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 atheroscelerosis, arthritis, multiple sclerosis, and diabetes.

Description:
CROSS-REFERENCE TO PRIOR APPLICATIONS  
       [0001]    This is a divisional of copending application Ser. No. 09/269,019, filed on Apr. 6, 1999, which was in turn a 35 USC 371 U.S. national stage application of international application number PCT/EP97/05079, filed on Sep. 17, 1997. 
     
    
     
       FIELD OF THE INVENTION  
         [0002]    The present invention relates to the use of proteins extractable from animal organs, particularly from livers of mammals, for the preparation of medicaments active against autoimmune diseases, in particular activity against atherosclerosis, arthritis, multiple sclerosis, and diabetes.  
         BACKGROUND OF THE INVENTION  
         [0003]    The administration of complete Freund&#39;s adjuvant has proved to be capable of inducing an experimental arthritis very similar to rheumatoid arthritis in rats. On the other hand, the administration of adjuvant to rabbits induces no arthritic pathology, but atherosclerosis. The studies carried out have evidenced that, in both lesions, immunoreactivity to an endogenous factor, which has been identified as the Heat Shock Protein 60 (HSP60), is present. Subsequent searches have confirmed these observations, proving that the administration of complete Freund&#39;s adjuvant can be replaced by the administration of HSP60, resulting in the same pathologies. Afterwards, pre-treatment of rat with adjuvant, HSP60 or fragments thereof has proved to prevent the onset of arthritis, with a still obscure mechanism, whereas the administration subsequent to the adjuvant worsens the progress of the disease.  
           [0004]    More recently, pre-treatment with adjuvant has been found to also prevent other experimental pathologies which can be defined, generally speaking, as autoimmune disease, such as diabetes or experimental allergic encephalomyelitis (EAE). Finally, HSP60 has been found to have structural analogies to a high number of autoantigens, therefore it is assumed to be related to pathologies more widely than what up to now observed.  
           [0005]    WO 92/10197 disclosed protein fractions extractable with perchloric acid from organs of mammals, and their use as anticancer agents. Within these fractions, three main components could be identified, having molecular weights 50, 14 and 10 KDa on gel electrophoresis. The purified extract containing these three components will be referred to as UK  101  in the following. The sequence of the 14 KDa protein component, which is the main, if not the only, responsible for the described activities, is reported in the Table hereinbelow and in WO 96/02567, and it has turned out to be related to that described by other authors (Levy-Pavatier, Eur. Biochem. 1903, 212 (3) 665-73) which have assumed that the novel identified sequences belong to the family of the proteins known as chaperoning, to which the HSPs themselves-belong.  
         SUMMARY OF THE INVENTION  
         [0006]    The proteins described in WO 92/10197 and those of WO 96/02567 (in the following referred to as UK  114 ) show anyhow properties never observed for chaperonins or analogous proteins. More specifically, it has been found that said proteins can be used in the prevention and in the treatment of autoimmune diseases, in particular atherosclerotic conditions, such as the atherosclerosis induced by organ transplants, arthritis, multiple sclerosis, and diabetes. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0007]    The invention relates preferably to the use of the purified proteins UK  101  and UK  114  for the preparation of medicaments for the prevention and the treatment of autoimmune diseases such as atherosclerosis following organ transplants, arthritis, multiple sclerosis, diabetes.  
         [0008]    Moreover the invention comprises the use of proteins showing a high homology degree to UK  114 , of at least 80%, preferably of at least 90%.  
       ANTIATHEROSCLEROTIC ACTIVITY  
       [0009]    It has been ascertained that nowadays the more frequent cause of failure of organ transplants in time is no more the rejection, but the formation of atherosclerotic plaques at the contact point between the vases of the transplanted organ and those of the host. This pathology is worsened by the usual immunosuppressors such as cyclosporin, whereas the use of AZT, which is however very toxic, appears to be useful.  
         [0010]    The activity of the proteins UK  101  and UK  114  has been evidenced using both a conventional atherosclerosis model, which is that of the rabbit pre-treated with complete Freund&#39;s adjuvant, and a transplant atherosclerosis model. In the first case, the subcutaneous treatment with adjuvant induces within 21 days the formation of atherosclerotic plaques at the iliac bifurcation and at the aortic arch. The pretreatment (7 days before) with UK  101  or UK  114  has significantly prevented the development of the pathology in a high percent of cases compared with the treatment with the only adjuvant, which has lead to the development of the disease in all of the animals.  
         [0011]    On the other hand, the experimental model of transplant atherosclerosis consists in the venous by-passes at the level of arteries in the rat. After a short time, the formation of atherosclerotic plaques at the level of the host vase, as it happens in the human pathology, has been observed. The pre-treatment (7 days before) with UK  101  or UK  114  has significantly prevented the development of the pathology in a high percent of cases, compared with what observed in the animals non pre-treated before the transplant.  
       ANTIARTHRITIS ACTIVITY  
       [0012]    This activity has been evidenced using a conventional arthritis model, which is the adjuvant-induced arthritis. In this model, Lewis rats are injected at the tail base with complete Freund&#39;s adjuvant: within 7 days, a pathology at the rear leg appears, characterized by swelling and joints alterations. The pathology reaches its peaks from the 14th to the 21st day, then decreasing until the leg returns to normal conditions. The pre-treatment (7 days before) with UK  101  or UK  114  has significantly prevented the development of the pathology in a high percent of cases compared with treatment with the only adjuvant, which has lead to the development of the pathology in 100% of the animals. The treatment with UK  101  or UK  114  after the administration of adjuvant has worsened the progress of the pathology.  
         [0013]    Therefore, it is considered that UK  101  and UK  114  are capable of modifying the progress of or of preventing pathological conditions such as arthritis and rheumatoid arthritis.  
       ACTIVITY AGAINST MULTIPLE SCLEROSIS  
       [0014]    This has been evidenced using a conventional multiple sclerosis model: the experimental allergic encephalomyelitis (EAE). The pathology is induced injecting subcutaneously Lewis rats with a Guinea-pig spinal cord homogenate together with complete Freund&#39;s adjuvant. The pathology appears as a progressive paralysis starting from the rear limbs, which begins at about the 12th day, reaches a maximum at about the 21st day and undergoes remission at about the 30th day from the administration of the immunogen. The pre-treatment (7 days before) with UK  101  or UK  114  has significantly prevented the development of the pathology in a high percent of cases and a less serious pathology has appeared, compared with treatment with the only marrow homogenate and adjuvant, which has lead to the development of the pathology in 100% of the animals.  
         [0015]    Therefore UK  101  and UK  114  are believed to be able of changing the progress of or preventing pathological conditions such as multiple sclerosis.  
       ANTIDIABETIC ACTIVITY  
       [0016]    This has been evidenced using a conventional diabetes model, represented by the BB rat which spontaneously develops diabetes around the 45th day of life. The animals have been treated at the 30th day of life with UK  101  or UK  114  and the development of the pathology has been observed, compared with untreated control animals. The pre-treatment has been found to decrease the incidence and the 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 a compassionate treatment with the substance. All of the patients treated, independently of the effect on the tumor pathology, have shown a remission of the diabetic pathology going so far as to quit the insulin therapy.  
         [0017]    Therefore UK  101  and UK  114  are believed to be capable of changing the course of diabetes or of preventing it.  
         [0018]    The antidiabetic activity has in fact been confirmed, although up to now in a limited number of cases, also in vivo in patients suffering from diabetes.  
       Administration  
       [0019]    The proteins of the invention can be administered using suitable formulations, mainly injectable.  
         [0020]    The pattern of the administration (doses, frequency of administration, etc.) will be determined according to the circumstances, depending on factors such as conditions of the patient, phase of the disease, etc., but usually a daily dosage ranging from 1 to 100 mg will be suitable.  
                           TABLE                                       Met Ser Glu Asn Ser Glu Glu Pro Val Gly Glu Ala               1            5                   10                       Lys Ala Pro Ala Ala Ile Gly Pro Tyr Ser Gln Ala                   15                    20                       Val Leu Val Asp Arg Thr Ile Tyr Tie Ser Gly Gln             25                30               35                       Leu Gly Met Asp Pro Ala Ser Gly Gln Leu Val Pro                    40                  45                       Gly Gly Val Val Glu Glu Ala Lys Gln Ala Leu Thr            50                  55                  60                       Asn Ile Gly Glu Ile Leu Lys Ala Ala Gly Cys Asp                        65                  70                       Phe Thr Asn Val Val Lys Ala Thr Val Leu Leu Ala                75                  80                       Asp Ile Asn Asp Phe Ser Ala Val Asn Asp Val Tyr           85                    90                  95                       Lys Gln Tyr Phe Gln Ser Ser Phe Pro Ala Arg Ala                       100                   105                       Ala Tyr Gln Val Ala Ala Leu Pro Lys Gly Gly Arg                 110            115                 120                       Val Glu Ile Glu Ala Ile Ala Val Gln Gly Pro Leu                        125                 130                       Thr Thr Ala Ser Val                135                      
 
         [0021]    [0021] 
     
       
       
         1 
         
           
             1  
           
           
             1  
             137  
             PRT  
             Unknown  
             
               Protein extracted with perchloric acid from 
      mammalian liver  
             
           
            1 

Met Ser Glu Asn Ser Glu Glu Pro Val Gly Glu Ala Lys Ala Pro Ala 
1               5                   10                  15 

Ala Ile Gly Pro Tyr Ser Gln Ala Val Leu Val Asp Arg Thr Ile Tyr 
            20                  25                  30 

Ile Ser Gly Gln Leu Gly Met Asp Pro Ala Ser Gly Gln Leu Val Pro 
        35                  40                  45 

Gly Gly Val Val Glu Glu Ala Lys Gln Ala Leu Thr Asn Ile Gly Glu 
    50                  55                  60 

Ile Leu Lys Ala Ala Gly Cys Asp Phe Thr Asn Val Val Lys Ala Thr 
65                  70                  75                  80 

Val Leu Leu Ala Asp Ile Asn Asp Phe Ser Ala Val Asn Asp Val Tyr 
                85                  90                  95 

Lys Gln Tyr Phe Gln Ser Ser Phe Pro Ala Arg Ala Ala Tyr Gln Val 
            100                 105                 110 

Ala Ala Leu Pro Lys Gly Gly Arg Val Glu Ile Glu Ala Ile Ala Val 
        115                 120                 125 

Gln Gly Pro Leu Thr Thr Ala Ser Val 
    130                 135