CA2153797A1 - Combination of a soluble complement receptor-1(scri) and an amidinophenyl or amidinonaphthyl-ester for treating inflammation - Google Patents

Abstract

A method of treating a 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, including pharmaceutically acceptable salts thereof.

Description

~ W O 94/16719 2 1 ~ 3 7 3 7 PCT/GB94/00122 COMBINATION OF A SOLU8LE COMPLEMENT RECEPTOR-1(5CRl) AND AN AMIDINOPHENYL OR
AMIDINONAPHTYL ESTER FOR TREATING INFLAMMAl-ION
The present invention relates to the.~e.~c compositions o~ protease inhibitors and human soluble complement receptor 1 which act synergi~Sic~lly to 5 inhibit activation of complement. Such compositions are useful in the therapy of infl:~mm~3tory or immllne disorders involving complement activation.
Complement lecep~or type 1 (CRl) is present on the membranes of erythrocytes, monocytes/macrophages, granulocytes, B cells, some T cells, splenic follicular den-lritic cells, and glomerular podocytes. CRl binds to the complement 10 components C3b and C4b and has also been referred to as the C3b/C4b l`~,C~ Ol. The structural org~ni~ation and primary sequence of one allotype of CRl is known (Klickstein et al., 1987, J. Exp. Med. 165: 1095-1112, Klicl~tein et al., 1988, J. Exp.
Med. 168:1699-1717; Hourcade et al.,l988, J. Exp. Med. 168:1255-1270, WO
89/09220, WO 91/05047). It is composed of 30 short consensus repeats (SCRs) thateach 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 ~limen~ional triple loop structure through disulphide linkages with the third and first and the fourth and second half-cystines in disulphide bonds. CRl is further arranged as 4 long homologous repeats (LHRs) of 7 SCRs each. Following a leader sequence, the CRl 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 tr~n~membrane region and a 43 residue cytoplasmic tail.
Several soluble fragments of CRl have been generated via recombinant DNA
procedures by elimin~ting the tr~n~memhrane region from the DNAs being expressed(WO 89/09220, WO 91/05047). The soluble CR1 friqpmPnt~ were functionally active7 bound C3b andlor C4b and demonstrated Factor I cofactor activity depending upon the regions they con~ine-l Such constructs inhibited in vitro complement-related functions such as neutrophil oxidative burst, complement meAi~tecl haemolysis, and C3a and C5a production. A particular soluble construct, sCR1/pBSCRlc, also demonstrated in vivo activity in a reversed passive Arthus reaction (WO 89/09220, WO 91/05047; Yeh etal., 1991, J. Tmmllnnl. 146:250-256), suppressed post-ischemic myocardial infl~mm~tion and necrosis (WO
89/09220, WO 91/05047; Weisman etal., Science, 1990, 249:146-151; Dupe, R. et al. Thrombosis & Haemostasis (1991) 65(6) 695.) and extended survival rates 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 el 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 21~3~7 Wo 94/16719 PCT/GB94/0012 myocardial infarction (Weisman et al,, 1990 Science 249:146-1~1, Dupe et al,1991(above)).
In a number of cases, the doses of sCR1 required for therapeutic effects in these models were large (>Smg/kg). Because sCRl is a bioph~...~e~llic~l produced5 by ~ n cell culture techniques, it is desirable to reduce the dose and hence the cost of therapy.
Certain ~mi-linophenyl and amidinonaphthyl esters of carboxylic acids are known to be inhibitors of complement activation as well as having antitrypsin, antiplasmin, ~ntik:~llikTein and antithrombin activity (GB 2095-239, GB 2083-818).
GB 2083818 discloses compounds of formula (A):
Rl ~l~Z-COO =3~H2 wherein Z lG~lesellts -(CH2)a-, -(CH2)b-CH(R3)-, -CH=C(R4)- or -O-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 R1 and R2, w hich may be the same or different, represent each a hydrogen atom, straight or branched chain alkyl group of 1 to 4 carbon atoms, -O-Rs, -S-Rs, -COORs, -COR6, -O-COR7, -NHCOR7, -(CH2)C-NRgRg, -SO2NRgRg, NO2, CN, halogen, CF3, methylenedioxy, NH

R8 NH F~ ~, R~o where c is 0, 1 or 2; Rs is a hydrogen atom, straight or branched chain alkyl ~roup of 1 to 4 carbon atoms, or benzyl group; R6 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; R8 and Rg, which may be the same or different, are each a hydrogen atom, straight or branched chain alkyl group of l to 4 carbon atoms, oramino radical protecting group; and R1o is a hydrogen atom, dimethyl or CF3.
GB 2095239 discloses compounds of the general formula (B):

~ WO 94/16719 215 3 7 9 7 PCT/GB94/00122 NH
Rl 1 ~C~ (B) ~12 wherein Rl 1 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, R13-(CH2)d-, R14-(CH2)e~~

R,4-CH2 {~ or R~s where R 13 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;
R1s and R16~ which may be the same or different, are each a hydrogen atom, a straight or branched alkyl group of 1 to 4 carbon atoms, -OR17, methylenedioxy group, -SR17~ -COOR17. -COR18- -OCORlg, -NHCOR1g, -(CH2)f-NR20R21 (f is 0, 1,2), -SO2NR20R21, a halogen atom, -CF3, NO2, CN, R.2 -r ~ or -NH ~3 R17 is a hydrogen atom, a straight or branched alkyl group of 1 tO 4 carbon atoms or a benzyl group; R1g is a hydrogen atom, a straight or branched alkyl group of 1 to 4 carbon atoms; R1g is a straight or branched alkyl group of 1 to 4 carbon atoms; R20 and R2 1~ 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=CR2s-;
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; R2s is a hydrogen 30 atom or a straight or branched alkyl group of 1 to 4 carbon atoms and the carbon WO 94/16719 PCTIGB94/00122 ~
2 1~ 3r~ 97 atom of the CR2s moiety is ~tt:3r-he~l to the COO group; and R12 l~-esellLs -R26, -OR26, -COOR27, one or two of the same halogen atoms, -NH2, -SO3H, -CO~ or-NHCO ~ Rz~

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 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 enzy~nes reversibly (US 4,285,932, US 4,507,283, EP 0,297,882, R.A.G.Smith et al, 1985 Progress in Fibrinolysis VII 227-231). US 4285932, US 4507283 and EP 0297882 disclose compounds of formula (C):

H2N~ oC~Rx wherein Rx is benzoyl optionally substituted with one or two substituents independently selected from halogen, Cl 6 alkyl, C2 6 aL~cenyl, Cl 6 alkoxy, Cl 6 aLkanoyloxy, C1 6 alkanoylamino, amino, dimethylamino or guanidino;
naphthoyl; or acryloyl optionally substituted with C1 6 alkyl, furyl or phenyl wherein the phenyl moiety is optionally substituted with C1 6 alkyl.
Thus this type of compound is not a specific inhibitor of the proteases of the complement system.
Synergistic compositions of CR1-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 disease or disorder associated with infl~mm~tion or inappropriate complement activation which method comprises admini~tering to a ~ """~l in need thereof an effective amount of a soluble CRl protein and an effective amount of an amidinophenyl or amidinonaphthyl ester of formula (I) having complement inhibitory activity:

~ WO 94/16719 2 1 5 3 7 ~ 7 PCT/Gs94/00122 ~ ~--OC~

wherein A is phenyl optionally substituteA with C1 4 alkyl, C1 4 alkoxy, Cl 4 alkoxyc~L,onyl, halo, NH2, sulphonyl, benzoyl or Cl 4 alkylbenzoylamino or naphthyl; and B is CH2=CH- optionally substituted by a group selected from C1 6 alkyl, phenyl and phenyl substituted with Cl 6 alkyl; phenyl optionally substituted with one or two substi~l-e~ts independently selected from halogen, C1 6 aL~cyl, C2 6 aLkenyl, Cl 6 aL~coxy, Cl 6 alkenoyloxy, Cl 6 aLIcanoylamino, amino, dimethylamino or guanidino;
or naphthyl, including pha~ aceulically acceptable salts thereof.
Preferably, A is phenyl optionally sllbstitl-teA 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 C1 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 ~mirline group.
Suitable examples of halo include chloro and bromo.
Pharmaceuti~lly acceptable salts may be formed with ph~rm~ceuti~lly acceptable acids, for example, maleic, hydrochloric, hydrobromic, phosphoric, acetic, fumaric, salicylic, citric, lactic, m~n~lelic, tartaric, methanesulphonic and oxalic acid.
In a preferred aspect, the soluble CRl 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, andis especially that obtainable from pBSCRlc/pTCSgpt, as described in WO 89/09220.
The amounts of each cc,lll~oulld are chosen such that the conce.lLIdLion of eachcomponent required to inhibit by 50% haemolysis of se )~ ~ erythrocytes in a standard complement assay is lowered colllp~ed with that required for the individual components in the same assay. This increase in potency is described by a synergyfactor which is defined in more detail below.
The invention also provides the use of a soluble CRl protein and an amidinophenyl or amidinonaphthyl ester having complement inhibitory activity in the manufacture of a medicament for the treatment of a disease or disorder associated with infi~mm~tion or in~plu~liate complement activation.
The compounds may be a~lmini~tered by standard routes, such as, for O example, intravenous infusion or bolus injection, and may be ~timini~tered together or sequentially, in any order.
When the compounds are administered together they are preferably given in the form of a pharmaceutical composition comprising both agents. Thus, in a further Wo 94/16719 PCT/GB94/00122~
2~3~7 aspect of the invention there is provided a pharmaceutical composition comprising a soluble CR1 protein and an amidinophenyl or amidinonaphthyl ester having complement inhibitory activity together with a pharmaceutically acceptable carrier.
In a preferred embodiment, the composition may be form~ te~l in accordance with routine procedures as a pharmaceutical composition adapted for intravenous ~rlminictTation to human beings.
In a further aspect, the invention therefor provides a method for the preparation of a pharm~ceutical composition of the invention, which method comprises admixing the combination of soluble CR1 protein and an amidinophenyl or amidinonaphthyl ester of formula (I), including pharm~cel-tic~lly acceptable salts thereof.
The present invention also provides a method of treating a disease or disorder associated uith infl~mm~tion or inappropriate complement activation comprising ?~Ciminictering 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 treatm~nt of a disease or disorder isin the dose range of 0.01-lOOmg/kg; preferably 0.1-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 toester 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 m~nnitol, and local anaesthetics for injectable compositions, including, for example, lidocaine.
A pharrn~eutic~l 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 condition, in particular acute myocardial infarction, in a human or non-human animal, said method comprising ~rlmini~tering to the patient a composition according to this inventlon.
This invention further provides a method for treating adult respiratory distresssyndrome (ARDS) in a human or non-human animal, said method comprises ~lministering to the patient a composition according to this invention.
The invention also provides a method of delaying hyperacute allograft or hyperacute :~enograft rejection in a human or non-human animal which receives a transplant b~ ~tlmini~tering a composition according to this invention.

The methods and co,lJ~osiuons of this invention are useful in the ~ nllf.nt of complement-me~ t~l or complement-related disorders, including but not limited tothose listed below.

5 Disease and Disorders Illvolvil~, Compl~ n~ont Neurological Disorders multiple sclerosis stroke 10 Guillain Barré Syndrome traumatlc bram lnJury Parkinson's disease allergic encephalitis 15 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 in~mm~tion of autoi.l...~rle ~ e~es 25 Crohn's Disease adult respiratory distress syndrome thermal injury including burns or frostbite uveitis 30 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 WO 94/16719 PCT/GB94/00122_ 2~ ~37~7 lmmllne Complex Disorders and Autoimmune Diseases rheumatoid arthritis systemic lupus erythem~tos-l~ (SLE) SLE nephritis proliferative nephritis glomerulonephritis hemolytic anemia myasthenia gravis Reproductive Disorders antibody- or complement-ms~ ted infertility MATERIALS
BRL 55730 - is the soluble complement lt;Cel~LOl type 1 derived from the expression of plasmid pBSCRlc/pTCSgpt in CHO cells (WO 89/09220).

BRL24894A (APAN) - 4-amidinophenyl 4'-methoxyben7O~te HCl (EP~009879) BRAPAN - 4-amidino-2-bromophenyl 4'-methoxyben7o~te HCI (Example 3).

METHODS
Anti-complement Activity Measured by the Haemolysis of Sheep Erythrocytes Functional activity of complement inhibitors was ~sessed by mea~-lring the 25 inhibition of complement mediated lysis of sheep erythrocytes sen~iti~e~l 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 essenti~lly as described in Dacie & Lewis, 1975 (Practical ~m~tology 5th Edition, Churchill Livingstone, 30 Edinburgh and New York, pp3-4). Briefly, blood was warmed to 37C for S
minlltes, the clot removed and the rem~ining serum clarified by centrifugation. The serum fraction was split into small aliquots and stored at -196C. Aliquots werethawed as required and diluted in the Hepes buffer immediately before use.
Inhibition of complement-me li~tecl lysis of sensitised sheep erythrocytes was 35 measured using a standard haemolytic assay using a v-bottom microtitre plate format as follows, es~enti~lly as described by Weisman et al 1990 (above).

~ WO 94/16719 21~ 3 7 9 7 PCT/GB94/00122 Standard assay 25 ~11 of a range of concentrations of inhibitor (typically in the region of 0.1,ug/ml - 0.00078~1g/ml final for BR~55730 and 100 - 0.1~M final of APAN or BRAPAN) diluted in Hepes (0.lM Hepes pH7.4/0.15M NaCl) buffer were inc~lb~ted -, S with 25 ,ul of buffer and 50 ,ul of the 1/125 diluted serum for 15 minutes at 37C.
1 of prewarmed sen~iti~e~ sheep erythrocytes were added for 1 hour at 37C in a final reaction volume of 200 ,~LI . Samples were spun at 300g at 4C for 15 minutes before transferring 150 ~Ll of supernatant to flat bottom microtitre plates and determining the absorption at 410 nm, which reflects the amount of lysis in each test solution. Maximum lysis was determined by incubating serum with erythrocytes in the absence of any inhibitor (E+S) from which the proportion of background Iysis had been subtracted (determined by incubating erythrocytes with buffer) (E). The background lysis by inhibitor was assessed by inr~lb~ing inhibitor with erythrocytes (E+I) and then subtracting that from test samples (E+I+S). Inhibition was expressed as a fraction of the total cell lysis such that IH50 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 37C.
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 IH ,o values were determined from the titration curve by reading off the concentration corresponding to IH=0.5.

Synergy Assays The assay was carried out in a similar manner to that described above except 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 ~11 of inhibitor 1 to 25 ~Ll of inhibitor 2 in the presence of serum and measuring the degree of lysis as described above.

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

WO 94tl6719 PCT/GB94/0012~
2~ 53~9~ _ Tnhihitor 1, BRL55730 was titrated on its own and in the p~esence of various concentrations of inhibitor 2, APAN. A plot was made of the tBRL55730] vs IH
with and without APAN (Fig.1). The IH50 of BRL55730 was ~stim~tetl at cach S 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 CA~ lt was estim~t~l 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 eshm~t~d from tne plot of [APANl vs IH (Fig.2) (column 2). The IH50 for BRL55730 was delelmi.led 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 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.

Isobolo~ram Analysis The IH50's of inhibitor 1, eg BRL55730 and inhibitor 2, eg APAN were determined separately. These experiment~lly determined values are plotted on theaxes of the isobologram and were connected by a straight line, te~rned the line of additivity (Fig. 3). This line represents combinations of the two inhibitors which, 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 infli~te antagonism and points below this curve indicate synergy. BRL55730 was titrated in the presence of fixed concentrations of APAN and the IH50 of BRL55730 determin~oA at each APAN
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 assayed. Then BRL55730[X] was assayed togeeher with APAN[y~ in the same haemolysis assay. The amount of inhibition was calculated for the two inhibitorswhen assayed separately and w hen assayed together.
If synergy occurs then the inhibition of the compounds assayed together should be greater than the sum of the two inhibitors separately ~WO g4/16719 215 ~ 7 9 7 PCT/GB94/00122 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 Standard Standard error si~e error of the of the mean mean squared a a n~ s.e.m.~, (s.e.m.)~2 b b nh s.e.m.h (s.e.m)h2 APAN
ab ab n~h s.e.mah (s.e.m.)ah2 s Null Hypothesis = H0 ab = a + b (ie effect is additive) Alternative Hypothesis = H1 ab ~ a + b (ie effect is not additive) t = ab - a - b Equation 1 ~((s.e.m.ab2) + (s.e.m.a2) + (s.e.mb2)}

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 Mnge of 100 ~lM - 0.78 ~M for APAN and 0.125 ~g/ml - 0.00098 ~Lg/ml for BRL55730. Two titMtion curves were performed for BRL55730 from which the mean IH50 was determined as 0.01 ~Lg/ml (Fig.1) and one curve for APANfrom which the IH50 was determined as 10 ~lM (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 ,uM for each titration (Fig.1). From the data the synergy factor was calculated as described above.

WO 94/16719 PCTIGB94/00122~
Table 1: Determirlation of the Synergy Factor for BRL55730 and APAN.

2 3 . 4 5 6 [APAN] IHof IHS0 of 0.5-IHof Adjusted Synergy ,uM APAN BRL55730 APAN rBRL55730] Factor ~Lg/ml ~g/ml 0 - 0.01 0.5 0.01 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 ~lM APAN with BRL55730 reduces the IHS0S by approximately 8 fold. The calculated synergy factor at each concentr~,tion of APAN is given in Table 1 and shows that the effect of APAN is more than additivesince 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 ~Lg/ml, was titrated on its own; the concentration at which no inhibition of complement activation occurred was found to be ~ 0.0004 ~g/ml. Titration of APAN on its own showed that a concentration of 4 ~lM gave an IH of 0.31 and 2 ~M gave an IH of 0.16. When BRL55730 was titrated in the presence of APAN at 4 and 2 ~LM, the inhibition at 0.0004 ,ug/ml of BRL55730 was greater than that could be accounted for by APAN
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 Figs. 1 & 2. The IHSO'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 in~lie~ting that the interaction issynergistic.

~WO 94/16719 215 3 7 9 7 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 S (i) 0.005 ~lg/ml BRL55730 4 ~LM APAN
(ii) 0.005 ~g/ml BRL55730 2 ~LM APAN
(iii) 0.002 ~g/ml BRL55730 4 ~LM APAN
(iv) 0.002 ~g/ml BRL55730 2 ~M APAN

10 The st~ti~tic~l parameters are given below for each concentration pair.

Table 2: Statistical Analysis of Concentration Pair (i) PARAMETER BRL55730 APAN BRL55730 + APAN
0.005 ,ug/ml 4 ,uM 0.005 ~Lglml + 4 ,uM
MEAN IH 0.312 0.297 0.676 SEM 0.0117 0.0092 0.0096 (SEM)2 0.0001360.000086 0.000092 n 14 14 14 df 39 t-value 3.822 At 39 df, probablility of 0.9995 t = 3.558 Calculated t > 3.558. Therefore a + b ~ ab 15 Table 3: Statistical Analysis of Concentration Pair (ii) PARAMETER BRL55730 APAN BRL55730 + APAN
0.005 ,ug/ml 2 !lM 0.005 ~Lg/ml + 2 ,uM
MEAN IH 0.251 0.124 0.509 SEM 0.0135 0.0183 0.00947 (SEM)2 0.0001830.000334 0.0000897 n 16 16 16 df 45 t-value 5.407 At 45 df, probablility of 0.9995 t = 3.550 Calculated t > 3.550. Therefore a + b ~ ab 2 1~ 3 7 9 ~able 4: Statistical Analysis of Concentration Pair (iii3 PARAMETER BRL55730 APAN BRL55730 + APAN
0.002 ,u~/ml4 ~M 0.002 ~/ml + 4 ~M
MEAN IH 0.123 0.337 0.564 SEM 0.00741 0.00972 0.00691 (SEM32 0.0000550.0000945 0 0000477 n 16 16 16 df 45 t-value 7.451 At 45 df, probablility of 0.9995 t = 3.550 Calculated t > 3.550. Therefore a + b ~ ab Table 5: Statistical Analysis of Concentration Pair (iv) PARAMETER BRL55730 APAN BRL55730+APAN
0.002 ,ug/ml2 ~M 0.002 ,ug/rnl + 2 ~M
MEAN IH 0.121 0.192 0.422 SEM 0.0104 0.0106 0.00886 (SEM)2 0.0001090.000112 0.0000784 n 16 16 16 df 45 t-value 6.267 At 45 df, probablility 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. Syner~y Effect of BRL55730 on APAN
The effect of BRL55730 on the IH50 of APAN was tested in the same way as described in Example 1 a but in this instance APAN was titrated over the range 0.78~LM to lOO~LM in the presence of fixed concentrations of BRL55730 between 0.001 - 0.006 ~lg/ml. The effect of BRL55730 on the IH50 of APAN is given in Table 2 and shows that addition of 0.006 ~Lg/ml of BRL55730 shifts the IH50 of APAN from 10 ~LM to 1 ~LM which is an improvement of 10 fold. The synergy factor was determined as described in the Methods and found to be > l (Table 6) ~WO 94/16719 2 ~ S 3 7 9 7 PCTlGB94/00122 indicating that the synergy process between APAN and BRL55730 isreversible.
Unlike the previously described Example la, the synergy factor apl)ea.~ to be dependent on the concentration of BRL55730.

S Table 6: Determination of the Synergy Factor for APAN and BRL5~730 [BRL55730] IHof IH50 of 0.5-IHof Adjusted Synergy g/ml BRL55730 APAN BRL55730 tAPANl Factor ~M ,uM
O - 10 0.5 10 0.001 0.025 5.3 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. Isobolo~ram Analysis of APAN with BRL55730 Isobologram analysis was performed as described above using data from Fig.
2 and Table 6. Data points fell below the line of additivity which inrliç~te~l the effect was synergistic.

f. Syner~y of APAN with BRL55730 in Serum Diluted 1/35.7 To test whether the synergy seen between APAN and BRL55730 is lS 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 Iysis of the sencisi~ed sheep erythrocytes, S0 ~11 of serum at various dilutions from 1/10 to 1/lS0 were preincubated with S0 ~11 of 0.1 M Hepes pH
7.4/ 0.15 M NaCl buffer at 37C for lS mins. 100 ~l of erythrocytes were added and samples were incubated for either lS mins or 30 mins at 37C. Unlysed cells werespun down at ~ 300 g at 4C for lS mins and then lS0 ~11 of supen ~t~nt were 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 dilution for lS 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 thatmore concentrated serum was used and incubation times were reduced to 15 mins.
Titration of BRL55730 in the more concentrated serum gave an IHS0 of ~
0.14 ~Lg/ml (mean of two determinations3 which is 14 fold greater than in 1/125 WO 94/16719 PCT/GB94/00122~
2~53~9~
diluted serum. Similarly the IH50 of APAN was found to be 52 ~ (mean of two determinations) which is about five fold greater than in the more di lute serum. The synergy e~. ;n ..o.nt~ were carried out by titrating BRL55730 over ;~ concentration range of 1 - 0.0078 ~lg/ml in the presence of APAN at concentrations ranging from 2 5 - 18 ~lM. The s....~ of the data is given in Table 7 and demonstrates that thesynergy effect can be extended to more concentrated serum. The synergy factor inthis instance appears to be dependent on the concentration of APA:~.

Table 7: Synergy Value Between BRL55730 and APAN in more concentrated 10 Serum [APANl IHofAPAN IH50 of 0.5 - IHof Adjusted Synergy M BRL55730 APAN [BRL55730] Factor ,ug/ml ~g/ml 0 - 0.14 0.5 0.14 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 ~LM fall below the addivitity line indicating synergy. The 2 ~lM 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 HCI (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 ~WO 94/16719 21~ 3 7 9 7 PCT/GB94/00122 IH50 of BRAPAN was 3 ,uM. A single titration curve of BRL55730 from 0.1 -0.00078 ~Lg/ml was determined which gave an IH50 of 0.021 ~g/ml. To test for synergy BRL55730 was titrated over the same concentration range but in the presence of BRAPAN ranging from 0.1 ~lM to O.9~1M. Table 8 demonstrates the effect and S synergy potential of BRAPAN on BRL55730. As with APAN at the same semm dilution the synergy factor is >1 indicating that BRAPAN synergises with the BRL55730. The synergy factor remains fairly constant over the concenlldtion 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

~BRAPAN] IHof IH50 of 0.5-IHof Adjusted Synergy M BRAPAN BRL55730 BRAPAN rBRL55730] Factor ~g/ml ~g/ml 0 0 0.021 0.5 0.021 .

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 Anal~sis of the effect of BRAPAN on BRL55730 Using the data described in Example lh for BRAPAN, an isobologram was constructed. All the data points fell below the line of additivity in(lic~ting synergy was occurring.

Co-formulation of sCR1 (BRL 55730) and APAN (BRL 24894A) D-Mannitol (Sigma.UK, 60mg) was dissolved in water for injection (9.Sml).
APAN was disssolved in HPLC-grade methanol to a final concentration of 6 mg/ml by stiring at ambient temperature (20-25C) for 5 min. The solution (0.5ml) was added IMMEDIATELY to the mannitol solution and mixed by shaking. A solution of Wo 94/16719 pcTlGs94lool2~ -2~3797 BRL 55730 (Smg/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 ~ J~.cl~ture of -60C for 20 hours. The white solid had the following composition and was stored desiccated at -70C:
BRL 55730: lmg; B~L24894A: 3mg; D-Mannitol: 60mg; sodiumphos~h~te trace.

Preparation of 4-Amidino 2-bromophenyl 4'-methoxyben70~te HCI (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. Af~er 2 hours, the bubbling was stopped and the solid in solution placed at 4C for two days. The white solid was isolated by filtration. This was rapidly suspended in ethanol solution (50 ml) and a satulal~;d solution of ammonia in ethanol (75 ml) was added. The suspension went clear almost immefli~tely 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).
Addition of concentrated hydrochloric acid (5 ml) led to the rapid formation of a white crystalline mass which was isolated by filtration and recryst~ e l from ethanol (20 ml) and diethyl ether (100 ml). Yield: 860 mg (50%). mp: 279-80.

lH nmr (CDC13-d6DMSO) o: 9.2 (4H, br d, amidine), 8.2 (lH, d, k2Hz;aryl-~).

7.8 (lH, m, aryl-H), 7.25 (lH, 1=9Hz. aryl-~) Infrared (nujol): 3325, 3125, 2300-3400, 1670, 1610, 1585, 1410, 1300, 1175, 1045, 880, 835, 725, 620 cm~l 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 rec;ystallised twice from S:l diethyl ether/ethanol (~ 100 ml) to leave the white title compound. Yield: 225 mg (37%).mp 212-3C.

~WO 94/16719 PCTlGB94/00122 lH nmr (d6DMSO-trace CDCl3) ~: 9.55 (4H, br, amidinophenol NH), 6.95-8.25 (7H, m, aryl-H), 3.9 (lH, s, OCH3) Infrared (nujol): 2500-3400, 1735, 1665, 1605, 1580,1260, 1230, 1170, 1070, 1020, 5 and 830 cm~l Analysis: C 46.66, H 3.74, N 7.19%
Cls,H12,N2,Br,Clrequires C46.72%, H3.66%, N7.26%

10 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 (7)

1. A method of treating a 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:

(I) 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 C1-6 alkyl, phenyl and phenyl substituted with C1-6 alkyl; phenyl optionally substituted with one or two substituents independently selected from halogen, C1-6 alkyl, C2-6 alkenyl, C1-6alkoxy, C1-6 alkenoyloxy, C1-6 alkanoylamino, amino, dimethylamino or guanidino;or naphthyl, including pharmaceutically acceptable salts thereof.

2. The use of a soluble CR1 protein and an amidinophenyl or 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.

3. A pharmaceutical composition comprising a soluble CR1 protein and an amidinophenyl or amidinonaphthyl ester of formula (I) as defined in claim 1 having complement inhibitory activity together with a pharmaceutically acceptable carrier.

4. 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 claim 3.

5. A pharmaceutical pack comprising one or more containers filled with one or more of the ingredients of the pharmaceutical composition of claim 3.

6. A method for the preparation of a pharmaceutical composition according to claim 3, which method comprises admixing the combination of soluble CR1 protein and anamidinophenyl or amidinonaphthyl ester of formula (I) as defined in claim 1.

7. A method, use, composition, method, pack or method according to claim 1, 2, 3, 4, 5 or 6, respectively, wherein the soluble CR1 protein is that encoded by the nucleic acid vector pBSCR1c/pTCSgpt and the ester is 4-amidinophenyl 4'-methoxybenzoate HCl or 4-amidino-2-bromophenyl 4'-methoxybenzoate HCl.