NZ259737A - Complement-inactivating composition comprising soluble cr-1 protein and an amido-(phenyl or naphthyl) ester - Google Patents

Description

New Zealand No. 259737 International No. PCT/GB94/00122 ■■■I ,|l n I Priority Dat^s): ColTJfF)U>d: ,G!?.l.'.i..5^/r '-fcca* tf).

ZTKni"WS7 ?uWiG*-<>K>n Darts:.... P.O. Mo: NO DRAWINGS NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Combination of a soluble complement receptor-1 (SCR1) and an amidinophenyl or amidino naphthyl-ester for treating inflammation Name, address and nationality of applicant(s) as in international application form: SMITHKLINE BEECHAM PLC, a British company of New Horizons Court, Brentford, Middlesex TW8 9EP, England 5P 7 WO 94/16719 PCT/GB94/00122 COMBINATION OF A SOLUBLE COMPLEMENT RECEPT0R-1(5CR1) AND AN AMIDINOPHENYL OR AMIDINONAPHTYL ESTER FOR TREATING INFLAMMATION The present invention relates to therapeutic compositions of protease inhibitors and human soluble complement receptor 1 which act synergistically to 5 inhibit activation of complement. Such compositions are useful in the therapy of inflammatory or immune disorders involving complement activation.

Complement receptor type 1 (CR1) is present on the membranes of erythrocytes, monocytes/macrophages, granulocytes, B cells, some T cells, splenic follicular dendritic cells, and glomerular podocytes. CR1 binds to the complement 10 components C3b and C4b and has also been referred to as the C3b/C4b receptor. The structural organisation and primary sequence of one allotype of CR1 is known (Klickstein etal., 1987, J. Exp. Med. 165:1095-1112, Klickstein etal., 1988, J. Exp. Med. 168:1699-1717; Hourcade et al., 1988, J. Exp. Med. 168:1255-1270, WO 89/09220, WO 91/05047). It is composed of 30 short consensus repeats (SCRs) that 15 each contain around 60-70 amino acids. In each SCR, around 29 of the average 65 amino acids are conserved. Each SCR has been proposed to form a three dimensional triple loop structure through disulphide linkages with the third and first and the fourth and second half-cystines in disulphide bonds. CR1 is further arranged as 4 long homologous repeats (LHRs) of 7 SCRs each. Following a leader sequence, the CR1 20 molecule consists of the N-terminal LHR-A, the next two repeats, LHR-B and LHR-C, and the most C-terminal LHR-D followed by 2 additional SCRs, a 25 residue putative transmembrane region and a 43 residue cytoplasmic tail.

Several soluble fragments of CR1 have been generated via recombinant DNA procedures by eliminating the transmembrane region from the DNAs being expressed 5 (WO 89/09220, WO 91/05047). The soluble CR1 fragments were functionally active, bound C3b and/or C4b and demonstrated Factor I cofactor activity depending upon the regions they contained. Such constructs inhibited in vitro complement-related functions such as neutrophil oxidative burst, complement mediated haemolysis, and C3a and C5a production. A particular soluble construct, 30 sCRl/pBSCRlc, also demonstrated in vivo activity in a reversed passive Arthus reaction (WO 89/09220, WO 91/05047; Yeh et al., 1991, J. Immunol. 146:250-256), suppressed post-ischemic myocardial inflammation and necrosis (WO 89/09220, WO 91/05047; Weisman et al., Science, 1990,249:146-151; Dupe, R. et al. Thrombosis & Haemostasis (1991) 65(6) 695.) and extended survival rates 35 following transplantation (Pruitt & Bollinger, 1991, J. Surg. Res 50:350; Pruitt et al., 1991 Transplantation 52; 868), as well as demonstrating therapeutic inhibition of complement activation in several animal models of disease such as lung injury (Rabinovici et al, 1992 J. Immunol. 149:1744-1750; Mulligan et al, 1992 J. Immunol. 148:3086-3092), intestinal ischaemia (Hill et al, 1992 FASEB J. 6:A1049) and acute myocardial infarction (Weisman et al, 1990 Science 249:146-151, Dupe et al, 1991 (above)).

In a number of cases, the doses of sCRl required for therapeutic effects in these models were large (>5mg/kg). Because sCRl is a biopharmaceutical produced 5 by mammalian cell culture techniques, it is desirable to reduce the dose and hence the cost of therapy.

Certain amidinophenyl and amidinonaphthyl esters of carboxylic acids are known to be inhibitors of complement activation as well as having antitrypsin, antiplasmin, antikallikrein and antithrombin activity (GB 2095-239, GB 2083-818). 10 GB 2083818 discloses compounds of formula (A): wherein Z represents -(CH2)a-. -(CH2)b-CH(R3)-, -CH=C(R4)- or 15 -0-CH(R4)-* where a is 0,1,2 or 3, b is 0,1 or 2, R3 is a straight or branched chain alkyl group of 1 to 4 carbon atoms or a cycloalkyl group of 3 to 6 carbon atoms, and R4 is a hydrogen atom or a straight or branched chain alkyl group of 1 to 4 carbon atoms and wherein the -CH(R3)-, = C(R4)- or -CH(R4)- moiety is bonded to the -COO group; and R^ and R2, which 20 may be the same 01 different, represent each a hydrogen atom, straight or branched chain alkyl group of 1 to 4 carbon atoms, -O-R5, -S-R5, -COOR5, -CORfr -O-COR7, -NHCOR7, -(CH2)c-NR8R9, -SO2NR8R9, NO2, CN, halogen, CF3, methylenedioxy, where c is 0,1 or 2; R5 is a hydrogen atom, straight or branched chain alkyl group of 1 to 4 carbon atoms, or benzyl group; Rg is a hydrogen atom or straight or branched chain alkyl group of 1 to 4 atoms; R7 is a straight or branched chain alkyl group of 1 30 to 4 carbon atoms; Rg and R9, which may be the same or different, are each a hydrogen atom, straight or branched chain alkyl group of 1 to 4 carbon atoms, or amino radical protecting group; and Rjq is a hydrogen atom, dimethyl or CF3.

(A) NH GB 2095239 discloses compounds of the general formula (B): NH Ru-c wherein Rl J represents a straight or branched chain alkyl group 1 to 6 carbon atoms, a straight 5 or branched chain alkenyl group of 2 to 6 carbon atoms having 1 to 3 double bonds, where R \ 3 is a cycloalkyl group of 3 to 6 carbon atoms or a cycloalkenyl group of 3 to 6 atoms having 1 or 2 double bonds; d is 0, 1,2 or 3; R14 is an amino or guanidino group or a protected amino or guanidino group; e is a number from 1 to 5; Rl5 and R^j, which may be the same or different, are each a hydrogen atom, a straight or branched alkyl group of 1 to 4 carbon atoms, -OR 17, methylenedioxy group, -SR17, -COOR17, -CORjg, -OCOR19, -NHCOR19, -(CH2)f-NR20^21 (f is 0, 1,2), -SO2NR20^21» a halogen atom, -CF3, N02, CN, Rl7 is a hydrogen atom, a straight or branched alkyl group of 1 to 4 carbon atoms or a benzyl group; Rig is a hydrogen atom, a straight or branched alkyl group of 1 to 4 carbon atoms; R19 is a straight or branched alkyl group of 1 to 4 carbon atoms; R20 and R21, which may be the same or different, are each a hydrogen atom, a straight or 25 branched alkyl group of 1 to 4 carbon atoms, or an amino-protecting group; R22 is O, S or NH; R23 is a 2\3'-dimethyl or 3'-CF3 group; Y is -(CH2)g-(g is 0, 1, 2 or 3), -(CH2)h"CHR24- (h is 0, 1 or 2), or -CH=CR25-; R24 is a straight or branched alkyl group of 1 to 4 carbon atoms and the carbon atom or the CHR24 moiety is attached to the COO group; R25 is a hydrogen 30 atom or a straight or branched alkyl group of 1 to 4 carbon atoms and the carbon Rl3-(GH2)d-.Rl4-(CH2)e-. atom of the CR25 moiety is attached to the COO group; and R12 represents -R26>" OR26. -COOR27. one or two of the same halogen atoms, -NH2. -SO3H, wherein R26 is a straight or branched alkyl group of 1 to 4 carbon atoms; R27 is a hydrogen atom or a straight or branched alkyl group of 1 to 4 carbon atoms; and R28 is a hydrogen atom or a guanidino group.

Other amidinophenyl esters of carboxylic acids are also known to inhibit 10 proteases of the coagulation pathway (A.D.Turner et al, 1986 Biochem. 25:4929-35) and have also been employed to acylate the active centres of fibrinolytic enzymes reversibly (US 4,285,932, US 4,507,283, EP 0,297,882, R.A.G.Smith etal, 1985 Progress in Fibrinolysis VII227-231). US 4285932, US 4507283 and EP 0297882 disclose compounds of formula (C): HN W )-0CO*.

H,N ^—" (C) wherein Rx is benzoyl optionally substituted with one or two substituents independently selected 20 from halogen, Cj.g alkyl, C2-6 alkenyl, Cj.g alkoxy, Cj.g alkanoyloxy, Cj_6 alkanoylamino, amino, dimethylamino or guanidino; naphthoyl; or acryloyl optionally substituted with Cj.g alkyl, furyl or phenyl wherein the phenyl moiety is optionally substituted with Cj.g alkyl.

Thus this type of compound is not a specific inhibitor of the proteases of the complement system.

Synergistic compositions of CRl-related polypeptides with certain organic compounds have been described (W0 92/10205).

According to the present invention there is provided a method of treating a 30 disease or disorder associated with inflammation or inappropriate complement activation which method comprises administering to a mammal in need thereof an effective amount of a soluble CR1 protein and an effective amount of an amidinophenyl or amidinonaphthyl ester of formula (I) having complement inhibitory activity: 259737 HN 1 H,N \ II y— ®—oc—® (i) wherein A is phenyl optionally substituted with Cj.4 alkyl, Cj.4 alkoxy, C1.4 alkoxycarbonyl, halo, NH2, sulphonyl, benzoyl or C 1.4 alkylbenzoylamino or naphthyl; and B is CH2=CH- optionally substituted by a group selected from Cj.g alkyl, phenyl and phenyl substituted with Cj.g alkyl; phenyl optionally substituted with one or two subsrituents independently selected from halogen, Cj.g alkyl, C2-6 alkenyl, Cj.6 alkoxy, C\.£ alkenoyloxy, Ci_6 alkanoylamino, amino, dimethylamino'or guanidino; or naphthyl, including pharmaceutical^ acceptable salts thereof.

Preferably, A is phenyl optionally substituted in the 2- or 3- position by halogen and the amidine substituent is in the 4-position of the phenyl ring. B is preferably phenyl 4-substituted by Cj_4 alkoxy and optionally further substituted by halogen. Most preferably, B is 4-methoxyphenyl and A is phenyl or 2-bromophenyl, 4-substituted by the amidine group.

Suitable examples of halo include chloro and bromo.

Pharmaceutical^ acceptable salts may be formed with pharmaceutically acceptable acids, for example, maleic, hydrochloric, hydrobromic, phosphoric, acetic, 20 fumaric, salicylic, citric, lactic, mandelic, tartaric, methanesulphonic and oxalic acid.

In a preferred aspect, the soluble CR1 component used in combination therapy is encoded by a nucleic acid vector selected from the group consisting of pBSCRlc, pBSCRls, pBM-CRlc, pBSCRlc/pTCSgpt and pBSCRls/pTCSgpt, and-is especially that obtainable from pBSCRlc/pTCSgpt, as described in WO 89/09220. 25 The amounts of each compound are chosen such that the concentration of each component required to inhibit by 50% haemolysis of sensitized erythrocytes in a standard complement assay is lowered compared with that required for the individual components in the same assay. This increase in potency is described by a synergy factor which is defined in more detail below.

The compounds may be administered by standard routes, such as, for example, intravenous infusion or bolus injection, and may be administered together or sequentially, in any order.

N.7. >' ■ 2 0 FEB 1997 h ... i . '. 2by/37 When the compounds are administered together they are preferably given in the form of a pharmaceutical composition comprising both agents.

Thus, in a principal aspect of the invention there is provided a pharmaceutical composition comprising a soluble CR1 protein and an amidinophenyl or amidinonaphthyl ester of formula (I) having complement inhibitory activity together with a pharmaceutically acceptable carrier, wherein the soluble CR1 protein and the ester of formula (I) act synergistically to inhibit activation of complement.

In a preferred embodiment, the composition may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.

The invention also provides the use of a soluble CR1 protein and an amidinophenyl or amidinonaphthyl ester of formula (I) having complement inhibitory activity in the manufacture of a medicament for the treatment of a disease or disorder associated with inflammation or inappropriate complement activation, wherein the soluble CR1 protein and the ester of formula (I) act synergistically to inhibit activation of complement.

In a further aspect, the invention therefor provides a method for the preparation of a pharmaceutical composition of the invention, which method comprises admixing the combination of soluble CR1 protein and an amidinophenyl or amidinonaphthyl ester of formula (I), including pharmaceutically acceptable salts thereof.

The present invention also provides a method of treating a disease or disorder associated with inflammation or inappropriate complement activation comprising administering to a subject in need of such treatment a therapeutically effective amount of a composition of the invention.

In the above methods, the subject is preferably a human.

An effective amount of the protein for the treatment of a disease or disorder is in the dose range of O.Ol-lOOmg/kg; preferably O.l-lOmg/kg.

An effective amount of the ester for the treatment of a disease or disorder is in the dose range 0.05-100 mg/kg; preferably 0.05-10 mg/kg. The ratio of protein to ester is preferably in the range 1:1 to 1:20 by weight.

The composition typically contains a therapeutically active amount of the protein and ester and a pharmaceutically acceptable excipient or carrier such as saline, buffered saline, dextrose, or water. Compositions may also comprise specific stabilising agents such as sugars, including mannose and mannitol, and local anaesthetics for injectable compositions, including, for example, lidocaine.

A pharmaceutical pack comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition is also within the scope of the invention.

The present invention also provides a method for treating a thrombotic 5 condition, in particular acute myocardial infarction, in a human or non-human animal, said method comprising administering to the patient a composition according to this invention.

This invention further provides a method for treating adult respiratory distress syndrome (ARDS) in a human or non-human animal, said method comprises administering to the patient a composition according to this invention.

The invention also provides a method of delaying hyperacute allograft or hyperacute xenograft rejection in a human or non-human animal which receives a transplant by administering a composition according to this invention. - 6a - WO 94/16719 PCT/GB94/00122 The methods and compositions of this invention are useful in the treatment of complement-mediated or complement-related disorders, including but not limited to those listed below.

Disease and Disorders Involving Complement Neurological Disorders multiple sclerosis stroke Guillain Barrd Syndrome traumatic brain injury Parkinson's disease allergic encephalitis Disorders of Inappropriate or Undesirable Complement Activation hemodialysis complications hyperacute allograft rejection corneal graft.rejection xenograft rejection 20 interleukin-2 induced toxicity during IL-2 therapy paroxysmal nocturnal haemoglobinuria Inflammatory Disorders inflammation of autoimmune diseases 25 Crohn's Disease adult respiratory distress syndrome thermal injury including burns or frostbite uveitis Post-Ischemic Reperfusion Conditions myocardial infarction balloon angioplasty post-pump syndrome in cardiopulmonary bypass or renal hemodialysis renal ischemia 35 hepatic ischemia Infectious Diseases or Sepsis multiple organ failure septic shock Immune Complex Disorders and Autoimmune Diseases rheumatoid arthritis systemic lupus erythematosus (SLE) SLE nephritis proliferative nephritis glomerulonephritis hemolytic anemia myasthenia gravis Reproductive Disorders antibody- or complement-mediated infertility MATERIALS BRL 55730 - is the soluble complement receptor type 1 derived from the expression of plasmid pBSCRlc/pTCSgpt in CHO cells (WO 89/09220).

BRL24894A (APAN) - 4-amidinophenyl 4'-methoxybenzoate HCl (EP-0009879) BRAPAN - 4-amidino-2-bromophenyl 4'-methoxybenzoate HCl (Example 3).

METHODS Anti-complement Activity Measured by the Haemolysis of Sheep Erythrocytes Functional activity of complement inhibitors was assessed by measuring the 25 inhibition of complement mediated lysis of sheep erythrocytes sensitised with rabbit antibodies (obtained from Diamedix Corporation, Miami, USA). Human serum diluted 1:125 or 1/35.7 in 0.1 M Hepes pH 7.4/ 0.15 M NaCl buffer was the source of complement and was prepared from a pool of volunteers essentially as described in Dacie & Lewis, 1975 (Practical Haematology 5th Edition, Churchill Livingstone, 30 Edinburgh and New York, pp3-4). Briefly, blood was wanned to 37°C for 5 minutes, the clot removed and the remaining serum clarified by centrifugation. The serum fraction was split into small aliquots and stored at -196°C. Aliquots were thawed as required and diluted in the Hepes buffer immediately before use.

Inhibition of complement-mediated lysis of sensitised sheep erythrocytes was 35 measured using a standard haemolytic assay using a v-bottom microtitre plate format as follows, essentially as described by Weisman et al 1990 (above).

Standard assay p.1 of a range of concentrations of inhibitor (typically in the region of O.ljig/ml - 0.00078|ig/ml final for BRL55730 and 100 - O.lpM final of APAN or BRAPAN) diluted in Hepes (0.1M Hepes pH7.4/0.15M NaCl) buffer were incubated 5 with 25 |J.l of buffer and 50 (il of the 1/125 diluted serum for 15 minutes at 37°C. 100 Hi of prewarmed sensitised sheep erythrocytes were added for 1 hour at 37°C in a final reaction volume of 200 |il. Samples were spun at 300g at 4°C for 15 minutes before transferring 150 |il of supernatant to flat bottom microtitre plates and determining the absorption at 410 nm, which reflects the amount of lysis in each test 10 solution. Maximum lysis was determined by incubadng serum with erythrocytes in the absence of any inhibitor (E+S) from which the proportion of background lysis had been subtracted (determined by incubating erythrocytes with buffer) (E). The background lysis by inhibitor was assessed by incubating inhibitor with erythrocytes (E+I) and then subtracting that from test samples (E+I+S). Inhibition was expressed 15 as a fraction of the total cell lysis such that EH50 represents the concentration of inhibitor required to give 50% inhibition of lysis. For experiments in which serum had been diluted 1/35.7, the incubation time was reduced to 15 mins at 37°C. Otherwise conditions were the same.

Maximum Lysis: A max = (E+S) - (E) Lysis in presence of inhibitor: Ao = (E+I+S) - (E+I) Amount of inhibition: IH = (Amax-Ao^ Amax Plots were made of [inhibitor] vs IH and IH50 values were determined from the titration curve by reading off the concentration corresponding to 1H=0.5.

Synergy Assays The assay was carried out in a similar manner to that described above except 30 that inhibitor 1 eg BRL55730 was titrated in the presence of a fixed concentration of inhibitor 2 eg APAN. This was carried out by adding 25 p.1 of inhibitor 1 to 25 p.1 of inhibitor 2 in the presence of serum and measuring the degree of lysis as described above.

Determination of the Synergy Factor For each synergy experiment both inhibitors were titrated on their own as well as together.

PCT / GB94/00122 EXAMPLE 1 Inhibitor 1, BRL55730 was titrated on its own and in the presence of various concentrations of inhibitor 2, APAN. A plot was made of the [BRL55730] vs IH with and without APAN (Fig.l). The IH50 of BRL55730 was estimated at each 5 APAN concentration. A second plot of [APAN] vs IH was made (Fig.2) from which the IH corresponding to the concentration of APAN used in the synergy experiment was estimated. The results were then tabulated (Table 1).

Column 1 refers to the concentration of APAN. The proportion of inhibition that a particular concentration of APAN contributes was estimated from the plot of 10 [APAN] vs IH (Fig.2) (column 2). The IH50 for BRL55730 was determined at each concentration of APAN (column 3). The contribution that a particular concentration of APAN made to the IH50 of BRL55730 was subtracted ie 0.5 - IH (ApAN) (column 4). This value was used to read off the concentration of BRL55730 which alone would have provided this level of inhibition (column 5). The adjusted BRL55730 15 concentration was divided by the measured IH50 to give the synergy factor i.e. column 5/column 3 = column 6. If the effect of APAN was additive, the synergy factor would be 1; values greater than 1 represent a synergistic effect and the greater the value, the greater the degree of synergy.

Isobologram Analysis The IH50's of inhibitor 1, eg BRL55730 and inhibitor 2, eg APAN were determined separately. These experimentally determined values are plotted on the axes of the isobologram and were connected by a straight line, termed the line of additivity (Fig. 3). This line represents combinations of the two inhibitors which, 25 when used together, would result in 50% inhibition (Tallarida (1992) Pain 49: 93-97, Miaskowski & Levine, (1992) 51: 383-387). Hence points falling on the line of additivity indicate an additive effect, points above this line indicate antagonism and points below this curve indicate synergy. BRL55730 was titrated in the presence of fixed concentrations of APAN and the IH50 of BRL55730 determined at each APAN 30 concentration. This data was plotted on the isobologram (Fig. 3).

Statistical Analysis of Synergy using Fixed Concentration Pairs.

BRL55730 at concentration x which was below its IH50 was assayed in the standard assay. APAN at concentration y which was below its IH50 was also 35 assayed. Then BRL55730[X] was assayed together with APANjyj in the same haemolysis assay. The amount of inhibition was calculated for the two inhibitors when assayed separately and when assayed together.

If synergy occurs then the inhibition of the compounds assayed together should be greater than the sum of the two inhibitors separately ie BRL55730[x]/APAN[y] > BRL55730[X] + APAN[y] The data was analysed statistically by t-test according to the formula listed below.

Group Mean Sample size Standard error of the mean Standard error of the mean squared a BRL55730 a "a s.e.m.a (s.e.m.)a2 b APAN b nh s.e.m.h (s.e.m)h2 ab BRL55730/APAN ab nah s.e.mab (s.e.m.)ab2 Null Hypothesis = HO ab = a + b (ie effect is additive) Alternative Hypothesis = HI ab * a + b (ie effect is not additive) t = * ab - a - b Equation 1 V{(s.e.m.ab2) + (s.e.m.a2) + (s-e-m.b2)) t was compared with critical levels in t-tables where degrees of freedom (df) df = na + nb + nab - 3 a. Synergy of APAN with BRL55730 in Serum Diluted 1/125 BRL 24894A (APAN) molecular weight 324.24 was made 50 mM in dimethylsulphoxide (DMSO). BRL55730 (in lOmM sodium phosphate pH7.2 buffer) was at 5.3 mg/ml. Both inhibitors were titrated in the standard assay over the concentration range of 100 p.M - 0.78 |iM for APAN and 0.125 p.g/ml - 0.00098 20 (ig/ml for BRL55730. Two titration curves were performed for BRL55730 from which the mean IH50 was determined as 0.01 Hg/ml (Fig.l) and one curve for APAN from which the IH50 was determined as 10 |i.M (Fig.2). To test for synergy, BRL55730 was titrated over the same concentration range but in the presence of fixed concentrations of APAN from 1-6 |iM for each titration (Fig.l). From the data the 25 synergy factor was calculated as described above.

Table 1: Determination of the Synergy Factor for BRL55730 and APAN. 1 2 3 4 6 [APAN] IH of IH50 of 0.5 - IH of Adjusted Synergy \iM APAN BRL55730 APAN [BRL55730] Factor |ig/ml Hg/ml 0 - 0.01 0.5 0.01 1 1 0.03 0.007 0.47 0.009 1.3 2 0.09 0.004 0.41 0.008 2.0 3 0.16 0.0027 0.34 0.006 2.2 4 0.21 0.0022 0.29 0.004 1.8 0.27 0.0019 0.23 0.003 1.6 6 0.31 0.0013 0.19 0.0022 1.7 From the data in Table 1, inclusion of 6 (J.M APAN with BRL55730 reduces the IH50 5 by approximately 8 fold. The calculated synergy factor at each concentration of APAN is given in Table 1 and shows that the effect of APAN is more than additive since the synergy factor is > 1. The synergy factor also remains fairly constant across the range of concentrations used with a mean value of 1.8.

BRL55730, concentration range 0.04 - 0.000039 jig/ml, was titrated on its 10 own; the concentration at which no inhibition of complement activation occurred was found to be ~ 0.0004 |ig/ml. Titration of APAN on its own showed that & concentration of 4 (iM gave an IH of 0.31 and 2 |iM gave an IH of 0.16. When BRL55730 was titrated in the presence of APAN at 4 and 2 |iM, the inhibition at 0.0004 |ig/ml of BRL55730 was greater than that could be accounted for by APAN 15 only showing that APAN potentiates the activity of BRL55730 below the no-effect concentration. b. Isobologram Analysis BRL55730 with APAN The additivity line was constructed as described above taking the data from 20 Figs. 1 2. The IH50's of BRL55730 at each APAN concentration (columns 1 & 3 of Table 1 respectively) were then plotted on the isobologram as shown in Fig. 3. The points fall below the line of additivity indicating that the interaction is synergistic.

WO 94/16719 PCT/GB94/00122 c. Statistical Analysis of Synergy Between BRL55730 and APAN using Fixed Concentration Pairs The following concentration pairs were tested for synergy as described above (i) 0.005 p.g/ml BRL55730 4 jjlM APAN (ii) 0.005 |ig/ml BRL55730 2 \iM APAN (iii) 0.002 ng/ml BRL55730 4 \xM APAN (iv) 0.002 jig/ml BRL55730 2 ^M APAN The statistical parameters are given below for each concentration pair.

Table 2: Statistical Analysis of Concentration Pair (i) PARAMETER BRL55730 0.005 ng/ml APAN 4 jaM BRL55730 + APAN 0.005 |ig/ml + 4 M.M MEAN IH 0.312 0.297 0.676 SEM 0.0117 0.0092 0.0096 (SEM)2* 0.000136 0.000086 0.000092 n 14 14 14 df 39 t-value 3.822 At 39 df, probability of 0.9995 t = 3.558 Calculated t > 3.558. Therefore a + b ab Table 3: Statistical Analysis of Concentration Pair (ii) PARAMETER BRL55730 0.005 |ig/ml APAN 2 p.M BRL55730 + APAN 0.005 Jig/ml + 2 |iM MEAN IH 0.251 0.124 0.509 SEM 0.0135 0.0183 0.00947 (SEM)2 0.000183 0.000334 0.0000897 n 16 16 16 df 45 t-value .407 At 45 df, probability of 0.9995 t = 3.550 Calculated t > 3.550. Therefore a + b * ab Table 4: Statistical Analysis of Concentration Pair (iii) PARAMETER BRL55730 0.002 ng/ml APAN 4 |lM BRL55730 + APAN 0.002 p.g/ml + 4 jiM MEAN IH 0.123 0.337 0.564 SEM 0.00741 0.00972 0.00691 (SEM)2 0.000055 0.0000945 0.0000477 n 16 16 16 df 45 t-value. 7.451 At 45 df, probability of 0.9995 t = 3.550 Calculated t > 3.550. Therefore a + b * ab Table 5: Statistical Analysis of Concentration Pair (iv) PARAMETER BRL55730 0.002 |J.g/ml APAN 2 p.M BRL55730 + APAN 0.002 us/ml + 2 |iM MEAN IH 0.121 0.192 0.422 SEM 0.0104 0.0106 0.00886 (SEM;2 0.000109 0.000112 0.0000784 n 16 16 16 df 45 t-value 6.267 At 45 df, probability of 0.9995 t = 3.550 Calculated t > 3.550. Therefore a + b * ab At each of the tested concentration pairs, the alternative hypothesis was shown to be correct ie that the effect was not additive and since a + b < ab, synergy has been demonstrated. d. Synergy Effect of BRL55730 on APAN The effect of BRL55730 on the IH50 of APAN was tested in the same way as described in Example la but in this instance APAN was titrated over the range 0.78nM to IOOjiM in the presence of fixed concentrations of BRL55730 between 15 0.001 - 0.006 ng/ml. The effect of BRL55730 on the IH50 of APAN is given in Table 2 and shows that addition of 0.006 |ig/ml of BRL55730 shifts the IH50 of APAN from 10 jiM to 1 which is an improvement of 10 fold. The synergy factor was determined as described in the Methods and found to be > 1 (Table 6) indicating that the synergy process between APAN and BRL55730 is reversible. Unlike the previously described Example la, the synergy factor appears to be dependent on the concentration of BRL55730.

Table 6: Determination of the Synergy Factor for APAN and BRL55730 [BRL55730] (ig/ml IH of BRL55730 IH50 of APAN HM 0.5 - IH of BRL55730 Adjusted [APAN] |iM Synergy Factor 0 - 0.5 1 0.001 0.025 .3 0.475 9 1.7 0.003 0.13 1.8 0.37 7 3.9 0.006 0.34 1.0 0.16 3 3.0 e. Isobologram Analysis of APAN with BRL55730 Isobologram analysis was performed as described above using data from Fig. 10 2 and Table 6. Data points fell below the line of additivity which indicated the effect was synergistic. f. Synergy of APAN with BRL55730 in Serum Diluted 1/35.7 To test whether the synergy seen between APAN and BRL55730 is 15 reproducible in more concentrated serum, experiments were carried out in serum that had been diluted 1/35.7 which was 3.5 fold more concentrated than described in Example la. As a preliminary test to make sure that this concentration of serum did not produce maximum lysis of the sensitised sheep erythroc> .es, 50 |il of serum at various dilutions from 1/10 to 1/150 were preincubated with 50 p.1 of 0.1 M Hepes pH 20 7.4 / 0.15 MNaCl buffer at 37°C for 15 mins. 100^.1 of erythrocytes were added and samples were incubated for either 15 mins or 30 mins at 37°C. Unlysed cells were spun down at ~ 300 g at 4°C for 15 mins and then 150 Jil of supernatPii /ere transfered to a flat bottom microtitre plate before reading the absorbance at 410 nm. A plot of the serum dilution vs absorbance showed that incubation of serum at 1/35.7 25 dilution for 15 mins with erythrocytes gave ~ 90% of the maximum lysis. Since this concentration of serum proved suitable, synergy experiments of BRL55730 with APAN were carried out in a similar manner as described in Example la except that more concentrated serum was used and incubation times were reduced to 15 mins.

Titration of BRL55730 in the more concentrated serum gave an IH50 of ~ 30 0.14 |ig/ml (mean of two determinations) which is 14 fold greater than in 1/125 diluted serum. Similarly the IH50 of APAN was found to be 52 |i.M (mean of two determinations) which is about five fold greater than in the more dilute serum. The synergy experiments were carried out by titrating BRL55730 over a concentration range of 1 - 0.0078 (ig/ml in the presence of APAN at concentrations ranging from 2 5 - 18 H.M. The summary of the data is given in Table 7 and demonstrates that the synergy effect can be extended to more concentrated serum. The synergy factor in this instance appears to be dependent on the concentration of APAN.

Table 7: Synergy Value Between BRL55730 and APAN in more concentrated 10 Serum 1 2 3 4 6 [APAN] jiM IH of APAN IH50 of BRL55730 |ig/ml 0.5 - IH of APAN Adjusted [BRL55730] Hg/ml Synergy Factor 0 - 0.14 0.5 0.14 1 2 *0 0.135 0.5 0.14 1.03 4 0.025 0.11 0.475 0.135 1.23 6 0.035 0.055 0.465 0.125 2.27 9 0.055 0.045 0.445 0.11 2.44 12 0.100 0.032 0.4 0.105 3.28 0.16 0.025 0.34 0.1 4.00 18 0.2 0.02 0.3 0.085 4.25 • g. Isobologram Analysis of BRL55730 and APAN in More Concentrated Serum The IH50's of BRL55730 and APAN in serum diluted 1/35.7 were used to construct the additivity line. Data points from columns 1 and 3 of Table 7 were used to construct an isobologram. All the points except at 2 |iM fall below the addivitity line indicating synergy. The 2 |iM data point shows a synergy factor of 1.03 in Table 7 and falls on the line of additivity in the isobologram showing that at this 20 concentration the effect may be additive. h. Synergy Effect of BRAPAN on BRL55730 4-Amidino-2-bromophenyl 4'-methoxybenzoate HCl (BRAPAN) molecular weight 386 was made 10 mM in DMSO and titrated as described in the Methods 25 using serum diluted 1/125. From two separate determinations the mean value for the IH50 of BRAPAN was 3 pM. A single titration curve of BRL55730 from 0.1 -0.00078 |ig/ml was determined which gave an IH50 of 0.021 jig/ml. To test for synergy BRL55730 was titrated over the same concentration range but in the presence of BRAPAN ranging from 0.1 |iM to 0.9nM. Table 8 demonstrates the effect and synergy potential of BRAPAN on BRL55730. As with APAN at the same serum dilution the synergy factor is >1 indicating that BRAPAN synergises with the BRL55730. The synergy factor remains fairly constant over the concentration range giving a mean value of 1.7 which again is very similar to that seen for APAN.

Table 8: Synergy Value Between BRL55730 and BRAPAN 1 2 3 4 6 [BRAPAN] IH of IH50 of 0.5 - IH of Adjusted Synergy |iM BRAPAN BRL55730 BRAPAN [BRL55730] Factor jig/ml Hg/ml 0 0 0.021 0.5 0.021 1 0.1 0 0.015 0.5 0.021 1.4 0.2 0 0.013 0.5 0.021 1.62 0.3 0.01 0.011 0.49 0.02 1.82 0.4 0.03 0.013 0.47 0.019 1.46 0.5 0.04 0.012 0.46 0.018 1.5 0.6 0.06 0.008 0.44 0.016 2.00 0.7 0.09 0.007 0.41 0.015 2.14 0.8 0.13 0.007 0.37 0.013 1.86 0.9 0.16 0.0059 0.34 0.011 1.86 i. Isobologram Analysis of the effect of BRAPAN on BRL55730 Using the data described in Example lh for BRAPAN, an isobologram was 15 constructed. All the data points fell below the line of additivity indicating synergy was occurring.

EXAMPLE 2 Co-formulation of sCRl (BRL 55730) and APAN (BRL 24894A) 20 D-Mannitol (Sigma.UK, 60mg) was dissolved in water for injection (9.5ml).

APAN was disssolved in HPLC-grade methanol to a final concentration of 6 mg/ml by stiring at ambient temperature (20-25°C) for 5 min. The solution (0.5ml) was added IMMEDIATELY to the mannitol solution and mixed by shaking. A solution of BRL 55730 (5mg/ml in lOmM sodium phosphate pH 7.2,0.2ml) was added, shaken and immediately frozen in solid CO2. The material was lyophilised at an average pressure of 2-3 mbar and a condenser temperature of -60°C for 20 hours. The white solid had the following composition and was stored desiccated at -70°C: 5 BRL 55730: lmg; BRL 24894A: 3mg; D-Mannitol: 60mg; sodium phosphate: trace.

EXAMPLE 3 Preparation of 4-Amidino 2-bromophenyl 4'-methoxybenzoate HCl (BRAPAN) This material was prepared in two steps from 2-bromo 4-cyanophenol. a: Preparation of 2-bromo-4-amidinophenol hydrochloride 2-Bromo-4-cyanophenol (1.35 g, 6.8 mmole) was dissolved in ethanol (20 ml). Hydrogen chloride gas was passed through the cooled solution, a white precipitate forming after 45 mins. After 2 hours, the bubbling was stopped and the 15 solid in solution placed at 4°C for two days. The white solid was isolated by filtration. This was rapidly suspended in ethanol solution (50 ml) and a saturated solution of ammonia in ethanol (75 ml) was added. The suspension went clear almost immediately, and was stirred for 24 hours and allowed to stand for a similar period. All volatile material was removed and the white solid was taken up in water (20 ml). 20 Addition of concentrated hydrochloric acid (5 ml) led to the rapid formation of a white crystalline mass which was isolated by filtration and recrystallised from ethanol (20 ml) and diethyl ether (100 ml). Yield: 860 mg (50%). mp: 279-80°.

*H nmr (CDCl3-d6DMSO) 8: 9.2 (4H, br d, amidine), 8.2 (IH, d, I=2Hz; aryl-H), 25 7.8 (IH, m, aryl-H), 7.25 (IH, I=9Hz, aryl-H) Infrared (nujol): 3325, 3125, 2300-3400,1670, 1610,1585,1410, 1300, 1175,1045, 880, 835,725, 620 cm" 1 Analysis: C 33.45, H 3.28, N 10.95% C7HgN2OBrCl requires C 33.43, H 3.21, N 11.14% b. Preparation of the title compound To a solution of 4-methoxybenzoyl chloride (271 mg, 1.59 mmole) in dry pyridine (5 ml) was added 2-bromo-4-amidinophenol hydrochloride (400 mg, 1.59 mmole). The initial suspension became a clear solution and then a precipitate 35 reformed. After 1 hour stirring infrared analysis showed the formation of an ester. The pyridine was removed at reduced pressure the last traces by azeotrope with ethanol. The white solid obtained was recrystallised twice from 5:1 diethyl ether/ethanol (-100 ml) to leave the white title compound. Yield: 225 mg (37%). mp 212-3°C. nmr (d^DMSO-trace CDCI3) 8: 9.55 (4H, br, amidinophenol NH), 6.95-8.25 (7H, m, aryl-H), 3.9 (IH, s, OCH3) Infrared (nujol): 2500-3400,1735, 1665, 1605, 1580,1260, 1230,1170, 1070,1020, 5 and 830 cm' 1 Analysis: C 46.66, H 3.74, N7.19% Ci5,Hi2,N2,Br,CI requires C 46.72%, H 3.66%, N7.26% In the figures: Fig. 1 shows the effect of different concentrations of APAN on BRL 55730; Fig. 2 shows the inhibition of complement activation by APAN; and Fig. 3 is an isobologram of BRL 55730 and APAN in a standard assay.

Claims (6)

WHAT WE CLAIM IS: 25 9737

1. A pharmaceutical composition comprising a soluble CR1 protein and an amidinophenyl or amidinonaphthyl ester of formula (I) having complement inhibitory activity: HN. ° y— @—oc—(i) HjN wherein A is phenyl optionally substituted with C1.4 alkyl, C1.4 alkoxy, C1.4 alkoxycarbonyl, halo, NH2, sulphonyl, benzoyl or C1.4 alkylbenzoylamino or naphthyl; and B is CH2=CH- optionally substituted by a group selected from Cj.g alkyl, phenyl and 15 phenyl substituted with Cj.g alkyl; phenyl optionally substituted with one or two substituents independently selected from halogen, Cj.g alkyl, C2-6 alkenyl, C].^ alkoxy, C]_6 alkenoyloxy, Cj.6 alkanoylamino, amino, dimethylamino or guanidino; or naphthyl, including pharmaceutically acceptable salts thereof, together with a pharmaceutically acceptable carrier, wherein the soluble CR1 protein and the 20 ester of formula (I) act synergistically to inhibit activation of complement.

2. A pharmaceutical composition suitable for treating a disease or disorder associated with inflammation or inappropriate complement activation which treatment comprises administering to a mammal in need thereof an 25 effective amount of a soluble CR1 protein and an effective amount of an amidinophenyl or amidinonaphthyl ester of formula (I) as defined in claim 1 having complement inhibitory activity.

3. The use of a soluble CR1 protein and an amidinophenyl or 30 amidinonaphthyl ester of formula (I) as defined in claim 1 having complement inhibitory activity in the manufacture of a medicament for the treatment of a disease or disorder associated with inflammation or inappropriate complement activation, wherein the soluble CR1 protein and the ester of formula (I) act synergistically to inhibit activation of complement.

4. A method for the preparation of a pharmaceutical composition according to claim 1, which method comprises admixing the combination of soluble CR1 protein and an amidinophenyl or amidinonaphthyl ester of formula (I) as defined in claim 1. -20- 259737

5. A composition according to claim 1 or 2, the use according to claim 3 or a method according to claim 4, wherein the soluble CR1 protein is that encoded by the nucleic acid vector pBSCRlc/pTCSgpt and the ester is 4-amidinophenyl 4'-methoxybenzoate HCl or 4-amidino-2-bromophenyl 4'-methoxybenzoate HCl.

6. A composition according to claim 1 or 2, the use according to claim 3 or a method according to claim 4, wherein the ratio of protein to ester is in the range 1:1 to 1:20 by weight. P' '"C By the authorised agents ' A. J. PARK & SON Per END OF CLAIMS -21 -