Patent Application: US-93100901-A

Abstract:
a synthetic tetrapeptide having an amino acide sequence , glycine - proline - arginine - proline has pro - inflammatory effects on human fibroblastic cells , including synovial cells . an amide analog of gprp is ineffective in inducing , or effective in causing a loss of , pro - inflammatory effects .

Description:
the amino acid sequence of the pro - inflammatory peptide of the invention is gly - pro - arg - pro ( gprp ) ( seq id no : 2 ). this sequence is related to amino acids 17 through 20 of the a / alpha chains of fibrinogen ( fig1 ). amino acids 1 through 16 of the a / alpha chains are cleaved - off by thrombin or thrombin - like proteases in vivo and this now separated peptide is known as fibrinopeptide a . cleavage of fibrinopeptide a by thrombin is the initial cleavage in the formation of fibrin clots from its precursor , fibrinogen ( see fig2 ). this cleavage exposes new amino - termini of the a / alpha chains ( beginning with amino acid number 17 ) with the leading sequence gly - pro - arg . these newly exposed sequences , which are centrally located in a spatial model of fibrinogen have been referred to as “ knobs ”, and are able to bind to complementary “ holes ” in the gamma chains of the same or different fibrinogen molecules and thus facilitate the crosslinking of fibrin ( ogen ) into a meshwork , or clot ( see fig5 ). i believe that there exists another receptor - like “ hole ” on the surface of human synovial cells , through which the “ knobs ” can mediate pro - inflammatory effects . synthetic peptides modeled after these “ knobs ” are able to inhibit fibrin polymerization by covering up the “ holes ” on the gamma chains and are commercially available . the natural sequence of the new amino terminus following fibrinopeptide a cleavage is tripeptide consisting of gly - pro - arg or tetrapeptide comprising core sequence gly - pro - arg , and this peptide functions well as a polymerization inhibitor ( see page 1015 of laudano et al . reference , i . e ., reference 25 . it was found that the modified peptide gly - pro - arg - pro ( seq id no : 2 ) was an even more potent inhibitor of polymerization , although the reason why is not understood and is hypothesized to be due to steric constraints ( rigidity ) imposed by the terminal proline . many studies of fibrinogen polymerization make use of such synthetic peptides . i first used a commercial gprp to inhibit fibrin polymerization in studies of fibrin co - culture with endothelial cells and have published our results ( reference 19 ). i found gprp ( seq id no : 2 ) to be a good inhibitor of fibrin polymerization yet had no direct effects itself on endothelial cells . i also found gprp to have low to no effects on other cell types , such as foreskin fibroblasts . i derived several primary cell lines from the synovium of ra and osteoarthritis ( oa ) patients . i wanted to examine the effects , if any , of polymerized fibrin on synovial fibroblasts in vitro , to mimic the environment found in vivo in a rheumatoid joint . i was surprised to find that the inclusion of gprp ( seq id no : 2 ) along with fibrinogen ( to inhibit polymerization ) did not abrogate my initial findings of increased icam - 1 ( intercellular adhesion molecule - 1 ) expression ( caused by exposure to fibrin clots ) by the synovial fb . icam - 1 is considered to be such a key component in the inflammation of the rheumatoid joint , that ra therapeutic trials have been based on the inactivation of icam - 1 alone . indeed , i found that the gprp ( seq id no : 2 ) peptide alone was able to induce icam - 1 expression by the synovial fibroblasts as measured both by elisa and flow cytometry . by flow cytometry , gprp ( seq id no : 2 ) peptide increased icam - 1 expression in two ways : 1 ) increasing the percentage of cells expressing icam - 1 , and 2 ) increasing the amount of surface icam - 1 per cell . i conducted experiments with more than seven different ra fibroblast lines and obtained consistently increased expression of icam - 1 induced by gprp ( seq id no : 2 ) with all lines . my experience is that gprp is able to reliably induce the adhesion molecule icam - 1 about as well as any cytokine . the effects of gprp ( seq id no : 2 ) on icam - 1 induction are dose - dependent . a significant induction at 1 . 0 , 0 . 5 , and 0 . 1 mg / ml was observed . induction at 0 . 01 mg / ml was found not to be significant . table 1 illustrates an experiment using gprp ( seq id no : 2 ) to increase icam - 1 expression on different ra synovial cell lines assayed by flow cytometry , and compares those results with the failure of gprp ( seq id no : 2 ) to induce icam - 1 on human foreskin fibroblasts . table 2 illustrates an experiment using gprp ( seq id no : 2 ) to increase icam - 1 in different ra synovial lines assayed by elisa and compares those results with the failure of gprp ( seq id no : 2 ) to induce icam - 1 on human foreskin fibroblasts . i also assayed several lines for v - cam - 1 expression , and found that about 50 % of the lines tested upregulated v - cam - 1 expression in response to the peptide . i also found that vcam - 1 induction is not as robust as icam - 1 . i have confirmed an upregulation of functional adhesion molecules by gprp ( seq id no : 2 ) using a human in vitro t cell adhesion assay ( see table 5 below ). one consideration in evaluating the effects of any substance on my collection of ra synovial cell lines , is that the synovial tissues i used were collected sequentially as the patient surgeries were scheduled . therefore , i had no knowledge of the patients &# 39 ; disease history , or what medications they might have been taking or for how long . another effect of the peptide of the invention on synovial fb , is the induction of the chemotactic cytokines il - 8 and gro - alpha , each of which is intimately involved in the recruitment of lymphocytes into inflamed tissues . i used commercial elisa kits to assay cell culture supernatants of ra fibroblasts that were cultured for both 24 and 48 hours with or without peptide . ra line 1 increased its production of il - 8 12 . 6 - fold at twenty - four hours and fifteen - fold at forty - eight hours . ra line 2 behaved similarly with ten - fold increases at both twenty - four and forty - eight hours . for comparison , monocytes , considered to be excessive il - 8 secreters , increase il - 8 production approximately three - fold when stimulated with phytohemagglutinin ( pha ). it is believed that a technical error in the absorbance readings only of our gro - alpha assay may have resulted in a very significant underestimation of the amount of gro - alpha secreted in response to peptide . however , in a visual observation of developed elisa plates , the fold increase was at least as much as was seen for il - 8 and probably is greater . using foreskin fb as control cells , i accurately measured only a 1 . 2 fold increase in gro - alpha secretion in response to the peptide both at twenty - four and forty - eight hours . i believe that not only does gprp ( seq id no : 2 ) participate in the chemotactic recruitment of lymphocytes via induction of il - 8 and gro - alpha , but it gives the lymphocytes a place to “ park ” once they arrive via the simultaneous induction of adhesion molecules such as icam - 1 and vcam - 1 . table 6 presents our elisa results for the chemotactic cytokines il - 8 and gro - alpha . in another experiment using rt - pcr , it was found that synovial cells exposed to gprp ( seq id no : 2 ) had significantly more il - 6 gene expression than controls . il - 6 has been demonstrated to be both a chemoattractant and pro - inflammatory cytokine in ra . the gprp ( seq id no : 2 ) peptide of the invention is believed to induce gene expression of icam - 1 through its stimulation of the intracellular signal nf - kappab which regulates the intensity of gene transcription . nf - kappab activation is already well known to affect a broad array of immediate - early gene products , such as tnf , interleukins , chemokines and colony stimulating factors ; genes that are tightly regulated during inflammation and wound healing . inclusion of the specific inhibitor of nf - kappab , pyrrolidinedithio - carbamate ( pdtc ), stopped the entire increase in icam - 1 expression induced not only by interleukin - 1 ( as expected ), but also by the gprp ( seq id no : 2 ) peptide . pdtc is an anti - oxidant that prevents nf - kappab activation and its translocation to the nucleus ( reference 26 ). as nf - kappab is also known to be involved in il - 8 transcription , it is expected that pdtc would also inhibit the il - 8 secretion induced by gprp ( seq id no : 2 ). table 7 shows the inhibitory results of ptdc on the ability of gprp ( seq id no : 2 ) to induce icam - 1 . most modifications of the gprp ( seq id no : 2 ) sequence ( amino acid substitutions ) are not effective polymerization inhibitors . for example , changing gly1 to anything else destroys its inhibitory action as there appears to be no extra “ room ” for a side chain in the gamma chain “ hole ” into which it must fit . however , as noted below , amino acid pro4 functions differently . actually , pro4 is not essential for activity , as gly - pro - arg alone can inhibit fibrinogen polymerization , although inhibition is much more effective when pro4 is included . changing the carboxyl group at the end of pro4 to an amide group ( oh to nh2 ) has been found to be approximately three and one - half times more effective at inhibiting polymerization as the original h - gly - pro - arg - pro - oh ( seq id no : 2 ) ( reference 20 ). interestingly , when i substituted the amide analog of gprp ( seq id no : 2 ), it was totally ineffective at inducing icam - 1 in synovial fibroblasts . although it is not yet shown that the amide form of gprp ( seq id no : 2 ) still binds to the synovial cell receptor , it is unlikely that this change in the number four position would compromise binding ability . further research is contemplated in using the amide analog of the peptide of the invention to determine if other pro - inflammatory responses of the synovial fb are also prevented . table 8 contains data comparing the activities of gprp - oh ( seq id no : 2 ) and gprp - nh2 ( seq id no : 2 ). this table also contains data showing that synovial cell incubation with other , coagulation - related peptides ( fibrinopeptides a and b , and an amino acid sequence which prevents platelet aggregation by binding to the fibrinogen receptor ) have no effect on icam - 1 induction . table 9 illustrates an analysis of the binding of biotinylated amino acid sequence 17 - 28 of human fibrinogen aα chain ( sac [ kbtn ]) to ra synovial fibroblasts by flow cytometry . the biotinylated control peptide ( kree - seq id no : 3 ) represents corresponding sequences derived from the b / beta chain of fibrinogen following removal of fibrinopeptide b . these data suggest that unlabeled fibrin , unlabeled gprvverhqsac ( sac ) ( seq id no : 4 ) and unlabeled gprp ( seq id no : 2 ) can compete with biotin - labeled sac for binding to human synovial fibroblasts . this competition further suggests that the labeled peptide competitively binds to a specific cell surface receptor of synovial cells . thus , labeled peptide could facilitate the isolation , identification and characterization of its receptor , through standard immunological techniques such as immunohistochemistry , flow cytometry and immunoprecipitation . the data collected thus far leads me to believe that there exist on the surface of human synovial cells , a receptor that can be stimulated when fibrinogenesis is ocurring in the synovial joint . the ligand for this receptor is created when newly exposed amino acid sequences become available due to fibrinopeptide a release from the precursor fibrinogen . this newly exposed “ knob ” has two options : 1 ) it can bind to fibrinogen gamma chains thereby promoting cross - links and the formation of fibrin clots , or 2 ) given the right environment ( synovial cells bearing the correct receptor ) can also act as ligand for these cellular receptors . binding of the ligand stimulates intracellular signaling mechanisms some of which require nf - kappa b and results in the simultaneous expression and production of pro - inflammatory mediators , such as an array of adhesion molecules and chemotactic cytokines . further research is contemplated to determine what further activities synovial fb might produce under the influence of this ligand ( il - 1 , chemoattractant il - 16 , mcp - 1 , prostaglandin production , matrix metalloprotease production , and collagenase - 1 and cathepsin production , for example ). it is believed that in vivo , fibrin - induced activation of synovial fb results in the induction of a phenotypic “ pro - inflammatory fb ” leading to the recruitment , activation , attachment and retention of lymphocytes , all of which occur to a tremendous degree in the chronically inflamed rheumatoid joint . it is well known that the recruitment and attachment of lymphocytes to synovial fb results in further amplification of inflammation . most recent therapies directed at biologically alleviating inflammation in the rheumatoid joint have focused on inhibiting one or another facets of the inflammatory response , i . e ., using anti - icam - 1 monoclonals , monoclonal antibodies or engineered proteins directed against tnf , etc ., and are very costly . a recent review by lorenz , et . al ( reference 27 ) discusses the advantages and disadvantages of current and emerging ra therapies and describes further efforts to reduce inflammation via antibodies to specific cytokines . it is becoming apparent that studies targeting a single biological entity at a time are being replaced by studies in which they are combined with more traditional anti - inflammatory medications . also contained in this review is a discussion of emerging results for targeting the membrane urokinase - type plasminogen activator of human synoviocytes . my findings could advance such approaches . a double blind clinical trial of stanozolol ( enhances both systemic and intra - articular fibrinolytic activity ) in ra patients ( reference 29 ) resulted in clinical benefit , i . e ., decrease in erythrocyte sedimentation rate , improvement in articular index , decreased duration of morning stiffness , decrease in pain , and decreased plasma fibrinogen concentrations . it was believed that the clinical improvement likely could have been due to the induced reduction of synovial fibrin . it is believed that once we more fully understand the effects of the peptide of the invention on synovial fb , as compared with less specialized fb found elsewhere in the body , a modification thereof , including the amide form , could be used as a binding , but non - signalling ligand , which would specifically target synovial fibroblasts , and thereby prevent the natural ligand from provoking the fibroblasts towards the pro - inflammatory phenotype . since it is known that procoagulant activity occurs at a heightened level in a rheumatoid joint , this would represent an important approach to subduing the inflammatory component . it may also answer the question of exactly how fibrinogenesis promotes inflammation — a fact long known to be true , but its mechanism still unknown . i am not aware if the reported activities of all of the modified peptides that have been created in studies of fibrinogen polymerization , have any bearing on our observations . there is no evidence to suggest that the gamma chain “ hole ” is the same size or sequence as our putative receptor on synovial cells . modified peptides which reportedly do not bind the gamma chain “ hole ” may still bind to this receptor and any peptide derivatives ( previously known to inhibit fibrin polymerization or not ) would have to be re - tested using our cell system . looking in a new direction , extravascular fibrin deposition is also frequently observed in association with neoplastic tissues in vivo . many clinical and experimental findings ( references 21 - 23 ) support the hypothesis that fibrin facilitates tumor growth and metastasis , although the mechanisms are not yet known . in a recent study of fibrin deposition in head and neck tumors ( reference 24 ), there was evidence of in situ thrombin activation and fibrin formation , and it was noted that the fibrin deposition was almost exclusively localized to the connective tissue compartment immediately surrounding the tumors . in 10 / 25 laryngeal and 4 / 9 hypopharyngeal cancers , characteristic fibrin accumulation was seen around tumor cell nodules , at the interface of connective tissue and tumorous parenchyma . tumor cell clusters were observed embedded within connective tissue “ soaked with fibrin ”. fibrin was not detected in the histologically normal part of tissue surrounding the squamous cell carcinomas . we look to culture connective tissue fibroblasts originating from these areas surrounding the tumors with the expectation that gprp ( seq id no : 2 ) or other peptides like it may induce a release of factors from these specialized fb which in turn would have a “ feeder ”, or pro - cancerous effect on the tumor cells . the present invention also includes therapeutic or pharmaceutical compositions comprising a peptide or a peptide derivative of the invention in a form which can be combined with or in combination with a pharmaceutically acceptable carrier for any appropriate manner for administration , including , for example , oral , nasal , intravenous or intramuscular administration . appropriate dosages , duration and frequency of administration would be determined by known factors , such as the condition of the patient , the type and severity of the disease and the method of administration . the term “ carrier ” includes a diluent , adjuvant , excipient , or vehicle with which the peptide is administered . such pharmaceutical carriers can be sterile liquids , such as water and oils , including those of petroleum oil such as mineral oil , vegetable oil such as peanut oil , soybean oil , and sesame oil , animal oil , or oil of synthetic origin . saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers , particularly for injectable solutions . suitable pharmaceutical excipients include starch , glucose , lactose , sucrose , gelatin , malt , rice , flour , chalk , silica gel , sodium stearate , glycerol monostearate , talc , sodium chloride , dried skim milk , glycerol , propylene , glycol , water , ethanol and the like . the therapeutic compositions , if desired , can also contain minor amounts of wetting or emulsifying agents , or ph buffering agents . these compositions can take the form of solutions , suspensions , emulsion , tablets , capsules , powders , sustained - release formulations and the like . the composition can be formulated with traditional binders and carriers such as triglycerides . examples of suitable pharmaceutical carriers are described in “ remington &# 39 ; s pharmaceutical sciences ” by e . w . martin . such compositions contain a therapeutically effective amount of the therapeutic composition , together with a suitable amount of carrier so as to provide the form for proper administration to the patient . the formulation should suit the mode of administration . the composition may be formulated in accordance with routine procedures as a pharmaceutical composition adapted for local injection administration to human beings . typically , compositions for local injection administration are solutions in sterile isotonic aqueous buffer . where necessary , the composition may also include a solubilizing agent and a local anesthetic , such as lidocaine to ease pain at the site of the injection . generally , the ingredients are supplied either separately or mixed together in unit dosage form , for example , as a dry lyophilized powder or water free concentrate in a hermetically sealed container , such as an ampoule or sachette indicating the quantity of active agent . where the composition is administered by injection , an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration . the therapeutic or pharmaceutical compositions of the invention can be formulated as neutral or salt forms . pharmaceutically acceptable salts , include those formed with free amino groups , such as those derived from hydrochloric , phosphoric , acetic , oxalic , tartaric acids , etc ., and those formed with free carboxyl groups , such as those derived from sodium , potassium , ammonium , calcium , ferric hydroxides , isopropylamine , triethylamine , 2 - ethylamino ethanol , histidine , procaine , etc . the present invention also provides for the modification of the peptide or peptide derivatives such that it is more stable once administered to a subject , i . e ., once administered it has a longer time period of effectiveness as compared to unmodified peptide . such modifications are well known to those of skill in the art , e . g ., polyethylene glycol derivatization ( pegylation ), microencapsulation , etc . while this invention has been described as having preferred sequences , ranges , steps , materials , or designs , it is understood that it includes further modifications , variations , uses and / or adaptations thereof following in general the principle of the invention , and including such departures from the present disclosure as those come within the known or customary practice in the art to which the invention pertains , and as may be applied to the central features hereinbeforesefforth , and fall within the scope of the invention and of the limits of the appended claims . it is further understood that the present invention is not limited to the claims appended hereto . 1 . colvin r b , johnson r a , mihm m c , and dvorak h f . role of the clotting system in cell - 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