CN1118141A - Combination of a soluble complement receptor -1(SCR1) 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

Be used in combination soluble complement receptor-1 (SCR1) and amidino groups phenyl or amidino groups naphthyl ester for treating inflammation

The present invention relates to has the therapeutic combination of synergistic protease inhibitor and human soluble complement receptor 1 to suppressing complement activation.Described compositions can be used for treating inflammation relevant with complement activation or immune disease.

1 type complement receptors (CR1) is present on the film of various kinds of cell, divides dendritic follicular cells, the glomerule podocyte as erythrocyte, monocyte/macrophage, granular cell, B cell, some T cell, spleen.CR1 can combine with complement component C3 b and C4b, therefore also is known as the C3b/C4b receptor.The structure of a kind of abnormal shape of present known road CR1 is formed and elementary sequence (Klickstein etc., 1987, J.Exp.Med.165:1095-1112; Klickstein etc., 1988, J.Exp.Med.168:1699-1717; Hourcade etc., 1988, J.Exp.Med.168:1255-1270, WO89/09220, WO91/05047).It is made up of the equal repeated fragment (SCRs) of 30 weak points, and each repeated fragment comprises about 60-70 aminoacid.For each SCR, in its average 65 aminoacid, nearly 29 remain unchanged.Someone thinks, each SCR forms a three-dimensional tricyclic structure by disulfide bond, the 3rd and first cysteine group and the 4th and second cysteine group between form disulfide bond.And CR1 further arranges and forms four long homotype repeated fragments (LHRs), and per 7 SCR form one.According to a leading sequence, the formation of CR1 molecule is in proper order: N-holds LHR-A, is thereafter two repeated fragments, LHR-B and LHR-C are maximum C-end LHR-D below, and then are two other SCRs, 25 so-called transfer membrane zones that residue constitutes, 43 Cytoplasm afterbodys that residue constitutes.

Several solubility CR1 fragments by the DNA recombinant technique by from produced by excision transfer membrane zone the DNA that expresses (WO89/09220, WO91/05047).Solubility CR1 fragment has functional activity, can combine with complement component C3 b and/or C4b, and proof has the cofactor activity of the factor 1 of the zone decision that is comprised by it.This structure suppresses the function relevant with complement external, as neutrophil's oxidation set out, the haemolysis of complement-mediated and the generation of C3a and C5a.Also showed active (WO89/09220 in the specific reverse in vivo passive anaphylactic hypersensitivity of solubility structure sCR1/pBSCR1c, WO91/05047, Yeh etc., 1991, J.Immunol.146:250-256), to rear portion ischemic myocardial inflammation and the inhibited (WO89/09220 of myocardial necrosis, WO91/05047, Weisman etc., Science, 1990,249:145-151; Dupe.R. etc., Thrombosis﹠Haemostasis (1991) 65 (6) 695), can also improve the survival rate (Pruitt﹠amp of transplant organ tissue; Bollinger, 1991, J.Surg.Res.50:350, Pruitt etc., 1991 Transplantation52:868), in the animal model of several diseases, complement activation is shown the therapeutic inhibitory action, for example for injury of lung (Rabinovici etc., 1992J.Immunol.149:1744-1750, Mulligan etc., 1992, J.Immunol.148:3086-3092), intestinal ischemia (Hill etc., 1992 FASEBJ.6:A1049) and acute myocardial infarction (Weisman etc., 1990Science249:146-151, Dupe etc., 1991, the same) therapeutical effect all arranged.

In many cases, the dosage for the treatment of the needed sCR1 of these animal model diseases is big (greater than 5mg/kg).Because sCR1 is a kind of biological agent product that is produced by the mammalian cell culture technique, therefore wishes to reduce using dosage, thereby reduce medical expense.

The amidino groups phenyl of known some carboxylic acid and amidino groups naphthyl ester are the inhibitor of complement activation effect, and have antitrypsin, antifibrinolysin, anti-kallikrein and antithrombin activity (GB2095-239, GB2083-818).

GB2083818 discloses the chemical compound of formula (A):

Wherein, Z representative-(CH ₂) _a-,-(CH ₂) _b-CH (R ₃)-,-CH=C (R) ₄-or-O-CH (R ₄)-, a here is 0,1,2 or 3, and b is 0,1 or 2, R ₃Be straight or branched alkyl, or have the cycloalkyl of 3 to 6 carbon atoms, R with 1 to 4 carbon atom ₄Be hydrogen atom or straight or branched alkyl with 1 to 4 carbon atom, wherein-CH (R ₃)-,=C (R ₄)-or-CH (R ₄)-part is connected with-COO group; R ₁And R ₂Can be identical or inequality, they represent hydrogen atom separately, have the straight or branched alkyl of 1 to 4 carbon atom ,-O-R ₅,-S-R ₅,-COOR ₅,-COR ₆,-O-COR ₇,-NHCOR ₇,-(CH ₂) _c-NR ₈R ₉,-SO ₂NR ₈R ₉, NO ₂, CN, halogen, CF ₃, methylene-dioxy,

Or Wherein c is 0,1 or 2, R ₅Be hydrogen atom, have the straight or branched alkyl or a benzyl of 1 to 4 carbon atom, R ₆Be hydrogen atom or straight or branched alkyl with 1 to 4 carbon atom, R ₇Be straight or branched alkyl with 1 to 4 carbon atom, R ₈And R ₉Can be identical or different, each naturally hydrogen atom, have the straight or branched alkyl or the amino protecting group of 1 to 4 carbon atom, R ₁₀Be hydrogen atom, dimethyl or CF ₃

GB2095239 discloses the chemical compound of general formula (B): Wherein, R ₁₁Represent the straight or branched alkyl with 1 to 6 carbon atom, straight or branched alkenyl, R with 2 to 6 carbon atoms and 1 to 3 two key ₁₃-(CH ₂) _d-, R ₁₄-(CH ₂) _e-,

R wherein ₁₃Be to have the cycloalkyl of 3 to 6 carbon atoms or have 3 to 6 carbon atoms and the cycloalkenyl of 1 or 2 two key, d is 0,1,2 or 3, R ₁₄Be amino or guanidine radicals or protected amino or guanidine radicals, e is 1 to 5 number, R ₁₅And R ₁₆Can be identical or different, each naturally hydrogen atom, have 1 to 4 carbon atom the straight or branched alkyl ,-OR ₁₇, methylene-dioxy ,-SR ₁₇,-COOR ₁₇,-COR ₁₈,-OCOR ₁₉,-NHCOR ₁₉,-(CH ₂) _f-NR ₂₀R ₂₁(wherein f is 0,1,2) ,-SO ₂NR ₂₀R ₂₁, halogen atom ,-CF ₃, NO ₂, CN,

Or

Wherein, R ₁₇Be hydrogen atom, have the straight or branched alkyl or a benzyl of 1 to 4 carbon atom, R ₁₈Be hydrogen atom, have a straight or branched alkyl of 1 to 4 carbon atom, R ₁₉Be straight or branched alkyl with 1 to 4 carbon atom, R ₂₀And R ₂₁Can be identical or different, each is hydrogen atom, the straight or branched alkyl with 1 to 4 carbon atom or amido protecting group naturally, R ₂₂Be O, S or NH, R ₂₃Be 2 ', 3 '-dimethyl or 3 '-CF ₃Base, Y are-(CH ₂) _g-(wherein g is 0,1,2 or 3) ,-(CH ₂) _h-CHR ₂₄-(wherein h is 0,1 or 2) or-CH=CR ₂₅-, R ₂₄Be straight or branched alkyl with 1 to 4 carbon atom, and CHR ₂₄The carbon atom of part is connected R with the COO base ₂₅Be hydrogen atom or straight or branched alkyl with 1 to 4 carbon atom, and CR ₂₅The carbon atom of part is connected with the COO base; R ₁₂Representative-R ₂₅,-OR ₂₆,-COOR ₂₇, one or two identical halogen atom ,-NH ₂,-SO ₃H,

Or R wherein ₂₆Be straight or branched alkyl with 1 to 4 carbon atom, R ₂₇Be hydrogen atom or straight or branched alkyl with 1 to 4 carbon atom, R ₂₈Be hydrogen atom or guanidine radicals.

The amidino groups phenylester of also knowing other carboxylic acids can suppress the protease (A.D.Turner etc. in the solidification path; 1986Biochem.25:4929-35); and the active center (US4,285,932, US4 that also have been used for reversibility ground acyl group plasmin; 507; 282, EP0,297,882; R.A.G.Smitn etc., 1985Progressin Fibrinolysis VII227-231).US4,285,932, US4,507,283 and EP0,297,882 disclose formula (C) chemical compound:

R wherein _xBe the benzoyl that can be replaced arbitrarily by one or two substituent group that is selected from following radicals: halogen, C _1-6Alkyl, C _2-6Alkenyl, C _1-6Alkoxyl, C _1-6Alkanoyloxy, C _1-6Alkanoyl amido, amino, dimethylamino or guanidine radicals; Or can be by C _1-6The acryloyl group that alkyl, furyl or phenyl replace arbitrarily, wherein phenyl moiety can be by C _1-6Alkyl replaces arbitrarily.

Therefore, this compounds is not the specific inhibitor of complement protein enzyme system.

The someone has described the cooperative compositions (WO92/10205) of CR1 related polypeptide and some organic compound formation.

The invention provides a kind of method that is used for the treatment of with inflammation or unsuitable complement activation diseases associated or imbalance, this method comprises that the complement that has that the mammal of needs treatment is granted the solubility CR1 albumen of effective dose and effective dose suppresses the amidino groups phenylester of active formula (I) or amidino groups naphthyl ester and acceptable salt pharmaceutically thereof:

Wherein,

A is can be by any phenyl that replaces of following radicals: C _1-4Alkyl, C _1-4Alkoxyl, C _1-4Alkoxy carbonyl group, halogen, NH ₂, sulfonyl, benzoyl or C _1-4Alkylbenzene formamido group or naphthyl;

B is can be by any CH that replaces of one of following radicals ₂=CH-:C _1-6Alkyl, phenyl and by C _1-6The phenyl that alkyl replaces; Be selected from any phenyl that replaces of substituent group of following radicals separately by one or two: halogen, C _1-6Alkyl, C _2-6Alkenyl, C _1-6Alkoxyl, C _1-6Chain ene acyloxy, C _1-6Alkanoyl amido, amino, dimethylamino or guanidine radicals; Perhaps naphthyl.

A is preferably by the phenyl of halogen 2 or 3 any replacements, and the amidino groups substituent group is positioned at 4 of phenyl ring.B is preferably 4 by C _1-4The phenyl that alkoxyl replaces and can further be replaced arbitrarily by halogen.Most preferred situation is that B is the 4-methoxyphenyl, and A is 4 phenyl or the 2-bromophenyls that are replaced by amidino groups.

The example of the halogen that is suitable for comprises chlorine and bromine.

Pharmaceutically acceptable salt can form by acceptable acid pharmaceutically, for example, and maleic acid, hydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid, fumaric acid, salicylic acid, citric acid, lactic acid, mandelic acid, tartaric acid, methanesulfonic acid and oxalic acid.

In aspect preferred, the solubility CR1 composition that is used for combination therapy is encoded by the nucleic acid mediator, this nucleic acid mediator is selected from pBSCR1c, pBSCR1s, pBM-CR1c, pBSCR1c/pTCSgpt and pBSCR1s/pTCSgpt, particularly the mediator that is obtained by pBSCR1c/pTCSgpt (WO89/09220).

Select the consumption of each chemical compound so that in the complement algoscopy of standard, suppress the concentration of each required composition of 50% sensitized erythrocyte haemolysis and compare low with the concentration of needed single composition in same mensuration.The increase that this effect is renderd a service can be explained by the synergism factor that describes in detail below.

The present invention also provides solubility CR1 protein and has had complement and suppressed active amidino groups phenylester or amidino groups naphthyl ester and be used for the treatment of application in the medicine with inflammation or unsuitable complement activation diseases associated or imbalance in preparation.

These chemical compounds can be according to general administration, and for example, intravenous infusion or bolus injection also can lump together administration, perhaps according to any order administration successively.

When compounds together during administration, preferably to comprise the pharmaceutical compositions administration of two kinds of compositions.Therefore, on meaning further, the invention provides to contain solubility CR1 protein and have complement and suppress active amidino groups phenyl or amidino groups naphthyl ester and the pharmaceutical composition of acceptable carrier pharmaceutically.

In preferred embodiments, compositions can be mixed with the medicament that is used for people's intravenous administration according to conventional method.

Therefore, more furthermore, the present invention also provides the method for preparing pharmaceutical composition of the present invention, this method comprise with the amidino groups phenyl of solubility CR1 protein and formula (I) or amidino groups naphthyl ester and pharmaceutically the mixture of acceptable salt mix.

The present invention also provides the method for a kind of treatment and inflammation or unsuitable complement activation diseases associated or imbalance, and this method comprises that the patient to this treatment of needs grants the present composition that effective dose is gone up in treatment.

In above-mentioned Therapeutic Method, the preferred people of treatment target.

The proteinic effective dosage ranges that is used for the treatment of above-mentioned disease or imbalance is 0.01-100mg/kg, preferred 0.1-10mg/kg.

The effective dosage ranges that is used for the treatment of the ester of above-mentioned disease or imbalance is 0.05-100mg/kg, preferred 0.05-10mg/kg.Described protein to the part by weight of ester preferably in 1: 1 to 1: 20 scope.

Compositions comprises the protein of therapeutic activity dosage and ester and pharmaceutically acceptable excipient or carrier, for example saline, buffer saline, glucose or water usually.Described compositions can also contain special stabilizing agent such as sugar, comprises mannose and mannitol etc., and the local anesthetic (comprising for example lignocaine) that is used for injectable composition.

The kit that comprises one or more packing materials that one or more pharmaceutical composition compositions are housed is also included within the scope of the present invention.

The present invention also provides the method for treatment thrombosis disease, particularly treats the method for people or non-human animal's myocardial infarction, and this method comprises grants compositions of the present invention to patient.

The present invention goes back and then provides the method for the respiratory distress syndrome (ARDS) of a kind of adult of treatment or inhuman adults, and this method comprises uses compositions of the present invention to patient.

The present invention also provides a kind of people or non-human animal's the super acute homograft rejection effect or method of super acute xenograft rejection effect of accepting to transplant of delaying, and this method comprises uses compositions of the present invention.

Method and composition provided by the invention can be used for treating by complement-mediated or with the complement diseases associated, include but is not limited to following disease:

With complement diseases associated and the imbalance of imbalance nerve

Multiple sclerosis

Apoplexy

The multiple neuritis's syndrome of acute heat characteristic of disease

Plane wound property brain injury

Parkinson

The anaphylaxis encephalitis

The imbalance relevant with unsuitable or unusual complement activation

Complication of hemodialysis

Super acute homograft rejection

Corneal graft rejection

Xenograft rejection

In with the interleukin-2 therapeutic process by its toxicity of bringing out

Paroxysmal nocturnal hemoglobinuria

Inflammatory diseases

The autoimmune disease inflammation

Crohn disease

Adult property respiratory distress syndrome

The temperature damage comprises burn and chilblain

Uveitis

Perfusion property disease again after the ischemia

Myocardial infarction

The disease that balloon angioplasty brings out

Syndrome after the compression in cardiopulmonary bypass surgery or kidney hemodialysis

Renal ischemia disease

Hepatic ischemia disease

Infectious disease or sepsis

Multiple organ failure

Septic shock compound disease of immunity and autoimmune disease

Rheumatoid arthritis

Systemic lupus erythematosus (SLE)

The SLE nephritis

Proliferative glomerulonephritis

Glomerulonephritis

Hemolytic anemia

Myasthenia gravis reproduction disorder

The infertility of antibody or complement-mediated

Experiment material

BRL55730 is 1 type soluble complement receptor, derives from the expression product (WO89/09220) of the plasmid pBSCR1c/pTCSgpt in the Chinese hamster ovary celI.

BRL24894A (APAN) is 4 '-methoxybenzoic acid 4-amidino groups phenyl ester hydrochlorate (EP-0009879).

BRAPAN is 4 '-methoxybenzoic acid 4-amidino groups-2-bromobenzene ester hydrochloride (example 3).

Experimental technique is measured anticomplementary activity by the sheep red blood cell hemolytic reaction

The functional activity of complement inhibitor can be estimated the hemolytic inhibitory action of the sheep red blood cell of complement-mediated by measuring, and the prior palpus rabbit antibody of used sheep red blood cell (from DiamedixCorporation, Miami, USA obtains) sensitization.Human serum is diluted to 1: 125 or 1: 35.7 with the Hepes buffer (containing 0.15M NaCl) of 0.1M, pH7.4, human serum is the source of complement, is actually to prepare from the pooled serum of volunteer method such as Dacie﹠amp; Lewis 1975 described (Practical Haematology 5th Edition, Churchill Livingstone, Edinburgh and New York, PP3-4).In brief, blood is warming to 37 ℃ earlier and kept 5 minutes, remove blood clot, with the serum clarification of centrifuging remainder.Then serum is distributed into five equilibrium in a small amount, stores down at-196 ℃.Earlier wait branch serum to thaw in a small amount this when needing, face with preceding with the dilution of Hepes buffer.

Can use standard set haemolysis algoscopy to measure to the hemolytic inhibitory action of sensitization sheep red blood cell of complement-mediated, the mensuration process is to carry out on the microtitration plate of a V-arrangement bottom surface, the mensuration program is as follows, in fact is nineteen ninety (on seeing) described method such as Weisman.The assay method of standard

(the typical concentrations scope is: the final concentration of BRL55730 is 0.1 μ g/ml-0.00078 μ g/ml earlier inhibitor to be diluted to certain concentration range with Hepes buffer (0.1M Hepes pH7.4/0.15M NaCl), the final concentration of APAN or BRAPAN is 100-0.1 μ M), then with inhibitor and the 25 μ l buffer and the 50 μ l1 of 25 these concentration ranges of μ l: the serum of 125 dilutions are together 37 ℃ of incubations 15 minutes.Add the sheep red blood cell 100 μ l through the sensitization of preheating again, making last reactant volume was 200 μ l, 37 ℃ of following incubations 1 hour.With sample under 4 ℃ with the rotating speed of 300g centrifugal 15 minutes, then 150 μ l supernatant are moved in the flat microtitration plate behind the incubation, measure the absorption at 410nm place, institute's value can reflect the haemolysis amount of each mensuration solution.Maximum haemolysis can be by measuring serum and the common incubation of erythrocyte (E+S) when not adding any inhibitor, and therefrom deduct background haemolysis amount (E) (by erythrocyte and the common incubation of buffer are measured).The background haemolysis of inhibitor can be by measuring (E+I) with inhibitor and the common incubation of erythrocyte, then from the result of working sample (E+I+S) with its deduction.Inhibitory action is expressed as the percent of the total haemolysis of cell, so IH50 then represents the required inhibitor concentration of generation 50% hemolysis inhibition.Some the experiment in, serum is diluted into 1: 35.7, the incubation time reduce to 37 ℃ following 15 minutes.Other conditions are all identical.The haemolysis of maximum haemolysis: Amax=(E+S)-when (E) having inhibitor: Ao=(E+I+S)-(E+I) amount of suppression: $\mathrm{IH}=\frac{A\max -A0}{A\max }$ Inhibitor concentration is mapped to IH, and the IH50 value then can be measured by reading corresponding to the concentration value of IH=0.5 on the titration curve from then on.The synergism determination method

Assay method and the method for narrating above are similar, and difference is that inhibitor 1 (for example BRL55730) carries out titration in the presence of the inhibitor 2 (for example APAN) of fixed concentration.Operation sequence is in the presence of serum 25 μ l inhibitor 1 to be added in the 25 μ l inhibitor 2, measures its haemolysis degree then as stated above.The mensuration of cofactor

For each synergism test, two all titration and common titration separately of inhibitor.

Embodiment 1

Independent titration of inhibitor 1BRL55730 and titration under the situation that has various concentration inhibitor 2APAN.Under the situation that has and do not exist APAN, use [BRL55730] to IH mapping (Fig. 1).Can under each APAN concentration, evaluate the IH50 of BRL55730.Second figure maps (Fig. 2) to IH with [APAN], assesses out in view of the above and the corresponding IH value of APAN concentration that is used for the synergism test.Then these results are made table (table 1).

Table 1 the 1st hurdle is the concentration of APAN.The inhibiting size that a certain specific APAN concentration causes by [APAN] to assessing out (the 2nd hurdle) on the figure (Fig. 2) that IH did.Measure the IH50 value (the 3rd hurdle) of BRL 55730 under each APAN concentration.A certain specific APAN concentration will be deducted the effect of the IH50 generation of BRL55730, i.e. 0.5-IH (APAN) (the 4th hurdle).Read out in the concentration (the 5th hurdle) that BRL55730 makes the BRL55730 of this inhibition level that the time spent produces separately with this numerical value.Divided by the IH50 that has recorded, draw the synergism factor, i.e. the 5th hurdle value/the 3rd hurdle value=the 6th hurdle value with the concentration of the BRL55730 that regulates.If the effect of APAN is an additivity, this synergism factor is 1 so, and this numerical value has the concertedness effect greater than 1 expression, and this numerical value is big more, and synergistic degree is also strong more.Isobologram (isobologram) is analyzed

Measure the IH50 of inhibitor 1 (for example BRL55730) and inhibitor 2 (for example APAN) respectively.With the mapping on the axle of equivalent line chart of these measured value of experiment, and, be called additivity straight line (Fig. 3) with the straight line connection.50% inhibitory action (Tallarida (1992) Pain 49:93-97, Miaskowski﹠amp that this straight line representative produces when two inhibitor are used in combination; Levine, (1992) 51:383-387).Therefore, the point that is positioned on this additivity straight line shows that superposition is arranged, and be positioned at this point above straight line and show that antagonism is arranged, and the point that is positioned at below this straight line shows that synergism is arranged.Titration BRL55730 under the situation of fixing APAN concentration, and under each APAN concentration, measure the IH50 value of BRL55730.The also mapping (Fig. 3) on the coordinate of equivalent line chart of this data.The synergistic statistical analysis of doing with fixed paired concentration

According to the assay method of standard BRL55730 is measured being lower than under the concentration x of its IH50.Equally APAN is measured being lower than under the concentration y of its IH50.Then, with same hemolysis determination method, the BRL55730 of x concentration and the APAN of y concentration are measured jointly.In the time of can calculating these two inhibitor and measure separately and survey periodic inhibitory action amount jointly.

If the generation synergism, the inhibitory action of the chemical compound of being measured jointly should be greater than these two summations that inhibitor acts on respectively, promptly so

BRL55730 _[x]/APAN _[y]＞BRL55730 _[x]＋APAN _[y]

According to following formula, with t-check to the data analysis that takes statistics.

Group	Meansigma methods	Sample size	The standard error of meansigma methods	The meansigma methods standard error square
					???????a ????BRL55730	????a	????na	????s.e.m.a	?????(s.e.m.)a 2
???????b ?????APAN	????b	????nh	????s.e.m.b	??????(s.e.m)b 2
					??????ab ?BRL55730/APAN	????ab	????nah	????s.e.mab	??????(s.e.m.)ab 2

Null hypothesis=HO ab=a+b (promptly effect is an additivity)

Alternative hypothesis=H1 ab ≠ a+b (promptly effect is not an additivity)

The t value is compared with the critical level in the t check list, wherein, degree of freedom (df)=n _a+ n _b+ n _Ab-3a. serum dilution is the synergism of 1/125 o'clock APAN and BRL55730

The molecular weight of BRL24894A (APAN) is 324.24, and it is made into the solution of 50mM in dimethyl sulfoxine (DMSO).BRL55730 is made into the solution of 5.3mg/ml in the sodium phosphate buffer of 10mM pH7.2.Assay method by standard carries out titration to two inhibitor, and the concentration range of APAN is 100 μ M-0.78 μ M, and the concentration range of BRL55730 is 0.125 μ g/ml-0.00098 μ g/ml.BRL55730 has been made two titration curves, and recording its average IH50 by curve is 0.01 μ g/ml (Fig. 1), and APAN has been made a titration curve, and recording its IH50 by curve is 10 μ M (Fig. 2).In order to measure synergism, titration BRL55730 in identical concentration range, but each titration is all carried out (Fig. 1) in the presence of the APAN of different fixing concentration (1 μ M to 6 μ M).Go out the synergism factor by these data computation as stated above.The mensuration of the synergism factor of table 1:BRL55730 and APAN

?1	?2	3	4	?5	?6
						?[APAN] ?μM	?IH (APAN)	IH50 (BRL55730) μg/ml	?0.5-IH (APAN)	[BRL55730] μ g/ml that adjusts	The synergism factor
?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
?5	?0.27	?0.0019	?0.23	?0.003	?1.6
						?6	?0.31	?0.0013	?0.19	?0.0022	?1.7

From the data of table 1 as seen, 6 μ MAPAN and BRL55730 combined effect make IH50 reduce about 8 times.Listed in the table 1 under each APAN concentration, calculate the synergism factor, the synergism factor is greater than 1, the effect that shows APAN is greater than superposition.The synergism factor keeps quite constant in used concentration range, and meansigma methods is 1.8.

In 0.04-0.000039 μ g/ml concentration range, BRL55730 is carried out independent titration, find that not producing the inhibiting concentration of any complement activation approximately is 0.0004 μ g/ml.APAN is carried out independent titrating result show, when concentration was 4 μ M, the IH value was 0.31, and when concentration was 2 μ M, the IH value was 0.16.In the presence of 4 μ M and 2 μ MAPAN, BRL55730 is carried out titration, the concentration of finding BRL55730 is that inhibitory action is bigger than the inhibitory action that is caused separately by APAN under the situation of 0.0004 μ g/ml, shows that APAN can strengthen the activity of BRL55730 below invalid concentration.B. in the presence of APAN to the isobologram analysis of BRL55730

From Fig. 1 and Fig. 2, obtain data as stated above, draw the additivity straight line.IH50 (seeing Table 1 the 1st hurdle and the 3rd hurdle respectively) with the BRL55730 under each APAN concentration maps (as shown in Figure 3) on the coordinate of equivalent line chart then.Be positioned at the following point of additivity straight line and show that its interaction is synergitic.C. use fixed paired concentration to synergistic statistical analysis between BRL55730 and APAN

As previously described, with following its synergism of paired concentration determination.(ⅰ) below 0.005 μ g/ml BRL, 55,730 4 μ M APAN (ⅱ), 0.005 μ g/ml BRL, 55,730 2 μ M APAN (ⅲ), 0.002 μ g/ml BRL, 55,730 4 μ M APAN (ⅳ), 0.002 μ g/ml BRL, the 55730 2 μ M APAN the right statistical parameter of each concentration is shown.Table 2: concentration is to the statistical analysis of (ⅰ)

Parameter	????BRL55730 ????0.005μg/ml	????APAN ????4μM	????BRI55730＋APAN ????0.005μg/ml＋4μM
				On average	??????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
The t-value	3.822
					At degree of freedom df=39, probability=0.9995 o'clock, t value＞3.558 that t=3.558 calculates, so a+b ≠ ab

Table 3: concentration is to the statistical analysis of (ⅱ)

Parameter	????BRL55730 ????0.005μg/ml	????APAN ????2μM	????BRL55730＋APAN ????0.005μg/ml＋2μM
				Average 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
The t-value	5.407
					At degree of freedom df=45, probability=0.9995 o'clock, t value＞3.550 that t=3.550 calculates, so a+b ≠ ab

Table 4: concentration is to the statistical analysis of (ⅲ)

Parameter	???BRL55730 ??0.002μg/ml	????APAN ????4μM	????BRL55730＋APAN ????0.002μg/ml＋4μM
				Average 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
The t-value	7.451
					At degree of freedom df=45, probability=0.9995 o'clock, t value＞3.550 that t=3.550 calculates, so a+b ≠ ab

Table 5: concentration is to the statistical analysis of (ⅳ)

Parameter	????BRL55730 ????0.002μg/ml	????APAN ????2μM	????BRL55730＋APAN ????0.002μg/ml＋2μM
				Average 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
The t-value	6.267
					At degree of freedom df=45, probability=0.9995 o'clock, t value＞3.550 that t=3.550 calculates, so a+b ≠ ab

Above data show that for each determined paired concentration, the hypothesis of selecting property fully of statistical analysis is correct, i.e. their interaction is not an additivity, because a+b＜ab has shown synergism.D.BRL55730 is to the synergism of APAN

Measure the influence of BRL55730 by the method identical, but APAN carries out titration in the scope of 0.78 μ M to 100 μ M in the presence of the BRL55730 of fixed concentration (0.001-0.006 μ g/ml) here to the IH50 of APAN with the foregoing description 1a.BRL55730 is shown in Table 2 the influence of the IH50 of APAN, shows that the IH50 of APAN when BRL55730 concentration is 0.006 μ g/ml becomes 1 μ M from 10 μ M, has improved 10 times.Measure the synergism factor as stated above, its value is greater than 1 (table 6), shows that the synergism process between APAN and the BRL55730 is reversible.1a is different with previous examples, and this synergism factor be it seems the concentration that depends on BRL55730.The mensuration of the synergism factor of table 6:APAN and BRL55730

[BRL55730] μg/ml	?IH (BRL55730)	IH50 (APAN) μM	?0.5-IH (BRL55730)	[APAN] μ M that adjusts	The synergism factor
						?0	?-	?10	?0.5	?10	?1
						?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. in the presence of BRL55730 to the isobologram analysis of APAN

Carry out the isobologram analysis as stated above with the data among Fig. 2 and the table 6.Data point all is positioned under the additivity straight line, and the effect of showing is synergitic.F. in serum dilution the synergism of 1/35.7 o'clock APAN and BRL55730

Whether the synergism between the APAN and BRL55730 can reproduce in the denseer serum in order to be determined at, and experiment is to carry out in 1/35.7 o'clock in serum dilution, and this concentration is 3.5 times of the serum-concentration described in the example 1a.In order to determine that this serum-concentration can not make the sheep red blood cell of sensitization produce maximum haemolysis, makes a pilot study earlier.With 50 μ l dilution factors is that each serum of 1/10 to 1/150 and 50 μ l 0.1M Hepes pH7.4/0.15M NaCl buffer were 37 ℃ of precincubation 15 minutes.Add 100 μ l erythrocyte, with sample 37 ℃ of incubations 15 minutes or 30 minutes.Removed undissolved cell in centrifugal 15 minutes at 4 ℃ of rotating speeds, draw 150 μ l supernatant then and move into flat-bottom microtiter plates, read absorbance at 410nm wavelength place then with about 300g.With serum dilution absorbance is mapped, show 1/35.7 dilution serum and the common incubation of erythrocyte 15 minutes, cause about 90% maximum haemolysis.Be fit to because proved this serum-concentration, so the synergism of BRL55730 and APAN experiment can be by carrying out with example 1a similar methods, different is to have adopted denseer serum, and the incubation time reduces to 15 minutes.

Titration BRL55730 in denseer serum, the IH50 that obtains are 0.14 μ g/ml (meansigma methods of twice mensuration), and this IH50 value is to be 14 times of the IH50 that obtained in 1/125 o'clock in serum dilution.Similar therewith, the IH50 of APAN is 52 μ M (meansigma methodss of twice mensuration) in denseer serum, approximately is 5 times of IH50 value in rarer serum.The synergism experiment is undertaken by following condition: in the presence of APAN BRL55730 is carried out titration, the concentration range of APAN is 2 μ M to 18 μ M, and the concentration range of BRL55730 is 1 μ g/ml to 0.0078 μ g/ml.Data are summarised in the table 7, show that synergism can expand to denseer serum.The synergism factor in this example be it seems the concentration that depends on APAN.Table 7: the synergism value in denseer serum between BRL55730 and APAN

1	?2	?3	?4	?5	?6
						[APAN] μM	?IH(APAN)	IH50 (BPL55730) μg/ml	?0.5-IH (APAN)	[BRL55730] μ g/ml that adjusts	The synergism 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
						15	?0.16	?0.025	?0.34	?0.1	?4.00
18	?0.2	?0.02	?0.3	?0.085	?4.25

G. the isobologram analysis of BRL55730 and APAN in denseer serum

With serum dilution is that the IH50 of 1/35.7 o'clock BRL55730 and APAN draws the additivity straight line.Data point with the 1st hurdle in the table 7 and the 3rd hurdle is drawn equivalent line chart.Except that 2 μ M, other all points all are positioned under the additivity straight line, and showing has synergism.In table 7, the synergism factor at the data point place of 2 μ MAPAN concentration is 1.03, and in equivalent line chart, this point is positioned on the additivity straight line, and the effect that shows APAN under this concentration may be an additivity.H.BRAPAN is to the synergism of BRL55730

With molecular weight be 386 4 '-methoxybenzoic acid 4-amidino groups-2-bromobenzene ester hydrochloride (BRAPAN) is mixed with the solution that concentration is 10mM in DMSO, and to adopt dilution factor according to method described in the experimental technique be that 1/125 serum carries out titration.The meansigma methods that obtains the IH50 of BRAPAN from twice independent mensuration is 3 μ M.Measuring concentration earlier is the independent titration curve of the BRL55730 of 0.1 to 0.00078 μ g/ml, and the IH50 that draws is 0.021 μ g/ml.In order to measure synergism, in same concentration range, BRL55730 is carried out titration, but titration is to carry out in the presence of the BRAPAN of 0.1 μ M to 0.9 μ M in concentration range.Table 8 has shown influence and the synergism of BRAPAN to BRL55730.Identical with the APAN of identical serum dilution, the synergism factor＞1 shows that BRAPAN and BRL55730 have synergism.In above-mentioned concentration range, it is quite constant that the synergism factor keeps, and its meansigma methods is 1.7, and also the numerical value that obtains with APAN is very approaching.Synergism value between table 8:BRL55730 and BRAPAN

????1	????2	????3	????4	????5	????6
						?[BRAPAN] ?μM	?IH ?(BRAPAN)	?IH50 ?(BRL55730) ?μg/ml	?0.5-IH ?(BRAPAN)	[BRL55730] μ g/ml that adjusts	The synergism factor
?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.BRAPAN is to the isobologram analysis of the influence of BRL55730

Data with BRAPAN among the example 1h are drawn equivalent line chart.All data points all be positioned at additivity collinear below, show to have produced synergism.The combination preparation of embodiment 2 sCR1 (BRL55730) and APAN (BRL24894A)

(Sigma.UK 60mg) is dissolved in the water for injection (9.5ml) with the D-mannitol.APAN is dissolved in the HPLC level methanol again, stirred 5 minutes down in room temperature (20-25 ℃), making last concentration is 6mg/ml.At once this solution (0.5ml) is added in the mannitol solution jolting mixing then.Add BRL55730 solution 0.2ml (concentration is 5mg/ml, is dissolved in the sodium phosphate of 0.2ml 10mM pH 7.2) again, jolting is also freezing in solid CO2 immediately.Again average pressure 2-3 millibar, condenser temperature-60 ℃ following lyophilization 20 hours.The white solid that obtains composed as follows, with it at-70 ℃ of following kept dry: BRL55730:1mg; BRL24894A:3mg; D-mannitol: 60mg; Sodium phosphate: trace.The preparation of embodiment 34 '-methoxybenzoic acid 4-amidino groups-2-bromobenzene ester hydrochloride (BRAPAN)

This chemical compound is made through two steps by 2-bromo-4-cyanophenol.A.2-the preparation of bromo-4-amidino groups phenolate hydrochlorate

(1.35g 6.8mmol) is dissolved in the 20ml ethanol with 2-bromo-4-cyanophenol.Then hydrogen chloride gas is fed this through refrigerative solution, form white precipitate after 45 minutes.Stop ventilation after 2 hours, the solid in the solution was placed 2 days in 4 ℃.White solid is told in filtration.This solid is suspended in rapidly in the 50ml alcoholic solution, and adds the saturated solution 75ml of ammonia in ethanol.This suspension almost becomes clarification immediately, and it was stirred 24 hours, leaves standstill the similar time again.All volatile material are removed, white solid is dissolved in the 20ml water.Add concentrated hydrochloric acid 5ml, cause rapid formation white crystal, filter and tell crystal, use 20ml ethanol and 100ml ether recrystallization then.Output: 860mg (50%), fusing point: 279-80 ℃.

¹H nuclear magnetic resoance spectrum (CDCl ₃-d ⁶DMSO) 6:9.2 (4H, brd, amidine),

8.2 (1H, d, J=2Hz, aryl-H),

7.8 (1H, m, aryl-H), 7.25 (1H, J=9Hz, the infrared spectrum (liquid paraffin method) of aryl-H): 3325,3125,2300-3400,1670,1610,

1585,1410,1300,1175,1045,880,835,725,620cm ^-1。Elementary analysis: C33.45, H3.28, N10.95%

Theoretical value: C ₇H ₈N ₂OBrCl C33.43, H3.21, the preparation of N11.14%b. title compound

To the 4-methoxy benzoyl chloride (271mg, in anhydrous pyridine 1.59mmol) (5ml) solution, add 2-bromo-4-amidino groups phenolate hydrochlorate (400mg, 1.59mmol).Initial suspension becomes clear solutions, and then generates precipitation.Stir after 1 hour, infrared analysis shows that ester forms.Pyridine is removed in decompression, and last micro-pyridine is by removing with the ethanol azeotropic.The white solid of gained obtains white title compound with 5: 1 ether (about 100ml) recrystallization twice.Output: 225mg (37%).Fusing point: 212-3 ℃.

¹H nuclear magnetic resoance spectrum (d ⁶DMSO-trace CDCl ₃) δ:

(9.55 4H, br, amidino groups phenol NH)

6.95-8.25 (7H, m, aryl-H), 3.9 (1H, s, OCH ₃) infrared spectrum (liquid paraffin method): 2500-3400,1735,1665,1605,1580,

1260,1230,1170,1070,1020 and 830cm ^-1Elementary analysis: C46.66, H3.74, N7.19%

Theoretical value: C ₁₅H ₁₂N ₂BrCl C46.72%, H3.66%, N7.26%

In the accompanying drawing:

Fig. 1 represents the effect of the APAN of variable concentrations to BRL55730;

Fig. 2 represents the inhibitory action of APAN to complement activation;

Fig. 3 is by the BRL55730 of standard determination method drafting and the equivalent line chart of APAN.

Claims (7)

1. the method for a treatment and inflammation or unsuitable complement activation diseases associated or imbalance, this method comprises that the complement that has that the mammal of needs treatments is granted the solubility CR1 protein of effective dose and effective dose suppresses the amidino groups phenylester of active formula (I) or amidino groups naphthyl ester and acceptable salt pharmaceutically thereof

Wherein, A is can be by any phenyl that replaces of following radicals: C _1-4Alkyl, C _1-4Alkoxyl, C _1-4Alkoxy carbonyl group, halogen, NH ₂, sulfonyl, benzoyl or C _1-4Alkylbenzene formamido group, perhaps naphthyl: B is can be by any CH that replaces of one of following radicals ₂=CH-base: C _1-5Alkyl, phenyl and C _1-6The phenyl that alkyl replaces; Be selected from any phenyl that replaces of substituent group of following radicals separately by one or two: halogen, C _1-6Alkyl, C _2-6Alkenyl, C _1-6Alkoxyl, C _1-6Chain ene acyloxy, C _1-6Alkanoyl amido, amino, dimethylamino or guanidine radicals; Perhaps naphthyl.

2. solubility CR1 protein and have complement and suppress the amidino groups phenylester of active formula as claimed in claim 1 (I) or amidino groups naphthyl ester and be used for the treatment of application in the medicine with inflammation or unsuitable complement activation effect diseases associated or imbalance in preparation.

3. pharmaceutical composition, said composition contain solubility CR1 protein and have complement and suppress the amidino groups phenylester of active formula as claimed in claim 1 (I) or amidino groups naphthyl ester and acceptable carrier pharmaceutically.

4. the method for a treatment and inflammation or unsuitable complement activation effect diseases associated or imbalance, this method comprises that the patient to this treatment of needs grants the compositions that the claim 3 of effective dose is gone up in treatment.

5. kit that comprises one or more packing materials is equipped with the composition of the described pharmaceutical composition of one or more claim 3 in the described packing material.

6. method for preparing the described pharmaceutical composition of claim 3, this method comprises that the mixture with the amidino groups phenylester of solubility CR1 protein and the described formula of claim 1 (I) or amidino groups naphthyl ester mixes.

7. according to claim 1,2,3,4, the described Therapeutic Method of 5 or 6 difference, purposes, compositions, Therapeutic Method, kit and preparation method, wherein solubility CR1 protein is by nucleic acid mediator pBSCR1c/pTCSgpt encoded protein matter, ester is 4 '-methoxybenzoic acid 4-amidino groups phenyl ester hydrochlorate or 4 '-methoxybenzoic acid 4-amidino groups-2-bromobenzene ester hydrochloride.

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