Patent Publication Number: US-2012028985-A1

Title: Combinational therapy for treating autoimmune disease

Description:
FIELD OF THE INVENTION 
     This invention relates to the field of therapeutics more in particular it relates to a pharmaceutical composition. It also relates to a method for treating autoimmune diseases by administration of methotrexate or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof or a tautomer thereof and a compound according to formula (I). 
     BACKGROUND OF THE INVENTION 
     Autoimmune diseases are caused by a misguided immune response regarding parts of the body as foreign. For instance in rheumatoid arthritis parts of the joints are attacked by the immune system, whereas multiple sclerosis is characterized by loss of the myelin sheath due to autoimmune attack. 
     Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and degeneration of joints. During the course of the disease irreversible joint destruction takes place in addition to extra-articular manifestations. This disorder leads to severe disability and significant reduction in quality of life. 
     RA is a disease which is quite common especially among the elderly. Its treatment with conventional medications such as non-steroid anti-inflammatory agents is not satisfactory. In view of the increasing aging of the population, especially in the developed Western countries and in Japan the development of new medications for the treatment of RA is urgently required. 
     In general most cells rely on the salvage pathway to receive the nucleotides required for cell division. But in cases of rapid proliferation this source is not adequate. These rapidly dividing cells have to produce nucleotides de novo. This occurs by two independent pathways for purines and pyrimidines. The most important example for rapid cell proliferation is the clonal expansion of lymphocytes during an immune response. Evidently, a selective inhibitor of this process can be of great therapeutically value for treating RA, and other autoimmune diseases, and for oncological applications. 
     Dihydroorotate dehydrogenase (DHODH) is the rate limiting enzyme for the de novo synthesis of pyrimidines. This enzyme is a highly promising target for the treatment of the above mentioned diseases. 
     In fact, various inhibitors have already been identified. Some of them have been clinically tested. The most important example is leflunomide that is currently being used in the routine treatment of rheumatoid arthritis. However, for other compounds like brequinar of FK778 the development has been discontinued in the clinical phase (D V Cramer (1996): Transplant Proc 28, 960 to 963; A Ma and H Chen (2002): Curr Drug Targets Cardiovasc Haematol Disord 2, 57-71). 
     Experiences of the recent year clearly showed that combination therapy frequently yields improved efficacy compared to monotherapy, especially in early RA. This is in particular true for combinations of disease modifying antirheumatic drugs (DMARDs) with biological agents but also for co-administration of two or more DMARDs (J M Kremer et al. (2002): Ann Intern Med 137, 726-733). 
     It would thus be advantageous to have a combination therapy at hand that yields improved efficacy compared to monotherapy. 
     Methotrexate is currently one of the most widely prescribed DMARDs for the treatment of rheumatoid arthritis. Combination therapy of methotrexate with other DMARDs does increase the clinical success of low-dose methotrexate treatment. This has been demonstrated and published e.g. for leflunomide (Ann Intern Med. 2002 Nov. 5; 137(9):142, Clin Exp Rheumatol. 1999 November-December; 17(6 Suppl 18):566-8 and Arthritis Rheum. 1999 July: 42(7)1322-8). 
     The synergistic effect can be explained by the different and apparently complementary biochemical mechanisms of action of the two compounds (Semin Arthritis Rheum. 1999 August; 29(1):14-26). Low-dose methotrexate inhibits cytokine production, purine biosynthesis, and, in an animal model, caused the release of adenosine, a potent anti-inflammatory agent. Leflunomide, through inhibition of de novo pyrimidine biosynthesis, can regulate lymphocyte proliferation. 
     The drawback of the combination of methotrexate and leflunomide lies in the toxicity of both compounds to the liver. Severe liver-toxicity has been observed in patients treated with the two compounds (Arthrit. Rheum. 2000; 43(11):2609-11), resulting in the need for careful monitoring in all patients treated with this combination. Therefore, due to the observed additive or even potentiated liver toxicity a combination therapy of methotrexate and leflunomide in RA patients is generally contra-indicated. 
     Consequently there is a need for safer compositions in this field, displaying less liver-toxicity while maintaining the positive synergistic clinical effect. 
     DESCRIPTION OF THE INVENTION 
     It has now unexpectedly been found that by administering methotrexate in a combination therapy together with DHODH inhibitors of formula (I) as described herein, not only the efficacy of the treatment is increased, but also toxic side effects of methotrexate are diminished. This particular effect is not observed when leflunomide is applied in combination with methotrexate, where actually a potentiation of toxic side effects is observed. This surprising result indicates that liver toxicity caused by leflunomide is a compound-specific effect and that DHODH inhibition in general is not mandatorily linked to liver toxicity as demonstrated by a compound of formula (I). 
     In a first aspect this invention relates to a kit comprising a first pharmaceutical composition comprising a compound according to formula (I) or a pharmaceutically acceptable salt of formula (I), or a prodrug of formula (I), or a physiologically functional derivative of formula (I), or a stereoisomer of formula (I) or a tautomer of formula (I) and a second pharmaceutical composition comprising methotrexate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a tautomer thereof, 
     
       
         
         
             
             
         
       
         
         
           
             wherein 
             A is an aromatic or non-aromatic 5- or 6-membered hydrocarbon ring wherein optionally one or more of the carbon atoms are replaced by a group X, wherein X is independently selected from the group consisting of S, O, N, NR 4 , SO 2  and SO; 
             L is a single bond or NH; 
             D is O, S, SO 2 , NR 4 , or CH 2 ; 
             Z 1  is O, S, or NR 5 , 
             Z 2  is O, S, or NR 5 ; 
             R 1  independently represents H, halogen, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, —CO 2 R″, —SO 3 H, —OH, —CONR*R″, —CR″O, —SO 2 —NR*R″, —NO 2 , —SO 2 —R″, —SO—R*, —CN, alkanyloxy, alkenyloxy, alkynyloxy, alkanylthio, alkenylthio, alkynylthio, aryl, —NR″—CO 2 —R′, —NR″—CO—R*, —NR″—SO 2 —R′, —O—CO—R*, —O—CO 2 —R*, —O—CO—NR*R″; cycloalkyl, heterocycloalkyl, alkanylamino, alkenylamino, alkynylamino, hydroxyalkanylamino, hydroxyalkenylamino, hydroxyalkynylamino, —SH, heteroaryl, alkanyl, alkenyl or alkynyl; 
             R* independently represents H, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aminoalkanyl, aminoalkenyl, aminoalkynyl, alkanyloxy, alkenyloxy, alkynyloxy, —OH, —SH, alkanylthio, alkenylthio, alkynylthio, hydroxyalkanyl, hydroxyalkenyl, hydroxyalkynyl, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, aryl or heteroaryl; 
             R′ independently represents H, —CO 2 R″, —CONR″R′″, —CR″O, —SO 2 NR″, —NR″—CO-haloalkanyl, haloalkenyl, haloalkynyl, —NO 2 , —NR″—SO 2 -haloalkanyl, haloalkenyl, haloalkynyl, —NR″—SO 2 -alkanyl, —NR″—SO 2 -alkenyl, —NR″—SO 2 -alkynyl, —SO 2 -alkanyl, —SO 2 -alkenyl, —SO 2 -alkynyl, —NR″—CO-alkanyl, —NR″—CO-alkenyl, —NR″—CO-alkynyl, —CN, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aminoalkanyl, aminoalkenyl, aminoalkynyl, alkanylamino, alkenylamino, alkynylamino, alkanyloxy, alkenyloxy, alkynyloxy, -cycloalkyloxy, —OH, —SH, alkanylthio, alkenylthio, alkynylthio, hydroxyalkanyl, hydroxyalkenyl, hydroxyalkynyl, hydroxyalkanylamino, hydroxyalkenylamino, hydroxyalkynylamino, halogen, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, aryl, aralkyl or heteroaryl; 
             R″ independently represents hydrogen, haloalkanyl, haloalkenyl, haloalkynyl, hydroxyalkanyl, hydroxyalkenyl, hydroxyalkynyl, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aminoalkanyl, aminoalkenyl or aminoalkynyl; 
             R′″ independently represents H or alkanyl; 
             R 2  is H or OR 6 , NHR 7 , NR 7 OR 7 ;
           or R 2  together with the nitrogen atom which is attached to R 8  forms a 5 to 7 membered, preferably 5 or 6 membered heteroyclic ring wherein R 2  is —[CH 2 ] s  and R 8  is absent;   
         
             R 3  is H, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, alkanyloxy, alkenyloxy, alkynyloxy, —O-aryl; —O-cycloalkyl, —O-heterocycloalkyl, halogen, aminoalkanyl, aminoalkenyl, aminoalkynyl, alkanylamino, alkenylamino, alkynylamino, hydroxylamino, hydroxylalkanyl, hydroxylalkenyl, hydroxylalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, heteroaryl, alkanylthio, alkenylthio, alkynylthio, —S-aryl; —S-cycloalkyl, —S-heterocycloalkyl, aralkyl, haloalkanyl, haloalkenyl or haloalkynyl; 
             R 4  is H, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl; 
             R 5  is H, OH, alkanyloxy, alkenyloxy, alkynyloxy, O-aryl, alkanyl, alkenyl, alkynyl or aryl; 
             R 6  is H, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, alkanyloxyalkanyl, alkanyloxyalkenyl, alkanyloxyalkynyl, alkenyloxyalkanyl, alkenyloxyalkenyl, alkenyloxyalkynyl, alkynyloxyalkanyl, alkynyloxyalkenyl, alkynyloxyalkynyl, acylalkanyl, (acyloxy)alkanyl, (acyloxy)alkenyl, (acyloxy)alkynyl, non-symmetrical (acyloxy)alkanyldiester, non-symmetrical (acyloxy)alkenyldiester, non-symmetrical (acyloxy)alkynyldiester, or dialkanylphosphate, dialkenylphosphate or dialkynylphosphate; 
             R 7  is H, OH, alkanyl, alkenyl, alkynyl, aryl, alkanyloxy, alkenyloxy, alkynyloxy, —O-aryl, cycloalkyl, heterocycloalkyl, or —O-cycloalkyl, —O-heterocycloalkyl; 
             R 8  is hydrogen, alkanyl, alkenyl or alkynyl; 
             E is an alkanyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl or cycloalkyl group or a fused bi- or tricyclic ring system wherein one phenyl ring is fused to one or two monocyclic cycloalkyl or heterocycloalkyl rings or one bicyclic cycloalkyl or heterocycloalkyl ring, or wherein two phenyl rings are fused to a monocyclic cycloalkyl or heterocycloalkyl ring, wherein monocyclic and bicyclic cycloalkyl and heterocycloalkyl rings are as defined herein, and wherein all of the aforementioned groups may optionally be substituted by one or more substituents R′; 
             Y is hydrogen, halogen, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, alkanyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl or cycloalkyl group or a fused bi- or tricyclic ring system wherein one phenyl ring is fused to one or two monocyclic cycloalkyl or heterocycloalkyl rings or one bicyclic cycloalkyl or heterocycloalkyl ring, or wherein two phenyl rings are fused to a monocyclic cycloalkyl or heterocycloalkyl ring, and wherein all of the aforementioned groups may optionally be substituted by one or more substituents R′, or Y is 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein R 1 , X, A, Z 1 , Z 2 , R 8 , R 2 , E and p are as defined herein; 
                 m is 0 or 1; 
                 n is 0 or 1; 
                 p is 0 or 1; 
                 q is 0 or 1; 
                 r is 0 or 1; 
                 s is 0 to 2; and 
                 t is 0 to 3. 
               
             
           
         
       
    
     In another preferred embodiment, the present invention relates to a compound of formula (I), wherein
         A is an aromatic or non-aromatic 5- or 6-membered hydrocarbon ring wherein optionally one or more of the carbon atoms are replaced by a group X, wherein X is independently selected from the group consisting of S, O, N, NR 4 , SO 2  and SO;   L is a single bond;   D is O, S, SO 2 , NR 4 , or CH 2 ;   Z 1  is O, S, or NR 5 ;   Z 2  is O, S, or NR 5 ;   R 1  independently represents H, halogen, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, —CO 2 R″, —SO 3 H, —OH, —CONR*R″, —CR″O, —SO 2 —NR*R″, —NO 2 , —SO 2 —R″, —SO—R*, —CN, alkanyloxy, alkenyloxy, alkynyloxy, alkanylthio, alkenylthio, alkynylthio, aryl, —NR″—CO 2 —R′, —NR″—CO—R*, —NR″—SO 2 —R′, —O—CO—R*, —O—CO 2 —R*, —O—CO—NR*R″; cycloalkyl, heterocycloalkyl, alkanylamino, alkenylamino, alkynylamino, hydroxyalkanylamino, hydroxyalkenylamino, hydroxyalkynylamino, —SH, heteroaryl, alkanyl, alkenyl or alkynyl;   R* independently represents H, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aminoalkanyl, aminoalkenyl, aminoalkynyl, alkanyloxy, alkenyloxy, alkynyloxy, —OH, —SH, alkanylthio, alkenylthio, alkynylthio, hydroxyalkanyl, hydroxyalkenyl, hydroxyalkynyl, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, aryl or heteroaryl;   R′ independently represents H, —CO 2 R″, —CONR″R′″, —CR″O, —SO 2 NR″, —NR″—CO-haloalkanyl, haloalkenyl, haloalkynyl, —NO 2 , —NR″—SO 2 -haloalkanyl, haloalkenyl, haloalkynyl, —NR″—SO 2 -alkanyl, —NR″—SO 2 -alkenyl, —NR″—SO 2 -alkynyl, —SO 2 -alkanyl, —SO 2 -alkenyl, —SO 2 -alkynyl, —NR″—CO-alkanyl, —NR″—CO-alkenyl, —NR″—CO-alkynyl, —CN, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aminoalkanyl, aminoalkenyl, aminoalkynyl, alkanylamino, alkenylamino, alkynylamino, alkanyloxy, alkenyloxy, alkynyloxy, -cycloalkyloxy, —OH, —SH, alkanylthio, alkenylthio, alkynylthio, hydroxyalkanyl, hydroxyalkenyl, hydroxyalkynyl, hydroxyalkanylamino, hydroxyalkenylamino, hydroxyalkynylamino, halogen, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, aryl, aralkyl or heteroaryl;   R″ independently represents hydrogen, haloalkanyl, haloalkenyl, haloalkynyl, hydroxyalkanyl, hydroxyalkenyl, hydroxyalkynyl, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aminoalkanyl, aminoalkenyl or aminoalkynyl;   R′″ independently represents H or alkanyl;   R 2  is H or OR 6 , NHR 7 , NR 7 OR 7 ;
           or R 2  together with the nitrogen atom which is attached to R 8  forms a 5 to 7 membered, preferably 5 or 6 membered heteroyclic ring wherein R 2  is —[CH 2 ] s  and R 8  is absent;   R 3  is H, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, alkanyloxy, alkenyloxy, alkynyloxy, —O-aryl; —O-cycloalkyl, —O-heterocycloalkyl, halogen, aminoalkanyl, aminoalkenyl, aminoalkynyl, alkanylamino, alkenylamino, alkynylamino, hydroxylamino, hydroxylalkanyl, hydroxylalkenyl, hydroxylalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, heteroaryl, alkanylthio, alkenylthio, alkynylthio, —S-aryl; —S-cycloalkyl, —S-heterocycloalkyl, aralkyl, haloalkanyl, haloalkenyl or haloalkynyl;   
           R 4  is H, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;   R 5  is H, OH, alkanyloxy, alkenyloxy, alkynyloxy, O-aryl, alkanyl, alkenyl, alkynyl or aryl;   R 6  is H, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, alkanyloxyalkanyl, alkanyloxyalkenyl, alkanyloxyalkynyl, alkenyloxyalkanyl, alkenyloxyalkenyl, alkenyloxyalkynyl, alkynyloxyalkanyl, alkynyloxyalkenyl, alkynyloxyalkynyl, acylmethyl, (acyloxy)alkanyl, (acyloxy)alkenyl, (acyloxy)alkynyl, non-symmetrical (acyloxy)alkanyldiester, non-symmetrical (acyloxy)alkenyldiester, non-symmetrical (acyloxy)alkynyldiester, or dialkanylphosphate, dialkenylphosphate or dialkynylphosphate;   R 7  is H, OH, alkanyl, alkenyl, alkynyl, aryl, alkanyloxy, alkenyloxy, alkynyloxy, —O-aryl, cycloalkyl, heterocycloalkyl, or —O-cycloalkyl, —O-heterocycloalkyl;   R 8  is hydrogen, alkanyl, alkenyl or alkynyl;   E is an alkanyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl or cycloalkyl group or a fused bi- or tricyclic ring system wherein one phenyl ring is fused to one or two monocyclic cycloalkyl or heterocycloalkyl rings or one bicyclic cycloalkyl or heterocycloalkyl ring, or wherein two phenyl rings are fused to a monocyclic cycloalkyl or heterocycloalkyl ring, wherein monocyclic and bicyclic cycloalkyl and heterocycloalkyl rings are as defined herein, and wherein all of the aforementioned groups may optionally be substituted by one or more substituents R′;   Y is hydrogen, halogen, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, alkanyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl or cycloalkyl group or a fused bi- or tricyclic ring system wherein one phenyl ring is fused to one or two monocyclic cycloalkyl or heterocycloalkyl rings or one bicyclic cycloalkyl or heterocycloalkyl ring, or wherein two phenyl rings are fused to a monocyclic cycloalkyl or heterocycloalkyl ring, and wherein all of the aforementioned groups may optionally be substituted by one or more substituents R′, or Y is       

     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein R 1 , X, A, Z 1 , Z 2 , R 8 , R 2 , E and p are as defined herein; 
                 m is 0 or 1; 
                 n is 0 or 1; 
                 p is 0 or 1; 
                 q is 0 or 1; 
                 r is 0 or 1; 
                 s is 0 to 2; and 
                 t is 0 to 3. 
               
             
           
         
       
    
     In another preferred embodiment, the present invention relates to a compound of formula (I), wherein
         A is an aromatic or non-aromatic 5- or 6-membered hydrocarbon ring wherein optionally one or more of the carbon atoms are replaced by a group X, wherein X is independently selected from the group consisting of S, O, N, N R 4 , SO 2  and SO;   L is NH;   D is O, S, SO 2 , NR 4 , or CH 2 ;   Z 1  is O, S, or NR 5 ;   Z 2  is O, S, or NR 5 ;
           R 1  independently represents H, halogen, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, —CO 2 R″, —SO 3 H, —OH, —CONR*R″, —CR″O, —SO 2 —NR*R″, —NO 2 , —SO 2 —R″, —SO—R*, —CN, alkanyloxy, alkenyloxy, alkynyloxy, alkanylthio, alkenylthio, alkynylthio, aryl, —NR″—CO 2 —R′, —NR″—CO—R*, —NR″—SO 2 —R′, —O—CO—R*, —O—CO 2 —R*, —O—CO—NR*R″; cycloalkyl, heterocycloalkyl, alkanylamino, alkenylamino, alkynylamino, hydroxyalkanylamino, hydroxyalkenylamino, hydroxyalkynylamino, —SH, heteroaryl, alkanyl, alkenyl or alkynyl;   
           R* independently represents H, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aminoalkanyl, aminoalkenyl, aminoalkynyl, alkanyloxy, alkenyloxy, alkynyloxy, —OH, —SH, alkanylthio, alkenylthio, alkynylthio, hydroxyalkanyl, hydroxyalkenyl, hydroxyalkynyl, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, aryl or heteroaryl;   R′ independently represents H, —CO 2 R″, —CONR″R′″, —CR″O, —SO 2 NR″, —NR″—CO-haloalkanyl, haloalkenyl, haloalkynyl, —NO 2 , —NR″—SO 2 -haloalkanyl, haloalkenyl, haloalkynyl, —NR″—SO 2 -alkanyl, —NR″—SO 2 -alkenyl, —NR″—SO 2 -alkynyl, —SO 2 -alkanyl, —SO 2 -alkenyl, —SO 2 -alkynyl, —NR″—CO-alkanyl, —NR″—CO-alkenyl, —NR″—CO-alkynyl, —CN, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aminoalkanyl, aminoalkenyl, aminoalkynyl, alkanylamino, alkenylamino, alkynylamino, alkanyloxy, alkenyloxy, alkynyloxy, -cycloalkyloxy, —OH, —SH, alkanylthio, alkenylthio, alkynylthio, hydroxyalkanyl, hydroxyalkenyl, hydroxyalkynyl, hydroxyalkanylamino, hydroxyalkenylamino, hydroxyalkynylamino, halogen, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, aryl, aralkyl or heteroaryl;   R″ independently represents hydrogen, haloalkanyl, haloalkenyl, haloalkynyl, hydroxyalkanyl, hydroxyalkenyl, hydroxyalkynyl, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aminoalkanyl, aminoalkenyl or aminoalkynyl;   R′″ independently represents H or alkanyl;   R 2  is H or OR 6 , NHR 7 , NR 7 OR 7 ;
           or R 2  together with the nitrogen atom which is attached to R 8  forms a 5 to 7 membered, preferably 5 or 6 membered heteroyclic ring wherein R 2  is —[CH 2 ] s  and R 8  is absent;   
           R 3  is H, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, alkanyloxy, alkenyloxy, alkynyloxy, —O-aryl; —O-cycloalkyl, —O-heterocycloalkyl, halogen, aminoalkanyl, aminoalkenyl, aminoalkynyl, alkanylamino, alkenylamino, alkynylamino, hydroxylamino, hydroxylalkanyl, hydroxylalkenyl, hydroxylalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, heteroaryl, alkanylthio, alkenylthio, alkynylthio, —S-aryl; —S-cycloalkyl, —S-heterocycloalkyl, aralkyl, haloalkanyl, haloalkenyl or haloalkynyl;   R 4  is H, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl;   R 5  is H, OH, alkanyloxy, alkenyloxy, alkynyloxy, O-aryl, alkanyl, alkenyl, alkynyl or aryl;   R 6  is H, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, alkanyloxyalkanyl, alkanyloxyalkenyl, alkanyloxyalkynyl, alkenyloxyalkanyl, alkenyloxyalkenyl, alkenyloxyalkynyl, alkynyloxyalkanyl, alkynyloxyalkenyl, alkynyloxyalkynyl, acylmethyl, (acyloxy)alkanyl, (acyloxy)alkenyl, (acyloxy)alkynyl, non-symmetrical (acyloxy)alkanyldiester, non-symmetrical (acyloxy)alkenyldiester, non-symmetrical (acyloxy)alkynyldiester, or dialkanylphosphate, dialkenylphosphate or dialkynylphosphate;   R 7  is H, OH, alkanyl, alkenyl, alkynyl, aryl, alkanyloxy, alkenyloxy, alkynyloxy, —O-aryl, cycloalkyl, heterocycloalkyl, or —O-cycloalkyl, —O-heterocycloalkyl;   R 8  is hydrogen, alkanyl, alkenyl or alkynyl;   E is an alkanyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl or cycloalkyl group or a fused bi-or tricyclic ring system wherein one phenyl ring is fused to one or two monocyclic cycloalkyl or heterocycloalkyl rings or one bicyclic cycloalkyl or heterocycloalkyl ring, or wherein two phenyl rings are fused to a monocyclic cycloalkyl or heterocycloalkyl ring, wherein monocyclic and bicyclic cycloalkyl and heterocycloalkyl rings are as defined herein, and wherein all of the aforementioned groups may optionally be substituted by one or more substituents R′;   Y is hydrogen, halogen, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, alkanyl, alkenyl, alkynyl, aryl, heteroaryl, heterocycloalkyl or cycloalkyl group or a fused bi- or tricyclic ring system wherein one phenyl ring is fused to one or two monocyclic cycloalkyl or heterocycloalkyl rings or one bicyclic cycloalkyl or heterocycloalkyl ring, or wherein two phenyl rings are fused to a monocyclic cycloalkyl or heterocycloalkyl ring, and wherein all of the aforementioned groups may optionally be substituted by one or more substituents R′, or Y is       

     
       
         
         
             
             
         
       
         
         
           
             
               
                 wherein R 1 , X, A, Z 1 , Z 2 , R 8 , R 2 , E and p are as defined herein; 
                 m is 0 or 1; 
                 n is 0 or 1; 
                 p is 0 or 1; 
                 q is 0 or 1; 
                 r 1; 
                 s is 0 to 2; and 
                 t is 0 to 3. 
               
             
           
         
       
    
     In another preferred embodiment, the present invention relates to a compound of formula (I), wherein:
         A is an aromatic or non-aromatic 5- or 6-membered hydrocarbon ring wherein optionally one or more of the carbon atoms are replaced by a group X, wherein X is independently selected from the group consisting of S, O, N, N R 4 , SO 2  and SO;   L is a single bond or NH;   D is O;   Z 1  is O;   Z 2  is O;   R 1  independently represents H, halogen, haloalkanyl, haloalkanyloxy, —CO 2 R″, —OH, —CN, alkanyloxy, cycloalkyl, heterocycloalkyl, alkanylamino, heteroaryl, alkanyl;   R* independently represents H, alkanyl, R′ independently represents H, cycloalkyl, hydroxyalkanyl, halogen, haloalkanyl, haloalkanyloxy;   R″ independently represents hydrogen, alkanyl;   R 2  is H or OR 6 ;   R 3  is H;   R 4  is H, alkanyl, cycloalkyl;   R 6  is H, alkanyl;   R 8  is hydrogen, alkanyl;   E is an alkanyl, aryl, heteroaryl, heterocycloalkyl or cycloalkyl group, wherein all of the aforementioned groups may optionally be substituted by one or more substituents R′;   Y is aryl, heteroaryl, heterocycloalkyl or cycloalkyl group wherein all of the aforementioned groups may optionally be substituted by one or more substituents R′;
           m is 0 or 1;   n is 0 or 1;   q is 0 or 1;   r is 0 or 1;   and   t is 0 or 1.   
               

     Preferably, in the compounds of Formula (I) L=single bond, and/or Z 1 ═O (thus r=1), and/or Z 2 ═O, and/or q=0, and/or t=1, and/or R 2 ═OH, and/or R 8 ═H. 
     Preferably, in the compounds of Formula (I) E is phenylene which is either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and Y is phenyl which is also either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and A is an aromatic hydrocarbon ring, wherein a carbon is replaced by S. 
     In a further particularly preferred embodiment, in compounds of formula (I), L=single bond, Z 1 ═O (thus r=1), Z 2 ═O, q=0, t=1, R 2 ═OH, R 8 ═H and E is phenylene which is either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and Y is phenyl which is also either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and A is an aromatic hydrocarbon ring, wherein a carbon is replaced by S. 
     In a further particularly preferred embodiment, in compounds of formula (I), L=single bond, Z 1 ═O (thus r=1), Z 2 ═O, q=0, t=1, R 2 ═OH, R 8 ═H and E is phenylene which is either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and Y is phenyl which is also either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and A is a non-aromatic hydrocarbon ring, wherein a carbon is replaced by S. 
     In a further particularly preferred embodiment, in compounds of formula (I), L=single bond, Z 1 ═O (thus r=1), Z 2 ═O, q=0, t=1, R 2 ═OH, R 8 ═H and E is phenylene which is either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and Y is phenyl which is also either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and A is a non-aromatic hydrocarbon ring. 
     In a further particularly preferred embodiment, in compounds of formula (I), L=single bond, Z 1 ═O (thus r=1), Z 2 ═O, q=0, t=1, R 2 ═OH, R 8 ═H and E is phenylene which is either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and Y is phenyl which is also either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and A is an aromatic hydrocarbon ring, wherein a carbon is replaced by O. 
     In a further particularly preferred embodiment, in compounds of formula (I), L=NH, Z 1 ═O (thus r=1), Z 2 ═O, q=0, t=1, R 2 ═OH, R 8 ═H and E is phenylene which is either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and Y is phenyl which is also either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and A is an aromatic hydrocarbon ring. 
     In a further particularly preferred embodiment, in compounds of formula (I), L=single bond, r=0, Z 2 ═O, q=0, t=1, R 2 ═OH, R 8 ═H and E is heteroaryl which is either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and Y is phenyl which is also either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and A is an aromatic hydrocarbon ring. 
     In a further particularly preferred embodiment, in compounds of formula (I), L=single bond, r=0, Z 2 ═O, q=0, t=1, R 2 ═OH, R 8 ═H and E is phenylene which is either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and Y is phenyl which is also either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and A is an aromatic hydrocarbon ring, wherein a carbon is replaced by N. 
     In a further particularly preferred embodiment, in compounds of formula (I), L=single bond, Z 1 ═O (thus r=1), Z 2 ═O, D=O, m=1, n=1, q=1, t=1, R 2 ═OH, R 3 =H, R 8 ═H and E is phenyl which is either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and Y is phenyl which is also either unsubstituted or substituted preferably with Cl, F and/or CF 3 , OCH 3 , OCH 2 CH 3 , or OCF 3 , and A is a non-aromatic hydrocarbon ring. 
     Another further particular embodiment of the compounds of formula (I) according to this invention is a compound of formula (II) or a pharmaceutically acceptable salt, or a prodrug, or a physiologically functional derivative, or a stereoisomer or a tautomer thereof. 
     
       
         
         
             
             
         
       
     
     Other further particular embodiments of the compounds of formula (I) according to this invention are compounds selected from the following:
         1. 3-(2,3,5,6-Tetrafluoro-3′-trifluoromethoxy-biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylic acid;   2. 4-(2′-Chloro-3,5-difluoro-biphenyl-4-ylcarbamoyl)-2,5-dihydro-thiophene-3-carboxylic acid;   3. 2-[3-Chloro-4-(2-chloro-6-fluoro-benzyloxy)-phenylcarbamoyl]-cyclopent-1-enecarboxylic acid;   4. 2-(2,3,5,6-Tetrafluoro-3′-trifluoromethoxy-biphenyl-4-ylcarbamoyl)-cyclopent-1-enecarboxylic acid;   5. 2-[4-(2-Chloro-6-fluoro-benzyloxy)-3-fluoro-phenylcarbamoyl]-cyclopent-1-enecarboxylic acid;   6. 2-(3-Fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl)-cyclopent-1-enecarboxylic acid;   7. 2-(3-biphenyl-4-ylureido)benzoic acid;   8. 2-(2,3,5,6-tetrafluoro-3′-methoxybiphenyl-4-ylcarbamoyl)furan-3-carboxylic acid;   9. 4-(3′-ethoxy-3,5-difluorobiphenyl-4-ylcarbamoyl)thiophene-3-carboxylic acid;   10. 2-(2,3,5,6-tetrafluoro-3′-methoxybiphenyl-4-ylcarbamoyl)cyclopent-1-enecarboxylic acid;   11. 2-(2,3,5,6-tetrafluoro-2′-methoxybiphenyl-4-ylcarbamoyl)cyclopent-1-enecarboxylic acid;   12. 2-(3,5-difluoro-3′-(trifluoromethoxy)biphenyl-4-ylcarbamoyl)cyclopent-1-enecarboxylic acid;   13. 3-hydroxy-2-(2,3,5,6-tetrafluoro-3′-(trifluoromethoxy)biphenyl-4-ylcarbamoyl)cyclopent-1-enecarboxylic acid;   14. 2-(2-chloro-4′-methoxybiphenyl-4-ylcarbamoyl)cyclopent-1-enecarboxylic acid;   15. 4-(3,5-difluoro-3′-(trifluoromethoxy)biphenyl-4-ylcarbamoyl)thiophene-3-carboxylic acid;   16. 2-[4-(2-Chloro-6-fluoro-benzyloxy)-3-fluoro-phenylcarbamoyl]-cyclopent-1-enecarboxylic acid;   17. 3-(3,5-difluoro-3′-(trifluoromethoxy)biphenyl-4-ylcarbamoyl)thiophene-2-carboxylic acid;   18. 2-(3-fluoro-3′-methoxybiphenyl-4-ylamino)nicotinic acid;   19. 2-(3,5-difluoro-3′-methoxybiphenyl-4-ylamino)nicotinic acid;   20. 5-cyclopropyl-2-(5-methyl-6-(3-(trifluoromethoxy)phenyl)pyridin-3-ylamino)benzoic acid;   21. 2-[4-(2-Chloro-6-fluoro-benzyloxy)-3-fluoro-phenylcarbamoyl]-cyclopent-1-enecarboxylic acid;   22. 2-[3,5-Dichloro-4-(2-chloro-6-fluoro-benzyloxy)-phenylcarbamoyl]-cyclopent-1-enecarboxylic acid;   23. 2-(2-Chloro-4′-dimethylamino-biphenyl-4-ylcarbamoyl)-cyclopent-1-enecarboxylic acid;   24. 3-(3-Fluoro-3′-methoxy-biphenyl-4-ylcarbamoyl)-thiophene-2-carboxylic acid;   25. 4-(2,3,5,6-Tetrafluoro-3′-trifluoromethoxy-biphenyl-4-ylcarbamoyl)-2,5-dihydro-thiophene-3-carboxylic acid;
 
or a pharmaceutically acceptable salt, or a prodrug, or a physiologically functional derivative, or a stereoisomer or a tautomer thereof.
       

     An alkanyl group, if not stated otherwise, denotes a linear or branched C 1 -C 6 -alkanyl, preferably a linear or branched chain of one to five carbon atoms; an alkenyl group, if not stated otherwise, denotes a linear or branched C 2 -C 6 -alkenyl group comprising one or more carbon-carbon double bonds and which may further comprise one or more carbon-carbon single bonds within its hydrocarbon chain; an alkynyl group, if not stated otherwise, denotes a linear or branched C 2 -C 6 -alkynyl group comprising one or more carbon-carbon triple bonds and which may further comprise one or more carbon-carbon double and/or single bonds within its hydrocarbon chain, wherein the alkanyl, alkenyl and alkynyl groups can optionally be substituted by one or more substituents R 9 , preferably by halogen. 
     The C 1 -C 6 -alkanyl, C 2 -C 6 -alkenyl and C 2 -C 6 -alkynyl residue may preferably be selected from the group comprising —CH 3 , —C 2 H 5 , —CH═CH 2 , —C≡CH, —C 3 H 7 , —CH(CH 3 ) 2 , —CH 2 —CH═CH 2 , —C(CH 3 )═CH 2 , —CH═CH—CH 3 , —C≡C—CH 3 , —CH 2 —C≡CH, —C 4 H 9 , —CH 2 —CH(CH 3 ) 2 , —CH(CH 3 )—C 2 H 5 , —C(CH 3 ) 3 , —C 5 H 11 , —C 6 H 13 , —C(R 9− ) 3 , —C 2 (R 9 ) 5 , —CH 2 —C(R 9 ) 3 , —C 3 (R 9 ) 7 , —C 2 H 4 —C(R 9 ) 3 , —C 2 H 4 —CH═CH 2 , —CH═CH—C 2 H 5 , —CH═C(CH 3 ) 2 , —CH 2 —CH═CH—CH 3 , —CH═CH—CH═CH 2 , —C 2 H 4 —C≡CH, —C≡C—C 2 H 5 , CH 2 —C≡C—CH 3 , —C≡C—CH═CH 2 , —CH═CH—C≡CH, —C≡C—C≡CH, —C 2 H 4 —CH(CH 3 ) 2 , —CH(CH 3 )—C 3 H 7 , —CH 2 —CH(CH 3 )—C 2 H 5 , —CH(CH 3 )—CH(CH 3 ) 2 , —C(CH 3 ) 2 —C 2 H 5 , —CH 2 —C(CH 3 ) 3 , —C 3 H 6 —CH═CH 2 , —CH═CH—C 3 H 7 , —C 2 H 4 —CH═CH—CH 3 , —CH 2 —CH═CH—C 2 H 5 , —CH 2 —CH═CH—CH═CH 2 , CH═CH—CH═CH—CH 3 , —CH═CH—CH 2 —CH═CH 2 , —C(CH 3 )═CH—CH═CH 2 , —CH═C(CH 3 )—CH═CH 2 , —CH═CH—C(CH 3 )═CH 2 , —CH 2 —CH═C(CH 3 ) 2 , —C(CH 3 )═C(CH 3 ) 2 , —C 3 H 6 —C≡CH, —C≡C—C 3 H 7 , —C 2 H 4 —C≡C—CH 3 , —CH 2 —C≡C—C 2 H 5 , —CH 2 —C≡C—CH═CH 2 , —CH 2 —CH═CH—C≡CH, —CH 2 —C≡C—C≡CH, —C≡C—CH═CH—CH 3 , —CH═CH—C≡C—CH 3 , —C≡C—C≡C—CH 3 , —C≡C—CH 2 —CH═CH 2 , —CH═CH—CH 2 —C≡CH, —C═C—CH 2 —C≡CH, —C(CH 3 )═CH—CH═CH 2 , —CH═C(CH 3 )—CH═CH 2 , —CH═CH—C(CH 3 )═CH 2 , —C(CH 3 )═CH—C≡CH, —CH═C(CH 3 )—C≡CH, —C≡C—C(CH 3 )═CH 2 , —C 3 H 6 —CH(CH 3 ) 2 , —C 2 H 4 —CH(CH 3 )—C 2 H 5 , —CH(CH 3 )—C 4 H 9 , —CH 2 —CH(CH 3 )—C 3 H 7 , —CH(CH 3 )—CH 2 —CH(CH 3 ) 2 , —CH(CH 3 )—CH(CH 3 )—C 2 H 5 , —CH 2 —CH(CH 3 )—CH(CH 3 ) 2 , —CH 2 —C(CH 3 ) 2 —C 2 H 5 , —C(CH 3 ) 2 —C 3 H 7 , —C(CH 3 ) 2 —CH(CH 3 ) 2 , —C 2 H 4 —C(CH 3 ) 3 , —CH(CH 3 )—C(CH 3 ) 3 , —C 4 H 8 —CH═CH 2 , —CH═CH—C 4 H 9 , —C 3 H 6 —CH═CH—CH 3 , —CH 2 —CH═CH—C 3 H 7 , —C 2 H 4 —CH═CH—C 2 H 5 , —CH 2 —C(CH 3 )═C(CH 3 ) 2 , —C 2 H 4 —CH═C(CH 3 ) 2 , —C 4 H 8 —C≡CH, —C≡C—C 4 H 9 , —C 3 H 6 —C≡C—CH 3 , —CH 2 —C≡C—C 3 H 7 , —C 2 H 4 —C≡C—C 2 H 5 , wherein in all of the above-mentioned groups, one or more of the hydrogen atoms can be replaced by a substituent R 9 , preferably by halogen. 
     R 9  independently represents H, —CO 2 R 10 , —CONR 10 R 11 , —CR 10 O, —SO 2 NR 10 , NR 10 —CO-haloalkanyl, haloalkenyl, haloalkynyl, —NO 2 , —NR 10 —SO 2 -haloalkanyl, haloalkenyl, haloalkynyl, —NR 10 —SO 2 -alkanyl, —NR 10 —SO 2 -alkenyl, —NR 10 —SO 2 -alkynyl, —SO 2 -alkyl, —SO 2 -alkenyl, —SO 2 -alkynyl, —NR 10 —CO-alkanyl, —NR 10 —CO-alkenyl, —NR 10 —CO-alkynyl, —CN, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aminoalkanyl, aminoalkenyl, aminoalkynyl, alkanylamino, alkenylamino, alkynylamino, alkanyloxy, alkenyloxy, alkynyloxy, cycloalkyloxy, —OH, —SH, alkanylthio, alkenylthio, alkynylthio, hydroxyalkanyl, hydroxyalkenyl, hydroxyalkynyl, hydroxyalkanylamino, hydroxyalkenylamino, hydroxyalkynylamino, halogen, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, aryl, aralkyl or heteroaryl. 
     R 10  independently represents hydrogen, haloalkanyl, haloalkenyl, haloalkynyl, hydroxyalkanyl, hydroxyalkenyl, hydroxyalkynyl, alkanyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aminoalkanyl, aminoalkenyl or aminoalkynyl. 
     R 11  independently represents H or alkanyl. 
     A cycloalkyl group denotes a monocyclic non-aromatic hydrocarbon ring containing three to eight carbon atoms, preferably four to eight carbon atoms, or a bicyclic non-aromatic hydrocarbon ring system containing seven to ten carbon atoms, preferably eight to ten carbon atoms, wherein the cycloalkyl group optionally comprises one or more double bonds, and wherein the cycloalkyl group is optionally substituted by one or more residues R 9  as defined above, and wherein in the cycloalkyl group one or two non-consecutive methylene groups may be replaced by a C═O or C═NR 7  group; non-limiting examples of the cycloalkyl group are cyclopropanyl, cyclobutanyl, cyclopentanyl, cyclohexanyl, cycloheptanyl and cyclooctanyl, preferably cyclopentanyl, cyclohexanyl or cycloheptanyl, wherein in the afore-mentioned groups optionally one or more of the hydrogen atoms is replaced by a residue R 9  as defined above. 
     A heterocycloalkyl group denotes a monocyclic non-aromatic hydrocarbon ring containing three to eight carbon atoms, preferably four to eight carbon atoms, or a bicyclic non-aromatic hydrocarbon ring system containing seven to ten carbon atoms, preferably eight to ten carbon atoms, wherein in the heterocycloalkyl group one or more of the carbon atoms of the in the hydrocarbon ring or ring system is replaced by a group selected from the group comprising —N(R 7 )—, —O—, —S—, —S(O)—, —S(O) 2 —; wherein the heterocycloalkyl group optionally comprises one or more double bonds, and wherein the heterocycloalkyl group is optionally substituted by one or more residues R′ as defined above, and wherein in the heterocycloalkyl group one or two methylene groups may be replaced by a C═O or C═NR 7  group; non-limiting examples of the heterocycloalkyl group are azepan-1-yl, piperidinyl, in particular piperidin-1-yl and piperidin-4-yl, piperazinyl, in particular N-piperazinyl and 1-alkylpiperazine-4-yl, morpholine-4-yl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, tetrahydropyranyl, tetrahydrothiophen, sulfolanyl, sulfolenyl, oxazolinyl, isoxazolinyl, oxazolidinyl, oxazolidinon-yl, wherein in the afore-mentioned groups optionally one or more of the hydrogen atoms is replaced by a residue R 9  as defined above. 
     An alkanyloxy, alkenyloxy or alkynyloxy group denotes an —O-alkanyl, —O-alkenyl or —O-alkynyl group, the alkanyl, alkenyl or alkynyl group being as defined above; the alkanyloxy group is preferably a methoxy, ethoxy, isopropoxy, t-butoxy or pentoxy group. 
     An alkanylthio, alkenylthio or alkynylthio group denotes an —S-alkanyl, —S-alkenyl or —S-alkynyl group, the alkanyl, alkenyl or alkynyl group being as defined above. 
     A haloalkanyl, haloalkenyl or haloalkynyl group denotes an alkanyl, alkenyl or alkynyl group which is substituted by one to five halogen atoms, the alkanyl, alkenyl or alkynyl group being as defined above; the haloalkanyl group is preferably a —C(R 12 ) 3 , —C 2 (R 12 ) 5 , —CH 2 —C(R 12 ) 3 , —CH 2 —C(R 12′ ) 3 , —CH(CH 2 (R 12 )) 2 , —C 3 (R 12 ) 7  or —C 2 H 4 —C(R 12 ) 3 , wherein instances of R 12  may the same or different and each R 12  is independently selected from F, Cl, Br or I, preferably F. 
     A hydroxyalkanyl, hydroxyalkenyl or hydroxyalkynyl, group denotes an HO-alkanyl, HO-alkenyl or HO-alkynyl group, the alkanyl, alkenyl or alkynyl group being as defined above. 
     A haloalkanyloxy, haloalkenyloxy or haloalkynyloxy group denotes an alkanyloxy, alkenyloxy or alkynyloxy group which is substituted by one to five halogen atoms, the alkanyl, alkenyl or alkynyl group being as defined above; the haloalkanyloxy, haloalkenyloxy or haloalkynyloxy group is preferably a —OC(R 12 ) 3 , —OC 2 (R 12 ) 5 , —OCH 2 —C(R 12 ) 3 , —OCH(CH 2 (R 12 )) 2 , —OC 3 (R 12 ) 7  or —OC 2 H 4 —C(R 12 ) 3 , wherein instances of R 12  may the same or different and each R 12  is independently selected from F, Cl, Br or I, preferably F. 
     A cycloalkyloxy group denotes an —O-cycloalkyl group; the cycloalkynyloxy group is preferably cyclopropoyx, cyclobutoxy and cyclopentoxy. 
     A hydroxyalkanylamino, hydroxyalkenylamino or hydroxyalkynylamino group denotes an (HO-alkanyl) 2 -N—, (HO-alkenyl) 2 -N— or (HO-alkynyl) 2 -N— group or HO-alkanyl-NH—, HO-alkenyl-NH— or HO-alkynyl-NH— group, the alkanyl, alkenyl or alkynyl group being as defined above. 
     An alkanylamino, alkenylamino or alkynylamino group denotes an HN-alkanyl, HN-alkenyl or HN-alkynyl or N-dialkanyl, N-dialkenyl or N-dialkynyl group, the alkanyl, alkenyl or alkynyl group being as defined above. 
     A halogen group is chlorine, bromine, fluorine or iodine, fluorine being preferred. 
     An aryl group preferably denotes a mono-, bi-, or tricyclic, preferably monocyclic aromatic hydrocarbon group having six to fourteen carbon atoms, wherein the aryl group is optionally substituted by one or more substituents R′, where R′ is as defined above; the aryl group is preferably-o-C 6 H 4 —R′, -m-C 6 H 4 —R′, -p-C 6 H 4 —R′, or phenyl, 1-naphthyl, 2-naphthyl, anthracenyl, in particular 1-anthracenyl and 2-anthracenyl group which may optionally be substituted by one or more R′, more preferably a phenyl group, -o-C 6 H 4 —R′, -m-C 6 H 4 —R′, -p-C 6 H 4 —R′. 
     A heteroaryl group denotes an aromatic 5-membered monocyclic aromatic hydrocarbon group wherein at least one of the carbon atoms is replaced by a heteroatom like O, N, S, or a- or a 6-membered monocyclic aromatic hydrocarbon group wherein at least one of the carbon atoms is replaced by an N-atom, S, and wherein the aromatic monocyclic 5- or 6-membered cyclic hydrocarbon group is optionally fused to a further monocyclic 5- to 7-membered, preferably 5- or 6-membered, aromatic or non-aromatic hydrocarbon ring, wherein in the further monocyclic aromatic or non-aromatic hydrocarbon ring one or more, preferably one or two carbon atoms may be replaced by a heteroatom like O, N, S; non-limiting examples of heteroaryl groups are thiazol-2-yl, thiazol-4-yl, thiazol-5-yl, isothiazol-3-yl, isothiazol-4-yl, isothiazol-5-yl, 1,2,4-oxadiazol-3-yl, 1,2,4-oxadiazol-5-yl, 1,2,4-thiadiazol-3-yl, 1,2,4-thiadiazol-5-yl, 1,2,5-oxadiazol-3-yl, 1,2,5-oxadiazol-4-yl, 1,2,5-thiadiazol-3-yl, 1-imidazolyl, 2-imidazolyl, 1,2,5-thiadiazol-4-yl, 4-imidazolyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-pyrimidinyl, 3-pyridazinyl, 4-pyridazinyl, 2-pyrazinyl, 1-pyrazolyl, 3-pyrazolyl, 4-pyrazolyl, 1H-tetrazol-2-yl, 1H-tetrazol-3-yl, tetrazolyl, indolyl, indolinyl, benzo[b]furanyl, benzo[b]thiophenyl, benzimidazolyl, benzothiazolyl, quinazolinyl, quinoxazolinyl, or preferably quinolinyl, tetrahydroquinolinyl, isoquinolinyl, tetrahydroisoquinolinyl group; the heteroaryl group can optionally be substituted by one or more substituents R 9 , where R 9  is as defined above; the skilled person will acknowledge that in the above definition the replacement of a “carbon atom” by a heteroatom includes any hydrogen atoms bound to said carbon atom. 
     An aralkyl group denotes an aryl group as defined above which is connected to the molecule of the present invention via an alkanyl, alkenyl or alkynyl bridge, wherein alkanyl, alkenyl or alkynyl is as defined above; preferred aralkyl groups are —CH 2 —C 6 H 5  (benzyl), —CH 2 —CH 2 —C 6 H 5  (phenylethyl), —CH═CH—C 6 H 5 , —C≡C—C 6 H 5 , -o-CH 2 —C 6 H 4 —R′, -m-CH 2 —C 6 H 4 —R′, -p-CH 2 —C 6 H 4 —R′; the aralkyl group can optionally be substituted on the aryl and/or alkanyl, alkenyl or alkynyl part by one or more substituents R 9 , wherein R′ is as defined above. 
     The meaning of E includes alkanyl alkenyl or alkynyl groups optionally substituted by one or more substituents R 9 , wherein alkanyl alkenyl or alkynyl is defined as above and the meaning of E further includes a cycloalkyl group optionally substituted by one or more substituents R 9  such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl or carbocyclic aromatic groups such as phenyl, 1-naphthyl, 2-naphthyl, anthracenyl, in particular 1-anthracenyl and 2-anthracenyl, and heteroaromatic groups such as N-imidazolyl, 2-imidazolyl, 2-thienyl, 3-thienyl, 2-furanyl, 3-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 2-pyranyl, 3-pyranyl, 4-pyranyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 2-pyrazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl and 5-oxazolyl. E also includes fused polycyclic aromatic ring systems such as 9H-thioxanthene-10,10-dioxide in which a carbocyclic aromatic ring or heteroaryl ring is fused to at least one heteroaryl ring. 
     The meaning of Y includes is hydrogen, halogen, alkanyl, alkenyl, alkynyl, cycloalkyl or O-aralkyl, wherein all of the aforementioned groups may optionally be substituted with one or more R′ as defined herein, or alternatively Y is E or —O-E, wherein E is as defined herein; in the aforementioned groups, it is furthermore preferred that optional substituents R′ are halogen. Y can also be 
     
       
         
         
             
             
         
       
     
     wherein A, X, R 1 , R 2 , R 8 , Z 1 , Z 2  and p have the meaning as defined above. 
     Specific Embodiments 
     In certain embodiments A is a 5-membered aromatic hydrocarbon ring wherein one or more, preferably one or two of the carbon atoms are replaced by a group X, wherein X is independently selected from the group consisting of S, O, N or NR 4 . 
     In certain embodiments A is selected from the group comprising the following: 
     
       
         
         
             
             
         
       
       
         
         
             
             
         
       
     
     wherein R 1  and R 4  are defined as above. 
     In certain embodiments A is a 6-membered aromatic hydrocarbon ring wherein one or more, preferably one or two of the carbon atoms are replaced by a group X, wherein X is independently selected from the group consisting of S, O or N. 
     In certain embodiments A is selected from the group comprising the following: 
     
       
         
         
             
             
         
       
     
     wherein R 1  and R 4  are defined as above. 
     In certain embodiments R 1  is H, OH, alkanyl, cycloalkyl, halogen, haloalkanyl CO 2 H or SO 3 H or tetrazole. 
     In certain embodiments R 2  is OH, NH 2 , NHOH, NHR 7 , NR 7 OR 7  or OR 6 . 
     In certain embodiments R 6  is benzoyloxymethyl, isobutyryloxymethyl, 4-aminobutyryloxymethyl, butyryloxymethyl, 1-(butyryloxy)ethyl, 1-(butyryloxy)-2,2-dimethylpropyl, 1-diethylphosphonooxyethyl, 2-(2-methoxyethoxy)-acetyloxymethyl, p-aminobenzoylmethyl, nicotinyloxymethyl, pivalyloxymethyl, glutaryloxymethyl, [2-(2-methoxyethoxy)ethoxy]-acetyloxymethyl, 2-(morpholine-4-yl)-ethyl, 1-diethylphosphonooxymethyl. 
     In certain embodiments R 3  is H, alkanyl, alkenyl, alkynyl, cycloalkyl, aryl, alkanyloxy, alkenyloxy, alkynyloxy, O-aryl; O-cycloalkyl, halogen, aminoalkanyl, aminoalkenyl, aminoalkynyl, akanylamino, akenylamino, akynylamino, hydroxylamino, haloalkanyl, haloalkenyl, haloalkynyl, hydroxylalkanyl, hydroxylalkenyl, hydroxylalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, heteroaryl, alkanylthio, alkenylthio, alkynylthio, S-aryl; S-cycloalkyl, aralkyl, preferably H. 
     In certain embodiments R 4  is H, alkanyl, alkenyl, alkynyl, cycloalkyl, aryl or heteroaryl, preferably H. 
     In certain embodiments R 8  is H or alkanyl, alkenyl, alkynyl, preferably H or methyl. 
     In certain embodiments Z 1  and Z 2  are both O. 
     In certain embodiments Y is hydrogen, halogen, alkanyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, heterocycloalkyl or O-aralkyl, wherein all of the aforementioned groups may optionally be substituted with one or more R 9  as defined herein, or alternatively Y is E or —O-E, wherein E is as defined herein; in the aforementioned groups, it is furthermore preferred that optional substituents R 9  are halogen. Y can also be: 
     
       
         
         
             
             
         
       
     
     wherein A, X, R 1 , R 2 , R 8 , Z 1 , Z 2  and p have the meaning as defined above. Preferably Y is E as defined herein below and more preferably Y is an optionally substituted phenyl. 
     In certain embodiments, the fused bi- or tricyclic ring system is a bicyclic ring system wherein one phenyl ring is fused to a 5- or 6-membered cycloalkyl or heterocycloalkyl ring or alternatively a tricyclic ring system wherein two phenyl rings are fused to a 5- or 6-membered cycloalkyl or heterocycloalkyl ring, wherein in the tricyclic ring system preferably the 5- or 6-membered cycloalkyl or heterocycloalkyl ring is placed between the two phenyl rings, more preferably the tricyclic ring system is 9H-thioxanthene-10,10-dioxide, wherein all of the aforementioned groups are optionally substituted by one or more substituents R 9 . 
     In certain embodiments E is an alkyl or cycloalkyl group, preferably selected from the group comprising methyl, ethyl, propyl, butyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl, wherein all of the aforementioned groups are optionally substituted by one or more substituents R 9 . 
     In certain embodiments E is an aryl or heteroaryl group selected from the group comprising phenyl, 1-naphthyl, 2-naphthyl, anthracenyl, in particular 1-anthracenyl and 2-anthracenyl, N-imidazolyl, 2-imidazolyl, 2-thienyl, 3-thienyl, 2-furanyl, 3-furanyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 2-pyranyl, 3-pyranyl, 4-pyranyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 2-pyrazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-oxazolyl, 4-oxazolyl and 5-oxazolyl, wherein all of the aforementioned groups are optionally substituted by one or more substituents R 9 . 
     In certain embodiments E is a fused bi- or tricyclic ring system, which is optionally substituted by one or more substituents R 9 , preferably a 9H-thioxanthene-10,10-dioxide group, which is optionally substituted by one or more substituents R 9 . 
     In certain embodiments R 9  is selected from the group comprising cyano, nitro, halogen, alkanyloxy, alkenyloxy, alkynyloxy, cycloalkyloxy, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, cycloalkyl, hetreocycloalkyl, heteroaryl, alkanyl, alkenyl, alkynyl or aryl, preferably R 9  is Br, F, Cl, CF 3 , OCF 3 , —CN, cyclopropoxy, cyclobutoxy, isopropoxy, ethoxy or methoxy. 
     In certain embodiments the heteroaryl group is selected from the group comprising imidazolyl, thienyl, furanyl, pyridyl, pyrimidyl, pyranyl, pyrazolyl, pyrazinyl, thiazolyl, 1H-tetrazol-2-yl, 1H-tetrazol-3-yl, or oxazolyl. 
     In certain embodiments t is 0, 1 or 2. 
     In certain embodiments s is 0 or 1. 
     In certain embodiments m=1 and D is O, S, SO 2 , NR 4 , or CH 2 , preferably S or O, more preferably O. 
     In certain embodiments m=0. 
     In certain embodiments q=0. 
     In certain embodiments n=0. 
     In certain embodiments r is 0 or 1. 
     In certain embodiments L is a single bond. 
     In certain embodiments q=1, m=1 and n=1, wherein preferably D=O and/or R 3 ═H). 
     In certain embodiments R 8 ═H. 
     In certain embodiments A is cyclopenten, thiophen, thiaziol or dihydrothiophen. 
     In certain embodiments —(C═Z 1 )—R 2  is COOH. 
     In certain embodiments R 1 ═H. 
     In certain embodiments Y is hydrogen, halogen, haloalkanyl, haloalkenyl, haloalkynyl, haloalkanyloxy, haloalkenyloxy, haloalkynyloxy, alkanyl, alkenyl, alkynyl, cycloalkyl or E, preferably F, CF 3 , OCF 3 , or phenyl, optionally substituted by one or more substituents R 9 , more preferably phenyl, optionally substituted by one or more F, Cl, methoxy, CF 3 , or OCF 3    
     In certain embodiments q=1 and n=0 or 1 and m=1 and D is preferably O. 
     In a preferred embodiment of the kit, the weight ratio of a compound according to formula (I) or a pharmaceutically acceptable salt of formula (I), or a prodrug of formula (I), or a physiologically functional derivative of formula (I), or a stereoisomer of formula (I) or a tautomer of formula (I) in the first pharmaceutical composition, to methotrexate or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof or a tautomer thereof in the second pharmaceutical composition is between 0.05 and 20, preferably between 0.1 and 10, more preferably between 0.2 and 5, even more preferably between 2 and 4 and most preferably between 2.3 and 3.5. 
     It is also preferred that the content of a compound according to formula (I) or a pharmaceutically acceptable salt of formula (I), or a prodrug of formula (I), or a physiologically functional derivative of formula (I) in the first pharmaceutical composition is between about 2 and 60 mg, preferably between about 5 and 50 mg. 
     It is further preferred that the content of methotrexate or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof or a tautomer thereof in the second pharmaceutical composition is between about 5 and 30 mg, preferably between about 10 and 25 mg. 
     In a particularly preferred embodiment of the kit, for each unit of said second pharmaceutical composition seven units of said first pharmaceutical composition are present in the kit. 
     Said kits may be used for the treatment or prevention of immunological and inflammatory disorders. Said disorder is preferably rheumatoid arthritis, psoriasis, atopic dermatitis, transplant rejection, systemic lupus erythematosus, inflammatory bowel disease, lupus nephritis or multiple sclerosis, most preferably rheumatoid arthritis. 
     Preferably the first pharmaceutical composition of the kit is administered once daily and the second pharmaceutical composition is administered once weekly. 
     It is also preferred that both pharmaceutical compositions of the kit are administered orally. 
     Methotrexate is herein also abbreviated as MTX. 
     The compositions described herein are administered via any conventional route. The administration may be carried out, for example, orally, intravenously, intraperitoneally, intramuscularly, subcutaneously or transdermally. 
     The compositions herein described can be administered orally, e.g. in the form of pills, tablets, coated tablets, sugar-coated tablets, hard and soft capsules, powders, granulates, solutions, syrups or suspensions. Administration can also be carried out rectally, e.g. in the form of suppositories, or parentally, e.g. in the form of injections or infusions. 
     The compositions described herein can be preferably administered transdermally, e.g. in the form of transdermal therapeutic systems (e.g. patches) or topical formulations (e.g. liposomes, crèmes, ointment, lotion, gels, dispersion, suspension, spray, solution). Topical formulations can also be administered via the pulmonary or nasal route. 
     In another preferred embodiment the pharmaceutical composition according to the invention is administered orally. 
     In a further aspect the present invention relates to a compound according to formula (I) or a pharmaceutically acceptable salt of formula (I), or a prodrug of formula (I), or a physiologically functional derivative of formula (I), or a stereoisomer of formula (I) or a tautomer of formula (I) for the use in the treatment or prevention of immunological and inflammatory disorders in a patient, wherein the treatment or prevention additionally comprises the application of methotrexate or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof or a tautomer thereof to the patient. 
     The present invention also relates to the use of a compound according to formula (I) or a pharmaceutically acceptable salt of formula (I), or a prodrug of formula (I), or a physiologically functional derivative of formula (I), or a stereoisomer of formula (I) or a tautomer of formula (I) for the manufacture of a pharmaceutical composition for the treatment or prevention of immunological and inflammatory disorders in a patient, wherein the treatment or prevention additionally comprises the application of methotrexate or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof or a tautomer thereof to the patient. 
     The invention further pertains to a method of treating or preventing immunological and inflammatory disorders in a patient comprising administering to the patient a therapeutically effective and tolerable amount of a compound of formula (I) simultaneously, sequentially or separately with a therapeutically effective and tolerable amount of methotrexate or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof or a tautomer thereof. 
     In a preferred embodiment of a compound, the use of a compound and the method of the invention, the a pharmaceutical composition comprising a compound according to formula (I) or a pharmaceutically acceptable salt of formula (I), or a prodrug of formula (I), or a physiologically functional derivative of formula (I), or a stereoisomer of formula (I) or a tautomer of formula (I) is administered once daily. 
     It is further preferred that the daily dosage of a compound according to formula (I) or a pharmaceutically acceptable salt of formula (I), or a prodrug of formula (I), or a physiologically functional derivative of formula (I), or a stereoisomer of formula (I) or a tautomer of formula (I) is in the range of from about 2 mg to about 60 mg, preferably in the range of from about 5 mg to about 50 mg. 
     Preferably, the pharmaceutical composition comprising methotrexate or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof or a tautomer thereof is administered once weekly. 
     In a particularly preferred embodiment, the weekly dosage of methotrexate or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof or a tautomer thereof is in the range of from about 5 mg to about 30 mg, preferably in the range of from about 10 mg to about 25 mg. 
     It is further preferred that the pharmaceutical composition comprising a compound according to formula (I) or a pharmaceutically acceptable salt of formula (I), or a prodrug of formula (I), or a physiologically functional derivative of formula (I), or a stereoisomer of formula (I) or a tautomer of formula (I) is administered orally. 
     It is also preferred that the pharmaceutical composition comprising methotrexate or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof or a tautomer thereof is administered orally. 
     In a preferred embodiment of a compound, the use of a compound and the method of the invention the disorder is rheumatoid arthritis, psoriasis, atopic dermatitis, transplant rejection, inflammatory bowel disease, systemic lupus erythematosus, lupus nephritis or multiple sclerosis, most preferably the disorder is multiple sclerosis or rheumatoid arthritis. 
     Preferably, the weight ratio of a compound according to formula (I) or a pharmaceutically acceptable salt of formula (I), or a prodrug of formula (I), or a physiologically functional derivative of formula (I), or a stereoisomer of formula (I) or a tautomer of formula (I) in the pharmaceutical composition that is preferably administered daily, to methotrexate or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof or a tautomer thereof in the second pharmaceutical composition that is preferably administered weekly is between 0.05 and 20, preferably between 0.1 and 10, more preferably between 0.2 and 5, even more preferably between 2 and 4 and most preferably between 2.3 and 3.5. 
     A further embodiment of the invention is the kit, use or compound of the present invention for the treatment or prevention of a disorder in a patient, the disorder is a disorder wherein the administration of methotrexate to a patient is medically indicated. Thus, the combination therapy presented herein may be applied to treat or prevent any disease which may be treated by methotrexate alone or in combination with further pharmaceutical agents than the ones of formula (I) as described herein. Thereby, the liver-toxicity-diminishing effect is obtainable for the treatment or prevention of all of said diseases. This embodiment encompasses, where applicable, all variations described herein in the further embodiments of the kit, use or compound according to the invention, for instance regarding dosage schemes. 
     Another specific embodiment of the invention is the kit, compound or use according to any of the preceding claims, wherein the decrease of liver enzyme levels (e.g. ALAT) measured in a spectrophotometric assay, by which the liver toxicity is determined, is 20% or higher, compared with liver enzyme levels (e.g. ALAT) measured in a spectrophotometric assay upon administration of a comparable dose of methotrexate alone (e.g. 5 to 30 mg/week), wherein the spectrophotometric assay comprises the following parameters: spectrophotometer (e.g. KONELAB 30i instrument, Thermo Fisher Scientific, 63303 Dreieich, Germany), sample type=serum, sample volume=15 μl, reagent=ALT reagent, reagent volume=115 μl, incubation time=90 sec., measure time=120 sec., result unit=U/L, wavelength (at which absorption is measured)=340 nm. 
     ALT reagent comprises 0.2 M L-alanine, 2.0 mM α-ketoglutarate, 100 mM phosphate buffer at pH 7.4 and ALT color reagent, the color reagent comprising 1.0 mM 2,4-dinitrophenylhydrazine in 1N Hydrochloric Acid. 
     In this context, “a comparable dose of methotrexate alone” means that (within measurement accuracy) the same dose of methotrexate (e.g. 5 to 30 mg/week) is compared to the amount of methotrexate administered in a combination therapy. 
     In certain embodiments of the invention, a compound according to formula (I) or a pharmaceutically acceptable salt of formula (I), or a prodrug of formula (I), or a physiologically functional derivative of formula (I), or a stereoisomer of formula (I) or a tautomer of formula (I) together with methotrexate or a pharmaceutically acceptable salt thereof or a stereoisomer thereof or a tautomer thereof may be comprised in one pharmaceutical composition. These embodiments encompass, where applicable, all variations described herein in the further embodiments of the kit, use or compound according to the invention. 
     In the context of the invention a compound and the composition and their pharmacologically acceptable salts can be administered to animals, preferably to mammals, and in particular to humans, dogs and chickens as therapeutics per se, as mixtures with one another or in the form of pharmaceutical preparations which allow enteral or parenteral use and which as active constituent contain an effective dose of the composition of the invention, or a salt thereof, in addition to customary pharmaceutically innocuous excipients and additives. 
     This invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, H. G. W, Nagel, B. and Kölbl, H. eds., Helvetica Chimica Acta 1995; CH-4010 Basel, Switzerland). 
     Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer&#39;s specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the applicant is not entitled to antedate such disclosure by virtue of prior invention. 
     The terms “treatment”, “treating” or the like are used herein to generally mean obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing the symptoms of a disease or may be therapeutic in terms of a partial or complete cure of a disease. “Treatment” as used herein also covers any treatment of a disease in a mammal, particularly a human. 
     The synthesis of these compounds is disclosed in U.S. patent application Ser. Nos. 10/193,526 and 10/736,711, which are hereby fully incorporated by reference. 
     The present invention relates to compositions with salts of a compound according to formula (I). The salts are preferably cations, most preferably selected from the group consisting of ammonia, arginine, benethamine, benzathine, calcium, choline, deanol, diethanolamine, diethylammonium, ethanolamine, ethylendiamine, meglumine, hydrabamine, imidazole, lysine, magnesium, hydroxyethylmorpholine, piperazine, potassium, epolamine, sodium, trolamine, tromethamine and zinc [Handbook of Pharmaceutical Salts, Ed. P. H. Stahl, C. G. Wermuth, Zurich 2002]. 
     A preferred salt of methotrexate is the di-sodium salt of methotrexate. 
     Preferred compositions described herein may comprise a carrier material or an excipient, including but are not limited to a lipophilic phase (as for example Vaseline, paraffines, triglycerides, waxes, polyalcylsiloxanes) an oil (olive oil, peanut oil, castor oil, triglyceride oil), an emulsifier (as for example lecithin, phosphatidylglyceroles, alkyl alcohols, sodium lauryl sulfate, polysorbats, Cholesterol, sorbitan fatty acid ester, polyoxyethylene fatty acid glycerol and -ester, poloxamers), a preservative (for instance benzalkonium chloride, chlorobutanol, parabene or thiomersal), a flavouring agent, a buffer substance (for example salts of acetic acid, citric acid, boric acid, phosphoric acid, tatric acid, trometamole or trolamine), a solvent (for instance polyethylenglycols, glycerol, ethanol, isopropanol or propyleneglycol), a solubilizer, an agent for achieving a depot effect, a salt for modifying the osmotic pressure, a carrier material for patches (for instance polypropylene, ethylene-vinylacetate-copolymer, polyacrylates, silicon), an antioxidant (for example ascorbate, tocopherol, butylhydroxyanisole, gallic acid esters or butylhydroxytoluol). 
     Suitable carrier materials or excipients may further include but are not limited to fillers and extenders (for example lactose, sucrose, mannitol, starch, cellulose, calcium hydrogenphosphate, calciumcarbonate), disintegrants (e.g. starch, cross-linked polyvinylpyrrolidone), binders (for example polyvinylpyrrolidone, mannitol, starch, tragacanth, cellulose, carboxymethylcellulose sodium, gelatine), gliders (for instance talcum, calcium behenate, stearic acid or magnesium stearate), wetting agents (for example sorbitol or glycerol), stabilizers (for example polyacrylic acids, bentonite), emulsifiers (for example hypromellose, hydoxypropylcellulose), preservatives (for instance benzalkonium chloride, chlorobutanol, parabene or thiomersal), sweetening flavouring or aromatizing agents, buffer substances (for example salts of acetic acid, citric acid, boric acid, phosphoric acid, tatric acid, trometamole or trolamine), solvents (for instance polyethylenglycols, glycerol, ethanol, isopropanol or propyleneglycol) or solubilizers, agents for achieving a depot effect, salts for modifying the osmotic pressure, or coating agents (for instance methylcellulose, hypromellose, hydoxypropylcellulose, carboxymethylcellulose sodium, ethylcellulose, methylhydroxypropylcellulosephthalate, celluloseacetate-phthalate, polyvinylpyrrolidone or copolymers of methacrylic acid and acrylate) or antioxidants (for example ascorbate, tocopherol, butylhydroxyanisole, gallic acid esters or butylhydroxytoluol). 
     The following are examples of the compounds according to the present invention: 
     
       
         
           
               
               
               
             
               
                   
               
               
                 Compound 
                 Structure 
                 UPAC name 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 3-(2,3,5,6-Tetrafluoro-3′- trifluoromethoxy-biphenyl-4- ylcarbamoyl)-thiophene-2-carboxylic  acid 
               
               
                   
               
               
                 2 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 4-(2′-Chloro-3,5-difluoro-biphenyl-4- ylcarbamoyl)-2,5-dihydro-thiophene-3- carboxylic acid 
               
               
                   
               
               
                 3 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-[3-Chloro-4-(2-chloro-6-fluoro- benzyloxy)-phenylcarbamoyl]- cyclopent-1-enecarboxylic acid 
               
               
                   
               
               
                 4 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-(2,3,5,6-Tetrafluoro-3′- trifluoromethoxy-biphenyl-4- ylcarbamoyl)-cyclopent-1- enecarboxylic acid 
               
               
                   
               
               
                 5 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-[4-(2-Chloro-6-fluoro-benzyloxy)-3- fluoro-phenylcarbamoyl]-cyclopent-1- enecarboxylic acid 
               
               
                   
               
               
                 6 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-(3-Fluoro-3′-methoxy-biphenyl-4- ylcarbamoyl)-cyclopent-1- enecarboxylic acid 
               
               
                   
               
               
                 7 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-(3-biphenyl-4-ylureido)benzoic acid 
               
               
                   
               
               
                 8 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-(2,3,5,6-tetrafluoro-3′- methoxybiphenyl-4- ylcarbamoyl)furan-3-carboxylic acid 
               
               
                   
               
               
                 9 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 4-(3′-ethoxy-3,5-difluorobiphenyl-4- ylcarbamoyl)thiophene-3-carboxylic  acid 
               
               
                   
               
               
                 10 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-(2,3,5,6-tetrafluoro-3′- methoxybiphenyl-4- ylcarbamoyl)cyclopent-1- enecarboxylic acid 
               
               
                   
               
               
                 11 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-(2,3,5,6-tetrafluoro-2′- methoxybiphenyl-4- ylcarbamoyl)cyclopent-1- enecarboxylic acid 
               
               
                   
               
               
                 12 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-(3,5-difluoro-3′- (trifluoromethoxy)biphenyl-4- ylcarbamoyl)cyclopent-1- enecarboxylic acid 
               
               
                   
               
               
                 13 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 3-hydroxy-2-(2,3,5,6-tetrafluoro-3′- (trifluoromethoxy)biphenyl-4- ylcarbamoyl)cyclopent-1- enecarboxylic acid 
               
               
                   
               
               
                 14 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-(2-chloro-4′-methoxybiphenyl-4- ylcarbamoyl)cyclopent-1- enecarboxylic acid 
               
               
                   
               
               
                 15 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 4-(3,5-difluoro-3′- (trifluoromethoxy)biphenyl-4- ylcarbamoyl)thiophene-3-carboxylic acid 
               
               
                   
               
               
                 16 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-[4-(2-Chloro-6-fluoro-benzyloxy)-3- fluoro-phenylcarbamoyl]-cyclopent-1- enecarboxylic acid 
               
               
                   
               
               
                 17 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 3-(3,5-Difluoro-3′-trifluoromethoxy- biphenyl-4-ylcarbamoyl)-thiophene-2- carboxylic acid 
               
               
                   
               
               
                 18 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-(3-fluoro-3′-methoxybiphenyl-4- ylamino)nicotinic acid 
               
               
                   
               
               
                 19 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-(3,5-difluoro-3′-methoxybiphenyl-4- ylamino)nicotinic acid 
               
               
                   
               
               
                 20 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 5-cyclopropyl-2-(5-methyl-6-(3- (trifluoromethoxy)phenyl)pyridin-3- ylamino)benzoic acid 
               
               
                   
               
               
                 21 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-[4-(2-Chloro-6-fluoro-benzyloxy)-3- fluoro-phenylcarbamoyl]-cyclopent-1- enecarboxylic acid 
               
               
                   
               
               
                 22 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-[3,5-Dichloro-4-(2-chloro-6-fluoro- benzyloxy)-phenylcarbamoyl]- cyclopent-1-enecarboxylic acid 
               
               
                   
               
               
                 23 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 2-(2-Chloro-4′-dimethylamino- biphenyl-4-ylcarbamoyl)-cyclopent-1- enecarboxylic acid 
               
               
                   
               
               
                 24 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 3-(3,5-Difluoro-3′-trifluoromethoxy- biphenyl-4-ylcarbamoyl)-thiophene-2- carboxylic acid 
               
               
                   
               
               
                 25 
                 
                   
                     
                     
                         
                         
                     
                   
                 
                 4-(2,3,5,6-Tetrafluoro-3′- trifluoromethoxy-biphenyl-4- ylcarbamoyl)-2,5-dihydro-thiophene-3- carboxylic acid 
               
               
                   
               
            
           
         
       
     
    
    
     
       FIGURE DESCRIPTION 
         FIG. 1 : Effect of treatment with formula (II)+MTX (methotrexate) on disease development 
     
    
    
     EXAMPLES 
     Collagen Induced Arthritis in Mice 
     Preparation: 
     Bovine Type II collagen solution is prepared by dissolving at 4 mg/ml in 0.01 M acetic acid at 4-8° C. with stirring overnight. Immunogen is prepared by emulsifying a 1:1 vol:vol combination of collagen solution and Complete Freund&#39;s Adjuvant (CFA) ( M. tuberculosis  H37Ra suspension: 4 mg/ml). 
     Immunization: 
     DBA1/J mice (male, 7-8 weeks) are weighed. The animals are anesthetized, injected subcutaneously into the shaved base of the tail with collagen/CFA (0.050 ml/mouse; 100 microgram/mouse collagen in CFA) using a 1 ml syringe fitted with a 25 G needle and returned to the cages. 
     Boost: 
     After three weeks (21 d) the procedure is repeated, though now with incomplete Freund&#39;s adjuvant (IFA) instead of CFA. 
     Disease Development: 
     Animals are observed for one week and macroscopic signs of arthritis are scored 3-times weekly (Monday, Wednesday, Friday: M, W, F). Each paw receives a score:
         a) 0=no visible effects of arthritis;   b) 1=edema and/or erythema of 1 digit;   c) 2=edema and/or erythema of 2 digits;   d) 3=edema and/or erythema of more than 2 digits;   e) 4=severe arthritis of entire paw and digits.       

     Calculate Arthritic Index (AI) by addition of individual paw scores and record. (Maximum AI=16) 
     After one week, mice are allocated to groups (10 mice per group) to give similar mean AI values and similar ranges of individual AI values in each treatment group. 
     Dosing: 
     Dosing is started, PO (per oral) once daily for 14 days. 
     
       
         
           
               
            
               
                   
               
               
                 Treatment Schedule 
               
            
           
           
               
               
               
               
               
            
               
                 Group No. 
                   
                   
                   
                   
               
               
                 ROA 
                 No. Mice 
                 Test Material 
                 Dose (mg/kg) 
               
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 1 
                 10 
                 PEG300 (vehicle) 
                 N/A 
                 PO 
               
               
                   
                   
                 non-immunized 
               
               
                   
                   
                 mice 
               
               
                 2 
                 10 
                 PEG300 (vehicle) 
                 N/A 
                 PO 
               
               
                   
                   
                 immunized mice 
               
               
                 3 
                 10 
                 MTX 
                 2.5 
                 PO 
               
               
                   
                   
                 Positive Control 
               
               
                 4 
                 10 
                 formula (II) 
                 50 
                 PO 
               
               
                 5 
                 10 
                 formula (II) 
                 50 
                 PO 
               
               
                   
                   
                 +MTX 
                 2.5 
                 PO 
               
               
                   
               
            
           
         
       
     
     AI scores determined 3× weekly. 
     Results: 
     
       
         
           
               
             
               
                   
               
               
                 Effect of Disease and Treatment on Arthritic Index 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 STUDY DAY 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Group 
                 Mouse 
                 22 
                 23 
                 24 
                 25 
                 26 
                 27 
                 28 
                 30 
                 33 
                 35 
                 37 
                 40 
                 41 
                 42 
               
               
                   
               
               
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Non- 
                 2 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 diseased 
               
               
                 PEG300 po 
                 3 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 4 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 5 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 6 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 7 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 8 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 9 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 10 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 2 
                 11 
                 0 
                 0 
                 0 
                 0 
                 0 
                 6 
                 11 
                 12 
                 12 
                 14 
                 12 
                 12 
                 12 
               
               
                 diseased 
                 12 
                 0 
                 0 
                 0 
                 0 
                 0 
                 6 
                 8 
                 12 
                 16 
                 16 
                 16 
                 16 
                 16 
               
               
                 PEG300 
                 13 
                 0 
                 0 
                 0 
                 0 
                 1 
                 4 
                 8 
                 10 
                 12 
                 11 
                 11 
                 11 
                 11 
               
               
                 po 
                 14 
                 0 
                 0 
                 0 
                 0 
                 2 
                 3 
                 7 
                 9 
                 9 
                 10 
                 10 
                 9 
                 9 
               
               
                   
                 15 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 4 
                 9 
                 12 
                 12 
                 12 
                 12 
                 12 
               
               
                   
                 16 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3 
                 7 
                 8 
                 10 
                 14 
                 14 
                 16 
                 16 
               
               
                   
                 17 
                 0 
                 0 
                 0 
                 0 
                 3 
                 5 
                 10 
                 12 
                 16 
                 15 
                 16 
                 16 
                 16 
               
               
                   
                 18 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 3 
                 9 
                 8 
                 10 
                 8 
                 8 
                 8 
               
               
                   
                 19 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 3 
                 4 
                 5 
                 5 
                 5 
                 8 
                 8 
               
               
                   
                 20 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3 
                 3 
                 6 
                 7 
                 10 
                 9 
                 12 
                 12 
               
               
                 3 
                 21 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 2 
                 9 
                 11 
                 10 
                 12 
                 12 
                   
                 12 
               
               
                 MTX, po 
                 22 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                 9 
                 12 
                 10 
                 11 
                 12 
                   
                 12 
               
               
                 2.5 mg/kg 
                 23 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 2 
                 5 
                 8 
                 8 
                 8 
                 9 
                   
                 9 
               
               
                   
                 24 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 3 
                 7 
                 9 
                 8 
                 9 
                 9 
                   
                 9 
               
               
                   
                 25 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 3 
                 11 
                 12 
                 12 
                 11 
                 14 
                   
                 14 
               
               
                   
                 26 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3 
                 11 
                 14 
                 13 
                 14 
                 14 
                   
                 14 
               
               
                   
                 27 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 5 
                 6 
                 11 
                 9 
                 8 
                 12 
                   
                 13 
               
               
                   
                 28 
                 0 
                 0 
                 0 
                 0 
                 4 
                 6 
                 6 
                 4 
                 4 
                 4 
                 4 
                 5 
                   
                 5 
               
               
                   
                 29 
                 0 
                 0 
                 0 
                 0 
                 5 
                 6 
                 6 
                 4 
                 5 
                 5 
                 5 
                 6 
                   
                 6 
               
               
                   
                 30 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 4 
                 4 
                 7 
                 6 
                 9 
                 12 
                   
                 12 
               
               
                   
               
            
           
           
               
               
            
               
                   
                 STUDY DAY 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Group 
                 Mouse 
                 22 
                 23 
                 24 
                 25 
                 26 
                 27 
                 28 
                 29 
                 30 
                 31 
                 32 
                 33 
                 34 
                 35 
               
               
                   
               
               
                 4 
                 31 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                   
                 1 
                 2 
                 5 
                 6 
                   
                 5 
               
               
                 Formula (II) 
                 32 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                   
                 0 
                 3 
                 6 
                 11 
                   
                 12 
               
               
                 50 mg/kg, po 
                 33 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                   
                 0 
                 1 
                 2 
                 2 
                   
                 4 
               
               
                   
                 34 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                   
                 1 
                 1 
                 2 
                 5 
                   
                 3 
               
               
                   
                 35 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                   
                 1 
                 3 
                 4 
                 6 
                   
                 8 
               
               
                   
                 36 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                   
                 0 
                 3 
                 3 
                 5 
                   
                 4 
               
               
                   
                 37 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                   
                 1 
                 1 
                 3 
                 5 
                   
                 4 
               
               
                   
                 38 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                   
                 2 
                 2 
                 2 
                 2 
                   
                 5 
               
               
                   
                 39 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 2 
                   
                 3 
                 4 
                 6 
                 8 
                   
                 8 
               
               
                   
                 40 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                   
                 0 
                 2 
                 3 
                 2 
                   
                 6 
               
               
                 5 
                 41 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 Formula (II) 
                 42 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3 
               
               
                 50 mg/kg, po 
                 43 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 44 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 MTX, po 
                 45 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 2.5 mg/kg 
                 46 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 47 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
               
               
                   
                 48 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 49 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 50 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
               
               
                   
               
            
           
           
               
               
               
            
               
                   
                 STUDY DAY 
                   
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Group 
                 Mouse 
                 37 
                 40 
                 42 
                 44 
                 47 
               
               
                   
                   
               
               
                   
                 4 
                 31 
                 8 
                 8 
                 8 
                 8 
                 8 
               
               
                   
                 Formula (II) 
                 32 
                 14 
                 14 
                 14 
                 14 
                 14 
               
               
                   
                 50 mg/kg, po 
                 33 
                 6 
                 6 
                 6 
                 6 
                 10 
               
               
                   
                   
                 34 
                 5 
                 4 
                 4 
                 4 
                 3 
               
               
                   
                   
                 35 
                 8 
                 8 
                 8 
                 8 
                 12 
               
               
                   
                   
                 36 
                 4 
                 4 
                 5 
                 5 
                 5 
               
               
                   
                   
                 37 
                 5 
                 5 
                 6 
                 6 
                 6 
               
               
                   
                   
                 38 
                 6 
                 6 
                 6 
                 6 
                 7 
               
               
                   
                   
                 39 
                 8 
                 8 
                 7 
                 8 
                 6 
               
               
                   
                   
                 40 
                 5 
                 6 
                 6 
                 6 
                 5 
               
               
                   
                   
               
            
           
           
               
               
            
               
                   
                 STUDY DAY 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Group 
                 Mouse 
                 36 
                 37 
                 38 
                 39 
                 40 
                 41 
                 42 
                 43 
                 44 
                 45 
                 47 
                 49 
                 51 
                 54 
               
               
                   
               
               
                 5 
                 41 
                 0 
                 0 
                 2 
                 4 
                 4 
                   
                 5 
                   
                 4 
                   
                 4 
                 4 
                 4 
                 4 
               
               
                 Formula (II) 
                 42 
                 2 
                 2 
                 3 
                 4 
                 4 
                   
                 4 
                   
                 4 
                   
                 4 
                 4 
                 5 
                 4 
               
               
                 50 mg/kg, po 
                 43 
                 0 
                 0 
                 1 
                 1 
                 2 
                   
                 4 
                   
                 4 
                   
                 4 
                 4 
                 5 
                 5 
               
               
                   
                 44 
                 0 
                 0 
                 5 
                 5 
                 5 
                   
                 5 
                   
                 5 
                   
                 5 
                 5 
                 5 
                 3 
               
               
                 MTX, po 
                 45 
                 1 
                 1 
                 4 
                 5 
                 5 
                   
                 2 
                   
                 4 
                   
                 5 
                 5 
                 5 
                 5 
               
               
                 2.5 mg/kg 
                 46 
                 0 
                 0 
                 2 
                 2 
                 3 
                   
                 4 
                   
                 3 
                   
                 5 
                 5 
                 4 
                 5 
               
               
                   
                 47 
                 1 
                 1 
                 3 
                 3 
                 4 
                   
                 3 
                   
                 4 
                   
                 4 
                 4 
                 4 
                 4 
               
               
                   
                 48 
                 0 
                 0 
                 2 
                 2 
                 5 
                   
                 4 
                   
                 4 
                   
                 4 
                 4 
                 7 
                 8 
               
               
                   
                 49 
                 0 
                 0 
                 5 
                 5 
                 4 
                   
                 4 
                   
                 5 
                   
                 4 
                 4 
                 6 
                 8 
               
               
                   
                 50 
                 1 
                 1 
                 4 
                 4 
                 5 
                   
                 5 
                   
                 4 
                   
                 8 
                 8 
                 fd 
               
               
                   
               
            
           
         
       
     
     Statistical Analysis: 
     
       
         
           
               
             
               
                   
               
             
            
               
                 Effect of Disease and Treatment on Average Arthritic Index: 
               
               
                   
               
            
           
           
               
               
            
               
                   
                 STUDY DAY 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Group 
                 Statistic 
                 22 
                 23 
                 24 
                 25 
                 26 
                 27 
                 28 
                 30 
                 31 
                 32 
                 33 
                 34 
               
               
                   
               
               
                 1 
                 Mean 
                 0 
                 0 
                 0 
                 0 
                 0.0 
                 0.0 
                 0.0 
                 0.0 
                   
                   
                 0.0 
               
               
                   
                 SD 
                 0 
                 0 
                 0 
                 0 
                 0.0 
                 0.0 
                 0.0 
                 0.0 
                   
                   
                 0.0 
               
               
                 2 
                 Mean 
                 0 
                 0 
                 0 
                 0 
                 0.6 
                 3.6 
                 6.4 
                 9.1 
                   
                   
                 10.7 
               
               
                   
                 SD 
                 0 
                 0 
                 0 
                 0 
                 1.1 
                 1.6 
                 3.0 
                 2.6 
                   
                   
                 3.6 
               
               
                 3 
                 Mean 
                 0 
                 0 
                 0 
                 0 
                 1.0 
                 1.6 
                 3.6 
                 7.0 
                   
                   
                 9.3 
               
               
                   
                 SD 
                 0 
                 0 
                 0 
                 0 
                 1.8 
                 2.3 
                 1.6 
                 3.3 
                   
                   
                 4.1 
               
               
                   
                 p value 
                   
                   
                   
                   
                 0.11 
                 0.24 
                 1.00 
                 0.10 
                   
                   
                 0.38 
               
               
                 4 
                 Mean 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0.7 
                 0.9 
                 2.2 
                 3.6 
                 5.2 
               
               
                   
                 SD 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0.8 
                 1.0 
                 1.0 
                 1.6 
                 2.9 
               
               
                   
                 p value 
                   
                   
                   
                   
                   
                   
                 0.02 
                 0.01 
                 0.003 
                 1.00 
                 0.37 
               
               
                 5 
                 Mean 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 SD 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 p value 
               
               
                   
               
            
           
           
               
               
            
               
                   
                 STUDY DAY 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                 Group 
                 Statistic 
                 35 
                 36 
                 37 
                 38 
                 39 
                 40 
                 41 
                 42 
                 43 
                 44 
               
               
                   
               
               
                 1 
                 Mean 
                 0.0 
                   
                 0.0 
                   
                   
                 0.0 
                 0.0 
               
               
                   
                 SD 
                 0.0 
                   
                 0.0 
                   
                   
                 0.0 
                 0.0 
               
               
                 2 
                 Mean 
                 11.7 
                   
                 11.3 
                   
                   
                 12.0 
                 12.0 
               
               
                   
                 SD 
                 3.2 
                   
                 3.5 
                   
                   
                 3.2 
                 3.2 
               
               
                 3 
                 Mean 
                 8.5 
                   
                 9.1 
                   
                   
                 10.5 
                   
                 10.6 
               
               
                   
                 SD 
                 3.7 
                   
                 4.0 
                   
                   
                 4.3 
                   
                 3.4 
               
               
                   
                 p value 
                 0.88 
                   
                 0.15 
                   
                   
                 0.30 
                   
                 0.34 
               
               
                 4 
                 Mean 
                 5.9 
                   
                 6.9 
                   
                   
                 6.9 
                   
                 7.0 
                   
                 7.1 
               
               
                   
                 SD 
                 2.7 
                   
                 2.9 
                   
                   
                 2.9 
                   
                 2.7 
                   
                 2.8 
               
               
                   
                 p value 
                 0.004 
                   
                 0.02 
                   
                   
                 0.01 
                   
                 0.01 
                   
                 0.04 
               
               
                 5 
                 Mean 
                 0.5 
                 0.5 
                 0.5 
                   
                   
                 4.0 
                   
                 4.1 
               
               
                   
                 SD 
                 1.0 
                 0.7 
                 0.7 
                   
                   
                 0.9 
                   
                 0.6 
               
               
                   
                 p value 
                 0.12 
                 0.04 
                 0.04 
                   
                   
                 2 × 10 −5   
                   
                 2 × 10 −5   
               
               
                   
               
            
           
           
               
            
               
                 2. Effect of Disease and Treatment on Average Arthritic Index: 
               
            
           
           
               
               
            
               
                   
                 STUDY DAY 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Group 
                 Statistic 
                 45 
                 47 
                 49 
                 51 
                 54 
                 56 
                 58 
                 59 
               
               
                   
               
               
                 4 
                 Mean 
                   
                 7.6 
               
               
                   
                 SD 
                   
                 3.4 
               
               
                   
                 p value 
                   
                 0.01 
               
               
                 5 
                 Mean 
                 4.7 
                   
                 4.7 
                 5.0 
                 5.1 
               
               
                   
                 SD 
                 1.3 
                   
                 1.3 
                 1.0 
                 1.8 
               
               
                   
                 p value 
                 1 × 10 −5   
                   
                 2 × 10 −5   
                 1 × 10 −5   
                 2 × 10 −5   
               
               
                   
               
            
           
         
       
     
     Histological Results: 
     Examination of the livers from these animals revealed that co-therapy with MTX appeared to prevent the toxicity associated with formula (II) treatment. The data shown indicate that co-administration of formula (II) and MTX has a beneficial effect not only on disease development, but also on liver toxicity in terms of reversing liver toxicity findings caused by administration of formula (II) or MTX alone. 
     
       
         
           
               
            
               
                   
               
               
                 Effect of Disease and Treatment on Liver Histology 
               
            
           
           
               
               
               
               
            
               
                   
                 Group* 
                 Mouse 
                 Observations 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 1 
                 1 
                 Normal 
               
               
                   
                   
                 2 
                 Normal 
               
               
                   
                   
                 3 
                 Normal 
               
               
                   
                   
                 4 
                 Normal 
               
               
                   
                   
                 5 
                 Normal 
               
               
                   
                   
                 6 
                 Normal 
               
               
                   
                   
                 7 
                 Normal 
               
               
                   
                   
                 8 
                 Mild multifocal subacute inflammation 
               
               
                   
                   
                 9 
                 Normal 
               
               
                   
                   
                 10 
                 Normal 
               
               
                   
                 2 
                 11 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 12 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 13 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 14 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 15 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 16 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 17 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 18 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 19 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 20 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                 3 
                 21 
                 Minimal glycogen depletion, hepatocyte 
               
               
                   
                   
                 22 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 23 
                 Normal 
               
               
                   
                   
                 24 
                 Normal 
               
               
                   
                   
                 25 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 26 
                 Normal 
               
               
                   
                   
                 27 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 28 
                 Minimal multifocal subacute inflammation 
               
               
                   
                   
                 29 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 30 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                 4 
                 31 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 32 
                 Mild centrilobular hypertrophy, glycogen 
               
               
                   
                   
                   
                 depletion, hepatocyte 
               
               
                   
                   
                 33 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 34 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 35 
                 Mild centrilobular hypertrophy, glycogen 
               
               
                   
                   
                   
                 depletion, hepatocyte 
               
               
                   
                   
                 36 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 37 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 38 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 39 
                 Mild glycogen depletion, hepatocyte 
               
               
                   
                   
                 40 
                 Mild centrilobular hypertrophy, glycogen 
               
               
                   
                   
                   
                 depletion, hepatocyte 
               
               
                   
                 5 
                 41 
                 Normal 
               
               
                   
                   
                 42 
                 Normal 
               
               
                   
                   
                 43 
                 Normal 
               
               
                   
                   
                 44 
                 Mild centrilobular hypertrophy, hepatocyte 
               
               
                   
                   
                 45 
                 Mild centrilobular hypertrophy, hepatocyte 
               
               
                   
                   
                 46 
                 Mild centrilobular hypertrophy, hepatocyte 
               
               
                   
                   
                 47 
                 Mild centrilobular hypertrophy, hepatocyte 
               
               
                   
                   
                 48 
                 Mild centrilobular hypertrophy, hepatocyte 
               
               
                   
                   
                 49 
                 Normal 
               
               
                   
                   
               
               
                   
                 *Group 1: Non-diseased, PEG300, po 
               
               
                   
                 Group 2: diseased, PEG300, po 
               
               
                   
                 Group 3: MTX, 2.5 mg/kg, po 
               
               
                   
                 Group 4: Formula (formula II),, 50 mg/kg, po 
               
               
                   
                 Group 5: Formula (formula II),, 50 mg/kg, po + MTX, 2.5 mg/kg, po 
               
            
           
         
       
     
     Experimental Design of 13-Week Repeat Dose Toxicity in Beagle Dogs—Combination with Methotrexate 
     The aim of this study was to obtain information on the interactive toxicity of formula (II) given simultaneously in combination with methotrexate (MTX) by repeated oral administration to Beagle dogs for 13 weeks and to assess the reversibility of any effect at the end of a 4-week recovery period. The animals were randomly allocated to five test groups employing a pseudo-random body weight stratification procedure that yielded groups with approximately equal mean body weights. Formula (II) was administered daily by oral administration, MTX was administered concomitantly once weekly by oral administration. A special focus of the study was to study the influence of the combination treatment on liver toxicity. 
     
       
         
           
               
             
               
                 TABLE I 
               
             
            
               
                   
               
               
                 Group allocation in combination toxicity study 
               
            
           
           
               
               
               
               
            
               
                   
                 Formula (II) dose 
                 Methotrexate (MTX) dose 
                   
               
               
                   
                 [mg/kg b.w./day, 
                 [mg/kg b.w./week, 
                 No. and sex of 
               
               
                   
                 p.o. 
                 p.o. 
                 animals 
               
               
                 Group 
                 once daily] 
                 once weekly] 
                 MS + RP 
               
               
                   
               
               
                 1 
                  0 
                 0 
                 4 + 2 m 
               
               
                   
                 (control) 
                 (control) 
                 4 + 2 f 
               
               
                 2 
                 25 
                 0 
                 4 m 
               
               
                   
                 (high dose) 
                 (control) 
                 4 f 
               
               
                 3 
                  0 
                 10/2.5# 
                 4 m 
               
               
                   
                 (control) 
                   
                 4 f 
               
               
                 4 
                 10 
                 10/2.5# 
                 4 m 
               
               
                   
                 (low dose) 
                   
                 4 f 
               
               
                 5 
                 25 
                 10/2.5# 
                 4 + 2 m 
               
               
                   
                 (high dose) 
                   
                 4 + 2 f 
               
               
                   
               
               
                 MS Main study 
               
               
                 RP Recovery period 
               
               
                 m Male 
               
               
                 f Female 
               
               
                 #The methotrexate dose was reduced to 2.5 mg/kg body weight as of test week 5 for the male animals and test week 4 for the female animals due to a single premature death in group 3. 
               
            
           
         
       
     
     For laboratory examinations blood samples were taken from the vena cephalica or vena saphena magna from animals fasted overnight. Serum samples were collected into tubes for biochemical tests. Clinical biochemistry parameters from venous blood were determined at the times listed below: 
     Prior to study start: all animals of a pool of 52 dogs 
     On test day 37 (approx. 24 hours after 6 th  Methotrexate administration): all 48 animals included in this study 
     At main study termination (test day 93): all 48 animals included in this study. 
     At the end of the recovery period: all 8 recovery animals included in this study 
     Clinical biochemistry parameters (e.g. bile acids, alanine aminotransferase=ALAT, alkaline phosphatise=aP, aspartate aminotransferase=ASAT, gamma-glutamyl-transferase=Gamma-GT, glutamate dehydrogenase=GLDH, creatine kinase=CK) were measured using KONELAB 30i instrument (Thermo Fisher Scientific, 63303 Dreieich, Germany) as U/L (unit/liter) serum. 
     Results of 13-Week Repeat Dose Toxicity in Beagle Dogs—Combination with Methotrexate 
     Oral treatment with 25 mg formula (II)/kg body weight/day alone caused slight signs of systemic intolerance mainly in form of emesis, defecation and soft faeces. One female dog treated with 10 mg MTX/kg body weight/week was found dead in the morning of test day 20. As a result, the MTX dose was reduced to 2.5 mg/kg body weight. Oral treatment with 10/2.5 mg MTX/kg body weight/week alone caused signs of systemic intolerance mainly in form of emesis, defecation and soft faeces. 
     Oral treatment with 10 or 25 mg formula (II)/kg body weight/day in combination with 10/2.5 mg MTX/kg body weight/week caused increased signs of systemic intolerance mainly in form of emesis, defecation and soft faeces, the intensity of effects caused by the two compounds was additive. 
     Oral treatment with formula (II) alone revealed no influence on the body weight, food and drinking water consumption and haematological parameters. Treatment with MTX alone or combined treatment with formula (II) and MTX caused comparable reductions in the body weight and food consumption. 
     Combined treatment of formula (II) and MTX led to several changes in the biochemical parameters identical to MTX alone. In general, oral treatment with formula (II) alone revealed changes in the biochemical parameters in form of slight increased enzyme activities of ALAT, ASAT and GLDH as well as slight increases of the bile acid concentration in serum (GLDH and bile acid data not shown). However, none of these increased values were statistically significant (at p≦0.01). Treatment with MTX alone caused pronounced and statistically significant (at p≦0.01) changes in the biochemical parameters of ALAT, ASAT, CK, GLDH as well as bile acids in comparison to formula (II) alone. For most clinical biochemistry parameters such as ASAT combined treatment with formula (II) and MTX caused similar effects to MTX alone indicating a simple additive toxic response. However, for the key liver enzyme ALAT it has been observed that the combination of MTX and formula (II) caused a significant decrease of ALAT concentration (table II) when compared to MTX or formula (II) given alone indicating a highly beneficial interaction regarding toxic response between these two drugs. 
     
       
         
           
               
             
               
                 TABLE II 
               
             
            
               
                   
               
               
                 Test item-related changes in selected biochemical parameters 
               
               
                 as compared to the control group 1 [%] 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Group 2 
                   
                   
                 Group 5 
               
               
                   
                 25 mg 
                   
                 Group 4 
                 25 mg 
               
               
                   
                 Formula 
                 Group 3 
                 10 mg Formula 
                 Formula (formula 
               
               
                   
                 (formula 
                 10/2.5 mg 
                 (formula 
                 II)/kg + 
               
               
                   
                 II)/kg 
                 MTX/kg 
                 II)/kg + MTX #   
                 MTX #   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 Parameter 
                 males 
                 females 
                 males 
                 females 
                 males 
                 females 
                 males 
                 females 
               
               
                   
               
            
           
           
               
            
               
                 Test day 37 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 ALAT 
                 none 
                 None 
                 none 
                 +190   
                 −33 
                 −8  
                 −17 
                 −30 
               
               
                 ASAT 
                 none 
                 None 
                 none 
                 +47** 
                 +48 
                 +78  
                 +27 
                 +33 
               
            
           
           
               
            
               
                 Test day 93 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 ALAT 
                 None 
                 None 
                 +386   
                 +567   
                 none 
                 None 
                 +47 
                 +105  
               
               
                 ASAT 
                 None 
                 None 
                 +55** 
                 +34** 
                  +62** 
                 +76** 
                  +56** 
                  +44** 
               
               
                   
               
               
                 **statistically significant at p ≦ 0.01 
               
               
                   # 10/2.5 mg MTX/kg 
               
            
           
         
       
     
     No changes were noted for ECG parameters, blood pressure, at ophthalmological and auditory examination, for the organ weights or at macroscopic examination at any of the tested dose levels. Mild enteritis in form of a lympho-plasmacellular infiltration was observed in the gastro-intestinal mucosa of nearly all test item-treated dogs. The finding was most pronounced in the animals treated with MTX alone compared to the animals treated with formula (II) alone or the combination of formula (II) and MTX. 
     Mean toxicokinetic parameters of formula (II) and methotrexate after individual and concomitant administration to Beagle dogs are shown in the following table Ill. A clear dose response relationship was noted for formula (II) plasma levels on test day 85, though not on test day 1, possibly caused by the severe toxicity of the 10 mg MTX/kg body weight/week, later reduced to 2.5 MTX/kg body weight/week. No accumulation with time was noted. Methotrexate did not appear to influence the formula (II) absorption and vice versa, therefore, no toxicokinetic interactions were observed. 
     
       
         
           
               
             
               
                 TABLE III 
               
             
            
               
                   
               
               
                 Mean toxicokinetic parameters of formula (II) and methotrexate after 
               
               
                 individual and concomitant administration to Beagle dogs 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                   
                 AUC 0-∞   
                 C max   
                 t 1/2   
               
               
                   
                 Dose 
                 Time of 
                 [μg * h/ml] 
                 [μg/ml] 
                 [h] 
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                 Group 
                 [mg/kg] 
                 sample 
                 male 
                 female 
                 male 
                 female 
                 male 
                 female 
               
               
                   
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                 Formula (II) 
                 2 
                 25 
                 day 1 
                 289 
                 773    
                 22.7 
                 56.3 
                 7.8 
                 8.6 
               
               
                   
                   
                   
                 week 13 
                 601 
                 1012    
                 56.9 
                 66.6 
                 6.1 
                 11.1 
               
               
                   
                 4 
                 10 
                 day 1 
                 322 
                 513    
                 30.3 
                 43.0 
                 6.3 
                 7.4 
               
               
                   
                 (+MTX) 
                   
                 week 13 
                 371 
                 598 2)     
                 35.7 
                 40.5 
                 6.3 
                 26.7 
               
               
                   
                 5 
                 25 
                 day 1 
                 858 
                 413    
                 60.0 
                 33.1 
                 8.4 
                 7.6 
               
               
                   
                 (+MTX) 
                   
                 week 13 
                 659 
                 1186    
                 53.6 
                 76.3 
                 7.9 
                 8.1 
               
               
                 Methotrexate 
                 3 
                 10 
                 day 1 
                 6.4 
                 5.3 
                 1.8 
                 1.1 
                 1.6 
                 2.4 
               
               
                   
                   
                 2.5 1)   
                 week 13 
                 3.0 
                 2.2 
                 0.7 
                 0.5 
                 2.6 
                 2.3 
               
               
                   
                 4 
                 10 
                 day 1 
                 6.6 
                 7.5 
                 1.7 
                 1.7 
                 1.6 
                 3.1 
               
               
                   
                 (+10SC) 
                 2.5 1)   
                 week 13 
                 2.0 
                 1.9 
                 0.4 
                 0.4 
                 2.0 
                 2.6 
               
               
                   
                 5 
                 10 
                 day 1 
                 5.9 
                 5.0 
                 1.2 
                 1.2 
                 2.2 
                 2.0 
               
               
                   
                 (+25SC) 
                 2.5 1)   
                 week 13 
                 2.5 
                 1.4 
                 0.6 
                 0.2 
                 2.1 
                 2.7 
               
               
                   
               
               
                   1)  The dose of methotrexate was reduced from 10 to 2.5 mg/kg as of test week 5 (M) and week 4 (F). 
               
               
                   2)  AUC 0-t  (+10SC) = in combination with 10 mg/kg formula (II);; (+25SC) = in combination with 25 mg/kg formula (II) 
               
            
           
         
       
     
     In conclusion, the effects of formula (II) and MTX given simultaneously were simply additive of their individual responses, but not greater than that expected by addition of their individual responses, i.e. no synergistic effects or potentiation was noted regarding increased toxicity. In addition, an increase of the formula (II) dose from 10 to 25 mg/kg body weight/day did not potentiate the toxicity profile. In contrast, the data shown indicate that co-administration of formula (II) and MTX has a beneficial effect on selected liver enzymes such as ALAT in terms of reversing enzyme elevation caused by MTX alone.