usually be established, using well-known techniques, from the lymph nodes taken from rats injected with the B chain (9-23). The lymph cell nodes can be isolated and cultured for 5 to 8 days with the B chain (9-23) and IL-2. Viable cells are recovered and a second round of stimulation can be carried out with the B chain (9-23) and splenocytes irradiated from a source of growth factors. After 5 to 6 passages in this manner, the proliferative potential of each cell line is determined. In order to carry out the proliferation analysis, the reactive T-cell lines of the B chain (9-23) can be cultured for three days with various concentrations of irradiated peptides and splenocytes derived from themselves. After three days, 0.5-1.0 μCi of [3 H] -thymidine is added over 12-16 hours. The crops were then harvested and incorporated certain accounts. The average CPM and the standard error of the average are calculated from the triplicate cultures. Peptide analogs that produce results that are less than three standard deviations of the average response with a comparable concentration of the B chain (9-23) are considered non-stimulatory. Peptide analogs that do not stimulate proliferation at concentrations less than equal to 20-50 μM are suitable for further selections. Candidate peptides that do not stimulate B-chain specific T cells (9-23), 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 can be immunized with 100-400 μM of the candidate peptides subcutaneously in mannitol acetate buffer, three times within a period of 10-15 days. After the last immunization, lymph node cells and / or spleen cells can be used in a proliferation assay, in which different concentrations of the immunizing peptide are cultured with the cells for 3-4 days. The last 18 hours of cultivation can be carried out with tritiated thymidine. The cells can then be harvested and counted in a scintillation counter, and the proliferative response can be expressed as CPM + SEM. Candidate peptides that induce a proliferation that is at least 2 times higher than the previous one (non-antigen) in 25 μM of the peptide, are considered immunogenic. Alternatively, the candidate peptide analog is considered immunogenic if it produces a proliferative response after immunization of the NOD mice in the complete Freund's adjuvant. Draining lymph node cells or spleen cells, when cultured in the presence of the immunizing analog, must induce a proliferation that is at least 2 times higher than the previous one (non-antigen) in 25 μM of the peptide. Candidate peptides that can inhibit proliferation by the B chain (9-23) are further tested for the ability to reduce the incidence of diabetes in NOD mice. Briefly, the peptides can be administered to the NOD mice in soluble form or emulsified with, for example, incomplete Freund's adjuvant (IFA). Typically, weekly administration of approximately 400 μg of peptide is sufficient. The incidence of diabetes in the treated mice, as well as in control or untreated 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 onset of diabetes. Peptide analogs should result in a statistically significant reduction in the percent of NOD mice suffering from diabetes within a monitoring period of up to about 25 weeks. As noted above, peptide analogs can also inhibit the response of specific human T cells of the B chain (9-23) in vitro. Such inhibition can be measured by a competition analysis, in which the candidate peptide analogs are tested for the ability to inhibit the proliferation of the T cell induced by the native B chain (9-23) (SEQ ID NO: 2). In such an analysis, the cells presenting the antigen are first irradiated and then incubated with the competitor peptide analogue and the peptide (9-23) of the native B chain. The T cells are then added to the culture. Various concentrations of the candidate peptide analogs are included in cultures that can be incubated for a total of 4 days. Following the incubation period, each culture is pulsed with, for example 1 μCi of [3 H] -thymidine for a further 12-18 hours. The cultures can then be collected in a fiberglass filter and counted as above. The average CPM and the average error standard can be calculated from the data determined in the triplicate crops. Peptide analogs that reduce proliferation by at least 25% at a concentration of 20-50 μM are preferred.

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 an insulin B chain peptide analogue as described herein. Patients with diabetes suitable for such treatment can be identified by accepted criteria in the field to establish a diagnosis of clinically defined diabetes. Such criteria may include, but not be limited to, low (less than 10 or 1 5 percentile controls) insulin secretion in the first phase after an intravenous glucose tolerance test (IVGTT9 or the persistence of elevated primary antibodies to antigens in islet such as insulin, GAD65 and / or ICA512.Patients without clinically defined diabetes who can benefit from prophylactic treatment can generally be identified by any predictable criteria accepted in the field.Patients who are not frankly diabetic can be predicted to develop diabetes in incoming years (1-5 years) based on 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 composition, that is, the presence or absence of an HLA allele that is associated with a high risk of diabetes (eg, DR3 / 4, DQ8); iii) presence or absence of the major autoantibodies raised in their blood to any or all of the antigens: insulin GAD65 and / or ICA 512; and iv) intravenous glucose tolerance test (IVGTT): low insulin secretion in the first phase, 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 can be considered. For example, the chances of developing diabetes in 5 years for a first degree in relation to an individual with diabetes are estimated to be: 100% for relatives with all 3 autoantibodies listed above; 44% for relatives with 2 antibodies; 1 5% for relatives with an antibody; and 0.5% for relatives without any antibody. Among the 50 first-degree relatives of patients with Type I diabetes followed by the onset of diabetes, 49/50 expressed one or more of the autoantibodies listed above. Effective diabetes treatments can be determined in several different ways. By satisfying any of the following criteria, or other accepted criteria in the matter, effective treatment is evidenced. The criteria may include, but not be limited to, delay in developing natural hyperglycemia, decreased frequency of hyperglycemic cases and / or prolongation of normal levels of C-peptide in the blood of patients. The peptide analogs of the present invention can be administered either alone, or as a pharmaceutical composition. Briefly, the pharmaceutical compositions of the present invention may comprise one or more of the peptide analogs described herein, in combination with one or more pharmaceutically and physiologically acceptable excipients, diluents or carriers. Such compositions may comprise regulators such as neutral regulated salt, regulated phosphate salt and the like, carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione , adjuvants (for example, aluminum hydroxide) and preservatives.

In addition, the pharmaceutical compositions of the present invention may also contain one or more active ingredients, such as, for example, sustained delivery systems or other immunopontoring agents. The compositions of the present invention may be formulated for the indicated manner of administration, including for example, for oral, nasal, venous, intracranial, intraperitoneal, subcutaneous or intramuscular administration. Within other embodiments of the invention, the compositions described herein may be administered as part of a sustained release implant. Within still other embodiments of the invention, the compositions of the present invention can be formulated as a lyophilisate; using appropriate excipients that provide stability as a lyophilizate and / or after rehydration. The pharmaceutical compositions of the present invention can be administered in a manner appropriate to the disease to be treated (or avoided). The amount 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 illness. Within particularly preferred embodiments of the invention, the peptide analog may be administered at a dose ranging from 0.1 to 100 mg / kg, although appropriate doses may be determined by clinical tests. Patients can be monitored for therapeutic effectiveness by delaying progression to natural diabetes and sustained insulin use to maintain normoglycemia, as described above. The following examples are offered by way of illustration and not by way of limitation.

EXAMPLE 1 Preparation of Peptides This Example illustrates the synthesis of representative peptide analogs. The peptides were synthesized by solid-phase methodology in a peptide synthesizer (Beckman model 990). Peptides with an amidated carboxyl terminus were prepared with a p-methylbenzylhydrylamine resin (MBHA resin); for peptides with a carboxyl-free term; a Merrifield resin coupled with the appropriately protected amino acid was used. Both resins were obtained from Bachem Fine Chemicasl (Torrance, CA). The derived amino acids (Bachem Fine Chemicals) used in the synthesis were the L configuration unless otherwise specified, and the N-alpha-amino function exclusively protected with the t-butyloxycarbonyl group. The 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 and 2-bromobenzyloxycarbonyl for tyrosine. The coupling of the carboxyl-terminal amino acid to the MBHA resin was carried out with dicyclohexylcarbodiimide and the subsequent amino acids were coupled with dicyclohexylcarbodiimide according to Ling er al. { Proc. Nati Acad. Sci, USA 81: 4302, 1984). After the last amino acid is incorporated, the t-butyloxycarbonyl protecting group is removed and the peptide-resin conjugate is treated with a mixture of 14 ml of hydrofluoric acid (HF), 1.4 ml of anisol, and 0.28 ml of Methylethyl sulfide per gram of resin conjugate at -20 ° C for 0.5 hr and 0 ° C for 0.5 hr. HF was removed under vacuum at 0 ° C, and the resulting peptide and the resin mixture was rinsed twice with diethyl ether and twice with chloroform and diethyl ether, alternatively. The peptide was extracted five times with 2 M acetic acid, and the extract was lyophilized. The lyophilized product was first purified on a thin Sephadex G-25 column (Pharmacia-LKB, Piscataway, NJ), grown in 30% acetic acid to remove the truncated fragments and inorganic salts (Ling et al., 1984). Then, the peptides were further purified by carboxymethylcellulose cation exchange chromatography CM-32 (Ling et al, 1984). The final purification was achieved by dividing chromatography on fine Sephadex G-23 (Ling et al., 1884). Alternatively, the crude peptide could be purified by preparative HPLC on a gradient HPLC system KP-100 from Biotage. The synthetic product was characterized by amino acid analysis, spectrometric analysis and reverse phase HPLC.

EXAMPLE 2 Long-Term T Cell Lines This Example illustrates the preparation of long-term insulin-specific NOD T cell lines. The insulin-specific NOD T cell lines are established by culturing lymphocytes isolated from populations that infiltrate the islet by in vitro stimulation with either porcine insulin at 25 μg / 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 carried out. { see Wegmann et al., Eur. J. Immunol 24: 1 853-1994): the NOD mouse pancreas was digested with collagenase and the individual islets were isolated manually. The infiltrating lymphocytes were then obtained by the digestion of mild trypsin. The insulin-specific T cell lines or clones were propagated by serial stimulation in the presence of NOD spleen cells, porcine insulin and lymphocytes. The colons were obtained by limiting the dilution of the T-cell lines specific for the B chain (9-23) in the presence of the cell presenting the antigen and the porcine insulin at 25 μg / ml. Cavities with a growing population of cells following the limiting dilution expand into the appropriate medium, and after a growth cycle were tested for their reactivity to the B chain (9-23) insulin when evaluating the proliferative response .

EXAMPLE 3 Effect of Peptide Analogues on Proliferation of Insulin Specific NOD T cell Clones This Example illustrates the effect of representative peptide analogues on T cell proliferation. T cell clones (NOD) of insulin specific chain B (9-23) (SEQ ID NO: 2) were isolated from the infiltrated islets as described in Example 2. Peptide analogs with unique alanine substitutions are prepared as described in Example 1 The effect of each analog on the proliferation of the T cell is then evaluated using an analysis performed on 96-well flat bottom microtitre plates . { see Daniel e al. Eur. J. Immunol. 25: 1 056, 1995). Briefly, ,000 T cell clones together with 1 million irradiated NOD spleen cells were cultured in the presence of 50 μg / ml of the peptide 9-23 of the insulin B chain or any of the substituted alanine peptides listed below in triplicate pools. The plates were incubated for a total of 72 hours in 7% carbon dioxide atmosphere with a pulse of 1 μCi / tritiated thymidine cavity for at least 6-8 hours of culture. The cells were harvested on a glass fiber filter and the associated radioactivity was counted in a liquid scintillation counter. The results are expressed as average per-minute counts of tripled cavities. The data obtained from five separate T cell clones show either a lack of proliferation or significantly reduced proliferation (relative to the native peptide 9-23 of the B chain of insulin; SEQ ID NO: 2) in the presence of the following analogs substituted of alanine: A12, A13, A15, A16, A17 and A18. These data are shown in Tables 1 and 2, below. Table 1 Response (cpm) of Clones of the T Cell of Specific NOD Table 2 Response (cpm) of Insulin Specific Murine NQD T Cell Clones Table 3 shows the response of four different T cell clones derived from NOD to the double alanine substituted peptide analog A16, A19 (NBI-6024; 16Y> A / 19C> A). NOD T cell clones are incubated in the presence of 50 μM of either the peptide (9-23) of the native B chain or NBI-6024. The data in Table 3 represent the average of the tripled + sample. standard error of the average. Within Table 3, S.l. (Stimulation index) = proliferation (cpm) in the presence of peptide / proliferation (cpm) in medium only. These data show a significant response when the cells are cultured with the peptide (9-23) of the native B chain, but little or no proliferation above the average only (previous) in the presence of NBI-6024 Table 3 EXAMPLE 4 T Cell Cell Proliferation Analysis Antagonism This Example illustrates the inhibition of response of the mouse T cell clones specific to the B chain (9-23) to the insulin chain B (9-23) peptide by representative peptide analogs. Peptide analogs of the B chain (9-23) containing alanine substitutions at residue 12, 13, 15 or 16 or the double-substituted peptide at positions 16 and 19 (A16 19¡ NBI-6024) are prepared as it is described in Example 1. Antagonism of the T cell is detected by evaluating the ability of the peptide analogs to inhibit the proliferation of the T cell induced by the native B chain (9-23) (SEQ ID NO: 2). In this analysis, cells that present the antigen first are irradiated and then incubated with the competitor peptide analog and the native B chain (9-23) peptide. The T cells were then added to the culture. Various concentrations of the candidate peptide analogs are included in cultures that are incubated for a total of 4 days. Following this incubation period, each culture was pulsed with μCi of [3 H] -thymidine for a further 12-18 hours. The crops are then collected in the glass fiber filters and counted as above. Average CPM and standard error of the average are calculated from the data determined in triplicate cultures. The results, shown in Figure 2, indicate that peptide analogs containing alanine substitutions at residue 12, 13, or 16 are capable of attenuating the T-cell response of the insulin B chain (9-23). 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 peptide, NBI-5096, is an unrelated peptide of the basic protein of myelin. The percent inhibition is calculated as: (1-experimental cpm / insulin peptide cpm) x 100%.

Table 4 Inhibition of the Peptide Response (9-23) of the B Chain of Insulin in Two Clones of the T Cell of Murine NOD by a Peptide Analogue N / A = Not applicable since no inhibition was observed The ability of NBI-6024 to block the stimulation induced by the peptide (9-23) of the B chain of T clones derived from NOD suggests that the alterations in the positions and 19 of the peptide (9-23) of the native insulin B chain did not alter the ability of the analogue to be recognized by the pathogenic T cells. In addition, these results indicate that the analog also binds to the MHC with sufficient affinity to allow recognition by the specific T cell of the B chain of insulin (9-23).

EXAMPLE 5 Effect of Peptide Analogues on Proliferation of T Cell Lines and Clones of Diabetic Patients This Example illustrates the lack of stimulation of T cell lines and clones derived from diabetic patients by representative peptide analogues. Peptide analogs of the B chain (9-23) containing alanine substitutions at residues 13, 15, 16 or 17 or the dually substituted alanine analogue A16 19 (NBI-6024) are prepared as described in Example 1 . The T cell lines of diabetic patients are prepared by isolating the lymphocytes from the patient's blood by subjecting the blood to a gradient density separation. The isolated lymphocytes are then cultured in the presence of peptide (9-23) of insulin B chain (10 μM) and recombinant human IL-2 in the presence of 5-1 0% of serum derived from itself and blood lymphocytes peripherals derived from itself, irradiated in culture medium. Four or five days later the cells are harvested and the cycle was repeated 2 or 3 times. Proliferation of the T cell line, in response to the peptide (9-23) of the native B chain (SEQ ID NO: 2) or peptide analogs, is measured by culturing 25,000 to 100,000 T cells in the presence of 50,000-200,000 of irradiated PBLs derived from themselves and different concentrations of the peptide (9-23) of the insulin B chain or a peptide analogue in triplicate cultures. After 4-5 days of culture, including the last 18 hours with radioactive thymidine, the cells are harvested and the associated radioactivity counted in a liquid scintillation counter. The results are expressed as average counts per minute for each of the peptide analogues tested. The results, shown in Figures 4-7, indicate that the T cell lines and the clones that proliferate in response to the peptide (9-23) of the insulin B chain (SEQ ID NO: 2) are not stimulated by peptide analogs. The results of these patients and others are summarized in Table 5. Table 5 Proliferative Response of Patient PBLs to Native Insulin Peptide or Analog NBI-6024 * Stimulation index = CPM with antigen / CPM with medium alone (without antigen) The results clearly demonstrate that cells from diabetic patients that are responsive to the peptide (9-23) of the B chain of insulin do not respond to the peptide binder altered, NBI-6024 having substitutions at position 16 and 19. We have also determined that APL NBI-6024 binds with similar affinity to DQ8 antigens. Thus, the absence of stimulation of the T cells of diabetic patients by NBI-6024 is not due to any incompatibility of the peptide with the MHC molecules present, but is more likely due to the recognition altered by the T cells specific to the B string (9-23).

EXAMPLE 6 Diabetes Incidence Reduction 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 et al., In Current Topics in Clinical and Experimental Aspects of Diabetes Mellitus)., Sakamoto eí al, eds. P 25-32 (Elsevier, Amsterdam, 1985). The disease is predicted by cellular infiltration in the T-cell pancreas that begins as early as one month of age. Soluble peptide analogs of the B chain (9-23) containing alanine substitutions at residues 12, 13 or 16 are administered subcutaneously to NOD mice at weekly intervals. 400 μg of each peptide are administered in each treatment to ten animals. After 9 treatments, the percent of mice in each treatment group that has become diabetic is evaluated by measuring blood glucose levels using a meter at weekly intervals. A reading of more than 200 mg / dl of blood glucose in two consecutive observations is considered indicative of natural diabetes. As shown in Figure 8, treatment with each of the substituted analogues of alanine resulted in a marked reduction in the incidence of diabetes. The data for the substituted analog A13 is also shown in Figure 9.

In another experiment, the B chain (9-23), the substituted analogue A1 3 or neurotensin (as a control) is administered subcutaneously to NOD mice at weekly intervals. 400 μg of each peptide are administered in each treatment to ten animals. After 13 treatments, the percent of mice in each treatment group that has become diabetic is evaluated as described above. As shown in Figure 10, the peptide (9-23) of the B chain reduces the incidence of diabetes. This reduction was more pronounced for the substituted analogue A13. To determine the ability of the dually substituted peptide A16 19 (NBI-6024) to control the development of diabetes in NOD mice, the peptide is administered to animals at an early age. In this way, female mice (n = 9, approximately 4 weeks old), are treated subcutaneously with 20 mg / kg (400 μg / mouse) of NBI-6024 for twelve weeks and then every other week until Week 35. Starting at 9-10 weeks of age, mice are monitored weekly for hyperglycemia, measuring blood glucose levels. As a control, the group of 10 female mice was left untreated. The results of this experiment are shown in Figure 11. As noted, treatment with NBI-6024 significantly reduced the incidence of diabetes by approximately 60-70% compared to the untreated group, (p <0.004). The observations are then confirmed and extended in a second experiment. Here, animals (n = 13-15) are treated with either NBI-6024 or unrelated peptide, neurotensin, NBI-6201, as described above. An additional group (n = 8) was left untreated. As shown in Figure 12, treatment with 20 mg / kg of the altered NBI-6024 peptide results in a reduced incidence of diabetes compared to either the untreated or the neurotensin-treated group. These results demonstrate that the altered NBI-6024 peptide ligand, designated around the peptide (9-23) of the B chain of inulin was able to confer protection to animals at risk of developing diabetes spontaneously. It is likely that T cells that recognize other pancreatic antigens are present in these animals, yet they seem to be regulated by insulin APL. The time of administration was approximately the same time that the autoreactive lymphocytes begin to infiltrate the pancreas and initiate the destructive 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 Immunogenicity of Representative Peptide Analogs This Example illustrates the immunogenicity of representative peptide analogs in NOD mice. Groups of 3-4 NOD mice are immunized with 100-400 μg of peptide analogs subcutaneously in mannitol acetate buffer three times within a period of 10-1 5 days. After the last immunization, the lymph node cells and / or spleen cells are used in a proliferation assay in which the different concentrations of the immunizing peptide are cultured with the cells for 3-4 days. The last 18 hours of the culture included tritiated thymidine. Cells are 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 doubly-substituted peptide NBI-6024 (A16 19) to induce a cellular immune response in the NOD mouse chain was then determined. Two female NOD mice are immunized with 10 mg / kg of NBI-6024 either as an aqueous suspension, or as a control, emulsified in complete Freund's adjuvant (CFA). On Day 8, three days after the last injection, the mice were sacrificed, the inguinal lymph node and spleen cells were removed and combined, and a single cell suspension was prepared. The cells are cultured in the presence of varying concentrations (0-25 μM) of NBI-6024. The ability of these lymph cells to proliferate in response to NBI-6024 is measured in vitro by the incorporation of [3 H] -thymidine. The results are presented in Table 6, in which the response is expressed as CPM + SEM average of triplicate cultures. The lymph node cells isolated from mice immunized with the analogue in CFA show a strong proliferative response to the change with the immunizing analog in a dose-dependent manner (Table 6). These results indicate that the alterations made in the sequence (9-23) of the native insulin B chain at positions 16 and 19 have not affected the ability of the peptide to bind the MHC halotype molecule associated with the NOD disease and, more particularly do not obstruct recognition by T cells. Table 6 Proliferative Response of Linf Node Cells to NBI-6024 of NOD Mice Immunized with NBI-6024 in CFA In addition, both the spleen cells and the inguinal lymph node cells isolated from the soluble administered peptide showed a strong proliferative response to APL when changed in vitro with NBI-6024 (Table 7 and Figures 17A and 17B). Even more impressive was the discovery that lymphocytes derived from NBI-6024 from mice immunized with the soluble peptide also respond to the insulin B chain (9-23). This cross-reactive characteristic is not observed with the emulsified peptide of CFA. This ability of the soluble peptide to induce a transverse reactive response may be desirable in controlling diabetes, as it may help mobilize protective NBI-6024-specific T cells to the pathogenic target tissue. TABLE 7 Proliferative Response to NBI-6024 or Chain (B) of Native Insulin (9-23) of the NOD Mice Immunized with NBI-6024 Soluble To determine the type of T cells produced after the soluble administration of NBI-6024, the culture supernatants of immune lymphoid cells were removed 48 hours after the start of the culture and the levels of several measured cytokines using standard ELISA technology. Impressively, the cytokine production profile of the T cells of mice immunized with soluble NBI-6024 produced the Th2 cytokines, interleukin-4 (Figure 18) and interleukin-5 (Table 8) and not interleukin-2 derived from Th 1 Within Table 8, the values are expressed in pg7ml as an average of triplicates + SEM. As a control, NBI-6024 emulsified with CFA did not induce the expected Th1 cytokine profile (IL-2) of immune T cells upon in vitro stimulation. Table 8 Tumor Cell Response of T-cells Induced with NBI-6024 Soluble The ability of soluble subcutaneous administration of NBI-6024 to induce cells such as Th2 is a desirable feature, since such cells are associated with the recovery of diabetes and other organ-specific autoimmune diseases (Sarvetnick, J. Exp. Med 184: 1597 -1600, 1996; Shaw et al, 1997; Balasa et al., J. Exp. Med. 86: 385-391, 1997). These cytokines derived from Th2 have strong anti-inflammatory activities that suppress the development of self-reactive Th1 cells that secrete pro-inflammatory cytokine mediating the disease. From the foregoing, it will be apparent that while the specific embodiments of the invention have been described herein for the purpose of illustrating the invention, various modifications may be made without departing from the spirit and scope of the invention. In accordance with the foregoing, the present invention is not limited except as by the appended claims.

Claims (27)

1. CLAIMS 1. A peptide analogue comprising residues 9 to 23 of the B chain of human insulin, wherein the peptide analogue differs in sequence from residues 9 to 23 of the B chain of native human insulin due to substitutions in between amino acid positions 1 and 4, and wherein at least one substitution occurs in a residue selected from the group consisting of residues 12, 13, 15 and 16.
2. 2. The peptide analogue according to claim 1, characterized in that the analogue of peptide has a sequence that differs from the B chain of native human insulin in two amino acid residues.
3. 3. The peptide analogue according to claim 1, characterized in that the peptide analogue has a sequence that differs from the B chain of native human insulin in three amino acid residues.
4. 4. The peptide analogue according to claim 1, characterized in that an amino acid substitution occurs at residue 19.
5. 5. The peptide analog according to claim 1, characterized in that at least one amino acid substitution is non-conservative.
6. The peptide analog according to claim 1, characterized in that the residue 12 is replaced.
7. 7. The peptide analogue according to claim 6, characterized in that residue 12 is a residue of alanine.
8. The peptide analogue according to claim 1, characterized in that the residue 13 is replaced.
9. 9. The peptide analog according to claim 8, characterized in that the residue 1 3 is a residue of alanine.
10. 10. The peptide analogue according to claim 1, characterized in that the residue 15.1 is replaced.
11. The peptide analog according to claim 10, characterized in that the residue 15 is an alanine residue.
12. The peptide analogue according to claim 1, characterized in that the residue 16 is replaced.
13. 13. The peptide analog according to claim 12, characterized in that residue 16 is an alanine residue.
14. 14. The peptide analogue according to claims 6-1 3, characterized in that the residue 19 is replaced.
15. 15. The peptide analogue according to claim 14, characterized in that the residue 19 is a residue of alanine
16. 16. The peptide analog according to claim 1, characterized in that it also comprises residue 24 of the B chain of human insulin.
17. 17. The peptide analogue according to claim 1, characterized in that the peptide analog comprises no more than 18 residues of the B chain of human insulin.
18. The peptide analog according to claim 1, characterized in that the peptide analog comprises no more than 16 residues of the B chain of human insulin.
19. 19. The peptide analog according to claim 1, characterized in that the peptide analog comprises no more than 15 residues of the B chain of human insulin.
20. 20. A peptide analog consisting essentially of residues 9 to 23 of the B chain of human insulin, wherein the peptide analogue differs in sequence from residues 9 to 23 of the B chain of native human insulin due to substitutions in between amino acid positions 1 and 4, and wherein at least one substitution occurs in a residue selected from the group consisting of residues 12, 13, 15 and
21. 21. A peptide analog consisting essentially of residues 9 to 24 of the B chain of human insulin, wherein the peptide analog differs in sequence from residues 9 to 23 of the B chain of native human insulin due to substitutions in between amino acid positions 1 and 4, and wherein at least one substitution occurs in a residue selected from the group consisting of residues 12, 13, 15 and 16.
22. 22. A pharmaceutical composition comprising a peptide analogue according to any of claims 1-19 in combination with a physiologically acceptable diluent or carrier.
23. 23. A method for inhibiting the development of diabetes, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition according to claim 22.
24. 24. A method for the treatment of diabetes, which comprises administering to a patient a therapeutically effective amount of a pharmaceutical composition according to claim 22.
25. 25. A peptide analogue comprising residues 9 to 23 of the human insulin B chain, wherein the peptide analog differs in sequence of residues 9 to 23 of the B chain of native human insulin due to substitutions at residues 16 and 19.
26. 26. A pharmaceutical composition comprising a peptide analogue according to claim 25 in combination with a diluent or carrier physiologically acceptable.
27. 27. A method for inhibiting the development of diabetes, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition according to claim 26.