Patent Application: US-84866401-A

Abstract:
matrices that support cell adhesion and growth are disclosed that deliver low heparin - binding affinity growth factor protein peptides in a controlled manner . these matrices comprise covalently or non - covalently bound heparin or heparin - like polymers , which serve to sequester the low heparin - binding affinity growth factor protein peptides within the matrix . the controlled release of some low heparin - binding affinity growth factor or peptides thereof occurs by degradation of some matrix component or dissociation of the low heparin - binding affinity growth factor protein peptides from the bound heparin . this differs from many controlled delivery devices in that release is not controlled solely by diffusion , and the rate of release may therefore be regulated by altering the rate of degradation of the matrix component or the amount of heparin bound within the matrix . the controlled release of such low heparin - binding affinity growth factor proteins such as ngf - β , nt - 3 and bdnf , is demonstrated . the invention also identifies basic domains that can be utilized to identify other low heparin - binding affinity growth factor protein peptides useful in delivery as part of the matrices described herein .

Description:
the present invention provides compositions useful in promoting the controlled release of low affinity - heparin - binding growth factors having a low heparin - binding domain of about 8 to about 30 basic amino acids in length . the following examples are included to demonstrate preferred embodiments of the invention . it should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention , and thus can be considered to constitute preferred modes for its practice . however , those of skill in the art should , in light of the present disclosure , appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention . heparin - affinity chromatography is a method commonly used to determine the relative affinity of heparin - binding proteins . if a protein elutes at nacl concentrations near or below physiological level ( approximately 140 mm ) it is not to be considered “ heparin - binding ” for purposes of the description of the present invention . this is because the growth factors would dissociate rapidly from heparin in vivo . the relative affinity for heparin of proteins was determined by heparin - affinity chromatography , using a tsk - gel heparin - 5pw ( 7 . 5 cm × 7 . 5 mm id ) column ( tosohass , stuttgart , germany ). samples of the protein were injected in 20 mm tris , ph 7 . 4 , 0 . 05 m nacl . elution was accomplished by running a gradient of nacl from 0 . 05 m to 2 . 0 m over 30 min , and the nacl concentration at which elution was observed was taken as a measure of the heparin - binding affinity of the protein . the relative heparin - binding affinity of proteins not previously reported in the literature to be heparin - binding was determined and compared with proteins such as bfgf and antithrombin iii , which are know to be strongly heparin - binding . the results are shown in table 1 . the two “ non - heparin - binding ” growth factors , tgf - β2 and ngfβ both eluted at sub - physiological nacl concentrations , suggesting that they have relatively low heparin - binding affinity , and will rapidly dissociate from heparin under physiological conditions . the two heparin - binding proteins , antithrombin iii and bfgf , elute at nacl concentrations that are much greater than physiological levels . others have also used heparin to increase the activity of and prevent the degradation of growth factors . schroeder et al . ( 20 ) used heparin to increase the stability of tgf - β1 and prevent loss of activity . they have also attached heparin to collagen and employed the heparin - tgf - β1 complex in collagen gels to show that such heparin - based systems can improve growth factor activity in vivo by controlled release ( 21 ). tgf - β1 is known to possess strong heparin binding affinity . however , such heparin - based release systems have not been tested previously with growth factors , which are considered to have low heparin - binding affinity ( those which are characterized by elution from heparin - affinity columns at sub - physiological nacl concentrations ). analysis of the primary sequence of proteins by the present inventors , including growth factors , has revealed that the primary sequence may contain regions that are basic in nature and as that the basic residues are commonly flanked by hydrophobic residues . these sequences are generally of a similar nature to the xbbxbx heparin - binding consensus ( where x is a hydropathic residue and b is a basic residue ) the heparin - binding consensus was described by cardin and weintraub ( 22 ). however , the exact sequence of the basic regions vary from protein to protein . the three - dimensional structure for many proteins is also available , which allows the location of basic regions to be determined . in order for basic regions to be useful for sequestration of a protein , they must be located on the surface of the protein . frequently , such regions are observed to occur in the amino or carboxy terminus of the protein . a three - dimensional fibrin gel in vitro model for assay neurite extension . fibrinogen was dissolved in water and dialyzed into tris - buffered saline ( tbs ) at ph 7 . 4 for 24 hr . the concentration of fibrinogen was determined by measuring the absorbance at 280 nm . fibrin gels were made containing a final concentration of 3 . 5 mg / ml fibrinogen , 2 . 5 mm ca ++ , and 2 nih units / ml thrombin in tbs . the polymerization mixture was incubated for 60 min at 37 ° c ., 95 % relative humidity , and 5 % co 2 . after polymerization , 1 ml tbs was added to each well . the gels were washed 5 times over 24 hr , with the first 4 wash solutions consisting of tbs and the last solution consisting of modified neural basal medium . dorsal root ganglia ( drgs ) were dissected from day 8 chick embryos and placed in hanks - buffered salt solution . the ganglia were placed inside the fibrin gels ( one per well ) and the gels were incubated for 60 min at 37 ° c ., 95 % relative humidity , and 5 % co 2 . after 60 min , 1 ml of modified neural basal medium was added to each well . the media was changed at 24 hr . bright field images of the ganglia were taken at 24 and 48 hr . the images were analyzed to determine the average length of neurite extension , which was calculated to be the area of an annulus between the drg body and the outer halo of extending neurites , as shown by herbert et al . ( 1996 ) ( 23 ). neurite extension for each experiment was normalized by the average neurite extension through unmodified fibrin gels from the same experiment and time point . results are shown in fig1 bar 1 . this study demonstrates the utility of the invention as a cell support and growth material that will enhance neurite extension in three - dimensions . this is also demonstrated of the utility the invention would have for promoting cell growth and neurite extension under conditions found in vivo . an example of a growth factor that is not considered to be heparin - binding growth factors , but that contains a basic sequence is nerve growth factor β ( ngfβ ) growth factor . this growth factor is considered to be non - heparin - binding and has even been used as a negative control for a heparin - binding protein in heparin - binding analysis ( see lee and lander ( 1991 ) ( 24 ). however , ngf contains a basic domain at its c - terminus amino acid position ( 230 - 241 of human ngf beta ) consisting of the following residues : cvlsrkavrra ( seq id no : 6 ). similar basic domains can be found in the carboxy termini of neurotrophin - 3 ( nt - 3 , calsrkigrt , 248 - 257 of human nt - 3 , seq id no : 7 ) and brain - derived neurotrophic factor ( ctltikrgr , 238 - 247 of human bdnf , seq id no : 8 ). the present example demonstrates that ngfβ , nt - 3 and bdnf can be delivered in a sustained manner from heparin - containing fibrin matrices that contain non - covalently immobilized heparin . this system consists of covalently immobilized heparin - binding peptides cross - linked to the fibrin matrix by factor xiiia ( see schense and hubbell ( 1999 ) ( 25 ) and heparin , which is non - covalently attached to these heparin - binding peptides . these materials have been shown by the present inventor to effectively deliver heparin - binding growth factors , such as basic fibroblast growth factor . ( u . s . ser . no . 09 / 141770 , specifically incorporated herein by reference ). with this system , a bi - domain peptide is synthesized to comprise a heparin - binding sequence in one domain and a substrate for the coagulation enzyme factor xiiia in the other domain ( schense and hubbell , ( 1999 ) ( 25 )). this enzyme covalently attaches the substrate domain of the bi - domain peptide to the fibrin network during coagulation , thereby also immobilizing the heparin - binding domain of the bi - domain peptide to the fibrin network . heparin is also included into the coagulation mixture , and the heparin is thereby immobilized to the fibrin network by binding to the immobilized peptide . the release system was first characterized with growth factors that demonstrate strong heparin binding affinity . the results of these studies are shown in fig1 . bar 1 shows the neurite extension through unmodified fibrin gels . bar 2 shows that the release system , consisting of heparin and incorporated heparin - binding peptide , does not promote neurite extension without the addition of growth factor . bars 3 , 4 and 6 show the dose - response effect of matrix - bound bfgf , which enhances neurite extension by up to 100 % ( bar 6 ). bars 5 and 7 show that when bfgf is added during polymerization of the fibrin gel in the absence of the release system , it does not enhance neurite extension , presumably because it diffuses out of the fibrin too quickly . bar 8 shows that the presence of bfgf in the culture media promotes neurite extension similar to that of matrix bound bfgf at the same concentration . bar 9 shows that vegf does not enhance neurite extension , demonstrating that the growth factor bound must also show bioactivity in neural models to promote neurite extension . bar 10 shows that when the amount of heparin - binding sites is decreased by 50 %, the release of growth factor is not significantly affected . this deviation supports the contention that a high excess of growth factor binding sites is present . bar 11 shows that when the peptide is cross - linked to the matrix , it will constitute a functional heparin - based delivery system . bar 12 demonstrates that heparin and bfgf without the immobilized heparin - binding peptide do not constitute a functional delivery system and that heparin and immobilized peptide will enhance and sustain the sustained release of growth factor . these results demonstrate that the delivery system is capable of sustained release of a heparin binding growth factor in an active form . the ability of heparin - containing fibrin matrices to deliver low - heparin - binding growth factors has been tested using ngf - β , nt - 3 and bdnf , all members of the neurotrophin family . fibrin gels were made as described in example 2 , except for the addition of peptide , heparin and neurotrophin described below . fibrin gels were made containing a final concentration of 3 . 5 mg / ml fibrinogen , 2 . 5 mm ca ++, 2 nih units / ml thrombin , 0 . 25 mm peptide , 0 . 125 mm heparin , and 0 . 1 μg / ml of the neurotrophin to be tested . otherwise , the assay was performed as described in example 2 . the results are shown in fig2 . the fibrin group represents the behavior of normal fibrin and was the control data set to which neurite extension was normalized . these ganglia were cultured in the presence of 20 ng / ml ngf . all other treatments contained no growth factors in the media . for each of the three neurotrophins tested , neurite extension was enhanced only when the heparin - based delivery system of peptide and heparin was present and bound to the matrix (“ bound ” bars ). the addition of neurotrophin alone (“ soluble ” bars ) without peptide or heparin did not enhance neurite extension presumably because the factor diffused out of the fibrin too quickly . the materials shown in fig2 differ from those in fig1 in that “ low - heparin - binding affinity ” growth factors were released in fig2 while the factors released in fig1 are considered strongly heparin - binding . this distinction is clearly demonstrated in example 1 , in which the heparin - binding growth factor ( bfgf ) eluted from heparin above physiological nacl concentrations , whereas the low and non - heparin - binding growth factors ( ngf - β , and tgf - β2 ) eluted at sub - physiological nacl concentrations . in both cases , the heparin - based delivery systems were able to deliver bioactive growth factors in a controlled manner . this demonstrates , surprisingly , that non - heparin - binding growth factors can be released in a sustained manner from heparin - containing matrices . to determine how long the release of low - affinity growth factors could be sustained , ngf - β - containing gels were washed for extended time periods prior to cell seeding . in each case , the gels were washed thoroughly with tbs . the results are shown in fig3 . the bioactivity of the growth factor present after 4 days is the same as after 1 day of washing . after 1 week of washing , the bioactivity of the growth factor released is decreased . these results demonstrate that ngf - β , nt - 3 and bdnf are being sequestered by heparin and that the increase in outgrowth versus unmodified fibrin is due to matrix bound growth factor , rather than free growth factor . this demonstrates that such heparin - based materials can be used to sequester proteins with low heparin - binding affinity , which contain exposed basic regions . in this three - dimensional fibrin matrix , heparin was immobilized at approximately 380 μg / ml . this material demonstrated ngf release over approximately one week . there are situations when release for as little as a day would be therapeutically useful . thus a useful amount of immobilized heparin would be at least 95 μg / ml , with higher amounts leading to release sustained over longer periods . the results shown here demonstrate that “ non - heparin - binding growth factors ”, such as ngfβ , bdnf and nt - 3 , can be released in a controlled manner from heparin - based drug delivery systems based on their low affinity for heparin . these proteins can be sequestered within three - dimensional materials containing immobilized heparin based on basic domains found in the proteins . furthermore , the growth factors released from these systems retain bioactivity in in vitro models as shown above . this demonstrates other low heparin - binding affinity growth factor proteins containing similar types of basic domains could be released from heparin - based delivery systems in a similar manner . the approach to sequence analysis described above can be applied to growth factors from other families as well . a list of growth factors and their sequences is shown in table 2 , which shows domains that may have low heparin - binding affinity and could be released from heparin - based delivery systems such as those described above . these factors include some members of the tgf - β family , namely tgf - β2 , tgf - β3 , and tgf - β4 . tgf - β2 , tgf - β3 and tgf - β4 are members of a family , which contains one strongly heparin - binding growth factor , tgf - β1 . however , other members of the tgfβ family have been reported in the literature to have lower heparin - binding affinity , such as tgf - β3 ( see lyon et al ( 1997 ) ( 26 ). tgf - β1 has been shown to be heparin - binding at physiological ionic strength , i . e . at 140 mm nacl . tgf - β3 lacks a key charge and has been demonstrated to be “ non - heparin - binding ” at physiological conditions . heparin - affinity chromatography on tgf - β2 by the present inventors demonstrates that it also possesses low - heparin - binding affinity under physiological conditions . the basic domain in each of these growth factors , which could potentially interact with heparin , is underlined in the list of sequences given in table 2 . some members of a growth factor family may be heparin - binding , while others are not , as is demonstrated by the tgf - β family . despite the low - heparin - binding affinity of such “ non - heparin - binding ” growth factors , they may still be released in a controlled manner from heparin - based delivery systems if they contain a basic domain and the number of heparin - binding sites present in the system is in relatively greater excess to the amount of growth factor to be bound . other families of growth factors also contain growth factors which are not reported in the literature to be heparin - binding , but which contain basic domains ( shown in table 2 . spd k qmavlp rr e r n r qaaaanp ens r g k g rr g q r g k n r gcvl taihlnvtdl glgyet k eel if r ycsgscd lga r pcgl r elev r vselglgya sdetvlf r yc agaceaaa r v ydlgl rr l r q rrr l rr e r v r aqp cc r ptay aaettyd k il k nls r n rr lv sd k vgqacc r piafdddlsf lddnlvyhil rk hsa kr cgc i ssshpifh r g efsvcdsvsv wvgd k ttatd i k g k evmvlg evninnsvf k qyffet k c r dp npvdsgc r gid hsdpa rr gel svcdsisewv taad kk tavd msggtvtvle k vpvs k gql k qyfyet k cnp mgyt k egc r gid yaeh k sh r gey svcdseslwv td k ssaidi r ghqvtvlge i k tgnspv k qyfyet r c k e a r pv k ngc r gid wvtd rr tavd l r g r evevlg evpaaggspl r qyffet r c k adnaeeggpg aggggc r gvd rr hwvsecvd s k hwnsyctt thtfv k altm dg k qaaw r f i r idtacv cv ls rk av rr a kr hwnsqc r t tqsyv r altm ds kkr igw r f i r idtscv ct lti kr g r d k hwnsqc k t sqtyv r alts enn k lvgw r w i r idtscv ca ls r kig r t rr hwvsec k a k qsyv r alta daqg r vgw r w i r idtacv ctl ls r tg r a gp etlcgaelvd alqfvcgd r g fyfn k ptgyg sss rr apqtg ivdeccf r sc dl rr lemyca plkpa k sa gp etlcgaelvd alqfvcgd r g fyfn k ptgyg sss rr apqtg ivdeccf r sc dl rr lemyca pl k pa k sa nsdsecplsh dgyclhdgvc myieald k ya cncvvgyige r cqy r dl k ww el r table 2 : sequences of low and high heparin - binding affinity growth factor proteins . basic domains of low heparin - binding affinity growth factor proteins are underlined and basic amino acid residues ( k or r ) analysis of the primary protein of growth factors , such as those shown in table 2 can be used to identity basic domains . by analyzing the sequences of the tgfβ family and the neurotrophin family , one skilled in the art can observe a pattern in the basic domains underlined in table 2 . from this pattern observed in the basic domains of low heparin - binding affinity proteins , an approximate formula was developed to identify similar basic domains in other proteins . the formula defines a basic domain to be of length about 8 to 30 amino acid residues , comprising at least 2 basic amino acid residues , with a ratio of basic to acidic amino acid residues to at least 2 , and a ratio of hydrophobic amino acid residues of at least 0 . 67 . secondary and tertiary protein structure also influences the heparin - binding affinity of a basic domain within a protein or peptide , and for this reason the formula is approximate . therefore , it is necessary to perform heparin - affinity chromatography or some other experimental technique to determine the relative heparin affinity of a protein or peptide . the term “ low heparin - binding affinity protein ” refers only to those proteins or peptides which elute from heparin - affinity chromatography at a nacl concentration of less than about 140 mm . the formula for sequence analysis described above was applied to the list of growth factors found in table 2 and used to identify basic domains which could potentially bind to heparin or heparin - like polymers . low heparin - binding affinity protein should dissociate rapidly from heparin under physiological conditions . however , surprisingly the results shown in fig3 demonstrate that such low heparin - binding affinity growth factor proteins can be released in a controlled manner from heparin - based delivery systems . this novel result suggests that other growth factors containing domains which meet the requirements of the formula described above may also be released in a controlled manner from heparin - based delivery systems . examples of such growth factors are shown in the sequence list in table 2 , and the basic domains of these growth factors are underlined . two members of the gdnf family , namely neurturin and persephin , both contain basic domains . gdnf is reported in the literature to be heparin - binding , but no reports have been made to date on the heparin - affinity of other members of the gdnf family ( 27 ). neurturin and persephin , based on the analysis presented above , would appear to have sufficient heparin binding affinity to be releasable by the methods and materials of this invention . igf - 1a and igf - 1b are members of the insulin - like growth factor family . although there are extensive reports of insulin - like growth factor binding proteins binding to heparin , there is no documentation in the literature of igf - 1a or igf - 1b binding to heparin . both of these proteins contain basic domains shown in table 2 . based on the analysis presented above , would appear to have sufficient heparin binding affinity to be releasable by the methods and materials of this invention . egf is another growth factor , which contains a basic domain , shown in table 2 , but which is not reported in the literature to be heparin - binding . in fact , the existence of a related growth factor specifically referred to as heparin - binding egf - like growth factor suggests that egf does not possess high heparin - affinity . the literature suggests egf is not heparin - binding based on sequence analysis ( 45 ). based on the analysis presented above , it appears to the present inventors to have sufficient heparin binding affinity to be releasable by the methods and materials of this invention . despite the basic domains found in each of these proteins , their heparin - binding affinity is still weak and characterized by elution from heparin - affinity columns at sub - physiological nacl concentrations ( i . e . growth factors which elute between about 25 mm and 140 mm nacl ). for example , heparin - affinity chromatography was performed for ngf - β and tgf - β2 . in both cases the proteins were found to elute at 50 mm nacl at ph 7 . 4 after several column volumes of buffer , which is well below the physiological nacl concentration . however , in vitro studies demonstrate that heparin - based release systems can still be used to release ngf - β in a controlled manner , in spite of its relatively low heparin - binding affinity . all of the growth factors described in the example contain basic domains , but lack any literature reports of heparin - binding affinity . however , based on comparison of the sequences and the results demonstrated with other non - heparin - binding growth factors of the neurotrophin family , sustained release from heparin - based systems should be possible with virtually any growth factor having a basic domain having the characteristics of basic domains described above . the following references are specifically incorporated herein by reference for the purposes indicated herein : 1 . presta , m ., satuto , m ., isacchi , a ., caccia , p ., pozzi , a ., gualandris , a ., rusnati , m ., bergonzoni , l ., and sarmientos , p . 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