Patent Application: US-80765107-A

Abstract:
this invention relates to the use of an antagonist of a g protein - coupled receptor in the prevention and / or treatment of fibrosis , such as the treatment of fibrosis associated with myocardial infarction or diabetes or certain pulmonary conditions . in a preferred embodiment , the antagonist is a c5a receptor antagonist , more preferably a cyclic peptide antagonist of the c5a receptor . in particular , the invention provides a method of prevention , treatment or alleviation of a fibrotic condition , comprising the step of administering an effective amount of an antagonist of a g protein - coupled receptor to a subject in need of such treatment .

Description:
for the purposes of this specification , the term “ fibrotic condition ” is to be taken to mean any fibrotic disorder , such as multiple sclerosis , retinal disorders including proliferative vitroretinopathy and macular degeneration , scleroderma , sclerosing peritonitis , fibrosis arising from trauma , burns , chemotherapy , radiation , infection or surgery and fibrosis of major organs such as the kidney , liver , heart or lungs . the term “ c5a receptor antagonist ” includes any compound which can reduce or inhibit effects mediated by the interaction between c5a and c5a receptor . thus the term includes polyclonal or monoclonal antibodies , peptides , peptidomimetics , and non - peptide compounds . the terms “ treating ,” “ treatment ,” and “ therapy ” as used herein refer to curative therapy , prophylactic therapy , and preventative therapy . for the purposes of this specification it will be clearly understood that the word “ comprising ” means “ including but not limited to ”, and that the word “ comprises ” has a corresponding meaning . as used herein , the singular forms “ a ”, “ an ”, and “ the ” include plural reference unless the context clearly dictates otherwise . thus , for example a reference to “ an enzyme ” includes a plurality of such enzymes , and a reference to “ an amino acid ” is a reference to one or more amino acids . unless defined otherwise , all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs . although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention , the preferred materials and methods are now described . for the purposes of this specification , the term “ alkyl ” is to be taken to mean a straight , branched , or cyclic , substituted or unsubstituted alkyl chain of 1 to 6 , preferably 1 to 4 carbons . most preferably the alkyl group is a methyl group . the term “ acyl ” is to be taken to mean a substituted or unsubstituted acyl of 1 to 6 , preferably 1 to 4 carbon atoms . most preferably the acyl group is acetyl . the term “ aryl ” is to be understood to mean a substituted or unsubstituted homocyclic or heterocyclic aryl group , in which the ring preferably has 5 or 6 members . a “ common ” amino acid is a l - amino acid selected from the group consisting of glycine , leucine , isoleucine , valine , alanine , phenylalanine , tyrosine , tryptophan , aspartate , asparagine , glutamate , glutamine , cysteine , methionine , arginine , lysine , proline , serine , threonine and histidine . an “ uncommon ” amino acid includes , but is not restricted to , d - amino acids , homo - amino acids , n - alkyl amino acids , dehydroamino acids , aromatic amino acids other than phenylalanine , tyrosine and tryptophan , ortho -, meta - or para - aminobenzoic acid , ornithine , citrulline , canavanine , norleucine , γ - glutamic acid , aminobutyric acid , l - fluorenylalanine , l - 3 - benzothienylalaine , and α , α - disubstituted amino acids . generally , the terms “ treating ”, “ treatment ” and the like are used herein to mean affecting a subject , tissue or cell to obtain a desired pharmacological and / or physiological effect . the effect may be prophylactic in terms of completely or partially preventing a disease or sign or symptom thereof , and / or may be therapeutic in terms of a partial or complete cure of a disease . “ treating ” as used herein covers any treatment of , or prevention of disease in a vertebrate , a mammal , particularly a human , and includes : preventing the disease from occurring in a subject who may be predisposed to the disease , but has not yet been diagnosed as having it ; inhibiting the disease , ie ., arresting its development ; or relieving or ameliorating the effects of the disease , ie ., cause regression of the effects of the disease . the invention includes the use of various pharmaceutical compositions useful for ameliorating disease . the pharmaceutical compositions according to one embodiment of the invention are prepared by bringing a compound of formula i , analogue , derivatives or salts thereof and one or more pharmaceutically - active agents or combinations of compound of formula i and one or more pharmaceutically - active agents into a form suitable for administration to a subject using carriers , excipients and additives or auxiliaries . frequently used carriers or auxiliaries include magnesium carbonate , titanium dioxide , lactose , mannitol and other sugars , talc , milk protein , gelatin , starch , vitamins , cellulose and its derivatives , animal and vegetable oils , polyethylene glycols and solvents , such as sterile water , alcohols , glycerol and polyhydric alcohols . intravenous vehicles include fluid and nutrient replenishers . preservatives include antimicrobial , anti - oxidants , chelating agents and inert gases . other pharmaceutically acceptable carriers include aqueous solutions , non - toxic excipients , including salts , preservatives , buffers and the like , as described , for instance , in remington &# 39 ; s pharmaceutical sciences , 20th ed . williams & amp ; wilkins ( 2000 ) and the british national formulary 43rd ed . ( british medical association and royal pharmaceutical society of great britain , 2002 ; http :// bnf . rhn . net ), the contents of which are hereby incorporated by reference . the ph and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art . see goodman and gilman &# 39 ; s the pharmacological basis for therapeutics ( 7th ed ., 1985 ). the pharmaceutical compositions are preferably prepared and administered in dosage units . solid dosage units include tablets , capsules and suppositories . for treatment of a subject , depending on activity of the compound , manner of administration , nature and severity of the disorder , age and body weight of the subject , different daily doses can be used . under certain circumstances , however , higher or lower daily doses may be appropriate . the administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals . the pharmaceutical compositions according to the invention may be administered locally or systemically in a therapeutically effective dose . amounts effective for this use will , of course , depend on the severity of the disease and the weight and general state of the subject . typically , dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition , and animal models may be used to determine effective dosages for treatment of the cytotoxic side effects . various considerations are described , eg . in l anger , science , 249 1527 , ( 1990 ). formulations for or al use may be in the form of hard gelatin capsules , in which the active ingredient is mixed with an inert solid diluent , for example , calcium carbonate , calcium phosphate or kaolin . they may also be in the form of soft gelatin capsules , in which the active ingredient is mixed with water or an oil medium , such as peanut oil , liquid paraffin or olive oil . aqueous suspensions normally contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions . such excipients may be suspending agents such as sodium carboxymethyl cellulose , methyl cellulose , hydroxypropylmethylcellulose , sodium alginate , polyvinylpyrrolidone , gum tragacanth and gum acacia ; dispersing or wetting agents , which may be ( a ) a naturally occurring phosphatide such as lecithin ; ( b ) a condensation product of an alkylene oxide with a fatty acid , for example , polyoxyethylene stearate ; ( c ) a condensation product of ethylene oxide with a long chain aliphatic alcohol , for example , heptadecaethylenoxycetanol ; ( d ) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol such as polyoxyethylene sorbitol monooleate , or ( e ) a condensation product of ethylene oxide with a partial ester derived from fatty acids and hexitol anhydrides , for example polyoxyethylene sorbitan monooleate . the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension . this suspension may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents such as those mentioned above . the sterile injectable preparation may also a sterile injectable solution or suspension in a non - toxic parenterally - acceptable diluent or solvent , for example , as a solution in 1 , 3 - butanediol . among the acceptable vehicles and solvents which may be employed are water , ringer &# 39 ; s solution , and isotonic sodiumchloride solution . in addition , sterile , fixed oils are conventionally employed as a solvent or suspending medium . for this purpose , any bland fixed oil may be employed , including synthetic mono - or diglycerides . in addition , fatty acids such as oleic acid may be used in the preparation of injectables . agents useful in the invention may also be administered in the form of liposome delivery systems , such as small unilamellar vesicles , large unilamellar vesicles , and multilamellar vesicles . liposomes can be formed from a variety of phospholipids , such as cholesterol , stearylamine , or phosphatidylcholines . dosage levels of the compounds of the present invention will usually be of the order of about 0 . 5 mg to about 20 mg per kilogram body weight , with a preferred dosage range between about 0 . 5 mg to about 10 mg per kilogram body weight per day ( from about 0 . 5 g to about 3 g per patient per day ). the amount of active ingredient which may be combined with the carrier materials to produce a single dosage will vary , depending upon the host to be treated and the particular mode of administration . for example , a formulation intended for oral administration to humans may contain about 5 mg to 1 g of an active compound with an appropriate and convenient amount of carrier material , which may vary from about 5 to 95 percent of the total composition . dosage unit forms will generally contain between from about 5 mg to 500 mg of active ingredient . it will be understood , however , that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed , the age , body weight , general health , sex , diet , time of administration , route of administration , rate of excretion , drug combination and the severity of the particular disease undergoing therapy . in addition , some of the compounds of the invention may form solvates with water or common organic solvents . such solvates are encompassed within the scope of the invention . the compounds of the invention may additionally be combined with other therapeutic compounds to provide an operative combination . it is intended to include any chemically compatible combination of pharmaceutically - active agents , as long as the combination does not eliminate the activity of the compound of this invention . for example , spironolactone , pirfenidone , gingko biloba extract ( welt et al , 1999 ), and tocopherol acetate ( rosen et al , 1995 ) are known in the art for treatment of fibrosis . inhibitors of prolyl hydroxylase , procollagen c - proteinase , also known as bone morphogenetic protein - 1 ( bmp - 1 ), or connective tissue growth factor 02450 , wo00 / are being investigated for this purpose by fibrogen , inc . see for example wo / 01 / 56996 , wo / 01 / 15729 , wo00 / 02450 , wo00 / 50390 , wo00 / 27868 , wo00 / 13706 , and wo / 9921860 . these compounds include prostacyclin and phenanthroline derivatives . the invention includes within its scope combinations of c5a inhibitors and such known agents . cyclic peptide compounds of formula i are prepared according to methods described in detail in our earlier applications no . pct / au98 / 00490 and no . pct / au02 / 01427 , the entire disclosures of which are incorporated herein by this reference . while the invention is specifically illustrated with reference to the compound acf -[ opdchawr ] ( pmx53 ), whose corresponding linear peptide is ac - phe - orn - pro - dcha - trp - arg , it will be clearly understood that the invention is not limited to this compound . compounds 1 - 6 , 17 , 20 , 28 , 30 , 31 , 36 and 44 disclosed in international patent application no . pct / au98 / 00490 and compounds 10 - 12 , 14 , 15 , 25 , 33 , 35 , 40 , 45 , 48 , 52 , 58 , 60 , 66 , and 68 - 70 disclosed for the first time in international patent application pct / au02 / 01427 have appreciable antagonist potency ( ic50 & lt ; 1 μm ) against the c5a receptor on human neutrophils . pmx53 and compounds 33 , 45 and 60 of pct / au02 / 01427 are most preferred . we have found that all of the compounds of formula i which have so far been tested have broadly similar pharmacological activities , although the physicochemical properties , potency , and bioavailability of the individual compounds varies somewhat , depending on the specific substituents . the general tests described in pct / au98 / 00490 and pct / au02 / 01427 may be used for initial screening of candidate inhibitor of g protein - coupled receptors , and especially of c5a receptors . the invention will now be described in detail by way of reference only to the following non - limiting examples and figures . example 1 effect of a c5a receptor antagonist on l - name - induced cardiac fibrosis male wistar rats ( 8 weeks old ) were obtained from the central animal breeding house of the university of queensland . the rats were administered a c5a receptor antagonist designated pmx53 , which has the formula : this agent was administered at a dosage of 1 mg / kg / day orally for 4 days before rats were additionally treated with nitro - l - arginine methyl ester ( l - name ) for 4 weeks , ie a total duration of treatment of antagonist of 32 days . l - name administration produces hypertension and cardiac remodelling as a result of inhibition of the production of nitric oxide ( no ). l - name was administered at a concentration of 400 mg / l in the drinking water for 4 weeks to give a mean daily intake of 18 . 7 ± 0 . 4 mg l - name ( 41 . 4 ± 0 . 8 mg / kg mean body weight ). body weight and food and water intakes were measured daily . neither l - name nor c5a receptor antagonist treatment altered water intake or growth rate , as shown in fig1 and 2 . systolic blood pressure was measured in selected unanaesthetised rats , using a tail - cuff method . as illustrated in fig3 , systolic blood pressure increased from 118 ± 3 mmhg to 160 ± 2 mmhg in l - name - treated rats without significantly altering heart rate or increasing left ventricular weight , as determined by echocardiograph or post - mortem examination , when compared to control rats . these results are shown in fig4 . similarly , right ventricular and other major organ weights were not significantly altered with l - name treatment . c5a receptor antagonist treatment of l - name rats significantly increased systolic blood pressure by 16 mmhg to 176 ± 3 mmhg , resulting in an increased left ventricular wet weight . additionally , c5a receptor antagonist treatment of control rats induced a non - significant increase in blood pressure . these results are summarised in fig3 and 4 . c5a receptor antagonist treatment of both control and l - name rats did not significantly alter wet weights of the remaining major organs . after 4 weeks of l - name treatment , heart function was determined in vivo by echocardiography and in vitro using the isolated langendorff heart preparation described below . collagen deposition was measured by image analysis using laser confocal microscopy of picrosirius red - stained cardiac slices , as described below . rats were euthanased with pentobarbitone ( 100 mg / kg ip ). blood was taken from the abdominal vena cava , centrifuged and the plasma frozen . plasma glucose was measured by precision plus blood glucose electrodes ( medisense , abbott laboratories ); plasma na + and k + were measured by flame photometry . collagen distribution was determined by image analysis of sections of heart and kidney stained with picrosirius red ( 0 . 1 % sirius red f3ba in picric acid ), which selectively stains fibrillar collagen . slides were left in 0 . 2 % phosphomolybdic acid for 5 minutes , washed , and left in picrosirius red for 90 minutes , then in 1 mm hcl for 2 minutes and 70 % ethanol for 45 seconds . the stained sections were analyzed with an image pro plus analysis program using an olympus bh2 microscope , with results expressed as a percentage of red area in each screen . at least 4 areas were examine in each heart . the results are presented in fig5 and 7 . image analysis showed an increase of 108 % in interstitial collagen and an 87 % increase in perivascular collagen in the left ventricle of l - name treated rats when compared to controls . similarly , a significant increase in collagen levels was observed in the right ventricle , where a 175 % increase in interstitial and a 37 % increase in perivascular collagen content occurred . l - name treatment also significantly increased the collagen content by 55 % in the tubulointerstitial areas of the kidneys with a smaller increase in glomerular spaces . c5a receptor antagonist treatment attenuated the increased collagen deposition . in c5a antagonist treated rats , l - name treatment produced 23 % and 43 % of the increase observed in rats treated with l - name only when comparing the left ventricular interstitial and perivascular areas respectively . similar results were observed in the right ventricle , where c5a receptor antagonist treatment of l - name restricted collagen deposition to 44 and 37 % in the interstitial and perivascular areas respectively . in the kidneys , c5a antagonist administration to l - name rats restricted collagen deposition to 30 % in the interstitium and normalized the increase in glomerular collagen concentrations observed in l - name treated rats . as illustrated in fig9 , l - name treatment resulted in a large inflammatory cell infiltration in both the left and right ventricles . a 30 - fold increase in inflammatory cell population was observed in the both left and right ventricular interstitial and perivascular areas following l - name treatment . c5a receptor antagonist treatment totally prevented inflammatory cell infiltration into left or right ventricles following l - name treatment . no information is so far available on inflammatory cell type or kidney infiltration . although l - name treatment did not significantly increase left ventricular weight , echocardiographic m - mode measurements showed that l - name treatment had triggered cardiac remodelling , increasing the left ventricular wall thickness and decreasing the left ventricular internal diameter in diastole . further l - name treatment significantly increased the ratio of early ( e ) to atrial ( a ) mitral valve inflow rates ( e / a ratio ), and significantly decreased diastolic volume and cardiac output . fractional shortening and ascending aortic flow rates were not significantly altered by l - name treatment . thus l - name treatment induces cardiac remodelling , with minor changes in systolic function and an improved diastolic function . c5a receptor antagonist treatment of control rats did not significantly alter any parameter measured by echocardiographic analysis . c5a receptor antagonist treatment of l - name rats normalised the increase in left ventricular wall thickness and decreased left ventricular internal dimensions . this treatment also significantly normalised the e / a ratio , diastolic volume and cardiac output . these results are presented in fig9 . the langendorff isolated heart preparation was used to determine the diastolic stiffness of the left ventricles ex vivo . rats were anaesthetised with sodium pentobarbitone ( 100 mg / kg ip ) and heparin ( 2000 iu ) was administered via the femoral vein . after allowing 2 minutes for the heparin to fully circulate , the heart was excised and placed in cooled ( 0 ° c .) crystalloid perfusate ( krebs - henseleit solution of the following composition in mm : nacl 118 , kcl 4 . 7 , mgso 4 1 . 2 , kh 2 po 4 1 . 2 , cacl 2 2 . 3 , nahco 3 25 . 0 glucose 11 . 0 ). the heart was then attached to the cannula with the tip of the cannula positioned immediately above the coronary ostia of the aortic stump , and perfused in a non - recirculating langendorff fashion at 100 cm of hydrostatic pressure . the buffer temperature was maintained at 35 ° c . the hearts were punctured at the apex to facilitate thebesian drainage and paced at 250 bpm . a balloon catheter was inserted in the left ventricle via the mitral orifice for measurement of left ventricular developed pressure . the catheter was connected via a three - way tap to a micrometer syringe and to a statham p23 pressure transducer . the outer diameter of the catheter was similar to the mitral annulus to prevent ejection of the balloon during the systolic phase . after a 10 minute stabilisation period , steady - state left ventricular pressure was recorded from isovolumetrically beating hearts . increments in balloon volume were applied to the heart until left ventricular end - diastolic pressure reached approximately 30 mmhg . to assess myocardial stiffness in isolated langendorff hearts , stress ( σ , dyne / cm2 ) and tangent elastic modulus ( e , dyne / cm2 ) for the midwall at the equator of the left ventricle were calculated by assuming spherical geometry of the ventricle and considering the midwall equatorial region as representative of the remaining myocardium : σ = vp w ⁢ ( 1 + 4 ⁢ ( v + w ) [ v 1 / 3 + ( v + w ) 1 / 3 ] 3 ) e = 3 ⁢ { vp w - σ + [ σ v + ( w ⁢ ⁢ σ - vp ) w ⁡ ( v + w ) + σ · ⅆ p p · ⅆ v ] × [ v 1 / 3 + ( v + w ) 1 / 3 ] [ v - 2 / 3 + ( v + w ) - 2 / 3 ] } where v is chamber volume ( ml ), w is left ventricular wall volume ( 0 . 943 ml / g ventricular weight ) and p is end diastolic pressure ( dyne / cm2 = 7 . 5 × 10 − 4 mmhg ). myocardial diastolic stiffness is calculated as the diastolic stiffness constant ( k , dimensionless ), the slope of the linear relation between e and a ( mirsky and parmley , 1973 ). to assess contractile function , maximal + dp / dt was calculated at a diastolic pressure of 5 mmhg . the results are shown in fig1 . all results are given as mean ± sem of at least 6 experiments . the negative log ec50 of the increase in either force of contraction in mn or rate of contraction in beats / min was determined from the concentration giving half - maximal responses in individual concentration - response curves . renal function results were corrected for kidney wet weight measured at the end of the experiment . these results were analysed by two - way analysis of variance followed by the duncan test to determine differences between treatment groups and by paired or unpaired t - tests as appropriate ; p & lt ; 0 . 05 was considered significant . at the end of the experiment , the atria and right ventricle were dissected away and the weight of the left ventricle plus septum was recorded . l - name treatment markedly increased the diastolic stiffness constant of the ventricles when compared to controls . developed pressure and contractility were not altered by l - name treatment . c5a receptor antagonist treatment prevented the increased diastolic stiffness constant of l - name rats without altering contractility or developed pressure . these results are presented in fig1 and 11 . the heart is removed under anaesthesia . the right atria and papillary muscles from the left ventricle are removed and suspended in organ baths a arresting tension of 5 - 10 mn adjusted to give the maximum twitch response . tissues are bathed in a modified tyrode &# 39 ; s solution , containing the following concentrations of salts in mm : nacl 136 . 9 , kcl 5 . 4 , mgcl 2 1 . 05 , cacl 2 1 . 8 , nahco 3 22 . 6 , nah 2 po 4 0 . 42 , glucose 5 . 5 , ascorbic acid 0 . 28 , sodium edetate 0 . 05 , bubbled with 95 % o 2 / 5 % co 2 , and stimulated at lhz at 35 ° c . as previously described ( brown et al , 1991a ). cumulative concentration - response curves are measured for noradrenaline and , following washout and re - equilibration , for calcium chloride . at the end of the experiment , papillary muscle dimensions are measured under the loading conditions of the experiment ; all tissues are blotted and weighed . thoracic aortic rings ( approximately 4 mm in length ) are suspended with a resting tension of 10 mn ( brown et al , 1991b ) and contracted twice with isotonic kcl ( 100 mm ). the presence of endothelium is demonstrated by addition of acetylcholine ( 1 × 10 − 5 m ). cumulative contraction responses to noradrenaline are measured . separate thoracic aortic rings are perfused with 10 % neutral buffered formalin , embedded in wax and stained with haemotoxylin and eosin . image analysis using a wild - leitz md30 + system is used to calculate the wall area of the thoracic aorta . both acute and chronic diseases which induce inflammation in the lung can lead to an irreversible process characterized by pulmonary fibrosis . the effect of pmx - 53 on a rat model of pulmonary fibrosis was assessed , using methods were adapted from taylor et al ( 2002 ). bleomycin is an antineoplastic agent which is a well - known cause of pulmonary fibrosis in humans ( thrall et al , 1978 ). bleomycin - induced pulmonary fibrosis in rats is a well - established model , which has a short experimental period and high success rates . bleomycn induces toxic injury to type i alveolar epithelial cells ( aec ), which causes release of tgf - β , pge 2 , granulocyte - macrophage colony stimulating factor ( gm - csf ), and insulin - like growth factors etc . this induces a massive activation of inflammatory cells such as pmns , macrophages and mesenchymal cells such as fibroblasts , which contribute to an overaggressive repair process , leading to fibrosis in the lung . pmx53 is a c5a receptor antagonist , which effectively inhibits the infiltration and the activation of inflammatory cells , such as pmns , monocytes , macrophages , and therefore reduces the release of reactive oxygen species and inflammatory mediators such as il - 1 and pla 2 . as a result , local tissue damage is prevented by a reduction of release of several factors , such as leukotrienes , and prostaglandins . we investigated whether pmx53 antagonists had any inhibitory effect on bleomycin - induced pulmonary fibrosis . male wistar rats , 6 weeks of age , were used . the rats were divided into 5 groups : group 3 : pmx53 at a dose rate of 10 mg / kg in 200 μl water p . o . ( gavage daily ) and bleomycin instillation ( n = 9 ) group 4 : pmx53 ( dose as for group 3 ) p . o . and saline instillation ( n = 3 ). group 5 : untreated rats maintained in the same environment as the other groups ( n = 3 ). drug - treated rats were given drug for 3 days before bleomycin administration . one intra - tracheal instillation of bleomycin at a dose of 0 . 5 mg / 100 g ( 0 . 7u / 100 g ) in 200 μl of saline was performed on day 1 , as described by taylor et al ., ( 2002 ). rats were anaesthetized by inhalation of 5 % or less halothane via a vaporizer . after a local spray of xylocalne to prevent airway spasm , the rats were intubated and a slow injection of bleomycin or sline control was completed . the rats were then rotated gently for about 1 - 2 minutes to allow the solution to diffuse evenly into both lungs ( christensen et al 2000 ). rats were kept in the fume cupboard until totally recovered , and then monitored for up to 18 days . body weight , food and water intake , and respiration were monitored daily . respiration was elevated as follows : score 0 , normal respiration ; score 1 , increased rate of breathing ; and score 2 , mouth open respiration . rats were euthanased before the end of the experimental period , if they consistently lost more than 10 % bodyweight for 48 hours , had score 2 respiration or had score 1 respiration for 48 hours . at the end of this period the rats were killed by exsanguination under anaesthesia , so that the lungs were clear of blood . for each rat , the left lung was immediately frozen in liquid nitrogen and stored at − 20 ° c . for quantitative collagen analysis using hydroxyproline assay . the right lung was fully inflated and fixed with 10 % formulated formalin by airway gravity fixation at a pressure of 30 cm water for 1 minute . haematoxylin and eosin ( h & amp ; e ) and picro sirius red ( pr ) staining for collagen were performed to assess collagen deposition in the lung . for quantitation of collagen stained with pr , polarized light images were converted to grey scale , and the total number of white pixels ( specific for collagen ) per image was determined as a percentage of the total pixel area . the procedure was applied to a total of four fields in the alveolar area and two fields in the peribronchial area and blood vessels per sample ( wang et al , 2000 ). the largest lobe of the right lung ( from 4 lobes ) in each rat was chosen . the data was analysed using the program “ sion image ”. hydroxyproline assay was performed by the method of christensen et al 1 ( 2000 ). lung tissue was excised , trimmed free of surrounding conducting airways , and homogenized in 2 mls saline . a 1 ml aliquot of lung homogenate was hydrolysed in 6n hcl ( 0 . 5 ml of homogenate and 0 . 5 ml of 12n hcl ) at 110 ° c . for 12 hours ; 50 μl aliquots were added to 1 ml of 14 % chloramine t , 10 % n - propanol , and 0 . 5m sodium acetate , ph 6 . 0 . after 20 min at 22 ° c ., 1 ml of ehrlich &# 39 ; s solution ( 1m p - dimethylaminobenzaldehyde in 70 % n - propanol and 20 % perchloric acid ) was added and allowed to incubate at 65 ° c . for 15 min . absorbance was measured at 550 nm , and the amount of hydroxyproline was determined against a standard curve generated with the use of known concentrations of reagent - grade hydroxyproline . data were compiled as the means ± se in the study . tests of significance were obtained by anova followed by student - newman - keuls post analysis . there were two stages involved in the bleomycin - induced pulmonary fibrosis in rat model . intra - tracheal instillation of bleomycin induced an acute lung inflammation in the rats , evident on day 2 - day 3 . four of the rats from the drug - treated group and four from the non - treated group were very ill , and had to be euthanased after 7 - 9 days . the lungs appeared swollen , with spreading white patchy lesions , as shown in fig1 and 13 . the lung weight to body weight ratio was significantly increased in bleomycin - treated rats , regardless of whether the rats were drug - treated or non - treated . the results are summarised in table 1 . under the microscope , numbers of inflammatory cells , including pmns , macrophages , lymphocytes etc . were observed in the alveolar spaces , with massive leakage of plasma and red blood cells ; this is illustrated in fig1 a . the size and number of type ii aecs in the alveolar spaces was clearly increased , as shown in fig1 b , while in normal lung , the type ii aecs covered only 5 - 10 % of the surface area of the alveoli , as shown in fig1 . there was no significant difference in histology between drug - treated and non - treated groups . collagen deposition in bleomycin instillation lungs showed a significant increase compared to normal lungs ( p & lt ; 0 . 01 , n = 3 ); saline instillation lungs ( p & lt ; 0 . 01 , n = 3 ); and saline instillation with pmx53 - treated lungs ( p & lt ; 0 . 01 , n = 3 ). however , there was no significant difference between the drug - treated group and non - treated group ( p & gt ; 0 . 01 , n = 4 ). these results are summarised in fig1 . eighteen days after intra - tracheal instillation of bleomycin , the degree of oedema was reduced in bleomycin - instilled lungs , and the lung / body weight ratio did not show a significant difference between either the bleomycin group and the non - bleomycin group , or between the drug - treated group and non - drug group ( data not shown ). as illustrated in fig1 , the inflammatory lesions in the lung became smaller and less dense in most of the rats compared with the acute inflammatory stage , whether or not the rats had received drug treatment . there were still numbers of inflammatory cells , many of which were alveolar macrophages , and red blood cells in the lung lesions , as shown in fig1 a . the thickness of the alveolar walls was increased , and there was some fibrinogen depositions in alveolar septa in some of the lungs , as shown in fig1 b . one drug - treated rat and one non - drug treated rat still had some obvious lung inflammatory lesions mixed with marked lung fibrosis lesions . it was difficult to assess the quantity of collagen deposition in the lung tissues from the h & amp ; e stained slides , because the amount of collagen and the spread of the collagen varied in each individual rat , and the number of the lesions in each lung was different . pr staining was more useful than h & amp ; e staining for assessment of collagen deposition in the lungs , as illustrated in fig2 to 30 and as summarised in table 2 . however , for the same reasons it was not an accurate measurement for comparison analysis of the collagen content . the hydroxyproline essay results are summarised in fig1 . bleomycin instillation significantly increased hydroxyproline levels in the rat lungs ( p & lt ; 0 . 01 , n = 3 , compared to normal rats ; p & lt ; 0 . 01 , n = 3 , compared to saline instilled rats ; p & lt ; 0 . 01 , n = 3 , compared to saline instilled with drug treated rats ). pmx53 significantly reduced the bleomycin - induced hydroxyproline levels ( p & lt ; 0 . 05 , n = 4 , compared to the rats with bleomycin instillation ). the failure of pmx53 to inhibit the toxic lung inflammation induced by bleomycin may indicate that the bleomycin - induced toxic inflammation was initiated through a different pathway or via a complicated inter - cellular reaction , rather than by a simple activation of the complement system . type i aec injury , type ii aec proliferation , fibroblast proliferation , and release of several cytokines , such as pge 2 , tgf - β 1 , and gm - csf , are considered to play major roles in pf . after 18 days , the lungs with bleomycin instillation showed some fibrosis , as demonstrated by the significantly increased hydroxyproline levels and collagen deposition as indicated by pr staining . we found that pmx53 significantly reduced the hydroxyproline levels , although this was difficult to confirm by histology or pr staining . it is possible that 18 days is too early for the histological changes to be evident , because most studies demonstrated that the dna and hydroxyproline changes occur between 14 - 21 days after bleomycin instillation , while histological evidence was present after 4 weeks . nevertheless , the significant reduction by pmx53 of bleomycin - induced hydroxyproline deposition indicates that the activation of the c5a cascade may be involved in the progression of fibrosis , although the role of c5a in bleomycin - induced pf is not fully understood . it will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding , various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification . references cited herein are listed on the following pages , and are incorporated herein by this reference . brown l , sernia c , newling r , fletcher p : comparison of inotropic and chronotropic responses in rat isolated atria and ventricles . clin exp pharmacol physiol 1991a ; 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