Patent Application: US-10197399-A

Abstract:
a process for determining complement activation due to contact between a biomaterial and a complement system , by incubating in vitro the biomaterial with the complement system and determining the formation of a complement convertase by using a substrate of the complement convertase and detecting substrate cleavage . the biomaterial after said incubation with the complement system may be separated therefrom and formation of a complement convertase may be determined with the separated biomaterial , the separated complement system , or both . the complement convertase may be factor b convertase , c3 convertase or c5 convertase , and the substrate may be a labeled oligopeptide comprising an amino acid sequence corresponding to the cleavage site of the complement convertase . suitable labels are dyes , fluorochromes , radioactive atoms or groups , and enzymes . either classical or alternative pathway complement activation , or both , are determined . the complement system may be a non - clotting derivative of blood , blood plasma or blood serum , including fibrinogen depleted forms , anticoagulated forms , and derivatives containing thrombin inhibitor . complement convertase substrates suitable for use in the process are also disclosed .

Description:
the subject invention provides an enzymatic technique to determine convertase activity towards synthetic peptides , which results in release of a labelling substance , such as a chromogenic , fluorescent , or radioactive label . although the complement system is activated through enzymatic cleavage of factors b , c3 and c5 by convertases , these enzymes have not yet been considered as useful markers for complement activation due to their intrinsic lability , short half life , but most of all due to their aspecific nature ( 39 - 42 ). therefore it was a surprise to observe remaining activity of these convertases on the surface of materials , as well as in the fluid phase , which could be ascribed to complement convertase activity by using inhibitors of other potential substrate cleaving enzymes . the amino acid sequence of the natural substrates for the complement convertases ( factors b , c3 , c5 ) is known . also the cleavage site of factor b by the factor b cleaving enzyme c3bd , of c3 by the c3convertases ( c4b2a and c3bbbp ) and of c5 by the c5 convertase ( c3bbb3bp ) is known ( 43 , 44 ). thus an artificial substrate resembling these structures could be used for these enzymatic processes . these small peptides substrates can be labeled in different ways ( 45 , 46 ) to measure the cleavage by the corresponding convertase activity . a suitable labeled peptide substrate for c3 - convertase would be leu - ala - arg - pna , for c5 - convertase leu - gly - arg - pna , and for factor b - convertase gly - lys - arg - pna . the nature of the label is not critical , and the amino group of the n - terminal amino acid may be free ( h - form ) or blocked ( e . g . by boc or bz ). the new enzymatic technique is based on the convertases formed on the surface of the test material and detects these convertases bound to the surface and / or free in plasma . specifically for the bound complement proteins this technique is most suitable , in contrast to the hemolytic and immunologic techniques . the new enzymatic technique shares the advantage of the hemolytic techniques in being relevant to the real activity of complement , since it measures important steps in the cascade . also the new technique can reach the discriminating sensitivity of the immunologic techniques , since the existing convertase activity in plasma and on biomaterials is low , while it increases 2 to 5 - fold after incubation of the biomaterial in plasma . since the convertase remains active during substrate conversion , the development of the colour can be extended until discrimination between the test samples is possible . another important aspect of the complement measurement on biomaterials with the presented in vitro technique is that substrate cleavage has been shown for human as well as various animal species ( 38 , 39 ). peptide substrates have been described in the past for the measurement of proteases . the synthesis of labeled substrates has been described ( 43 ). most of the activated plasma proteases are highly specific when compared with digestive enzymes , such as trypsin or chymotrypsin . in some cases , the plasma proteases cleave only one or two peptide bonds in their natural substrates . this high enzyme specificity apparently is due to the recognition of a specific amino acid sequence near the sensitive bond or a specific conformation of these amino acids near the sensitive bond . a number of studies of the effect of the various blood coagulation factors and complement enzymes have been published . substrates containing 4 - nitroaniline and amides of 7 - amino - 4 - methylcoumarin have been particularly useful . none of these substrates , however , are completely specific . other problems are the low rate of enzymatic hydrolysis and interference with other plasma proteins , such as albumin . these disadvantages have made these substrate techniques for detection of complement activation in vivo difficult to interprete , since complement activation in vivo is accompanied by activation of other blood cascade systems with some effect on substrate conversion . the methodology described in this document enables a high specificity , mainly due to the fact that the biocompatibility will be determined in vitro . in contrast to the measurements in blood samples from patients , during the in vitro measurement of biocompatibility , activation of cascades other than complement can be suppressed with commercially available inhibitors , allowing the process to be controlled ( 38 ). moreover , complement convertases have the specific property to bind to the target biomaterial surface . since the technique described herein is based on incubation of the test material in plasma followed by separation and washing of the test material , bound convertases on the material surface are separated from plasma prior to substrate conversion . the present invention provides a technique to determine one important aspect of the biocompatibility from biomaterials , being the extent of complement activation . the present invention allows in vitro determination of complement activation by incubating biological fluids , blood or blood products with biomaterials , allowing the complement system to be activated and complement convertases to bind to the biomaterials surface . subsequently , the biomaterials are washed to remove unbound blood proteins and cells and then the biomaterials are incubated in medium containing specific substrate , allowing cleavage of this substrate by complement convertases . this cleavage can then be measured due to release of colour , fluorochromes , radioactive label , etc . in general , substrates in the present invention are labeled tripeptides , although larger structures are not limited hereby . the substrates have or comprise an amino acid sequence resembling the cleavage site of the natural substrate for the complement convertase or any other amino acid sequence with low km and high specificity . the concentration of the substrate in the test procedure will normally be in the order of μm in order to have no substrate limitation during the test procedure . blood or blood plasma must be anticoagulated during incubation with the biomaterials . some anticoagulants , such as the ca ++ depleting agents citrate and edta , also affect the complement system . mg ++ may be used in these situations to allow alternative pathway complement activation . more preferably , serum or fibrinogen depleted plasma or blood / plasma with specific thrombin inhibitors is used to prevent clotting during incubation with biomaterials . a technique described in the present invention may be used in the screening of biomaterials to select the proper ones for construction of medical devices . it may also be used by test laboratories for blood biocompatibility testing . further it may be used for research purposes during clinical use of medical devices or inversily to test the ability of an unknown blood sample to react with biomaterials . the present invention also provides conditions to ensure specificity of the technique for complement convertases rather than other enzymes to cleave the substrates , by introduction of inhibitors for other enzymes during substrate conversion . the present invention comprises measurement of complement activation both on the material surface and in the fluid phase . dependent on the material surface , convertases are released in the biological fluid . the present invention provides measurement of complement activation in human or animal biological fluid . the present invention allows discrimination between activation of the alternative or classical pathway of the complement system , by employing ca ++ or mg ++ in the biological fluid during incubation with biomaterials , by means of heat treatment of the biological fluid or by inhibition of one of these pathways e . g . with antibodies . the present invention enables measurement of complement convertase binding to biomaterials during clinical use of these biomaterials by incubation of these used and washed biomaterials in the substrate medium . the present invention allows measurement of convertase activity with substrates labeled with chromophores or fluorogenes or other suitable markers which are released during peptide cleavage of substrate by the complement convertase . the present invention provides the possibility to characterize complement activation by any device , independent of three - dimensional structure or size . if needed the incubation time with substrate can be adjusted to the number of convertases formed during incubation which might be dependent on the surface area and material characteristics of the test material . the present invention can also be used to detect any deficiencies of the complement system to respond to standardized material with well defined characteristics with respect to complement activation . these standardized materials can be activators of the alternative or classical complement pathway . the advantage of this technique over the existing hemolytic or immunologic techniques is that screening for complement deficiency is possible without specialized laboratory techniques . the present invention can even be developed into a bedside monitoring of complement . the present invention can be performed at varying temperatures , such as at room temperature or at 37 ° c ., the main effect is the reaction speed . the present invention allows a great extent of standardization for both the measurement of biocompatibility from biomaterials with pooled normal serum and for the measurement of complement response from patients plasma to standard biomaterials . for the biocompatibility measurement one batch of serum can be stored deep frozen in aliquots , for complement testing one batch of uniform material can be used . the invention will be illustrated by the following examples which merely serve to illustrate the invention and not to limit it to the details shown . these examples demonstrate that we have evaluated a technique to detect the activation of the complement system , when a biomaterial is in contact with blood plasma . in our experiments it is shown that the biomaterial binds complement convertase and releases these convertases in plasma , which results in conversion of specific substrate . so activation of the complement system by biomaterials can now be measured directly in the fluid as well as on the surface . in these experiments we have also shown that by means of this technique a classification can be made between different biomaterials with regard to the complement - biocompatibility . therefore this test may become a powerful tool to access this biocompatibility of biomaterials in the selection and testing of materials to be used in medical devices . human blood was collected from a healthy volunteer by vena puncture . blood was mixed immediately with sodium citrate ( 0 . 316 %, final concentration ) to prevent clotting . then blood was centrifuged ( 1100 xg ) to obtain separation between plasma and blood cells . in our experiments we used this citrated human plasma after treatment with reptilase to coagulate fibrinogen . this plasma , deficient of fibrinogen ( def ppp ), contains the other coagulation components and can be mixed with ca ++ containing buffer to optimize complement activation . this def ppp was stored in aliquots at - 80 ° c . we diluted this in 50 mm tris buffer + 33 mm cacl 2 ( ph 7 . 4 ) to a percentage of 20 % prior to use . the biomaterial pdms ( polydimethyl - siloxane , silicon rubber ) was cut into 1 cm 2 pieces . for cleaning the biomaterial it was incubated at first in 70 % ethanol , and then rinsed in 0 . 9 % nacl . during the experiment the pieces of biomaterials were incubated in 500 μl diluted def ppp during 0 , 10 , 30 , or 60 min at room temperature . after incubation , the biomaterials were rinsed 3 times with saline . then the biomaterials were incubated in boc - leu - gly - arg - pna , diluted in tris buffer + 33 mm cacl 2 , during 60 hours at room temperature in the dark . simultaneously , also 100 μl def ppp , in which the biomaterials had been incubated , were mixed with 100 μl of h - leu - gly - arg - pna or bz - leu - gly - arg - pna , diluted in tris buffer , supplemented with 33 mm cacl 2 . after these 60 hours the od was measured at 405 nm in a spectrophotometer ( microplate reader 3550 uv ; biorad , richmond , calif ., usa ). these experiments showed colour formation releasing from pdms with substrate boc - leu - gly - arg - pna and in the incubated def ppp with substrates h - leu - gly - arg - pna and bz - leu - gly - arg - pna ( fig3 a ). then , the effect of the incubation time of the biomaterial in plasma was investigated . it is shown that substrate conversion , expressed as od 405 nm , increases when the biomaterial is for a longer time in contact with plasma ( fig3 b ), indicating more c5 - convertase formation . since in this experiment after an incubation time of 30 min there was sufficient conversion , this incubation time was chosen in further experiments as a standard time . to verify the specificity of complement convertase activity , pdms was incubated with diluted def ppp supplemented with ca ++ ( allowing convertases to be formed ) or with edta ( preventing convertase formation ). this experiment showed that edta completely prevented convertase formation , indicating some specificity of substrate for complement activation . in a second experiment the incubated def ppp was incubated in the presence of ca ++ as described above and 100 μl def ppp was collected after 30 minutes . then this collected def ppp was incubated with h - leu - gly - arg - pna and bz - leu - gly - arg - pna in the presence of ca ++ or in the absence of ca ++ with edta . the formed convertases remained active in edta , while additional convertase activity was formed in the presence of ca ++ during the incubation of activated def ppp with substrate h - leu - gly - arg - pna ( fig4 a ). this neoformation of convertases can be considered as an artifact . thus the second incubation of def ppp with substrate must be performed in ca ++ / mg ++ chelating medium , like edta . substrate bz - leu - gly - arg - pna is considered non - specific for complement activation , since edta did not inhibit further cleavage of this substrate . a third test for specificity was the incubation of pdms with def ppp heated during 30 min at 56 ° c ., in order to eliminate factor b from the alternative complement pathway . under these conditions convertase formation was considerably reduced , indicating the importance of functional factor b from the alternative complement pathway in convertase formation by pdms ( fig4 b ). we have investigated the effect of different types of biomaterial , since discrimination between materials is one of the major goals of this technique . we used the materials : pdms , pe ( polyethylene ) and ptfe ( polytetrafluoroethylene ), which are all frequently used for medical devices . we have tested the effects of convertase formation in the solutions and onto the biomaterials . the materials pdms , pe and ptfe activate the complement system , indicated by conversion of boc - 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