the practice of the present invention will employ , unless otherwise indicated , conventional techniques of molecular biology , microbiology , and recombinant dna technology which are within the skill of those working in the art . such techniques are explained fully in the literature . examples of texts for consultation include the following : sambrook molecular cloning ; a laboratory manual , third edition ( 2000 ). included within the scope of the present invention are those sequences and fragments of the polypeptides as described in seq id no : 1 to 3 , or functional equivalents thereof , which can be used for treating or preventing inflammatory conditions , especially in rheumatoid arthritis , and as potential anti - cancer drug . in yet another aspect , the invention contemplates a method of producing a polypeptide variant of a parent polypeptide comprising the sequences set forth in seq id no : 1 - 3 , or biologically active fragments thereof , comprising the steps of : ( a ) replacing at least one amino acid of the parent polypeptide , with a different amino acid to produce a modified polypeptide ; ( b ) deleting and / or adding the parent polypeptide , modifying the side chains , incorporating unnatural amino acids and / or their derivatives during peptide , polypeptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the polypeptides , fragments and variants of the invention ; ( c ) modifying the polypeptides , fragments or variants of the invention using ordinary molecular biology techniques to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as an immunogenic agent . the described features of the invention can be gathered from the following description of preferred embodiments in conjunction with the claims . the inventors have discovered the new peptide analogue with dual inhibitory activity against spla2 and mmps by using the following materials and methods : a family of peptides was synthesized based on the sequence of pip ( thwin et al . 2000 ; genbank accession nos . aaf73945 , ax175043 , cac43861 , cac43862 , cac43863 ) using the solid phase method with 9 - fluorenylmethoxy carbonyl chemistry . the peptides were purified by successive chromatography with gel filtration and reverse phase high pressure liquid chromatography to more than 95 % purity with yields between 10 - 30 %. the sequences were validated by matrix - assisted laser desorption / ionization time - of - flight mass spectrometry ( maldi - tof - ms ). this was carried out as described previously [ 10 ]. in brief , [ 3 h ] arachidonate - labeled e . coli membranes ( 0 . 005 mci / ml ; 5 . 8 μci / μmol , nen ) were used as substrate , and 100 mm tris - hcl — 25 mm cacl 2 , ph 7 . 5 solution as an assay buffer . the reaction mixture contained 20 μl of substrate , 0 . 85 pmole of purified human synovial spla 2 ( human type iia spla 2 , cayman chemicals , usa ), and an increasing concentrations of the family of synthetic peptides derived from the sequence of pip . the final volume of the reaction mixture was adjusted to 250 μl with an assay buffer . after incubation of the mixture ( 37 ° c ., 1 h ) and termination of the reaction with 750 μl of chilled pbs containing 1 % bsa , microfuge tubes containing the samples were centrifuged ( 10 , 000 g at 4 ° c . for 15 min ), and 500 μl aliquots of the supernatant taken to measure the amount of [ 3 h ]- labeled arachidonate released from the e . coli membrane using liquid scintillation counting ( multipurpose scintillation counter ls 6500 ; beckman , usa ). as controls , spla 2 was pre - incubated with the assay buffer alone . all samples , including controls , were performed in triplicate and plotted as the percentage inhibition relative to negative controls . ic 50 was determined by pre - incubating varying concentrations ( 1 - 250 μm ) of inhibitors in a constant volume , against a constant amount of enzyme as described earlier . a sigmoid dose response curve was generated to allow calculation of the ic 50 values . all samples were performed in triplicate . results were analyzed by nonlinear regression with graphpad prism ( version 2 . 01 ) and expressed as the percentage of inhibition relative to control values . gelatin zymography : human synovial fluid , purified human recombinant mmp - 2 and - 9 or apma - activated concentrated culture supernatant samples ( 10 μl ) was mixed ( 1 : 1 ) with laemmli sample buffer , loaded on 4 % acrylamide stacking gel and electrophoresed ( 120 v ) at 4 ° c . on a 10 % acrylamide - resolving minigel containing 0 . 1 % gelatin . after electrophoresis for 1 h , the gels were soaked in 2 . 5 % triton x - 100 on a shaker for 1 h , changing the solution after 30 min , to eliminate sds . after overnight incubation in zymogen activation buffer [ 50 mm tris - hcl , ph 7 . 5 , containing 10 mm cacl 2 and 5 mm zncl 2 ] at 37 ° c ., the gels were rinsed in distilled water , and stained for 3 hr with bromphenol blue , after which clear bands of digested gelatin were clearly visible . the gels were briefly rinsed in distilled water and scanned by the bio - rad gs - 710 calibrated imaging densitometer . collagenase activity assay : using collagen thin film as the substrate , a quantitative method [ 21 ] was established to measure the enzymatic activity of the mmps ( principally collagenase ). collagen type ii ( sigma ) solution ( 4 μl , 3 mg / ml in 0 . 012 n hcl ) was added to each well of 96 - well plates and precipitated by slowly increasing the ph with 4 μl of h 2 o and 8 μl of 0 . 012 n naoh . the plate was dried at 23 ° c . for 24 hr , washed with h 2 o , and dried again at 23 ° c ., resulting in a thin film containing 12 μg of collagen that was tightly bound to the bottom of the wells . enzymatic reactions of the collagenase i ( with and without peptide inhibitors ) against the collagen films were carried out in quadruplicate at 24 ° c . for 16 h . the digested portion of the film was washed away , while the undigested portion was fixed for 3 h with 0 . 1 % coomassie brilliant blue r in 10 % acetic acid and 45 % methanol , and the wells washed and dried . the undigested film was then dissolved in 100 μl of ethanol for 20 min to obtain a uniform colour distribution , and the od of each well read with a molecular devices emax precision microplate reader at 595 nm . inhibition of mmp - 1 enzymatic activity : enzyme inhibition by peptide analogues was assessed by using mmp - 1 colorimetric assay kit for drug discovery ( biomol ak - 404 quantizyme assay system ). briefly , human recombinant mmp - 1 catalytic domain in each well ( 15 . 3 u , 20 μl ) was allowed to react either with the candidate peptide ( test inhibitor ) dissolved in dmso ( final 0 . 5 % dmso in assay buffer ), or with the assay buffer alone ( control ) for 30 min at 37 ° c . immediately following incubation , the reaction was initiated by adding the thiopeptide substrate solution in assay buffer ( 10 μl of 100 μm ac - plg -[ 2 - mercapto - 4 - methyl - pentanoyl ]- lg - oc 2 h 5 ) into each well , and the absorbance at 412 nm continuously monitored for 10 min . 1 mm ellman &# 39 ; s reagent [ 5 , 5 ′- dithiobis ( 2 - nitrobenzoic acid ] in assay buffer ( 50 mm hepes , 10 mm cacl2 , 0 . 05 % brij - 35 ) was used as a colour developer in this assay system . the reaction velocity ( v ) was determined from the slope of the od versus time ( min ) plot for each test inhibitor using the linear regression analysis ( graph pad prism , version 2 ). inhibition (%) was then calculated using the equation , [ 100 ]−[( v inhibitor / control )× 100 ]. flsc culture and inhibition of extracellular mmp activity : all experiments were performed on hig - 82 rabbit fibroblast - like synoviocytes ( flscs ) obtained from american type culture collection ( manassas , va . ; catalogue no . crl - 1832 ). flscs were grown in ham &# 39 ; s f - 12 medium containing 10 % fetal bovine serum ( fbs ) and 1 % penicillin / streptomycin , as recommended by the supplier . for experimental purposes , flscs were seeded into 12 - well plates , grown to near confluence , and then serum starved ( ham &# 39 ; s f - 12 without fbs or penicillin / streptomycin ) for 16 h . after serum starvation of flscs , supernatants were removed and replaced with fresh ham &# 39 ; s f - 12 medium for 60 min at 37 ° c ./ 5 % co 2 before incubation for 2 h with various concentrations ( 1 - 10 μm ) of test inhibitor peptides : p - nt . ii , pip [ 59 - 67 ] monomer and dimer . flscs were then stimulated for 24 h with il - 1β ( 10 ng / ml ), and the supernatants concentrated by centrifugation in centricon centrifugal filter devices ( m . w . cutoff 30 , 000 ) for 35 min at 5000 rpm at 4 ° c . after activation with apma ( 1 mm final concentration ), the samples were measured for mmp - 1 activity using biomol ak - 404 quantizyme assay system , and for mmp - 2 and - 9 activities by gelatin zymography . cell viability assays : xtt ( sodium 3 ′-[ phenyl amine carboxyl )- 3 , 4 - tetrazolium ]- bis ( 4 - methoxy - nitro ) benzene sulfonic acid hydrate ) cell proliferation kit ii ( roche applied science ) was used to assess the possible cytotoxic effect of the peptides on the rabbit flscs . rna isolation of cultured flscs : flscs were grown to 80 % confluence on 12 - well culture dishes and starved in a serum - free medium with 5 μm pip [ 59 - 67 ] dimer for 2 h . the cells were then stimulated with 5 ng / ml il - 1β ( recombinant human , chemicon , usa .) for 24 h , washed with cold pbs and total rna isolated from peptide - treated ( n = 3 ) and untreated ( n = 3 ) flscs using rneasy ® mini kit . the rna samples were subsequently treated with rnase - free dnase - i at room temperature for 20 min and stored at − 80 ° c . until use . the quality and quantity of extracted rna was determined by spectrophotometry ( bio - rad , usa ). all rna samples used for qrt - pcr experiments were of highest purity with a 260 / a 280 ratios of 1 . 9 - 2 . 1 . the integrity and relative contamination with residual genomic dna was assessed by 1 % denaturing agarose gel electrophoresis . quantitative real time rt - pcr ( lightcycler ): quantitative fluorescence - based real - time pcr was carried out according to the manufacturer &# 39 ; s instructions using the lightcycler system with lightcycler faststart dna master plus sybr green i kit ( roche diagnostics , germany ) as an independent method for assessing relative gene expression . reverse transcription ( rt ) of rna and qrt - pcr were performed as follows : single stranded cdna was generated from 3 μg of total rna , 200 μm nucleotides , 500 units superscript ii reverse transcriptase ( invitrogen ) and 1 . 5 μm oligo ( dt ) 15 primers in 50 μl reactions . reverse transcription was stopped after 1 hr by heating to 95 ° c . for 5 min . the sense and anti - sense primers used for specific amplification and conditions for pcr cycles are shown in table 1 . the primers were designed by primer3 software : http :// bioinformatics . weizmann . ac . il / cgi - bin / primer / primer3 . cgi and subsequently checked for specificity using blast : http :// www . ncbi . nlm . nih . gov / genome / seq / hsblast . html . all primers and probes were synthesized by 1 st base pvt . ltd ., singapore . the expression of gapdh was used as an internal calibrator for equal rna loading and to normalize relative expression data for all other genes analyzed . the copy ratio of each analyzed cdna was determined as the mean of 3 experiments . the real - time quantitative rt - pcr data were quantified using relative quantification ( 2 − δδc t ) method as described [ livak , k . j , and schmittgen , t . d . ( 2001 ) analysis of relative gene expression data using real - time quantitative pcr and the 2 (- delta delta c ( t )) method [ 22 ]. based on in vitro inhibition assays against a purified human synovial spla 2 ( cayman , usa ), p - nt . ii analogue , pip [ 59 - 67 ] monomer , was identified as the shortest peptide possessing strongest spla 2 - inhibitory activity ( ic 50 3 . 8 μm ). displacing asp ( d ) with ala ( a ), ser ( s ) or glu ( e ) did not significantly enhance the inhibitory potency . however , a homodimer of pip [ 59 - 67 ] resulted in significant enhancement in spla 2 inhibitory activity ( ic 50 1 . 19 μm ). the results ( table 1 ) indicate that the dimerized peptide , pip [ 59 - 67 ] dimer , has the strongest inhibitory activity against the purified human synovial spla2 , while the other mutants are relatively less potent . the instability index shown in table 2 indicates that the peptide mutant d60 , 65e - pip [ 59 - 67 ] monomer may not be as stable as the other mutants , pip [ 59 - 67 ] monomer or the dimer , because of the former having an index greater than 40 . both the dimers of pip [ 59 - 67 ] and pip [ 57 - 67 ] appear to be more stable based on their instability indices ( i . e ., 6 . 46 and 3 . 2 , respectively ), but pip [ 57 - 67 ] dimer has a shorter half life compared to that of pip [ 59 - 67 ] dimer , and as a result pip [ 59 - 67 ] dimer may be the inhibitor of choice for therapeutic purposes . since the gelatinases ( mmp - 2 and mmp - 9 ) play an important role in inflammation , we first tested for inhibition of gelatinase activity using gelatin zymography ( fig1 a ). zymogram gels were scanned by the gs - 710 calibrated imaging densitometer ( bio rad , usa ). data representing mean ± sd ( n = 3 ) are not significantly different from control ( p & lt ; 0 . 05 ) using student &# 39 ; s t test ( fig1 b ). since the results indicate that inhibition of the peptides against the gelatinase activity is insignificant , the peptides were examined for inhibition against collagenase activity using purified collagenase i ( sigma ) and purified collagen type ii ( sigma ), in a collagen film assay with some modifications . [ note : as ic 50 values were calculated based on the results of our assay system using 3 h - labelled e coli membrane as substrate , they can vary depending on the type of method used for measuring the pla 2 activity . using a chromogenic isolated enzyme assay , ly315920 , the first potent and selective spla 2 inhibitor developed by eli lilly research laboratories , usa , inhibited spla2 activity with an ic 50 of 9 nm [ 23 ], whereas in our assay system , its ic50 was calculated as 2 . 29 μm ]. the results of collagenase i ( mmp - 1 ) inhibition assays ( table 3 ) indicate that the dimerized peptide mutant ( pip [ 59 - 67 ] dimer ) is the most potent inhibitor amongst the family of analogues examined for mmp - inhibitory activity . the half maximal inhibition concentration , ic50 ( mean ± sd ) was calculated as 8 . 43 ± 1 . 17 and 7 . 75 ± 1 . 11 μm , respectively , for p - nt . ii and pip [ 59 - 67 ] dimer ( fig2 ). specific test was subsequently used for further confirming the inhibitory activity of the most potent peptide ( pip [ 59 - 67 ] dimer ) against human recombinant mmp - 1 in comparison with that of the other mutant peptides . inhibition of human recombinant mmp - 1 enzyme activity by various peptide mutants is shown in fig3 . using the linear regression analysis , the reaction velocity ( v ) was determined from the slope of the od versus time ( min ) plot for each test inhibitor ( fig3 a ). mmp - 1 selective inhibitor nngh was included in the assay as a positive control . among those peptide analogues having stronger anti - collagenase i activity ( table 3b ), pip [ 59 - 67 ] dimer exhibited the strongest inhibition against the recombinant human purified mmp - 1 activity ( fig3 b , table 4 ), thus confirming the results obtained with the initial screening assays using collagen thin film as a substrate and collagenase 1 as an enzyme . since matrix metalloproteinases ( mmps ) play key roles in matrix remodeling processes that are essential for tumor growth and invasion [ 24 ], we examined the peptide candidate pip [ 59 - 67 ] dimer for inhibition against the activity of gelatinases ( mmp - 2 and mmp - 9 ) secreted by the rabbit synovial fibroblast cells upon stimulation by the cytokine il - 1β . gelatin zymography of the concentrated supernatants from the culture showed that increasing doses of pip [ 59 - 67 ] dimer ( 1 - 10 μm ) failed to inhibit the activity of mmp - 2 and mmp - 9 ( fig4 b ). further densitometric analysis of the zymograms indicated that the activity of secreted mmp - 2 and mmp - 9 in culture media was not significantly reduced by treatment with various doses ( 1 - 10 μm ) of pip [ 59 - 67 ] dimer as compared to untreated control fibroblast cells . these data clearly demonstrate that pip [ 59 - 67 ] dimer does not reduce the gelatinolytic activities of secreted mmp - 2 and mmp - 9 in treated fibroblast cells . in order to examine the potential suppression of spla2 and mmp gene expression by the candidate peptide , we used qrt - pcr for assessing relative mrna expression levels of il - 1β induced rabbit synovial fibroblasts in the presence and absence of the inhibitor peptide dimer . when the il - 1β - induced fibroblasts cells were treated with 5 μm of the peptide , the levels of the ( mmp - 1 , - 2 , - 9 , timp - 1 and spla2 ) gene transcripts were significantly suppressed ( fig5 ). since the levels of secreted mmp - 2 and mmp - 9 activity as measured by gelatin zymography are not significantly reduced ( fig4 b ), pip [ 59 - 67 ] dimer does not appear to directly inhibit secreted mmp - 2 or mmp - 9 activity . moreover , when the samples of human synovial fluid containing mmp - 2 and - 9 ( fig1 ) or the secreted mmp - 2 and - 9 in cultured synovial fibroblasts ( fig4 a ) were co - incubated in vitro with the peptides for 1 h and subjected to gelatin zymography , the results indicate that the inhibition of mmp - 2 and - 9 gelatinase activities by the peptide dimer is insignificant . from these results , it is evident that the peptide dimer acts against the gelatinases ( mmp - 2 and mmp - 9 ) mainly through the downregulation of the expression of genes encoding for mmp - 2 and mmp - 9 , rather than directly inhibiting the enzymatic activity of the gelatinases secreted by the fibroblasts ( fig5 ). in contrast , this peptide acts against collagenase 1 ( mmp - 1 ) through suppression of its gene expression ( fig5 ) and by directly inhibiting its enzyme activity ( table 4 ). these results provide strong in vitro evidence for control and modulation of enzyme activity and transcription of both mmp - 1 and spla 2 . since the initial cleavage of collagen by collagenase ( mmp - 1 ) is the critical irreversible step leading to the abnormalities observed in the disease state , this invention provides new therapeutic options in the treatment of arthritis and cancer . it will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention . 1 . murakami m , kudo i . ( 2002 ) phospholipase a2 . j . biochem . ( tokyo ). 131 , 285 - 92 . 2 . cirino g . et al . ( 1994 ) recombinant secreted nonpancreatic phospholipase a2 induces a synovitis - like inflammation in the rat air pouch . j rheumatol . 21 , 824 - 29 . 3 . jamal o s . et al . ( 1998 ) increased expression of human type iia secretory phospholipase a2 antigen in arthritic synovium . ann rheum dis . 57 , 550 - 58 . 4 . lin m k . et al . ( 1998 ) induction of secretory phospholipase a2 confirms the systemic inflammatory nature of adjuvant arthritis . inflammation 22 , 161 - 73 . 5 . jiang j , et al . ( 2002 ) expression of group iia secretory phospholipase a2 is elevated in prostatic intraepithelial neoplasia and adenocarcinoma . am j pathol . 160 ( 2 ): 667 - 71 . 6 . graff j r et al . ( 2001 ) expression of group iia secretory phospholipase a2 increases with prostate tumor grade . clin cancer res . 7 ( 12 ): 3857 - 61 . 7 . xu x c . ( 2002 ) cox - 2 inhibitors in cancer treatment and prevention , a recent development . anticancer drugs . 13 ( 2 ): 127 - 37 . review . 8 . gopalakrishnakone p ., thwin m m . et al . ( 2002 ) novel therapeutic and prophylactic agents and methods of using same . u . s . patent application ser . no . 10 / 163 , 499 ( us filing date : 7 jun . 2002 ). 9 . thwin m m et al . ( 2000 ) recombinant antitoxic and antiinflammatory factor from the nonvenomous snake python reticulatus : phospholipase a2 inhibition and venom neutralizing potential . biochemistry . 39 ( 31 ): 9604 - 11 . 10 . thwin m m et al . ( 2002 ) functional site of endogenous phospholipase a2 inhibitor from python serum . eur j biochem . 269 ( 2 ): 719 - 27 . 11 . thwin m m et al . ( 2004 ) effect of phospholipase a2 inhibitory peptide on inflammatory arthritis in a tnf transgenic mouse model : a time - course ultrastructural study . arthritis res ther . 6 ( 3 ): r282 - 94 . epub apr . 28 , 2004 . 12 . thwin m m et al . ( 2003 ) secretory phospholipase a2 activity in the normal and kainate injected rat brain , and inhibition by a peptide derived from python serum . exp brain res . 150 ( 4 ): 427 - 33 . epub apr . 18 , 2003 . 13 . gopalakrishnakone p , thwin m m , ong w y , sato k . ( 2004 ) phospholipase a2 - inhibitory peptide with anti - arthritic and neuroprotective activities . u . s . patent application ser . no . 10 / 836 , 825 intro ref . : gopal p 08 - us , filing date : 30 apr . 2004 . 14 . clark i m . et al . ( 2000 ) matrix metalloproteinase inhibitors in the treatment of arthritis . curr opin anti - inflammatory immunomodulatory invest drugs 2 , 16 - 25 . 15 . konttinen y t . et al . ( 1999 ) analysis of 16 different matrix metalloproteinases ( mmp - 1 to mmp - 16 ) in the synovial membrane : different profiles in trauma and rheumatoid arthritis . ann rheum dis . 58 , 691 - 97 . 16 . borkakoti n ( 2004 ) matrix metalloprotease inhibitors : design from structure . biochem . soc . trans . 32 , 17 - 20 . 17 . pardo a , selman m . ( 2005 ) mmp - 1 : the elder of the family . review . int j biochem cell biol . 37 ( 2 ), 283 - 8 . 18 . mook o r . et al . ( 2004 ) the role of gelatinases in colorectal cancer progression and metastasis . review . biochim biophys acta . 1705 ( 2 ), 69 - 89 . 19 . borkakoti . n . ( 2004 ) matrix metalloprotease inhibitors : design from structure . biochemical society transactions volume 32 , part 1 . 20 . bachmeier b e et al . ( 2005 ) matrix metalloproteinases in cancer : comparison of known and novel aspects of their inhibition as a therapeutic approach . expert rev anticancer ther . 5 ( 1 ), 149 - 63 . 21 . kolomytkin o v , et al . ( 2002 ) il - 1beta - induced production of metalloproteinases by synovial cells depends on gap junction conductance . j physiol cell physiol 282 ( 6 ): c1254 - c1260 . 22 . livak k j , schmittgen t d . ( 2001 ) analysis of relative gene expression data using real - time quantitative pcr and the 2 (- delta delta c ( t )) method . methods 25 ( 4 ): 402 - 8 . 23 . snyder d w . et al . ( 1999 ) pharmacology of ly315920 / s - 5920 , [[ 3 -( aminooxoacetyl )- 2 - ethyl - 1 -( phenylmethyl )- 1h - indol - 4 - yl ] oxy ] acetate , a potent and selective secretory phospholipase a2 inhibitor : a new class of anti - inflammatory drugs , spi . j pharmacol exp ther . 288 ( 3 ): 1117 - 24 . 24 . johansson n , ahonen m , kahari v m . ( 2000 ) matrix metalloproteinases in tumor invasion . cell mol life sci . 57 ( 1 ): 5 - 15 . review . 25 . bazan n g , colangelo v , lukiw w j . 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the present invention provides a new 18 - residue homodimerized peptide , designated pip dimer , which is a mutant of the optimized anti - inflammatory peptide p - nt . ii , the patent for which has recently been filed . p - nt . ii has the potential to modulate both the inflammatory and bone damaging components of rheumatoid arthritis , and was originally designed on the basis of the primary structure of the anti - inflammatory protein termed ‘ phospholipase inhibitor from python ’ . using solid phase chemistry , variants of p - nt . ii were designed and examined for inhibitory activity against secretory phospholipase a2 , a key enzyme involved in the inflammatory pathway , and matrix metalloproteinases that are involved in the remodeling and degradation of the extracellular matrix in rheumatoid arthritis and cancer . among the family of mutants tested , the dimerized peptide was found to be the most potent inhibitor against spla2 as well as the human recombinant mmp - 1 . this invention provides the utility of the peptide analogue pip dimer as a potential therapeutic agent for modulation of inflammatory diseases such as rheumatoid arthritis , and cancer . this invention relates to all the polypeptide analogues and polynucleotides , and to the use of those polypeptides and polynucleotides , their synthetic chemical analogues or variants that inhibit activity and synthesis of spla2 and mmps , in the diagnosis , study , prevention and treatment of rheumatoid arthritis and / or cancer .