Link:  Pharm/Biotech Resources

Title:  Methods for treatment of diabetes using a peptide analogues of insulin

United States Patent:  6,933,274

Issued:  August 23, 2005

Inventors:  Gaur; Amitabh (San Diego, CA); Ling; Nicholas (San Diego, CA); Conlon; Paul J. (Solana Beach, CA)

Assignee:  Neurocrine Biosciences, Inc. (San Diego, CA)

Appl. No.:  339160

Filed:  January 8, 2003

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 be 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.

BRIEF 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 residues 9 to 23 due to substitutions at between 1 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 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 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.

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.

DETAILED DESCRIPTION OF THE INVENTION

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 15 amino acids derived from insulin B chain.

As noted above, peptide analogues comprising any amino acid alteration(s) at the positions recited above are within the scope of this invention. Preferred peptide analogues contain non-conservative substitutions (i.e., alterations to 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 solid-phase 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 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. Following coupling, the t-butyloxycarbonyl 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 (a) 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; (b) should not stimulate insulin B chain (9-23) specific human T cells from patients; (c) should be immunogenic in the NOD mouse; (d) should reduce the incidence of diabetes in NOD mice and (e) 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 incidence of the disease in NOD mice. Certain representative assays for use in 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 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 (9-23). Lymph node cells may be isolated and cultured for 5 to 8 days with B chain (9-23) 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 (9-23)-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 μCi of [³H]-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-stimulatory. Peptide analogues which do not stimulate proliferation at concentrations of less than or equal to 20-50 μM 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 100-400 μg 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 34 days. The 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 25 μM 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 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 25 μM of the peptide.

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 μg 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.

As noted above, peptide analogues may also inhibit the response of B chain (9-23) specific human T cells in vitro. 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 μCi of [³H]-thymidine for an additional 12-18 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 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 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 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 (1-5 yrs) 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-i.e., the presence or absence of an HLA allele that is associated with a high risk of diabetes (e.g., DR3/4; DQ8); iii) presence or absence of high titer autoantibodies in their blood to any or all of the antigens: insulin, GAD65 and/or ICA 512; and iv) intravenous glucose tolerance test (IVGTT): low first-phase 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 with no antibodies. Among 50 first degree relatives of patients with Type I diabetes 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, 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, mannose, sucrose or dextrans, mannitol, proteins, polypeptides or amino acids such as glycine, antioxidants, chelating agents such as EDTA or glutathione, adjuvants (e.g., 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 compositions 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 excipients 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 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 maintaining normoglycemia as described above.
 

Claim 1 of 22 Claims

1. A peptide analogue comprising residues 9 to 23 of human insulin B chain of SEQ ID NO:1, wherein the peptide analogue differs in sequence from native human insulin B chain residues 9 to 23 due to amino acid substitutions at between 1 and 4 amino acid positions, wherein at least one substitution occurs at a residue selected from the group consisting of residues 12, 13, 15 and 16, wherein the peptide analogue is capable of reducing incidence of diabetes in non-obese diabetic (NOD) mice, and wherein the peptide analogue consists of no more than 18 residues of human insulin B chain.

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