USE OF AMINOTHIOPHENOLS IN THE TREATMENT OF DISORDERS ASSOCIATED WITH THE COMPLEMENT SYSTEM

Abstract: A compound having an aromatic or heterocyclic ring with at least one primary amine group NH2 and at least one sulphydryl group SH directly attached to the ring, or a pharmaceutically acceptable salt thereof, for use in the treatment or prophylaxis of a complement related disorder or for reducing the risk of graft rejection. Complement related disorders include autoimmune diseases such as arthritis and rheumatism, myocardial infarction and tissue damage associated with radiotherapy. Examples for the compounds are 2-aminothiophenol and 4-aminothiophenol.

Description: USE OF AMINOTHIOPHENOLS IN THE TREATMENT OF DISORDERS ASSOCIATED WITH THE COMPLEMENT SYSTEM The present invention relates to drugs for the treatment of conditions aggravated or caused by 5 complement as a source of inflammatory mediators. An area of particular importance is autoimmune diseases and especially a group of autoimmune diseases known as the Arthritides which includes arthritis and rheumatism. The compounds of the invention will also 10 be useful in treatment of myocardial infarction, as radioprotective agents in treatment of patients undergoing radiotherapy, and to help prevent graft rejection in transplant patients. Complement activation is known to have an adverse role in these 15 situations (25, 26) . Available drugs presently used to treat arthritis and rheumatism have toxic side effects that often prohibit their use after only a few weeks. Penicillamine, a commonly used drug, causes serious 20 proteinuria in up to 30% of patients. The toxic side effects can resemble the symptoms of the autoimmune disease Systemic Lupus Erythematosus (SLE) , which include vasculitis, joint pains, rash and nephritis. The symptoms of SLE can be understood not so much in 25 terms of specificities of the antibodies which are found but more in the formation of immune complexes between antibody and antigen. Other drugs are associated with SLE or "immune complex disease" as an adverse side-effect 30 (12). Hydralazine, administered for hypertension, procainamide for cardiac arrhythmia and phenytoin for epilepsy are all examples. Immune complex diseases, whether of the SLE type or drug-induced, are considered to be disorders 35 of immune complex handling. An individual becomes unable to deal successfully with an immune complex load and the result is immune complex deposition at inappropriate tissue sites such as small blood vessels and kidney glomeruli leading to tissue damage. Immune complexes are formed in all individuals but in healthy people they are removed through the reticuloendothelial system and removal is promoted through activation of the complement cascade (1). Subcellular components e.g. DNA and mitochondria, released from damaged tissue will also directly activate the classical complement pathway. When the complement cascade is activated through the classical pathway, components C1 , C2 and C4 become activated and a complex enzyme C3 convertase, consisting of C4b2a, is generated on the complement-activating surface. C3 which is activated, in turn becomes bound to the immune complex surface. Covalent binding of C3 and C4 to the surface of immune complexes occurs through reaction of the internal thiolester in C3 and C4 with amino or hydroxyl groups on the immune complex [2]. The role of activation of complement in clearance of immune complexes appears to be physiologically important because individuals who lack the early complement components are at increased risk of SLE [3] . Complement component C4 is encoded at two polymorphic loci, C4A and C4B within the class III region of the Major Histocompatibility Complex. The products of the two genes are very similar and the isotypic region has been located to 4 amino acids within a group of 6 out of the total of 1722 in the complete C4 sequence [4]. The C4A and C4B gene products differ in reactivity of the thiol ester site on activation. The C4A gene product has been shown to be more reactive than the C4B gene product with amino nucleophilic groups [5,6]. The physiological role of C4A and C4B may also differ because it has been observed that SLE is associated with non-functioning alleles at the C4A locus rather than the C4B locus [7] and the C4A protein has been demonstrated to be able to restore an immune response to C4 deficient guinea pigs which are immunologically comprised [8]. Guinea pig C4 has been shown in previous studies to have an activity profile like C4A [9]. It has been demonstrated that some of the above drugs interfere with the covalent binding reaction of C4 [2]. Both hydralazine [12] and penicillamine [13] are more inhibitory with C4A than with C4B. The inhibition of the covalent binding of C4A by penicillamine occurs over the therapeutic range and it had previously been observed that penicillamine causes a reduction in the deposition of C3 in arthritic joints [14]. On the basis of these results it has been proposed that inhibition of C4 covalent binding by penicillamine contributes to both therapy and toxicity with the drug [13]. It follows therefore that drugs having an inhibitory effect on C4 binding will almost certainly have a therapeutic effect on arthritis and rheumatism, although there may also be toxic side effects. Also, these drugs are expected to have an inhibitory effect on C3 binding, which will contribute to the therapeutic effect as well as to any side effects. The covalent binding reaction of C3 has been shown to be inhibited by penicillamine [27]. Captopril, an angiotensin-converting enzyme inhibitor, which is used as an anti-hypertensive, is another sulphydryl drug with immunotoxic side effects [16]. It is demonstrated also to be an inhibitor of the covalent binding reaction of C4. Penicillamine is an alpha amino beta thiol compound [15]. In order to investigate which aspects of penicillamine are important in promoting interaction of penicillamine with complement component C4 a range of chemically similar compounds has been investigated. These compounds show that it is not necessary for the amino and thiol groups to be on adjacent carbon atoms, as they are in penicillamine, to generate potent inhibition of C4. The importance of the sulphydryl group in inhibition of the covalent binding reaction of C4 is demonstrated. The present invention results from the discovery that aminothiophenols are effective inhibitors of C4 binding and of C3 binding and can therefore act as anti-inflammatory agents. The compounds described are more effective than penicillamine against C4B and against C3 and it is proposed that they will have an increased therapeutic effect and reduced toxic side effects compared with penicillamine. Aminothiophenols have previously been described in EP-A-0269434 as intermediates for the synthesis of pharmaceuticals such as antiarthritics but have not until now been suggested as agents for treatment in their own right. As intermediates, aminothiophenols are used to make two-ring compounds in which the original amino and sulphydryl groups are modified. Airiinothiophenols and related compounds are described herein as anti-inflammatory agents for the treatment or prophylaxis of a wide range of inflammatory conditions. The invention therefore provides a method for the treatment or prophylaxis of a complement related disorder in a patient comprising administering to the patient a compound having an aromatic or heterocyclic ring with at least one primary amine group H 2 and at least one sulphydryl group SH directly attached to the ring, preferably ortho or para to one another, or a pharmaceutically acceptable salt thereof. The complement-related disorder may be any inflammatory condition to which components of the complement system contribute, either directly or indirectly. The complement-related disorder may be an autoimmune disease such as arthritis or rheumatism, or the disorder may be myocardial infarction, or it may be tissue damage associated with radiotherapy. The compounds as defined will thus be useful as radioprotective agents, for reducing or minimising tissue damage resulting from radiotherapy treatment. The invention further provides a method of inhibiting the covalent binding of complement component C4 or C3 to a complement activating surface which method comprises treating the complement component C4 or C3 with a compound as defined herein. Examples of complement activating surfaces are immune complexes and damaged tissue. A complement activating surface may comprise complement component C1s. Also provided according to the invention is a method of reducing the risk of graft rejection which method comprises treating the graft or the recipient of the graft with a compound as defined herein. Further provided by the invention is a compound having an aromatic or heterocyclic ring with at least one primary amine group NH_ and at least one sulphydryl group SH directly attached to the ring, or a pharmaceutically acceptable salt thereof, for use in the treatment or prophylaxis of a complement related disorder or for reducing the risk of graft rejection. Another aspect of the invention is the use of a compound as defined herein for the treatment or prophylaxis of a complement related disorder or for reducing the risk of graft rejection. Preferred compounds are those having the formula where n is 0 to 4 and each R is selected from H, F, OH, SH, Cl, Br, I, N0 2 , NH 2 , mono or dialkylamino, S0 3 H, alkyl, carboxy alkyl, hydroxy alkyl, alkoxy or carboxy, or two groups R may together form a fused aromatic ring structure. Table 1 below identifies seven compounds by name and structure, including three according to the invention, and gives the concentration required for 50% inhibition of C4 binding (determined by methods described in the examples section below) . Figure 1 is a graph showing % inhibition of C4 binding against aminothiophenol concentration. As' shown by these results, the three aminothiophenols (compounds according to formula I where n is zero) have interesting properties, particularly o - aminothiophenol and p - aminothiophenol. It is anticipated that the presence of substituents in the aromatic ring (n is not zero) will alter the biodistribution properties and may enhance activity. An alkoxy eg. methoxy substituent may enhance activity. A hydrocarbon substituent, or the substitution of naphthol for phenol, will make the compounds more lipophilic and may permit localised administration. Compounds of formula II are of interest only insofar as they are capable of decomposing in vivo to yield a compound according to formula I. The presence of NO- or similar substituents may enhance decomposition in vivo. Results presented here show that inhibition of the covalent binding reaction of C4 by arylamine compounds is greatly increased by the presence of a sulphydryl group, as demonstrated when the inhibitory potency of aminophenol and aminothiophenol is compared (Table 1 ) . It has been observed previously that penicillamine is a potent inhibitor of the covalent binding reaction of C4 to a surface consisting of C1s bound to an antibody-antigen complex with erythrocytes as antigen [13]. Penicillamine is also a potent inhibitor of the covalent binding of C4 to C1s on agarose beads as the combined activating and binding surface. It has been suggested that inhibition of the covalent binding reaction of C4 by penicillamine results in formation of a thiazoline ring complex between C4 and penicillamine [13]. However, examples presented here show that formation of such a complex is unnecessary for potent inhibition of covalent binding of C4 to surfaces. Both o- and p- aminothiophenol are equally potent inhibitors and whilst o- aminothiophenol could form a thiazoline ring, it is most unlikely that p- aminothiophenol could form such a compound. The effect of the position of the sulphydryl substitution in the aromatic ring shows that it is likely to be the nucleophilicity of the sulphydryl group in the para position which is important in promoting interaction with the thiol ester in C4. Increasing inhibitory potency of inhibitors of the covalent binding reaction of C3 [22] and C4 [23] has already been noted. The effect of sulphydryl compounds on the covalent binding of the different isotypes of C4 shows that the difference in inhibition of C4A and C4B observed with arylaminothiol compounds is much less than the difference observed with the less hydrophobic aminothiols such as penicillamine and cysteine [13]. For penicillamine and cysteine, C4A is inhibited 8 to 9 times more effectively than C4B. With the arylaminothiophenols, the inhibitory potency towards C4A is 2-fold greater than the inhibitory potency of C4B. It has been observed that the difference between C4A and C4B which contributes to the binding specificity difference between the two isotypes resides in a sequence of 6 amino acids which is P-C-I-V-L-D in C4A but which is L-S-I-V-I-H in C4B [4]. It has been observed by site directed mutagenesis and by comparison of the active sites of C4 and its homologues from different species that the aspartate residue in the specificity conferring region promotes the increased interaction of C4A with amino nucleophilic groups [24]. It has been proposed that the cysteine/serine interchange promotes the interaction of C4A with sulphydryl groups [13]. However, it would seem that for more hydrophobic aminothiol compounds, the presence of a sulphydryl group in the specificity determining region of C4A is compensated for by the substitution of a proline in C4A for the more hydrophobic leucine in C4B. The inhibitory activity of aminothiophenols with C4B may prove to be beneficial in treatment according to the invention as compared to treatment with existing drugs. Inhibition of C4A binding is known to be important in the problems with handling of immune complexes in the periphery that lead to SLE and related symptoms. The role of C4B is not well defined and it is possible that it is more important in localised areas such as the joints. In addition, the reactivity of C4B is more like the complement component C3 and it is likely that aminothiophenols will also be potent inhibitors of C3. Results presented herein show that aminothiophenols are more active than penicillamine in inhibiting covalent binding of C3 in vitro. Another way in which drug-induced side effects might be reduced is by local administration of the drug to avoid systemic toxicity. The hydrophobic nature of compounds that come under the scope of the invention will render them particularly suitable for this purpose. EXAMPLES Example 1 Materials and Methods Coupling of proteins to Seoharose C1s was isolated from human serum and coupled to CNBr-Sepharose (Pharmacia, Milton Keynes, U.K.) as previously described [17]. Mouse monoclonal antibodies against C4c (L001 ) [18] and against C4d (L003) [19] were obtained as ascites fluid from the Commonwealth Serum Laboratories, Parkville, Australia. Antibodies were partially purified by ammonium sulphate precipitation [20] and were resuspended at a concentration of 15mg/ml then dialyεed into 500mM NaCl, 20mM sodium phosphate, pH7.5 for coupling to CNBr-Sepharose. The mixture of CNBr-Sepharose (1ml packed volume per 1Omg protein) and antibodies was tumbled at 20 C for 1 hour which resulted in greater than 90% of the protein binding to the resin. Longer coupling reactions at pH8.5 were found to reduce the binding capacity of the antibody affinity columns for C4. The derivatised Sepharose was used for affinity columns (2-5ml) after saturating the remaining binding sites on CNBr-Sepharose with 1M Tris-HCl, pH 7.5 (16 hours, 4 C) . Trypsin was coupled to Sepharose as described previously [22]. Purification of Human C4 Human C4 was purified by a 2 step procedure using affinity chromatography followed by ion-exchange on a Mono-Q HR5/5 column (Pharmacia) . All chromatography was performed at 4 C. C4A and C4B were purified together on a L00-1 monoclonal antibody column whereas C4A and C4B were separated using a L00- 3 monoclonal antibody column. Affinity columns were prepared and eluted essentially as described previously [19]. Pooled human plasma (2-3ml) containing 5mM EDTA was made 1mM with respect to phenylmethylsulphonyl- fluoride (PMSF) and centrifuged (13,000rpm, 5 minutes) n a Fisons microfuge at 4 C. The plasma was loaded onto the appropriate column, pre-equilibrated with 50mM Tris, 50mM Na-phosphate, 12.5mM Na-tetraborate, 2.5mM EDTA, -0.1mM PMSF, 0.01% Na-azide at pH 7.0. After 30 minutes at 4 C, the column was washed with 100mM Tris, 100mM Na-phosphate, 25mM Na-tetraborate, 5mM EDTA, 0.2mM PMSF, 0.02% Na-azide at pH 8.5 until the absorbance at 280nm of the eluate was zero. C4 was eluted with a linear gradient of 15ml of the pH 8.5 buffer and 15ml of the same buffer, adjusted to pH 11.5, finishing with a pH 11.5 wash. C4A elutes from the LOO-3 column at pH 9.0 to 9.5 whereas C4B elutes between pH 9.5 and 11. The appropriate C4 pools were then dialysed into 20mM Tris, 5mM EDTA, 0.02% Na- azide, 0.2mM PMSF at pH 7.5. The samples were then further purified by FPLC using ion-exchange on a Mono- Q column. The Mono-Q column was initially washed with 20mM Tris 5mM EDTA, 0.02% Na-azide, 0.2mM PMSF, 50mM Σ-amino caproate, 1M NaCl at pH 7.5 (buffer B) and then with the same buffer without NaCl (buffer A) . The sample was applied in 2ml aliquots at 1ml/min. The column was washed with 10% (v/v) buffer B until the absorbance at 280nm was zero. A linear gradient to 60% (v/v) buffer B was then applied in 30ml, followed by a wash (5ml) at 100% B (v/v) to complete the elution. Fractions of columns were monitored by SDS- PAGE as above, and C4 concentrations were estimated from their absorbance at 280nm. Binding of C4 to Sepharose-C1 s Pure C4, C4A or C4B (10-30μg) were radiolabelled with Na 125 I (500-700μCi) with 1 iodobead (Pierce, Poole, U.K.) as catalyst in a total volume of 100μl of 20mM sodium phosphate pH 7.5, containing 140mM NaCl, as previously described [17] Covalent binding of 125 IC4b, 125 IC4Ab or 125 I.C4Bb to Sepharose-C1 s was determined in triplicate [17] in a total volume of 150μl of 20mM sodium phosphate, 140mM NaCl containing 1% (v/v) dimethylsulphoxide. After 30 minutes at 37°C, the reaction was stopped by addition of 200μl of 200mM Tris-HCl, pH 6.8 containing 2% (w/v) sodium dodecylsulphate (SDS) and 8M urea. Sepharose 4B (Pharmacia) 200μl of a (50% (v/v) slurry in 20mM sodium phosphate, 140mM NaCl was then added and centrifugation and washing steps were performed as described previously [17]) . The final pellet was resuspended in 100mM NaCl (200μl) and was transferred to a clean tube for measurement of bound radioactivity. Complete inhibition of binding of 125I C4b binding was determined by including 20mM hydroxylamine, pH 7.5 in the initial incubation mixture with Sepharose-C1s. SDS-Polyacrylamide gel electrophoresis To distinguish between C4A and C4B, polyacrylamide gels were prepared with a ratio of acrylamide: biscarylamide of 1: 0.006 [21]. Sulphydryl compounds Aminothiophenol compounds were stored at 20 C in a dessicator containing a loosely-capped vial of 100mM dithiothreitol, to maintain a reducing environment. The aromatic compounds were initially dissolved as a stock solution in dimethyl sulphoxide and were then diluted 100 fold in 20mM sodium phosphate 140mM NaCl, pH 7.5. The stock solutions and dilutions were made fresh each day. Stock solutions of penicillamine and captopril were made freshly each day in 20mM sodium phosphate, 140mM NaCl, pH 7.5. Measurement' of reduction of C4 and C3 Pure C4 or C3, 50μg/ml in 20mM sodium phosphate pH 7.5 containing 140mM NaCl, was incubated (30μl) with the test compound for 30 minutes at 37°C. lodoacetamide was then added to a final concentration of 1OmM in the same buffer and the mixture was incubated for a further 10 minutes at 37 C. An equal volume of 200mM Tris-HCl, pH 6.8, containing 2% (w/v) sodium dodecyl sulphate and 8 M urea was then added and samples were incubated at 37 C for 20 minutes and then analysed by SDS-polyacrylamide gel electrophoresis. Results Effects on C4 and C1s cleavage of C4 No difference was observed between the extent of C4 cleavage by C1s in the presence or absence of any of the compounds tested. This confirms that the compounds have their effect by inhibiting the binding of C4 to C1s rather than simply preventing splitting of C4 to C4a and C4b. Reduction of interchain disulphide bridges was assessed for all of the sulphydryl compounds used. When C4 or C3 was incubated with o-aminothiophenol no reduction of interchain disulphide bridges was observed at up to 350μM. At 500μM approximately 30% of the C4 migrated as reduced C4 (that is as alpha, beta and gamma chains) whilst the rest of the C4 was unchanged and migrated as a 205kD band. Captopril and penicillamine caused no reduction of C4 at up to 5mM. Effect of sulphydryl compounds on covalent binding reaction of 'C4 to S-C1s The binding of 125I-labelled C4 to Sepharose- C1s as a combined activating and binding surface is inhibited in a dose-dependent manner by o- aminothiophenol, p-aminothiophenol and m- aminothiophenol (Fig. 1). The inhibition by the o- and p-substituted compounds is indistinguishable whilst the m-substituted compound is a less potent inhibitor with 50% inhibition of binding occurring at 510μM. The hydroxyl compound, aminophenol is a less potent inhibitor of the covalent binding reaction of C4 and ortho-aminiobenzoic acid is a very poor inhibitor (Table 1 ) . The inhibition of pooled C4 binding to Sepharose-C1s by penicillamine is 220μM, whilst the effect of captopril is less potent and 50% inhibition of binding occurs at 1mM. Effect on C4A and C4B Penicillamine has been shown previously to be a more potent inhibitor of the covalent binding of C4A to a complement-activating surface consisting of erythrocytes as antigen [17]. When the same experiment is carried out with Sepharose-C1s as the C4-activating surface, the same preference for inhibition of C4A is observed (Table 2) . With penicillamine as inhibitor, the inhibition of C4A covalent binding is 8 fold greater than the inhibition of C4B binding (table 2) but the difference between inhibition of C4A and C4B binding is only 2 fold with o-aminothiophenol as inhibitor. Example 2 In vitro studies with human C4 Human C4 (containing equal quantities of C4A and C4B) was purified as previously described above in Example 1 and inhibition of covalent binding to Sepharose-C1s was measured. The compounds tested were the hydrochloride salts of 4-methoxy, 2- aminothiophenol and of 2-aminothiophenol. The 4- raethoxy, 2-aminothiophenol inhibits 50% of the covalent binding of C4 at 95μM. The hydrochloride salt of 2-aminothiophenol inhibits 50% of the covalent binding of C4 at 20μM (Fig. 2). The hydrochloride salt has the additional advantage that it is highly water soluble and concentrated aqueous stock solutions are easy to prepare. Example 3 In vitro studies with human C3 Human C3 is the major component of the complement cascade and has the same covalent binding mechanism as C . C3 was prepared from human plasma [22] and inhibition of covalent binding of C3 to an activating surface (Sepharose-trypsin) was measured with the hydrochloride salt of 2-aminothiophenol. The results show that it is less effective as an inhibitor of the covalent binding reaction of C3 (Fig. 3). The 50% inhibition occurs at 154μM. However, it is a much better inhibitor of the covalent binding of C3 than is penicillamine which inhibits the covalent binding reaction of human C3 at greater than 1mM [27]. The increased activity of aminothiophenol on C3, compared with penicillamine, is not unexpected in view of the potency of aminiothiophenol with the C4B isotype of C4. This strengthens the evidence for aminothiophenol as a better anti-inflammatory agent than penicillamine which is likely to act as an anti-inflammatory agent in part through inhibition of the covalent binding reaction of C4. Example 4 Test of irritancy The hydrochloride salt of 2-aminothiophenol was dispersed to 100mM in "Hydrogel" (Trademark of East Riding Laboratories) and 2ml of the mixture was rubbed into human skin on the back of the hand. There was no indication of irritancy over a four hour period. Example 5 Studies were carried out with pigs. The reason for these studies was that Thiols have been shown to be radioprotective agents [28]. As already noted, minimising tissue damage can result from inhibition of complement activation and it is demonstrated here that inhibition via aminothiophenol could minimise tissue damage resulting from radiotherapy. Pigs are frequently used as test animals for radiotherapy. A) In vitro studies with Dig C4 C4 was prepared from pig plasma following a method previously used for other mammals [29]. The pure protein was shown to be activated with human C1s and the inhibition of covalent binding to sepharose- C1s was measured. The hydrochloride salt caused 50% inhibition at less than 50μM (Fig. 4) . B) In vivo studies 2-aminothiophenol was dispersed in "Hydrogel" at 100mM, 1OmM and 1mM of the aminothiophenol. Samples at each concentration were 2 applied to duplicate areas (5cm ) on the backs of pigs. The material was applied daily, for 7 days, to a least two distinct areas. There was no indication of any adverse reaction. Table 1. Inhibition of binding of 125I-C4 to Sepharose-C1s Structure Name Concentration for 50% inhibition of C4 binding Penicillamine 220μM o-aminothiophenol 90μM m-aminothiophenol 510μM p-aminothiophenol 88μM o-aminophenol 1.15mM o-aminobenzoic acid 37% at 5mM captopril 1mM SUBSTITUTE SBESΞT Table 2. Comparison of Inhibition of covalent binding of 1 5 IC4A and 25 IC4B to Sepharose-CΪs " . Concentration μM)for 50% binding to S-CT? Ratio Inhibitor C4A C4B C4B/C4A o-aminothiophenol 70 155 2.2 D-penicillamine 180 1500 8.3 9 - [1 ] Schifferli, J. A. & Peters, D. K. (1982) Lancet ii, 957-959. [2] Sim, E. (1991) Biochem. Soc. Trans. 19, 164- 170. [3] Colten, H. R. (1986) BioEssays 4, 249-272. [4] Yu, C. Y., Campbell, R. D. & Porter, R. R. (1988) Immunogenetics 27, 399-405. [5] Isenman, D. E. & Young, J. R. (1984) J. Immunol. 132, 3019-3027. [6] Law, S. K., Dodds, A. W. & Porter, R. R. (1984) EMBO J. 3, 1819-1823. [7] Hawkins, B. R. Wong, K. L., Wong, R. W. S., Chan, K. H., Dunckley, H., & Serjeantson, S. W. (1987) J. Rheumatol. 14, 1128-1131. [8] Finco, 0., Li, S., Cuccia, M., Rosen, F. S. & Carroll, M. C. (1992) J. Exp. Med. 175, 537-543. [9] Sim, E., Wood, M., Parker, K. E. & Jones, A. (1988) Comp. Immunol. Microbiol. Infect. Dis. 11, 163- 170. [10] Mongey, A. B. & Hess, E. V. (1989) Current Opinion in Rheumatol. 1, 353-359- [12] Sim, E. & Law, S. K. A. (1985) FEBS Letts 184, 323-327. [13] Sim, E., Dodds, A. W. & Goldin, A. (1989) Biochem. J. 259, 415-419. [14] Mellbye, 0. J. & Munthe, E. (1977) Ann. Rheum. Dis. 36, 453-458. [15] Howard-Lock, H. E., Lock, C. J. L., Mewa, A. & Kean, W. F. (1986) Sem. Arth. Rheum. 15, 261-281. [16] Hodsman, G. P. and Robertson, J. I. S., Brit. Med. J. (1983) 287, 851-852. [17] Sim, E., Gill, E. W. & Sim, R. B. (1984) Lancet ii, 422-424. [18] Hsiung, L. M. , Mason, D. W. & Dodds, A. W. (1987) Mol. Immunol. 24, 91-96. [19] Dodds, A.W., Law, S.K. & Porter, R.R. (1985) EMBO J. 4, 2239-2244. [20] Hsiung, L.M., Barclay, A.N., Brandon, M.R., Sim, E. & Porter, E. (1982) Biochem. J. 203, 293-298. [21] Roos, M.H., Mollenhauer, E., Demant, P. & Rittner, C.H., 1982, Nature 298, 854-856. [22] Sim, R.B., Twose, T.M., Paterson, D.S. & Sim, E. (1981) Biochem. J. 193, 115-127. [23] Sim, E., Parker & Jones, A. (1992) in Activators and Inhibitors of Complement (ed. R.B. Sim) pub. Kluwer, Netherlands in press. [24] Carroll, M.C., Falhallah, D.M., Berganaschini, L., Alicot, E.M. Isenman, D.E. (1991) Proc. Natl. Acad. Sci. USA 87, 6868-6872. [25] Weisman, H.F., Bartow, T., Leppo, M.K., Marsh, H.C., et al (1990) Science 249, 146-151. [26] Dalmasso, A.P., Platt, J.L. & Bach, F.H. (1991) Clin. Exp. Immunol. 86, s.1, 31-35. [27] Stanley, L. A. (1985), Part II thesis, University of Oxford. [28] Floersheim, G. L., (1992) Brit. J. Radiol. 65, 1025-1029. [29] Dόdds, A. & Law, S. K. A., (1990) Biochem. J. 265, 495-502. Figure Legends Figure 1. Inhibition of the covalent binding of human C4 to Sepharose-C1s by aminothiophenols. Ortho (•) Meta (+) and Para ( ) aminothiophenol were tested as inhibitors of covalent binding of 125I- labelled C4 as described in the text. The percentage of inhibition was determined by comparison with the inhibition of binding in the presence of 20mM hydroxylamine which was taken to be 100%. Figure 2. Inhibition of the covalent binding of human C4 to Sepharose-C1s by the hydrochloride salt of 2-aminothiophenol. Figure 3. Comparison of inhibitory potency of o-aminothiophenol and penicillamine on C3b binding. Figure 4. o-aminothiophenol activity with pig C4.

Claims: CLAIMS 1. A method for the treatment or prophylaxis of a complement related disorder in a patient comprising administering to the patient a compound having an aromatic or heterocyclic ring with at least one primary amine group NH_ and at least one sulphydryl group SH directly attached to the ring, or a pharmaceutically acceptable salt thereof. 2. A method as claimed in claim 1, wherein the disorder is an autoimmune disease such as arthritis or rheumatism. 3. A method as claimed in claim 1, wherein the disorder is myocardial infarction. 4. A method as claimed in claim 1, wherein the disorder is tissue damage associated with radiotherapy. 5. A method of inhibiting the covalent binding of complement component C4 or C3 to a complement activating surface which method comprises treating the complement component C4 or C3 with a compound having an aromatic or heterocyclic ring with at least one primary amiήe group NH„ and at least one sulphydryl group SH directly attached to the ring. 6. A method of reducing the risk of graft rejection which method comprises treating the graft or a recipient of the graft with a compound having an aromatic or heterocyclic ring with at least one primary amine group NH„ and at least one sulphydryl group SH directly attached to the ring, or a pharmaceutically acceptable salt thereof. 7. A method as claimed in any one of claims 1 to 6, wherein the compound has a primary amine group NH_ and a sulphydryl group SH which are directly attached to the ring and are ortho or para to one another. 8. A method as claimed in claims 1 to 7, wherein the compound has the formula where n is 0 to 4 and each R is selected from H, F, OH, SH, Cl, Br, I, N0 2 , NH 2 , mono or dialkylamino, SO.,H, alkyl, carboxy alkyl, hydroxy alkyl, alkoxy or carboxy, or two groups R may together form a fused aromatic ring structure. 9. A method as claimed in any one of claims 1 to 8, wherein the compound is 2-aminothiophenol. 10. A method as claimed in any one of claims 1 to 8, wherein the compound is 4-aminothiophenol. 11. The use of a compound having an aromatic or heterocyclic ring with at least one primary amine group NH_ and at least one sulphydryl group SH directly attached to the ring, or pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prophylaxis of a complement related disorder or for reducing the risk of graft rejection. 12. The use of a compound as claimed in claim 11. wherein the disorder is an autoimmune disease such as arthritis or rheumatism. 13. The use of a compound as claimed in claim 11, wherein the disorder is myocardial infarction. 14. The use of a compound as claimed in claim 11 , wherein the disorder is tissue damage associated with radiotherapy. 15. The use of a compound as claimed in any one of claims 11 to 14, wherein the compound has a primary amine group NH 2 and a sulphydryl group SH which are directly attached to the ring and are ortho or para to one another. 16. The use of a compound as claimed in any one of claims 11 to 15, wherein the compound has the formula where n is 0 to 4 and each R is selected from H, F, OH, SH, Cl, Br, I, NO * NH 2 ' mono or dialkylamino, SO_H, alkyl, carboxy alkyl, hydroxy alkyl, alkoxy or carboxy, or two groups R may together form a fused aromatic ring structure. 17. The use of a compound as claimed in any one of claims 11 to 16, wherein the compound is 2- aminothiophenol. 18. The use of a compound as claimed in any one of claims 11 to 16, wherein the compound is 4- aminiothiophenol. 19. A compound having an aromatic or heterocyclic ring with at least one primary amine group NH„ and at least one sulphydryl group SH directly attached to the ring, or a pharmaceutically acceptable salt thereof, for use in the treatment or prophylaxis of a complement related disorder or for reducing the risk of graft rejection. 20. A compound as claimed in claim 19, wherein the disorder is an autoimmune disease such as arthritis or rheumatism. 21. A compound as claimed in claim 19, wherein the disorder is myocardial infarction. 22. A compound as claimed in claim 19, wherein the disorder is tissue damage associated with radiotherapy. 23. A compound as claimed in any one of claims 19 to 22, wherein the compound has a primary amine group NH 2 and a sulphydryl group SH which are directly attached to the ring and are ortho or para to one another. 24. A compound as claimed in any one of claims 19 to 23, wherein the compound has the formula where n is 0 to 4 and each R is selected from H, F, OH, SH, Cl, Br, I, N0 2 , NH„, mono or dialkylamino, S0..H, alkyl, carboxy alkyl, hydroxy alkyl, alkoxy or carboxy, or two groups R may together form a fused aromatic ring structure. 2S. A compound as claimed in any one of claims 19 to 24, wherein the compound is 2-aminothiophenol. 26. A compound as claimed in any one of claims 19 to 24, wherein the compound is 4-aminothiophenol.

Inventors: EDMONDS STUART DAVID, GILL EDWARD WILLIAM, SIM EDITH, EDMONDS, STUART, DAVID, GILL, EDWARD, WILLIAM, SIM, EDITH
Applicants: EDMONDS STUART DAVID, GILL EDWARD WILLIAM, ISIS INNOVATION, SIM EDITH, ISIS INNOVATION LIMITED
Application year: 1993
Main classification: A61K31
IPC codes: A61K31, C07C323