MXPA99002571A

MXPA99002571A - Use of proteins as agents against autoimmune diseases

Info

Publication number: MXPA99002571A
Application number: MXPA/A/1999/002571A
Authority: MX
Inventors: Bartorelli Alberto; E Panerai Alberto
Original assignee: Zetesis Spa
Priority date: 1996-09-18
Filing date: 1999-03-17
Publication date: 1999-09-01

Abstract

The use of proteins extracted with perchloric acid from animal organs, for the preparation of medicaments active against autoimmune diseases, in particular with activity against atherosclerosis, arthritis, multiple sclerosis, diabetes.

Description

USE OF PROTEINS AS AGENTS AGAINST AUTOIMMUNITY DISEASES DESCRIPTION OF THE INVENTION The present invention relates to the use of proteins extracted from animal organs, particularly the liver of mammals, for the preparation of active drugs against autoimmune diseases, in particular, activity against atherosclerosis, arthritis, multiple sclerosis and diabetes. The administration of Freund's complete adjuvant has proven to be capable of inducing experimental arthritis very similar to rheumatoid arthritis in rats. On the other hand, the administration of the adjuvant to rabbits does not induce any arthritic pathology, but atherosclerosis. The studies carried out have shown that, in both lesions, an immunoreactivity against an endogenous factor is present, which has been identified as the Cardiac Shock Protein 60 (HSP60). Subsequent investigations have confirmed these observations, proving that the administration of Freund's complete adjuvant can be replaced by the administration of HSP60, resulting in the same pathologies. Subsequently, the pretreatment of rats with the adjuvant, HSP60 or fragments thereof, has proven to prevent the ref. 29758 arthritis, with a mechanism that is still unknown, while subsequent administration to the adjuvant aggravates the progress of the disease. More recently, it has been found that pretreatment with adjuvant also prevents other experimental pathologies that can be defined, in general terms, as autoimmune diseases, such as diabetes or experimental allergic encephalomyelitis (EAE). Finally, it has been found that HSP60 has structural analogies with a high number of autoantigens, therefore it is presumed that it is related to pathologies more widely than had been observed to date. WO 92/10197 discloses protein fractions extracted with perchloric acid from mammalian organs and their use as anticancer agents. Within these fractions, three main components have been identified, which have molecular weights of 50, 14 and 10 Da in gel electrophoresis. The purified extract containing these three components will be referred to as UK 101 hereafter. The sequence of the 14 kDa protein component, which is the main one if not the sole responsible for the described activities, is described in the Table presented below and in WO 96/02567, and it has been found that is related to that described by other authors (Levy-Favatier, Eur. Biochem. 1903, 212 (3) 665-73), which assume that the new sequences identified belong to the family of proteins known as chaperonins, to which belong the HSPs. The proteins described in the patent WO 92/10197 and those in the patent WO 96/02567 (hereinafter referred to as UK 114) show properties never observed in the chaperonins or in analogous proteins. More specifically, it has been found that such proteins can be used in the prevention and treatment of autoimmune diseases, in particular atherosclerotic conditions such as atherosclerosis induced by organ transplants, arthritis, multiple sclerosis and diabetes. The present invention relates preferably to the use of the purified proteins UK 101 and UK 114 for the preparation of medicaments for the prevention and treatment of autoimmune diseases such as atherosclerosis after organ transplants, arthritis, multiple sclerosis, diabetes. In addition, the present invention comprises the use of proteins that show a high degree of homology with UK 114, at least 80%, preferably at least 90%.

ACTIVID7? D ANTIATEROSCLEROTICA At present it is stated that the most frequent cause of failure in organ transplantation, with time is no longer the rejection but the formation of atherosclerotic plaques. at the point of contact between the vessels of the transplanted organ and those of the host. This pathology is aggravated by the usual immunosuppressants such as cyclosporine, while the use of AZT, which however is very toxic, seems to be useful. The activity of the UK 101 and UK 114 proteins has been demonstrated using both a conventional atherosclerosis model, which is that of rabbits pretreated with Freund's complete adjuvant, and with a model of atherosclerosis by transplantation. In the first case, the subcutaneous treatment with adjuvant induces in a period of 21 days, the formation of atherosclerotic plaques in the iliac bifurcation and in the aortic arch. The pretreatment (7 days before) with UK 101 or UK 114 has significantly prevented the development of the disease in a high percentage of cases, compared with treatment with only the adjuvant, which leads to the development of the disease in all animals. On the other hand, the experimental model of atherosclerosis by transplant consists of venous shunts at the artery level in rats. After a short time, the formation of atherosclerotic plaques at the level of the host vessels has been observed, as occurs in human pathology. The pretreatment (7 days before) with UK 101 or UK 114 has significantly prevented the development of the pathology in a high percentage of cases, in comparison with what was observed in animals not pretreated before transplantation. ACTIVITY ANTIARTHRITIS This activity has been evidenced using the conventional arthritis model, which is the arthritis induced by adjuvant. In this model, Lewis rats are injected at the base of the tail with Freund's complete adjuvant: in a period of 7 days, a pathology appears on the hind legs characterized by inflammation and alterations of the joints. The pathology reaches its maximum from day 14 to day 21, then decreases until the legs return to normal conditions. Pretreatment (7 days before) with UK 101 or UK 114 has significantly prevented the development of the pathology in a high percentage of cases, compared with treatment with only the adjuvant, which has led to the development of pathology in the 100% of the animals. Treatment with UK 101 or UK 114 after administration of the adjuvant has aggravated the progress of the pathology.

Therefore, it is considered that UK 101 and UK 114 are able to modify the progress or to prevent pathological disorders such as arthritis and rheumatoid arthritis. ACTIVITY AGAINST MULTIPLE SCLEROSIS This has been demonstrated using a conventional model of multiple sclerosis: experimental allergic encephalitis (EAE). The pathology is induced by injecting subcutaneously Lewis race rats with a guinea pig backbone homogenate together with complete Freund's adjuvant. The pathology appears in the form of a progressive paralysis that starts from the hind limbs, starting approximately on day 12, reaching a maximum approximately on day 21 and suffering a remission approximately on day 30 after administration of the immunogen. The pretreatment (7 days before) with UK 101 or UK 114, has significantly prevented the development of the pathology in a high percentage of cases and has presented a less severe pathology, compared with the treatment only with bone marrow homogenate and adjuvant, which has led to the development of pathology in 100% of animals. Therefore, the UK 101 and UK 114 proteins are believed to be capable of changing the progress or of preventing pathological disorders such as multiple sclerosis. ANTIDIABETIC ACTIVITY This has been demonstrated using a model of conventional diabetes represented by BB rats, which develop diabetes spontaneously around 45 days of life. The animals have been treated on the 30th day of their life with UK 101 or UK 114 and the development of the pathology has been observed, compared to untreated control animals. It has been found that pretreatment decreases the incidence and severity of the pathology in the experimental model. Some patients affected with tumors at different sites and also suffering from diabetes have been treated with UK 101 in the course of compassionate treatment with the substance. All patients treated, regardless of the effect on tumor pathology, have shown a reduction in diabetic pathology that has come to eliminate the need for insulin therapy. Therefore, UK 101 and UK 114 are believed to be able to change the course of diabetes or prevent it. The antidiabetic activity has in fact been confirmed, although to date in a limited number of cases, also in vivo in patients suffering from diabetes. The proteins of the present invention can be administered using suitable formulations, mainly injectables. The pattern of administration (dose, frequency of administration, etc.) will be determined in accordance with the circumstances, depending on factors such as the patient's condition, the stage of the disease, etc., but a daily dose is usually adequate. It varies from 1 to 100 mg. TABLE Met Ser Glu Asn Ser Glu Glu Pro Val Gl? Glu Wing TB Wing 1 5 10 Pro Wing Wing Ile Gly Pro Tyr Ser Gln Wing Val Leu Val Asp 15 20 25 ra Thr He Tyr He Ser Gly Gln Leu Gly Met Aßp Pro Wing 30 35 40 Ser Gly Gln Leu Val Pro Gly Gly Val Val ßlu Glu Ala Lys 45 50 55 Gln Wing Leu Thr Asn He Gly Glu He Leu Lys Wing Wing Gly 60 65 70 Cys Asp Phe Thr Asn Val Val Lys Wing Thr Val Leu Leu Wing 75 80 Asp He Aso Asp Phe Ser Wing Val Asn ? Val Tyr Lys Gln 65 90 95 Tyr Phe Gln Ser Ser Phe Pro Wing Ring Wing Wing Tyr Gln Val 100 105 110 Wing Wing Leu Pro Lys Gly Gly Wing Val Glu He ßlu Wing Wing 115 120 125 Wing Val Gln ßl and Pro Leu Thr Thr Ala Ser Val 130 135 LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) RECIPIENT: zetesis s.p.a. (B) STREET: Corso 2 Gallery (C) CITY: Milano (E) COUNTRY: Italy (F) POSTAL CODE: 20122 (ii) TITLE OF THE INVENTION: use of proteins as agents against autoimmune diseases (iii) NUMBER OF SEQUENCES : 1 (ív) LEGIBLE FORM IN COMPUTER (A) TYPE OF MEDIUM: floppy disk (B) COMPUTER: compatible with IBM PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: ( A) LENGTH: 137 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: Met Ser Glu Asn Ser Glu Glu Pro Val Gly Glu Ala Lys Ala 1 5 10 Pro Wing Wing He Gly Pro Tyr Ser Gln Wing Val Leu Val Asp 15 20 25 Ring Thr He Tyr He Ser Gly Gln Leu Gly Met Asp Pro Wing 30 35 40 Ser Gly Gln Leu Val Pro Gly Gly Val Val Glu Glu Ala Lys 45 50 55 Gln Ala Leu Thr Asn He Gly Glu lie Leu Lys Wing Ala ßly 60 65 70 Cys Asp Pbe Thr Asn Val Val Lys Ala Thr Val Leu Leu Ala 75 80? Sp He Asn Asp Phe Ser Wing Val Asn Asp Val Tyr Lys Gln 85 9 95 Tyr Phe ßln Ser Ser Phe Pro Wing Wing Wing 1 10000 105 Tyr ßln VI Wing Wing _Le_u_ Pro Lys ß_l_y. ß_l_y. Aír_o6 Val Glu He ßlu _A_l_a He Wing Val Gln GGllyy PPrroo LLeeuu TThhrr TThhrr AAllaa Ser Val 13300 135 It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is the conventional one for the manufacture of the objects to which it relates.

Claims

R E I V I N D I C A C I O N S

Having described the invention as above, the content of the following is claimed as property: 1. The use of proteins extracted with perchloric acid from the liver of mammals, for the preparation of active drugs against autoimmune diseases. 2. The use according to claim 1, characterized in that the protein has the following sequence:
Mßt Ser Glu Asn Ser Glu Glu Pro Val ßly ßlu Ala Lys Ala 1 5 10 Pro Wing Wing He ßly Pro Tyr Ser Gln Wing Val Leu Val Asp 15 20 25 Ring Thr He Tyr He Ser Gly Gn Leu ßly Met Met Asp Pro Wing 30 35 40 Ser ßly ßln Leu Val Pro ßly Gly Val Val Glu ßlu Ala Lys 45 50 55 Gln Ala Leu Thr? Sn He ßly ßlu He Leu Lys Ala Ala ßly 60 65 70 Cys Asp Phe Thr Asn Val Val Lys Ala Thr Val Leu Leu Ala 75 80 Asp He Asn Asp Phe Ser Wing Val Asn Asp Val Tyr Lys Gln 85 90 95 Tyr Phe ßln Ser Ser Phe Pro Ala Arg Ala Ala Tvr ßln Val 1 10000 105 110 Ala Ala Leu Pro Lys ßly ßly Ara Val ßlu He ßlu Ala He 1-15 1"2700 1-25 Wing Val Gln ßly Pro LLeeuu TThhrr TThhrr AAllaa Ser Val 1 133 D0 * 113355
3. The use according to claim 1, characterized in that the proteins used have a homology of at least 80% with the protein of claim 2.
4. Pharmaceutical compositions characterized in that they contain as an active ingredient the proteins according to any of claims 1 to 3, mixed with suitable excipients.
5. The use according to claim 1, for the preparation of drugs for the prevention and treatment of atherosclerosis after transplants.
6. The use according to claim 1, for the preparation of medicaments for the prevention and treatment of arthritis.
7. Use in accordance with the claim 1 for the preparation of medicines for the prevention and treatment of multiple sclerosis.
8. The use according to claim 1, for the preparation of medicaments for the prevention and treatment of diabetes.