Analogs of peptide p277 containing pharmaceutical compositions for the treatment and diagnosis of diabetes

 

(57) Abstract:

Describes a new polypeptide sequence I: where X1and X2each represent residues Cys or Val and X3is a remnant of Thr or Lys, but X1and X2both cannot be residues Cys, when X3represents Thr, and its salts and functional derivatives. Proposed new polypeptide has essentially the same biological activity as p277, but has much better stability. It can be used for any purpose for which you can use p277. Also describes the containing pharmaceutical compositions for the treatment and diagnosis of diabetes. 8 C. and 9 C.p. f-crystals, 5 Il., 9 table.

The present invention relates to new peptides that are variants of the epitope of human hitokage protein 60 KD (hsp 60), and to pharmaceutical compositions containing such peptides, and to methods of diagnosis and treatment of insulin-dependent diabetes mellitus (IDDM) with the use of such peptides.

Background of the invention

Diabetes type I or IDDM is an autoimmune disease caused by T-cells that attack and destroy producing insulin-cells located in aetsa without symptoms. The disease manifests clinically only when the total loss of the cells exceeds the capacity of the remaining cells to produce insulin. In fact, I believe that the collapse of glucose homeostasis and clinical IDDM take place only after the immune system inactivated 80-90% of the cells. Thus, patients who are identified as suffering from IDDM, certainly are in late-stage autoimmune destruction of their cells. In addition, the diagnosis of primary, pre-diabetes by detection of immunological markers-cell autoimmunity can be set only after the onset of the autoimmune process. Therefore, therapeutic search should find a safe, specific and effective way to minimize autoimmune process that has already started.

The authors present invention first studied this question by examining the development of spontaneous diabetes in NOD mice, which are considered to be a reliable model of human IDDM (Castano and Eisenbarth, 1990). In NOD mice at the age of about 4 weeks developing insult, which begins as colostomy infiltration and develops serious vnutriutrobnogo inflammation. Hyperglycemia, Kotor 35-40 weeks almost all NOD mice develop severe diabetes, and most die in the absence of insulin treatment. Male NOD mice rarely develop diabetes for unclear reasons. It is shown that diabetes in NOD mice is called autoimmune T-cells (Bendelac et al., 1987).

T-cell reactivity and antibodies to various antigens identified in patients with IDDM, as well as in NOD mice (Elias, 1994), and it is unclear whether the primary cause of the disease immunity to any one of the possible target antigens. In addition to the question about the causes of the disease, there is the question of treatment.

It is shown that the initiation of the autoimmune process in NOD mice can be prevented by various impacts on mice before the onset of diabetes, such as limiting the power of viral infection or nonspecific stimulation of the immune system (Bowman et al., 1994). Diabetes in NOD mice also prevented by the induction of immunological tolerance in prediabetic mice to antigen glutamic acid decarboxylase (GAD) (Kaufman et al., 1993; Tisch et ai., 1993).

The authors of the present invention previously found that diabetes in NOD mice can be prevented T-cell vaccination using T cells specific for the peptide p277 sequence of a molecule of the human hsp60 (Elias et al., 1991). This protein was first called hsp65, but is now called hsp60 with ucpte p277, being epitope of human hsp60 involved in IDDM and relevant positions 437-460 sequence of human hsp60, first described in the patent application of Israel N 94241 applicants of this application and in the work of Elias and Cohen, 1994, and has the following sequence:

Val-Leu-Gly-Gly-Gly-Cys-Ala-Leu-Leu-Arg-Cys-Ile-Pro-Ala-Leu-Asp-Ser-Leu-Thr-Pro-Ala-Asn-Glu-Asp (afterbirth. N 1).

It is shown that the introduction of the peptide p277 in early insolite also prevents the development of diabetes, possibly through negative negative modulation of immunity against p277 that is essential for NOD-diabetes (Elias et al., 1991; an application for a patent of Israel N 94241). Recent studies have shown that the peptide p277 can also be used to reverse-cell autoimmunity, which has progressed to a late stage (Elias and Cohen, 1994).

According to the latest laboratory research of the authors of the present invention, the autoimmune form of diabetes can be induced in mice C57BL/KsJ through the introduction of very-low-dose-cellular toxin streptozotocin (STZ) (Elias et al., 1994). While the standard low dose of STZ 40 mg/kg, administered daily for 5 days, usually causes clinical diabetes within 3 weeks, the introduction of 30 mg/kg for 5 days induces clinics is the pressure in the prodromal period of autoantibodies to insulin, antiidiotypic antibodies to antiinsulin antibodies and autoantibodies to hsp60. Mice also exhibit spontaneous T-cell reactivity to hsp60 and to its peptide p277 (Elias et al., 1994). Thus, it appears that the dose of STZ below the standard low dose initiates the autoimmune process, not dissimilar from the process observed in spontaneous diabetes developing in NOD mice (Elias et al., 1990).

The object of the present invention are variants of peptide p277, and these options can be used for the diagnosis and treatment of IDDM.

Summary of the invention

In the study of fragments and variants of the peptide p277 unexpectedly discovered that the peptides, in which a threonine residue is replaced by a lysine residue, and/or one or both of the cysteine residue substituted for a residue (remainder) of valine, also active in the treatment of diabetes, as p277. These results were even more unexpected, as the substitution of cysteine residues serine residues results in inactive peptides.

Thus, the present invention relates to a peptide containing the sequence I

< / BR>
where each of X1and X2is a remnant of Cys or Val, and X3is SS="ptx2">

In specific embodiments, the implementation of the present invention, the peptide consists of the sequence I, in which X1and X2are Cys and X3represents Lys and named here p277(Lys19) (afterbirth. N 3); or X1represents Val, X2represents Cys, and X3is Thr or Lys, and such peptides are named here p277(Val6) (afterbirth. N 4) and p277(Val6-Lys19) (afterbirth. N 5), respectively; or X1represents Cys, X2represents Val, X3is Thr or Lys, and such peptides are named here p277(Val11) (afterbirth. N 6) and p277(Val11-Lys19) (afterbirth. N 7), respectively; or X1and X2are Val and X3is Thr or Lys, and such peptides are named here p277(Val6-Val11) (afterbirth. N 8) and p277(Val6,11-Lys19) (afterbirth. N 9), respectively. Peptide p277(Val6-Val11) is called here also p277(V).

Also the aim of the present invention are methods and kits for early diagnosis of IDDM using the peptide sequence of the first invention. In the process of developing IDDM animals Express hsp60 molecule or molecules that are cross-reactive with them, which find their way into the blood the way, the presence of hsp60 (or molecules that are cross-reactive with him) or antibodies or T-cells specific for him, in the blood or urine is a method of analysis for the detection process IDDM to complete the destruction of beta cells, when the individual is doomed to a lifetime of diabetes.

The presence or occurrence of IDDM patients can be diagnosed through a blood test or urine specified patient for the presence of antibodies against hsp60 or T-cells that are immunologically reactive with human hsp60, using as antigen peptide sequence I of the present invention. In fact, any of the previously described procedures, which can be done using p277, such as are described in the application for international patent application published under N WO 90/10499, incorporated herein by reference, can also be used with the replacement p277 on the peptide with the sequence I of the present invention.

Accordingly, the present invention relates to a method of diagnosing the presence or occurrence of IDDM patients, including the test for the patient for the presence of antibodies to hsp60 or T-cells, which immunoreactive with hsp60, and a positive result of this test, n indicates the Oia or the occurrence of IDDM.

In this way the diagnosis of IDDM patient can be tested for presence of antibodies against hsp60, while this type of research may include radioimmunoassay or analysis using ELISA method.

The patient can also test for the presence of T-cells, which immunoreactive with hsp60. In one of the embodiments of the invention in this aspect of the research method includes a test for T-cell proliferation, containing stage:

(I) obtain a mononuclear cell fraction containing T cells from a blood sample taken from the specified patient;

(II) adding to the said mononuclear cell fraction antigen, selected from among peptides sequence I of the present invention;

(III) incubation of the indicated cell fraction in the presence of the indicated antigen within a reasonable period of time and under suitable culture conditions;

(IV) adding a labeled nucleotide to the incubated cell culture obtained in (III), at a suitable time before the end of the specified incubation period to ensure that the specified labeled nucleotide into the DNA of proliferating T cells; and

(V) determine the number proliferously above stage (IV) specified labeled nucleotide is a, preferably,3H-thymidine. The determination of the number of proliferating T-cells is carried out by calculating the index of stimulation of T-cells by standard methods.

In another embodiment of the invention in this aspect of the research method includes a test for T-cell cytokine response, in which stage (I)-(III) are the same as in the above-described test for T-cell proliferation, and on the fourth (IV) the stage is determined using standard methods with commercially available kits presence of the cytokine, such as interferon - IL-2, IL-4, IL-6, IL-10, TNF or TGF, secreted into the environment by responding lymphocytes.

In another aspect, the present invention relates to a method of in vivo, in which the antigen is selected from peptides with the sequence I, is injected to the patient subcutaneously and observe the appearance of detectable skin reaction (DTH).

The present invention also relates to the implementation of such analyses, and to kits for carrying out such analyses. Kits can be obtained for any of the many tests used to perform the present invention. Each kit includes all the materials needed for provetil against hsp60 may contain immobilized on a solid phase peptide sequences I and labeled antibody, able to learn nvariably region of antibodies against hsp60, which detects, as, for example, a labeled anti-human Fab. The kit can also contain instructions for use of kit and capacity for storage of materials set. You can use any plain label such as a radioisotope, an enzyme, a chromophore or a fluorophore. Typical radioisotope is iodine-125 or sulfur-35. Typical enzymes for these purposes are horseradish peroxidase, galactosidase and alkaline phosphatase.

A kit for diagnosing the presence of IDDM through research on the presence of antibodies against hsp60 includes:

(I) an antigen selected from the peptides of the sequence I;

(II) a labeled antibody capable of learn nvariably region of these antibodies against hsp60, which determine.

A kit for diagnosing the presence of IDDM through research on the presence of T-cells, which immunoreactive with hsp60, includes:

(I) an antigen selected from the peptides of the sequence I;

(II) a suitable environment for the cultivation of lymphocytes (T-cells);

(III) or labeled nucleotide testing T-cell proliferation, or set for cytokine analysis, for example on-interferon is only the peptide of sequence I in the form, suitable for injection.

The present invention also relates to a method of prevention or treatment of IDDM. Vaccination with antigenic peptide sequence I of the present invention can provide specific suppressive effect for autoimmunity to the antigen, and effectively creates resistance autoimmune process IDDM. The same is true in regard to vaccination to T-cells specific for these antigens in attenuating or avirulent form or after processing, to improve its antigenicity, or their fragments or active fractions. If you see that the patient is already in the initial stage preclinical IDDM, the injection of such antigen or T-cell (or fraction) can cause a suppressive effect on the autoimmune process for this antigen and, thus, to stop the autoimmune process before it will cause significant irreversible damage. The peptide can also be used as a therapeutic agent to stop the autoimmune process even after he is far advanced, as shown recently, the authors of the present invention in the treatment of NOD mice with peptide p277 (Elias and Cohen, 1994).

Accordingly, the present izopet is he T-cell product, selected from the group consisting of (a) human T-cells, which show specificity to the p277 sequence of human hsp60, and these cells are activated by incubation in the presence of the indicated peptide sequence I; (b) the indicated human T-cells in (a), which are irradiated or attenyerevan other way; (C) the indicated human T cells in (a), which are processed under pressure by means of hydrostatic pressure, processing chemical cross-linking agent and/or processing of cytoskeletal cross-linking agent; (g) fragments or surface proteins, derived from T-cells (a), (b) or (C); or (d) a peptide consisting essentially of the variable regions of the receptor (a), specific for a given protein, or its salt, functional derivative, or active fraction.

In a preferred embodiment of the present invention, the product contains autologous human T-cells derived from patients with IDDM, which are treated, and these T cells are activated by contact in vitro with the indicated peptide sequence I. Such specific and activated T-cells are then injected back into the patient, from which the La prevention or treatment of IDDM, containing a pharmaceutically acceptable carrier and, as active element, the effective amount of the peptide of sequence I, its salt or functional derivative. Pharmaceutically acceptable carrier is preferably an oil medium, such as an emulsion of mineral oil, known as incomplete adjuvant's adjuvant (IFA). However, IFA as adjuvant's adjuvant (CFA; drug containing various amounts of dead organisms Mycobacterium), does not apply to humans, because mineral oil is not converted during metabolism and can not be degraded by the body.

Currently, the authors of the present invention discovered that some of the fat emulsion, which for many years used for parenteral nutrition patients-people can also act as carriers for peptide therapy using the peptides of the present invention. For example, two such emulsions are commercially available fat emulsions, known as intralipid (Intralipid) and lipofundin (Lypofundin). Intralipid is a registered trade name of the company Kabi Pharma-cia, Sweden, for the fat emulsion for parenteral Pitanga, Germany. Both emulsions as fat contain soybean oil (100 or 200 grams in 1000 ml distilled water, respectively, 10% or 20%). As emulsifiers in the intralipid used phospholipids of egg yolk (12 g per 1 l of distilled water), and lipofundina used egg yolk lecithin (12 g per 1 l of distilled water). As in the intralipid and lipofundina isotonicity is achieved by adding glycerol (25 g/l).

The present invention also relates to a method for prevention or treatment of IDDM, which includes an introduction to a patient in need of such treatment, a pharmaceutical composition containing the peptide sequence I, or preparation containing T-cells, which are grown specifically to a specific peptide sequence I of the present invention.

Brief description of drawings

Fig. 1 is a graph showing the stability of p277 and p277(Val6-Val11). Shaded circles are results obtained with p277, where there's no shading and mugs to p277(Val6-Val11). Large circles show the results obtained after 1 week of storage, mugs sized results after 9 weeks, and the smallest circles - after 20 weeks.

6-Val11) (where there's no shading circles).

Fig. 3 shows that the T cells in the spleen of NOD mice proliferate in the presence of peptide p277(Lys19). We investigated the spleen 5 female NOD mice in each age group in T-cell proliferation in response to peptide p277(Lys19) (shaded circles), p277 (where there's no shading circles) and control peptide p278 (shaded squares).

Fig. 4 shows that the T cells diabetogenic clone C9 NOD mice proliferate in response to peptide p277(Lys19).

Fig. 5 is a graph showing the efficiency of pre-treatment p277(V) in IFA warning STZ-induced diabetes compared with the control treatment or processing GADp34.

Detailed description of the invention

Whenever the description of the invention uses the expression "peptide sequence I refer also its salts and functional derivatives, while preserving the biological activity of the peptide against diabetes.

"Salt" peptide I, the alleged invention are physiologically acceptable organic and inorganic salts.

"Functional derivative" of a peptide I, which is I in the side chains of the residues or the N - or C-terminal groups, methods known in the art, and are included in the invention, so long as they remain pharmaceutically acceptable, i.e., do not destroy the activity of the peptide, so far as it relates to its similarity to known activity p277, do not attach toxic properties on compositions that contain them, and do not render harmful influence on their antigenic properties. Such "functional derivatives" does not include changes that effectively convert one amino acid into another.

Taking into account the above limitations, these derivatives may, for example, include esters of aliphatic alcohols, carboxyl groups, amides obtained by reaction of carboxyl groups with ammonia or primary or secondary amines, N-acyl derivatives of free amino groups of amino acid residues, formed by reaction with acyl groups (for example, alkanolamine or carbocyclic rollname groups) or O-acyl derivatives of free hydroxyl group (for example, hydroxyl group ceilinga or traviling residues) formed by reaction with acyl groups.

The peptide sequence of the first invention can be used as an immunogen in pharmaceutical socialist compositions for the diagnosis of IDDM. These pharmaceutical and diagnostic compositions, which can be obtained by methods known in the art, are also part of the present invention. When the peptide of the present invention is used for therapeutic treatment or prophylactic vaccines, it is preferably injected with a biologically active media, which promotiom shift T-TN autoimmune T-cells. T cells CD4 helper type are divided into two groups according to the cytokines they secrete, when activated (Mosmann and Coffman, 1989): cell TN secrete IL-2 and interferon- , while cells IN secrete IL-4 and IL-10. This carrier preferably is a fat emulsion containing 10-20% of triglycerides of vegetable and/or animal origin, 1.2 to 2.4% of the phospholipids of vegetable and/or animal origin, of 2.25-4.5% osmoregulator, 0-0,5% of an antioxidant and sterile water to 100%. This media is, most preferably, intralipid or lipofundin. The use of such media are described in the patent application of Israel (indicated by the reference of the applicant 9523) registered in one day from the date of registration of the Israeli application for the present invention (ukazano links.

A therapeutic composition of the present invention may be administered orally or parenterally, for example subcutaneously, intramuscularly, intravenously, intranasally or intrarectal.

The examples here demonstrate the efficacy of therapeutic compounds and compositions of the present invention to scientifically recognized animal models of IDDM person.

Examples

Materials and methods

(I) Mouse. Inbred female mice NOD/Lt is obtained from the Centre for breeding animal Breeding Center) Weizmann Institute of Science, Rehovot, Israel. In these mice, aged 14-17 weeks spontaneously develops autoimmune diabetes that mimics IDDM in humans.

(II) Peptides. The peptides are synthesized by conventional methods Fmoc chemistry using a synthesizer ABIMED (Germany) and purified on columns for HPLC using chromatography with reversed phase. Sequence set using the analysis of amino acids. Synthesize the following peptides: peptide p277; control peptide p278 with the sequence Asn-Glu-Asp-Gln-Lys-Ile-Gly-Ile-Glu-Ile-Ile-Lys-Arg-Thr-Leu-Lys-Ile (afterbirth. N 10), corresponding to the positions 458-474 molecules of human hsp60; fragments p277: aminopropane p277 (p277. N), carboxypropyl p277 (p277.C) and p277C combined with p278 (p277.C-p278); the sequences of species with p277 bridge-SH groups (p277 (bridge Cys-Cys)); p277 with the replacement of both cystine residues with valine (p277(Val6-Val11)) or serine (p277(Ser6-Ser11)).

(III) Processing and control. Mice give emulsion p277, other peptides or bovine serum albumin (BSA purchased from Sigma, St. Louis, Missouri, USA) in a volume of 0.1 ml by subcutaneous injection in the back. Antigens diluted in SFR and emuleret in an equal volume of mineral oil (incomplete adjuvant's adjuvant (IFA; Sigma) or 10% of intralipid. In mice test glucose in the blood at 10 A. M. in negrodamus condition, as described with Elias al., 1991, during treatment (at the age of 7, 12, 15 or 17 weeks), and the mice that survived at the age of 40 weeks. Significant hyperglycemia is the concentration of glucose of 11.1 mmol/l or higher, because the concentration of glucose in the blood exceeds three standard deviations from the average level of glucose in the blood, measured in 100 healthy mice (not shown). Histological examination of the pancreatic islets carried out on sections stained with hematoxylin or eosin. The slices are evaluated independently by two observers, who are not aware of the identity of groups. To ensure statistical differences when different treatments are used to etok, obtained from female NOD mice, analyzed by T-cell proliferation as previously described (Elias et al., 1991). Briefly, 1105clonal cells/ml or 1106the splenocytes/ml incubated at fourfold repetition within 72 h in 0.2 ml of culture medium in the wells on the microtiter tablet in the presence or absence of 5 μg/ml of different antigens. Proliferation is measured, including [3H]-thymidine into DNA during the last 12 h of incubation. The results are calculated as stimulation index - relationship test medium Chim (the number of pulses per minute) in the presence of antigen to the average background Chim in the absence of antigen. The standard deviation between the four repetitions were always less than 10% of the average Chim. The background is < 1000 Chim in experiments with splenocytes and < 200 Chim in the case of cell C9. The spleen of each mouse are examined separately. The results obtained in each group of mice, below average STD.

Example 1

Treatment of NOD mice p277 or its fragments

To check if both halves p277 of 12 amino acids, alone or in combination, for NOD mice to the same extent as p277, female NOD mice at the age of 7 weeks treated by subcutaneous inocu are given in table. 1. You can see that the control peptide p278 hsp60 have no effect on the progression of the disease: of the 40 treated mice all have diabetes, and 90% die at the age of 40 weeks. In contrast, the peptide p277 completely prevents death and heals 60% of the 20 treated mice. Fragments p277 less effective: aminopropane p277 (p277. N), carboxypropyl p277 (p277.C) and their mixture (p277.N, p277.(C) act half as well as intact p277. Peptide p277.C attached to synthesize the peptide p278 to get a longer peptide; however, p277.C-p278 was no better than the one p277C. Therefore, we can conclude that, for the full therapeutic effect requires an intact peptide p277.

Example 2

Treatment of NOD mice with peptides with substitutions at p277

Experience in NOD mice following peptides: p277, p277 (bridge Cys-Cys), p277(Val6-Val11) and p277(Ser6-Ser11).

The NOD females treated with 100 µg peptide in 0.2 cm3emulsions of mineral oil (IFA), which is injected subcutaneously. Mice treated at the age of 12 weeks, and represent the number of cases of diabetes in 30 weeks.

As shown in the table. 2, p277(Val6-Val11) as effective in the treatment of diabetes, as p277, the frequency of diabetes in neobw diabetes is 22% and 23% respectively. On the other hand, neither p277 (bridge Cys-Cys), or p277(Ser6-Ser11) have no therapeutic effect. Two additional options p277, in which the cysteine residues in positions 6 and 11 are replaced by alanine residues or-aminobutyric acid, also ineffective when tested in vitro as a clone C9 and splenic T-cells of NOD. As these two peptides are not recognized by specific T-cells against p277 (not shown), further they had not experienced therapeutic effect in vivo.

Example 3

Sustainability p277(Val6-Val11)

As the reason for replacement cysteine is problematic stability of peptide p277, checked resistance p277(Val6-Val11) at different points in time after its synthesis. Peptide p277 unstable and collapses even in strictly aged conditions (liofilizovannye powder in vacuum, -20oC).

To compare the stability of p277 with its analogue p277(Val6-Val11), both peptides are synthesized simultaneously and stored as dry powders at -20oC. After 1, 9 and 20 days after the date of the synthesis weighed aliquots, dissolve and carry out tests. An indicator of the stability of the peptide is its ability to stimulate T-cell cast its effectiveness within 9 weeks and all its effectiveness within 20 weeks after synthesis, p277(Val6-Val11) does not change after 20 weeks of storage.

Since it was assumed that the instability p277 due to cysteine residues, it is expected that any analogs of the present invention, which retain biological activity p277, will have increased stability when compared with p277 by the method similar to that shown in this example to p277(Val6-Val11).

Example 4

Treatment of female mice NOD p277 or p277(Val6-Val11) reverses insult

Female NOD mice at the age of 12 weeks give subcutaneously with 100 μg/mouse unmodified p277 or p277(Val6-Val11) in 0.1 cm3emulsions of mineral oil (incomplete beta-blockers). Control mice receive the emulsion SFR and mineral oil. At the age of 6 months to 5 mice in each group kill, remove them pancreas and fixed in bouin, and sections stained with hematoxylin:eosin. Insult appreciate the blind. As the control mice developed severe diabetes, their death at the age of 5 months. At this time, the glucose content in the blood of control mice is in the range 29-48 mmol/L. the Results are shown in table. 3.

At the age of 12 weeks at the time will shave perestrojkiny insult and about 20% of the Islands show no insolita. It is believed that vnutritrekovye infiltration is a severe form of insulite associated with loss of function cells. As shown in the table. 3, shows a noticeable deviation in the appearance of Islands in the two treated groups compared with the control mice received the same oil. Mice treated with oil (control animals), show a progressive decrease in the number of intact islets, and you can only see 10% of normal islets at the age of 22 weeks - a time when all mice clearly suffering from diabetes. About 67% of the islets show nutristrategy insult at the age of 22 weeks, and the remaining 23% of the islets find colostomy insult. In contrast, mice treated with p277 or p277(Val6-Val11) demonstrate the increase in the number of normal islets (25 to 52%) and a fall in the number of islets with nutristrategy insulator (from 6% to 17%). The number of islets with colostranova insulator increases to 31-69%. Thus, the processing p277 or p277(Val6-Val11) is associated with histologic improvement, consistent with the change in the opposite direction extent of insulite, persisting more than 3 months after the treatment.

Additional rezultatov with 10 female NOD mice in each group, which gave p277(V) in oil or one oil, at the age of 12-15 weeks. Of the total number of 150 mice treated with a single oil, 90% developed diabetes and 85% died at the age of 32 weeks. In contrast, mice treated with p277(V), showed only 50% of cases of diabetes (p < 0.01) and only 20% died from severe disease (p < 0,01). Therefore, late processing p277(V) effective delayed development of lethal diabetes.

Example 5

Peptide therapy for diabetes type I (p277(Val6-Val11in lipid emulsions

Autoimmune destruction insulinproducing-cells in the pancreas mediated by T-lymphocytes. Inflammatory infiltrate develops near the islets of the pancreas at the age of 5-8 weeks, and the destruction of the cells, leading to insulin deficiency and overt diabetes, becomes apparent at the age of 14-20 weeks, and at the age of 35-40 weeks affects almost 100% of the female mice NOD.

Female NOD mice given 100 μg of peptide p277(Val6-Val11) mouse subcutaneously in 0.1 ml SPR or 10% lipid emulsion consisting of 10% soybean oil, 1.2% of egg phospholipids, 2.25% glycerol (intralipid, Kabi Pharmacia AB, Sweden).

Then check the frequency of diabetes in visibillity, the content of glucose in the blood greater than 11.1 mmol/l, measured at least twice at intervals of a week by a glucose analyzer II, Beckman. The success of peptide processing analyzed in maintaining normal glucose concentration in the blood (less than 11.1 mmol/l), remission vnutriutrobnogo inflammation of the pancreatic islets (insulate) and induction of antibodies to therapeutic peptide as an indicator of the immune response T-type. The results are given in table. 4.

As you can see from the table. 4, the treatment with the peptide of the present invention introduced into the intralipid, effectively to reduce the incidence of diabetes and mortality. On the other hand, treatment with the introduction of the peptide in SFR is ineffective.

Example 6

Peptides p277 and p277(Val6-Val11are immunologically cross-reactive

Cells from T-cell clone C9 isolated from NOD mice in pre-diabetic condition, incubated with peptides p277 (shaded circles), p277(Val6-Val11) (where there's no shading circles), p277(Ser6-Ser11) (shaded squares) and p277 (bridge Cys-Cys) (where there's no shading diamonds). The results are shown in Fig. 2. Found that the clone C9 shows the positive reaction of proliferation only pevnymi, and that peptides p277(Ser6-Ser11) and p277 (bridge Cys-Cys), which are therapeutically ineffective, are not immunologically cross-reactive.

Example 7

Patients with newly diagnosed IDDM demonstrate T-cell proliferative responses as to p277 and p277(Val6-Val11)

Patients with newly diagnosed IDDM (age 2 - 4 weeks), are under study for analysis of proliferation. The lymphocytes of the peripheral blood isolated from whole heparinized blood on ficoll-hipace and sceneroot in vitro on the proliferation, measured by including3H-thymidine induced by hsp60 peptide p277, p277(Val6-Val11) and a control peptide p278. The reaction of T-cell proliferation portrayed as stimulation index (SI) is a relationship-driven peptide inclusions thymidine to the background (without added antigen) inclusions thymidine in T cells.

As shown in the table. 5, patients with newly diagnosed IDDM answer p277(Val6-Val11) as well as to peptide p277. Therefore, p277(Val6-Val11) immunological equivalent p277. Because therapeutic effect p277 mediated immunological recognition that the factor is instead p277 in the treatment of diabetes.

Example 8

T-cell responses to hsp60 and peptide with IDDM and in healthy individuals

Twenty-one patients with IDDM and fourteen healthy blood donors sceneroot on anti-hsp60 and p277(V) through analysis of proliferation of example 7. Table. 6 and 7 show the proliferative response of T-cells (stimulation index; SI) of each individual in the control antigen (control AG), i.e., tetanus toxin and influenza virus a strain of Texas, to a recombinant hsp60 and p277(V). Values SI of 3 or more is considered positive.

Table. 8 is a summary table showing that 86% and 52% of patients respond to hsp60 and p277(V), compared with 21% and 14% of controls (p < 0,05). Thus, the group with IDDM is significantly positive for T-cell responses to hsp60 and p277(V). It follows that the number of individuals, reactive to hsp60 and p277(V), higher among patients with IDDM than in healthy people.

Example 9

T-Cell proliferation in NOD-mice on p277(Lys19)

Peptide p277(Lys19differs from p277 one amino acid. To confirm that p277(Lys19is autoantigens with IDDM in NOD mice, compared T-cell proliferation by a peptide p277 and peptide p277(Lys19). Fig. 3 shows that the T cells of the spleen septic p278.

The above results are confirmed when using clone C9 - diabetogenic clone of T-cells of NOD, which, as found, is responsible for the human peptide p277 (Elias et al., 1991). Discovered that cells C9 proliferate in response to peptide p277(Lys19in the same manner as peptide p277 (Fig. 4).

Example 10

Treatment of female mice NOD p277(Lys19)

The authors of the present invention previously reported that developed insult occurring in female NOD mice at the age of 3 months, you can stop by injection to mice a single injection of human p277; the treated mice is less than the number of cases of diabetes before clinically evident and the number of deaths from severe hyperglycemia. To test whether the peptide p277(Lys19), which represents the actual mouse peptide p277 as effective female NOD mice at 3 months of age, give 100 µg or human p277 or p277(Lys19in IFA.

Peptides p277 or p277(Lys18) at a concentration of 2 mg/ml in phosphate buffered saline (SFR) emuleret with an equal volume of incomplete adjuvant's adjuvant (IFA). Groups of 10 animals female NOD mice at the age of 3 months is injected subcutaneously with 0.1 ml (100 µg peptide) emulsion, steriade shall determine the level of glucose in blood using glucose analyzer II, Beckman. The frequency of hyperglycemia and mortality from diabetes is estimated at 6 months of age. Mice are considered to be in the condition of diabetes, if they have content in blood glucose greater than 11 mmol/L.

The results are given in table. 9. At the age of 6 months 90% of the control mice had severe hyperglycemia and 60% died from diabetes. In contrast, treatment or p277 or p277(Lys19prevents diabetes (40% and 50% of cases, respectively) and death (10% and 20% of cases, respectively). Thus, it appears that the actual mouse peptide p277(Lys19) as effective as alien p277 in the treatment of advanced insolita.

These results indicate that the proliferative response of T-cells of NOD mice, detected with the use of a molecule of the human hsp60 and its peptide p277, are the equivalent of autoimmune responses in murine and murine hsp60 sequence p277(Lys19). It is found in the case diabetogenic T-cell clone C9, and in the case of a spontaneous reaction that develops in the spleen of mice in a state of prediabetes. In addition, p277(Lys19) effective as p277, in the treatment of diabetes in NOD mice. Splenic T cells opposite by age and sex of the mice of other strains not PR is that the sequence of murine hsp60, targeted diabetogenic T-cells, confirms the General assumption that IDDM is caused by an autoimmune process.

Example 11

Treatment of STZ-induced diabetes with p277(V)

Streptozotocin is a beta-cell specific toxin that can cause toxic diabetes, destroying the beta cells within 24-48 h if injected a single dose of 200 mg/kg Obtained in small subtoxic doses, it induces in genetically susceptible mice insult, leading to diabetes. The inflammatory process may take 20-30 days, depending on dose STZ. The usual scheme of induction of STZ diabetes STZ dose is 200 mg per 1 kg of body weight, given in the form of 5 equal doses of 40 mg/kg / day after day (Like and Rossini, 1976), and the model is called low dose STZ. Found that if the total dose is reduced to 150 mg/kg (30 mg/kg 5), diabetes develops over 80-100 days. This slowly progressive form of STZ-induced diabetes, probably more like autoimmune diabetes in humans and has the same duration as the preclinical stage of spontaneous diabetes in NOD mice (Castano and Eisenbarth, 1990). In addition, when the dose is reduced acute toxic effect.

Itsokay protein 60 KD (hs who I previously showed,

as antitelomerase and T-cell proliferative response to hsp60 occur before overt diabetes. T-cell clones specific for hsp60 receive from NOD mice in a state of prediabetes and can read insult and hyperglycemia, with good digestion, young NOD mice (Elias et ai., 1991) or mice i NOD. Diabetogenic T-cell clones NOD recognize the peptide length 24 amino acid sequences of hsp60 437-460, which is called p277. Diabetes induced by STZ, also accompanied by a T-cell responses against p277 in the preclinical stage.

In the previous examples, it is shown that the peptide p277(Val6-Val11protects NOD mice. In the present experiment tested the effectiveness of treatment p277 (V) on models with low dose STZ.

Male mice, 10 in the group treated with doses of STZ 30 mg/kg daily for 5 days. A week later, the mice given 100 μg or p277 (V) in oil or peptide glutamic acid - decarboxylase (GADp34), described by Kaufman with al., 1993, as involved in diabetes in NOD mice, or one oil. The process is repeated on day 85. 100 day in mice take blood, and their blood tested for hyperglycemia (glucose concentration exceeds 15 nmole/l). The results are summarized in Fig. 5. You can see ponglikitmongkol interval. In contrast, the average level of blood glucose in mice treated with p277 (V), is within the normal range. Thus, p277 (V) may be effective in the treatment of diabetes induced by STZ in male mice C57BL/ksj and spontaneous diabetes in the developing genetically different female NOD mice. The applicability of the treatment p277 (V) is not limited to only one case of diabetes or diabetes only one genotype or sex.

All cited here references, including journal articles or abstracts, published or corresponding patent application U.S. or foreign patents, issued U.S. or foreign patents, or any other references, are fully included in the present as references, including all data, tables, drawings and text presented in the cited references. In addition, the entire contents of the references cited in the references cited here, is also fully included in the present as references.

Links at the stage known way, the stage conventional, known methods or conventional methods does not assume that any aspect of description or implementation of the present invention are disclosed, illustrated or predlagayutsya will show the General nature of the invention as fully that is, using the known information in the art (including the contents of the cited here for reference), and other specialists can easily modify and/or adapt such specific embodiments of the invention for various applications, without undue experimentation, without departing from the main idea of the present invention. Therefore, mean that such adaptations and modifications are within the objectives and a series of equivalents of the disclosed embodiments of the invention presented here is based on the explanations and instructions. It should also be borne in mind that used here the phraseology or terminology is for the purpose of description and not of limitation, so that the terminology or phraseology in the present description should be interpreted by specialists presented here in the light of the explanations and instructions, combined with specialist knowledge in this field of technology.

Links

Bendelac A, Carnaud C, Boitard C, Bach JF. (1987) Syngeneic transfer of autoimmune diabetes from diabetic NOD ice to healthy neonates. Requirement for both L3T4+ and Ly2 + T cells. J Exp Med. 166:823-32.

Bowman MA, Leiter EH and Atkinson MA. (1994) Prevention of diabetes in the NOD mouse: implications for therapeutic intervention in human disease. Immunology Today. 15:115-20.

Castano L, Eisenbarth GS. (1990) Type-I diabetes: a chsease Models. A Guidebook. pp 147-61.

Elias D, Markovits D, Reshef T, van Der Zee R and Cohen IR (1990) Induction and therapy of autoimmune diabetes in the non - obese diabetic (NOD/Lt) mouse by a 65-kDa heat shock protein. Proc Natl Acad Sci USA. 87:1576-80.

Elias D, Prigozin H, Polak N, Rapoport M, Lohse AW, Cohen IR (1994) Autoimmune diabetes induced by the b-cell toxin streptozotocin. Diabetes. 43: 992-98.

Elias D, Reshef T, Birk OS, van der Zee R, Walker MD, Cohen IR. (1991) Vaccination against autoimmune mouse diabetes with a T-cell an epitope of the human 65 kDa heat shock protein. Proc Natl Acad Sci USA. 88:3088-91.

Elias D. and Cohen IR. (1994) Peptide therapy for diabetes in NOD mice. The Lancet. 343:704-06.

Kaufman DL, Clare-Salzler M, Tian J, Forsthuber T, Ting GSP, Robinson P, Atkinson MA, Sercarz HER, Tobin AJ, Lehmann PV. (1993) Spontaneous loss of T-cell tolerance to glutamic acid decarboxylase in murine insulin-dependent diabetes. Nature. 366:69-72.

Like AA, Rossini AA (1976) Streptozotocin-induced pancreatic insulitis: new model of diabetes mellitus. Science. 193:415-17.

Mosmann, T. R. and Coffman, R. I. (1989) Ann Rev Immunol. 7:145-73.

Tisch R, Yang XD, Singer SM, Liblav RS, Fuggar L, McDevitt HO. (1993) Immune response to glutamic acid decarboxylase correlates with insulitis in non-obese diabetic mice. Nature. 366:72-75.

The list of sequences

(1) General information

(I) APPLICANT:

(A) NAME: YEDA RESEARCH AND DEVELOPMENT CO. LTD

(B) STREET: P. O. Box 95

(In) the CITY: Rehovot

(E) COUNTRY: Israel

(F) POSTAL CODE (ZIP): 76100

(A) NAME: lrun R. COHEN

(B) STREET: 11 Hankin Street

(In) the CITY: Rehovot

(E) COUNTRY: Israel

(F) POSTAL CODE (ZIP): 76354

(A) NAME; Dana ELIAS

(B) STREETS SHALL

(B) STREET: 23 Miller Street

(In) the CITY: Rehovot

(E) COUNTRY: Israel

(F) POSTAL CODE (ZIP): 76284

(II) TITLE of INVENTION: ANALOGS of PEPTIDE p277 CONTAINING PHARMACEUTICAL COMPOSITION FOR the TREATMENT OR DIAGNOSIS of DIABETES

(III) NUMBER OF SEQUENCES: 10

(IV) THE FORM OF COMPUTER-BASED READING:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: compatible with IDM PC

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, version #1.30 (EPO)

(VI) INFORMATION ABOUT THE PREVIOUS REQUEST:

(A) APPLICATION NUMBER: IL 112094

(B) REGISTRATION DATE: December 21, 1994

(VI) INFORMATION ABOUT THE PREVIOUS REQUEST:

(A) APPLICATION NUMBER: IL 114460

(B) REGISTRATION DATE: 05 July 1995

(2) INFORMATION ABOUT THE PLACENTA. N 1

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) single-Stranded

(D) TOPOLOGY: linear

(II) MOLECULE TYPE: peptide

(XI) SEQUENCE DESCRIPTION: PLACENTA. N 1

< / BR>
(2) INFORMATION ABOUT THE PLACENTA. N 2

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) single-Stranded

(D) TOPOLOGY: linear

(II) MOLECULE TYPE: peptide

(IX) FEATURES:
s or Val; Xaa in position. 19=Thr or Lys; Xaa in position. 6/11 both not equal Cys, when Xaa in position. 19 equal to Thr."

(XI) SEQUENCE DESCRIPTION: PLACENTA. N 2.

< / BR>
(2) INFORMATION ABOUT THE PLACENTA. N 3

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) single-Stranded

(D) TOPOLOGY: linear

(II) MOLECULE TYPE: peptide

(XI) SEQUENCE DESCRIPTION: PLACENTA. N 3

< / BR>
(2) INFORMATION ABOUT THE PLACENTA. N 4

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) single-Stranded

(D) TOPOLOGY: linear

(II) MOLECULE TYPE: peptide

(IX) FEATURES:

(A) NAME/KEY: Peptide

(B) LOCATION: 19

(D) OTHER INFORMATION:/note="Xaa in position. 19=Thr or Lys"

(XI) SEQUENCE DESCRIPTION: PLACENTA. N 4

< / BR>
(2) INFORMATION ABOUT THE PLACENTA. N 5

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) single-Stranded

(D) TOPOLOGY: linear

(II) MOLECULE TYPE: peptide

(XI) SEQUENCE DESCRIPTION: PLACENTA. N 5

< / BR>
(2) INFORMATION ABOUT THE PLACENTA. N 6

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) T IS RSNAKE:

(A) NAME/KEY: Peptide

(B) LOCATION: 19

(D) OTHER INFORMATION:/note="Xaa in position. 19 = Thr or Lys"

(XI) SEQUENCE DESCRIPTION: PLACENTA. N 6

< / BR>
(2) INFORMATION ABOUT THE PLACENTA. N 7

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) single-Stranded

(D) TOPOLOGY: linear

(II) MOLECULE TYPE: peptide

(XI) SEQUENCE DESCRIPTION: PLACENTA. N 7

< / BR>
(2) INFORMATION ABOUT THE PLACENTA. N 8

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) single-Stranded

(D) TOPOLOGY: linear

(II) MOLECULE TYPE: peptide

(IX) FEATURES:

(A) NAME/KEY: Peptide

(B) LOCATION: 19

(D) OTHER INFORMATION:/note="Xaa in position. 19 is Thr or Lys"

(XI) SEQUENCE DESCRIPTION: PLACENTA. N 8

< / BR>
(2) INFORMATION ABOUT THE PLACENTA. N 9

(I) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) single-Stranded

(D) TOPOLOGY: linear

(II) MOLECULE TYPE: peptide

(XI) SEQUENCE DESCRIPTION: PLACENTA. N 9

< / BR>
(2) INFORMATION ABOUT THE PLACENTA. N 10

(I) SEQUENCE CHARACTERISTICS:

(Is II) MOLECULE TYPE: peptide

(XI) SEQUENCE DESCRIPTION: PLACENTA. N 10

1. The polypeptide sequence I

< / BR>
where X1and X2each represent residues Cys or Val and X3is a remnant of Thr or Lys, but X1and X2both cannot be residues Cys, when X3represents Thr,

and its salts and functional derivatives.

2. The peptide under item 1, selected from peptides, designated here as p277 (Val11), where X1represents Cys, X2represents Val and X3represents Thr; p277 (Val6-Val11), where X1and X2are Val and X3represents Thr; p277 (Val11-Lys19), where X1represents Cys, X2is Val and X3represents Lys; p277 (Val6,11-Lys19), where X1and X2are Val and X3represents Lys.

3. The peptide under item 1 or 2 to obtain a composition for the diagnosis of insulin-dependent diabetes mellitus (hereinafter IDDM).

4. The diagnostic method developed or developing IDDM patients, including analysis of blood or urine specified patient using peptide under item 1 or 2 as antigen for the presence of antibodies to the influence of antibodies against hsp60, which immunoreactive with hsp60, indicates a high probability of the presence or the initial stage of IDDM.

5. The method according to p. 4, characterized in that the presence of antibodies against hsp60 explore using radioimmunoassay or ELISA method (enzyme-linked immunosorbent assay).

6. The kit for the diagnosis of IDDM by examining for the presence of antibodies against hsp60 by the method under item 4 or 5, comprising: (I) antigen, which is the peptide sequence (1) under item 1, and (II) labeled antibody capable of recognizing nvariably region of these antibodies against hsp60, which determine.

7. The diagnostic method developed or developing IDDM patients, including analysis of blood or urine specified patient using peptide under item 1 or 2 as antigen for the presence of T-cells that are immunologically reactive with human hsp60, which results showing the positive presence of T cells, which are immunoreactive with hsp60, indicates a high probability of the presence or the initial stage of IDDM.

8. The method according to p. 7, characterized in that the presence of T cells, which are immunoreactive with hsp60 checked with tests for T-cell proliferation, including the following Sogo patient, (II) adding to the said mononuclear cell fraction antigen, selected from among peptides under item 1; (III) incubation of the indicated cell fraction in the presence of the indicated antigen for a suitable period of time and under suitable cultivation conditions; (IV) adding a labeled nucleotide to inkubiruemykh cell culture (III) in a suitable time before the end of the specified incubation period to ensure that the specified labeled nucleotide into the DNA of proliferating T cells and (V) determination of the number of proliferating T cells by analyzing the number of labeled nucleotide incorporated into these T-cells.

9. The method according to p. 7, characterized in that the presence of T cells, which are immunoreactive with hsp60 checked with tests for T-cell cytokine reaction, comprising the following stages: (I) obtaining mononuclear cell fraction containing T cells from a blood sample taken from the specified patient; (II) adding to the said mononuclear cell fraction antigen, selected from among peptides under item 1; (III) incubation of the indicated cell fraction in the presence of the indicated antigen for a suitable period of time and when pthitami on Wednesday, when this cytokine is an interferon, IL-2, IL-4, IL-6, IL-10, TNF or TGF.

10. The kit for the diagnosis of IDDM by testing for the presence of T cells, which are immunoreactive with hsp60, according to the method under item 8, containing: (I) antigen, which is the peptide under item 1; (II) a labeled nucleotide, and (III) a suitable environment for the cultivation of lymphocytes.

11. The kit for the diagnosis of IDDM by testing for the presence of T cells, which are immunoreactive with hsh60, the method according to p. 9, containing: (I) antigen, which is the peptide under item 1; (II) a suitable environment for the cultivation of lymphocytes and (III) analytical kit for determination of the presence of the cytokine, secreted by responding lymphocytes in the environment.

12. A drug for prevention or treatment of IDDM, containing a T-cell product selected from the group including: a) human T-cells, which show specificity to the p277 sequence of human hsp60, where these cells are activated by incubation in the presence of the peptide sequence (I) under item 1; (b) the human T-cells, which are irradiated or attenyerevan other way; (C) these human T-cells, which are subjected to pressure treatment by means of hydrostatic pressure to the Ghent; (g) fragments or surface proteins formed from T-cells in (a), (b) or (C); or (d) a peptide consisting essentially of the variable regions of the receptor (a), specific for a given protein, or its salt, functional derivative, precursor or active fraction.

13. The drug under item 12, characterized in that the said human T-cells (a) are autologous T-cells obtained from IDDM patient under treatment, and the specificity of these T cells generated by contact in vitro with the indicated peptide sequence 1.

14. The drug under item 12 or 13, characterized in that these T cells have been created in vitro specificity to the peptide sequence of 1 under item 1.

15. Pharmaceutical composition for prevention or treatment of IDDM, containing the peptide sequence 1 p. 1 and a pharmaceutically acceptable carrier.

16. The pharmaceutical composition according to p. 15, where the specified peptide is a peptide in p. 2.

17. The peptide under item 1 or 2 as an active ingredient in pharmaceutical compositions for the prevention or treatment of IDDM.

Priority points:

21.12.94 on PP.1 - 17.

 

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