HK1022098A - Use of proteins as agents against autoimmune diseases - Google Patents

Description

Use of proteins as anti-autoimmune disease agents

The invention relates to the use of proteins extracted from animal organs, in particular from the liver of mammals, for the preparation of a medicament having activity against autoimmune diseases, in particular having activity against atherosclerosis, arthritis, multiple sclerosis, diabetes.

Administration of Freund's complete adjuvant has been shown to induce experimental arthritis in rats that closely resembles rheumatoid arthritis. On the other hand, administration of the adjuvant to rabbits induced not arthritis lesions but atherosclerosis. Studies have been conducted to show that there is immunoreactivity in both lesions for endogenous factors that have been identified as heat shock protein 60(HSP 60). Subsequent studies demonstrated that administration of HSP60 can be used in place of administration of freon's complete adjuvant and produce the same pathological changes, confirming the above observations. Later, it was again demonstrated that pretreatment of rats with adjuvant, HSP60 or a fragment thereof, prevented the onset of arthritis, the resistance of which is still unclear, while administration of the adjuvant followed by exacerbation of the disease process.

It has recently been found that pretreatment with adjuvants also prevents other experimental pathologies defined in general as autoimmune diseases such as diabetes or Experimental Allergic Encephalomyelitis (EAE). Finally, HSP60 was found to be structurally similar to many self-antigens, and it was therefore postulated to be associated with a wider variety of pathological changes than has been observed to date.

WO92/10197 discloses protein fractions extractable with perchloric acid from mammalian organs and their use as antigenic agents. Of these protein fractions, three major fractions were identified with molecular weights of 50, 14 and 10kDa on gel electrophoresis. The purified extract containing this component is hereinafter referred to as UK 101. The sequence of the 14kDa protein component, which is the major but not the only component having the above-mentioned activity, was reported in the following table and in WO96/02567 and was found to be related to proteins described by other authors [ Levy-Favatier, Eur. biochem.1903,212(3)665-73], and the newly identified sequence was presumed to belong to a family of proteins known as chaperones (chapronin) to which HSP itself also belongs.

The proteins described in WO92/10197 and 96/02567 (hereinafter UK114) exhibit certain properties not observed in chaperone proteins or analogues thereof. More particularly, it has been found that the protein can be used for the prevention and treatment of autoimmune diseases, in particular atherosclerotic diseases such as atherosclerosis induced by organ transplantation, arthritis, multiple sclerosis and diabetes.

Preferably, the present invention relates to the use of purified proteins UK101 and UK114 for the preparation of a medicament for the prevention and treatment of autoimmune diseases such as atherosclerosis, arthritis, multiple sclerosis and diabetes after organ transplantation.

Furthermore, the invention encompasses the use of proteins exhibiting a high degree of homology to UK114 of at least 80%, preferably at least 90%. Anti-atherosclerotic Activity

It has now been found that the more common cause of rapid failure of organ transplantation is no longer rejection, but rather atherosclerotic plaques formed at the point of contact between the transplanted organ vessel and the host vessel. The pathological changes are exacerbated by commonly used immunosuppressive agents such as cyclosporin, while the use of Azidothymidine (AZT), although highly toxic, appears to be beneficial.

The activity of the proteins UK101 and UK114 can be demonstrated in a conventional model of atherosclerosis, which is a rabbit pretreated with freund's complete adjuvant, and in a model of transplanted atherosclerosis. In the first case, subcutaneous treatment with adjuvant induced the formation of atherosclerotic plaques at the iliac bifurcation and the aortic arch within 21 days. Pretreatment with UK101 or UK114(7 days earlier) significantly prevented the development of pathological changes in a high proportion of cases compared to treatment with adjuvant alone, which resulted in the development of disease in all tested animals.

On the other hand, experimental models of graft atherosclerosis exist in venous bypass surgery at the level of the rat arteries. After a short time, atherogenesis is observed at the host vascular level, as occurs in human pathological changes. Pretreatment with UK101 and UK114(7 days earlier) prevented the development of pathological changes in a high proportion of cases compared to that observed in animals not pretreated prior to organ transplantation. Anti-arthritic activity

Anti-arthritic activity was demonstrated using a conventional arthritis model, i.e. adjuvant-induced arthritis. In this model, injection of complete freon adjuvant into the tail base of Lewis (Lewis) mice resulted in pathological changes characterized by swelling and joint deformity in the hind limbs within 7 days. Pathological changes peaked from day 14 to day 21 and then gradually declined until hind limbs returned to normal. Pretreatment with UK101 or UK114(7 days earlier) prevented the development of pathological changes significantly in a high proportion of cases compared to adjuvant treatment alone (which resulted in the development of pathological changes in 100% of animals). Treatment with UK101 or UK114 after administration of adjuvant worsens the course of pathological changes.

Therefore, UK101 and UK114 are believed to be able to improve the progression or prevent the development of pathological conditions such as arthritis and rheumatoid arthritis. Anti multiple sclerosis Activity

This activity was measured using a conventional multiple sclerosis model: experimental strain encephalomyelitis (EAE) was demonstrated. Lewy (Lewis) mice were injected subcutaneously with guinea pig spinal cord homogenate along with Freund's adjuvant to induce pathological changes. Progressive paralysis appeared around day 12 starting from the hind limb, reaching a maximum at around day 21 and remitting at around day 30 after administration of the immunogen. Pretreatment with UK101 or UK114(7 days ago) prevented the development of pathological changes significantly in a high proportion of cases and presented less severe signs than treatment with marrow homogenate and adjuvant alone, which resulted in the development of pathological changes in 100% of the tested animals.

UK101 and UK114 are thus believed to be capable of ameliorating the progression or preventing the occurrence of pathological conditions such as multiple sclerosis. Anti-diabetic Activity

This activity was demonstrated in a conventional model of diabetes as represented by BB mice, which spontaneously develop diabetes around day 45 of their growth, treated with UK101 or UK114 at day 30 of their growth and observed for the development of pathological changes, compared to untreated control animals. Pretreatment has been found to reduce the incidence of disease and the severity of pathological changes in experimental models. Some patients affected by tumors in different parts of the body and simultaneously suffering from diabetes were treated with UK101 during placebo treatment. All treated patients showed remission of the signs of diabetes, so that insulin treatment could be abandoned, independently of the effects on the neoplastic pathology.

It is therefore believed that UK101 and UK114 can alter the progression of diabetes or prevent its occurrence.

Indeed, anti-diabetic activity has been demonstrated in diabetic patients, although it has been demonstrated to date in only a limited number of cases.

The proteins of the invention may be administered using a suitable formulation, primarily an injectable formulation.

The administration form (e.g., administration dose, frequency, etc.) depends on factors such as the condition of the patient, the stage of the disease, etc., but generally a suitable daily dose range is 1 to 100 mg.

TABLE Met Ser Glu Asn Ser Glu Glu Pro Val Gly Glu Ala Lys Ala 1510 Pro Ala Ala Ala IlE Gly Pro Tyr Ser Gln Ala Val Leu Val Asp 152025 Arg Thr Ile Tyr Ile Ser Gly Gln Leu Gly Met Asp Pro Ala

30 35 40Ser Gly Gln Leu Val Pro Gly Gly Val Val Glu Glu Ala Lys

45 50 55Gln Ala Leu Thr Asn Ile Gly Glu Ile Leu Lys Ala Ala Gly

60 65 70Cys Asp Phe Thr Asn Val Val Lys Ala Thr Val Leu Leu Ala

75 80Asp Ile Asn Asp Phe Ser Ala Val Asn Asp Val Tyr Lys Gln85 90 95Tyr Phe Gln Ser Ser Phe Pro Ala Arg Ala Ala Tyr Gln Val

l00 105 110Ala Ala Leu Pro Lys Gly Gly Arg Val Glu Ile Glu Ala Ile

115 120 125Ala Val Gln Gly Pro Leu Thr Thr Ala Ser Val

130 135

Sequence table (1) basic information:

applicant (i) has come to

(A) Name: zetesis s.p.a.

(B) Street name: galleria del Corso 2

(C) City: milano

(E) The state is as follows: italy (chemical vapor deposition)

(F) Postal code (ZIP):20122

(ii) name of the invention: use of proteins as anti-autoimmune agents

(iii) number of sequences: 1

(iv) computer-readable form:

(A) type of medium: flexible disk

(B) A computer: IBM PC compatible machine

(C) Operating the system: PC-DOS/MS-DOS

(D) Software: PatentIn Release #1.0, version #1.30(EPO) (2) information on SEQ ID NO:1:

sequence characteristics:

(A) length: 137 amino acids

(B) Type (2): amino acids

(C) Chain type:

(D) topological structure: wire type

(ii) type of molecule: protein

(iii) suppose: is free of

(iv) antisense: is free of

(xi) sequence description: SEQ ID NO 1: Met Ser Glu Asn Ser Glu Glu Pro Val Gly Glu Ala Lys Ala 1510 Pro Ala Ala Ile Gly Pro Tyr Ser Gln Ala Val Leu Val Asp 152025 Arg Thr Ile Tyr Ile Ser Gly Gln Leu Gly Met Asp Pro Ala

30 35 40Ser Gly Gln Leu Val Pro Gly Gly Val Val Glu Glu Ala Lys

45 50 55Gln Ala Leu Thr Asn Ile Gly Glu Ile Leu Lys Ala Ala Gly

60 65 70Cys Asp Phe Thr Asn Val Val Lys Ala Thr Val Leu Leu Ala

75 80Asp Ile Asn Asp Phe Ser Ala Val Asn Asp Val Tyr Lys Gln85 90 95Tyr Phe Gln Ser Ser Phe Pro Ala Arg Ala Ala Tyr Gln Val

100 105 110Ala Ala Leu Pro Lys Gly Gly Arg Val Glu Ile Glu Ala Ile

115 120 125Ala Val Gln Gly Pro Leu Thr Thr Ala Ser Val

130 135

Claims (8)

1. Use of a protein extractable with perchloric acid from mammalian liver for the preparation of a medicament having activity against autoimmune diseases.

2. The use according to claim 1, wherein the protein has the following sequence: met Ser Glu Asn Ser Glu Glu Pro Val Gly Glu Ala Lys Ala 1510 Pro Ala Ala Ile Gly Pro Tyr Ser Gln Ala Val Leu Val AsP 152025 Arg Thr Ile Tyr Ile Ser Gly Gln Leu Gly Met Asp Pro Ala 303540 Ser Gly Gln Leu Val Pro Gly Gly Val Val Glu Glu Ala Lys 455055 Gln Ala Leu Thr Asn Ile Gly Glu Ile Leu Lys Ala Ala Gly 606570 Cys Asp Phe Thr Asn Val Val Lys Ala Thr Val Leu Leu Ala 7580 Asp Ile Asn Asp Phe Ser Ala Val Asn Asp Val Tyr Lys Gln 859095 Tyr Phe Gln Ser Ser Phe Pro Ala Arg Ala Ala Tyr Gln Val 100105110 Ala Ala Leu Pro Lys Gly Gly Arg Val Glu Ile Glu Ala Ile 115120125 Ala Val Gln Gly Pro Leu Thr Thr Ala Ser Val 130135

3. Use according to claim 1, wherein the protein used has at least 80% homology with the protein of claim 2.

4. A pharmaceutical composition comprising as active ingredient a protein according to claims 1-3 in admixture with suitable excipients.

5. Use according to claim 1, for the preparation of a medicament for the prevention and treatment of post-transplant atherosclerosis.

6. Use according to claim 1, for the preparation of a medicament for the prevention and treatment of arthritis.

7. Use according to claim 1, for the preparation of a medicament for the prevention and treatment of multiple sclerosis.

8. The use according to claim 1, for the preparation of a medicament for the prevention and treatment of diabetes.