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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

• the present invention relates to therapeutic compositions of protease inhibitors and human soluble complement receptor 1 which act synergistically to inhibit activation of complement. Such compositions are useful in the therapy of inflammatory or immune disorders involving complement activation.
• Complement receptor type 1 is present on the membranes of erythrocytes, monocytes/macrophages, granulocytes, B cells, some T cells, splenic follicular dendritic cells, and glomerular podocytes.
• CRl binds to the complement 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 CRl is known (Klickstein et ai, 1987, J. Exp. Med. 165:1095-1112, Vogelstein et al., 1988, J. Exp. Med. 168:1699-1717; Hourcade er ⁇ /.,1988, J. Exp. Med.
• SCR short consensus repeats
• LHRs long homologous repeats
• 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 transmembrane region and a 43 residue cytoplasmic tail.
• soluble fragments of CRl have been generated via recombinant DNA procedures by eliminating the transmembrane region from the DNAs being expressed (WO 89/09220, WO 91/05047).
• the soluble CRl 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.
• sCRl is a biopharmaceutical produced by mammalian cell culture techniques, it is desirable to reduce the dose and hence the cost of therapy.
• 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).
• GB 2083818 discloses compounds of formula (A):
• R4 is a hydrogen atom or a straight or branched chain alkyl group of 1 to 4 carbon atoms and wherein the -CH(R3)-,
• R and R2 which may be the same or different, represent each a hydrogen atom, straight or branched chain alkyl group of 1 to 4 carbon atoms, -O-R5,
• 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 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
• RI Q is a hydrogen atom, dimethyl or CF3.
• GB 2095239 discloses compounds of the general formula (B): wherein
• Ri 1 represents a straight or branched chain alkyl group 1 to 6 carbon atoms, a straight or branched chain alkenyl group of 2 to 6 carbon atoms having 1 to 3 double bonds,
• 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; R 14 is an amino or guanidino group or a protected amino or guanidino group; e is a number from 1 to 5; R15 and Rjg, 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, -CORig, -OCOR ⁇ , -NHCOR19, -(CH 2 )f-NR 2 oR21 ( f is 0, 1,2), -SO 2 NR oR2b a halogen atom, -CF3, NO2, CN,
• Rl7 is a hydrogen atom, a straight or branched alkyl group of 1 to 4 carbon atoms or a benzyl group
• Ri is a hydrogen atom, a straight or branched alkyl group of 1 to 4 carbon atoms
• Ri 9 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 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'-CF 3 group
• 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 atom or a straight or branched alkyl group of 1 to 4 carbon atoms and the carbon atom of the CR25 moiety is attached to the COO group;
• R12 represents -R26> ⁇ OR26.
• -COOR27 one or two of the same halogen atoms, -NH2' -SO3H,
• 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
• R2g is a hydrogen atom or a guanidino group.
• 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 enzymes 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):
• R x is benzoyl optionally substituted with one or two substituents independently selected from halogen, Ci .g alkyl, C2-6 alkenyl, C ⁇ .* alkoxy, C ⁇ . alkanoyloxy, C ⁇ . ⁇ alkanoylamino, amino, dimethylamino or guanidino; naphthoyl; or acryloyl optionally substituted with Ci _g alkyl, furyl or phenyl wherein the phenyl moiety is optionally substituted with Cj.g alkyl.
• this type of compound is not a specific inhibitor of the proteases of the complement system.
• a method of treating a disease or disorder associated with inflammation or inappropriate complement activation comprises administering to a mammal 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: HN O
• A is phenyl optionally substituted with Cj_4 alkyl, Cj_4 alkoxy, C1.4 alkoxycarbonyl, halo, NH2 > sulphonyl, benzoyl or C1.4 alkylbenzoylamino or naphthyl;
• 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 C 1.4 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.
• halo examples include chloro and bromo.
• Pharmaceutically acceptable salts may be formed with pharmaceutically acceptable acids, for example, maleic, hydrochloric, hydrobromic, phosphoric, acetic, fumaric, salicylic, citric, lactic, mandelic, tartaric, methanesulphonic and oxalic acid.
• 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, and is especially that obtainable from pBSCRlc/pTCSgpt, as described in WO 89/09220.
• each compound is 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 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 inflammation or inappropriate complement activation.
• 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.
• a pharmaceutical composition comprising both agents.
• a pharmaceutical composition comprising a soluble CRl protein and an amidinophenyl or amidinonaphthyl ester having complement inhibitory activity together with a pharmaceutically acceptable carrier.
• the composition may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
• the invention therefor provides a method for the preparation of a pharmaceutical composition of the invention, which method comprises admixing the combination of soluble CRl 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.
• 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 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 to ester is preferably in the range 1:1 to 1:20 by weight.
• 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 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.
• ARDS adult respiratory distress syndrome
• 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.
• 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.
• MATERIALS BRL 55730 - is the soluble complement receptor type 1 derived from the expression of plasmid pBSCRlc/pTCSgpt in CHO cells (WO 89/09220).
• BRAPAN 4-amidino-2-bromophenyl 4'-methoxybenzoate HC1 (Example 3).
• 25 ⁇ l of a range of concentrations of inhibitor typically in the region of O.l ⁇ g/ml - 0.00078 ⁇ g/ml final for BRL55730 and 100 - O.l ⁇ M final of APAN or BRAPAN
• a range of concentrations of inhibitor typically in the region of O.l ⁇ g/ml - 0.00078 ⁇ g/ml final for BRL55730 and 100 - O.l ⁇ M final of APAN or BRAPAN
• Hepes 0.1M Hepes pH7.4/0.15M NaCl
• 100 ⁇ l of prewarmed sensitised sheep erythrocytes were added for 1 hour at 37°C in a final reaction volume of 200 ⁇ l .
• 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 ⁇ l of inhibitor 1 to 25 ⁇ l of inhibitor 2 in the presence of serum and measuring the degree of lysis as described above.
• 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 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 [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 EH50's of inhibitor 1, eg BRL55730 and inhibitor 2, eg APAN were determined separately. These experimentally determined values are plotted on d e 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, 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 concentration. This data was plotted on the isobologram (Fig. 3).
• 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.
• BRL55730r x ⁇ was assayed together with APANryi in the same haemolysis assay. The amount of inhibition was calculated for the two inhibitors when assayed separately and when assayed together.
• 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 ⁇ M - 0.78 ⁇ M for APAN and 0.125 ⁇ g/ml - 0.00098 ⁇ g/ml for BRL55730.
• Two titration curves were performed for BRL55730 from which the mean IH50 was determined as 0.01 ⁇ g/ml (Fig.l) and one curve for APAN from which the IH50 was determined as 10 ⁇ M (Fig.2).
• BRL55730 was titrated over the same concentration range but in the presence of fixed concentrations of APAN from 1 - 6 ⁇ M for each titration (Fig.l). From the data the synergy factor was calculated as described above. Table 1: Determination of the Synergy Factor for BRL55730 and APAN.
• BRL55730 concentration range 0.04 - 0.000039 ⁇ g/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 ⁇ M 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 ⁇ M, the inhibition at 0.0004 ⁇ g/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.
• the additivity line was constructed as described above taking the data from 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.
• PARAMETER BRL55730 APAN BRL55730 + APAN 0.005 ⁇ g/ml 4 ⁇ M 0.005 ⁇ g/ml + 4 ⁇ M
• PARAMETER BRL55730 APAN BRL55730 + APAN 0.005 ⁇ g/ml 2 ⁇ M 0.005 ⁇ g/ml + 2 ⁇ M
• PARAMETER BRL55730 APAN BRL55730 + APAN 0.002 ⁇ g/ml 4 ⁇ M 0.002 ⁇ g/ml + 4 ⁇ M
• PARAMETER BRL55730 APAN BRL55730 + APAN 0.002 ⁇ g/ml 2 ⁇ M 0.002 ⁇ g/ml + 2 ⁇ M
• BRL55730 was tested in the same way as described in Example la but in this instance APAN was titrated over the range 0.78 ⁇ M to lOO ⁇ M in the presence of fixed concentrations of BRL55730 between 0.001 - 0.006 ⁇ g/ml.
• the effect of BRL55730 on the IH50 of APAN is given in Table 2 and shows that addition of 0.006 ⁇ g/ml of BRL55730 shifts the IH50 of APAN from 10 ⁇ M to 1 ⁇ M 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.
• BRAPAN 4-Amidino-2-bromophenyl 4'-methoxybenzoate HC1 (BRAPAN) molecular weight 386 was made 10 mM in DMSO and titrated as described in the Methods using serum diluted 1/125. From two separate determinations the mean value for the EH50 of BRAPAN was 3 ⁇ M. A single titration curve of BRL55730 from 0.1 - 0.00078 ⁇ g/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 ⁇ M to 0.9 ⁇ M.
• Table 8 demonstrates the effect and synergy potential of BRAPAN on BRL55730.
• 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.
• 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: BRL 55730: lmg; BRL 24894A: 3mg; D-Mannitol: 60mg; sodium phosphate: trace.
• Ci5,H 12 ,N ,Br,Cl requires C 46.72%, H 3.66%, N 7.26%
• Fig. 1 shows the effect of different concentrations of APAN on BRL 55730
• Fig. 2 shows the inhibition of complement activation by APAN
• Fig. 3 is an isobologram of BRL 55730 and APAN in a standard assay.

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