CA2124857A1 - Methods of inhibiting restenosis - Google Patents

Abstract

Polyionic derivatives of cyclodextrins and methods for preparing these derivatives are provided in which a polyionic derivative of cyclodextrin is combined with a growth factor, preferably a heparin binding growth factor. These compositions are of low solubility and are applied directly to the location of a wound. By virtue of the low solubility, the compositions remain in place at the site of application and slowly release growth factor. In an alternative embodiment, the cyclodextrin derivatives are administered in the absence of growth factor and are used to absorb growth factor present in the body at the location of the wound in order to prevent overstimulation of the wound response.

Description

2 ~2~7 PC~/US 9 2 / ~ 9 7 ~ 4 03 R2c ~ /P j ~ 1 4 ~C t~ ~3 ~T~0~8 O~ INHIBITIN~ R~8TENO~

xos~ x~ne~:to R~atQ~ A~ tie~
: Th:i~ is a continuation-in-part of:application Seria1 S5 No. 790,~20j filed November 12, 1991 ~p~nding)~ which is a cont nuation-in-part o~ application Serial No. 691~,168, filed :; Apri1 24, 19g1 (pending)~, which is a continuation:o~applica~ion Se~ial No. 397,559, filed ~ugust Z3, 1989 (now;~abandoned~ which in turn i :a continuation-in~part~o.f::app1icat1on~Serial No.
lO~434,6~59,~iled Nov~mbe:r 9, 1989~ (now U.S~Patent~No. 5~,01~9,5~2);
which~ a~con~inuat1~n of appli.cati~n Serial No. 07/~95:,68 : ~fil~ d~January lO, 1989 (now abandoned), which:in turn is~a continuat~on-in-par~ of applicat:ion Serial NO. 145,407, filed ~:
:~ January 1~ 1988 (now a~andoned~, all of ~aid::applications an~
l5~said~pa~ent:b~ny here~y~incorporated by reference. This is a~lso~a continuation-in-part~o~ application Serial~ No.:480,407, filèd F~bruary 15, l990, which is~hereby incorpo~ated by re~renc~
-:
F~ 1~ o~ X~v~n~
The pre~ent invention is direc~ed to compounds, composition~ and methods for healing wounded 1iving tissue, and parti~u1ar1y to sacch~ride-based compounds and compositions:
which remain 1Ocalized at the site:of ~ wound for ex~ended 25 periods af tim~
~:

, SU~3~TlTlJl-E SII~

W~ g3/09790 PCr~U~;92/~754 2 1 2 l,~,57 '"

The iniury o~E tissue initiates a serîes of events that result in tissue repz~ir and healing of the wound. During the firs~ severzal days following an injury, there is dir~ct~d 5 mi~ra~ion o~ neutrophil~;, mac:rophages and ~Eibroblasts to the site of the w~und . The mac:rophages and f ibrsblasts which migra~e to the wollnd sit~ are ac:tivated, thereby resulting in endogenolls growth fac:tor production, synthesis of a provisional extracPllular matrix, proliferation of fibroblasts and cc~llagen 10 synthe~;is. Finally from about two weeks to one year after infliction of the wound there i~ remodeling of the wound with active collagen turn over and cros~; linking (Pierc~ et al~, ~-~ 1991, J. Cell Bios::hem., 45:319 326~ . The manner in which :~his repair proc:ess is regulated is mo~;tly unkrlown; it is known, lS however, that cell prolif~ration, migration and protein synthesis carl be stimu~ a~ed ~y growth ~actors tha~ ac:t on ce~ ls ~aving receptt)rs f or th2se growth f actors .
In ~ivo studies have ~;hown that local applic:ation of ~:
exogenous singl~ growth factor~ or a combinatic:>n of gr~wth .
20 factors can ~nhance the healing process following experimental wounding in animals ~Antoniades et al., 1991, Proc~ Natl. Acad.
Sci . USA, 88: 565-56g) . The ability of these growth factors to promote wouncl healing has resulted in efforts to obtain these `~
f actors in purif ied f orm . It is known that a number of these 25 growth factors, known as heparin binding growth factors (HBGFs), have a s~rong a~f inity for heparin (reviewed in Lobb, 198~ l Eur.
J. Clin. Invest. ~ 8: 321~328 and Folkman and ~lagsbrlm, 1987, SciPns:e 235:~42~447). Aecordingly heparin af~inity chromatography has beerl used tc~ obtaln theee growth factors in 30 purified fonn. In addition the DN~ c:oding for a .numbe~ of these growth f actors has been isolated and the protein~ c:an be produced ~y recombinant DNA methods. These HBGFs hasre been shown to have mitogenic and non-mitogenic effects on virtually '''' ...... .......... ...... ...

~ ~ WO 93/09790 PC~r/US92/09754 ~12~,~S7 all mesoderm and neuroectodexm derived cells in vitro. ~BFGs are also known ~o promo~e ~he migration~, proliferation and differentiatic3n of these cells in vivo. It was suggested by Lobb (1988, Eur. ~. Clin~ Invest., 18:321-328) that HB~Fs could 5 therefore effect the repair o~ so~t tissue. It was ~urtheæ
suggested that HBGFs na;~y be us~d to e~ ct the repair of hard ti~sue such as bone and cartilage. In contrast to their benefic:ial ef~ec:~s, it i~ also Jcnown that gr~wth faztors may over-stimulate the wound healirlg respon~;e, re~;ulting in the 10 excessive smooth muscle cell prolifPration aa~d mi~ration which oc:cur, for example, in resltenosis following angiopla ty.
Knowledge o~ the af f inity of growth f actors f t)r heparin and the difficulty of ohtaining heparin il~ a pure, homogeneous ~orm has resulted in attempts to obtain a compound 15 which pc~;sesses hep~rin'~; affin:ity for growth fac:tors but whi::h cou:Ld be easily and reprodu~ibly manuf actured . P~s described in -~the parent applications ref erens~ed hereinabove, one group of compound~; meeting these requirements are cyclvdextrins, cy lic oligosaccharide~ consisting of up to at least six glucopyranose 2 0 units ~. .
U. 5 . Patent 5, 019, 562 to Folkman et al . (the Folkman et al. patent), which i~; in the lineage leading to the present application, is directed tc) 'he use of highly soluble cyclodextrin deriYatives to treat und~sirable cPll or tissue 25 growth~, The cyc:lc)dextrin derivatives disclosed in this patent axe c:ombined with growth inhibiting steroids or administered alone to absorb growth f actor~ pre~;ent in the bloocl stream . Th cyclodextrin derivativ~s discl9sed in tl~e Fc31kman et al. patent are highly hydrophilic and therefore high~y soluble. Th~ high 3 0 solubility of these dQrivatives is said to b2 an important factor which cooperatively interacts with the inher~nt complPxin~ ability of the cyclodextrin structure for exogenous --steroids. In additiont the high solubility of these compounds W0 ~3/~97~0 2 1 2 4 ~ PCr/U~g2/~7~

is said to facilitate introàuctlon o~ the c:ompounds into the body and to aid in dispersal via the blood stream.

The high solubility of the compounds disclosed in th~
Folkman et ~1. patent is deæirable f or systemic administration of these ~.olapo~itions to the body, on the other hand, howe~r, applicants have f ound that the high solubi~ity of these compound~; limits their ability to remairl localized in the ar~a 10 of a wound following adminis~ration. To maximize delivery c>f a given growth factor or fa;::tors to a wound site, applicants hav~
discc)~rered that it is desirable to obtain saccharide-based ~-^ compounds p~sgessing a high affini~y for gxow~h fa::tor and ~ry low solubility . According to one aspect of applicanks ' 15 discovery, such low solubility compounds are combined~ with a growth fa ::tor prior to administ:ration to the body and appli~d lo~::ally to the ite of a w~und. Due, at least in part, to th~ir low solubiliîy, su~h c:ompounds remaln at the ~it~ of application and slowly release the growth fac~or to optimlze the dosage of 20 growth factor~at the wound site. Applicants hava also ~ound th~t, alterna~:ively, a compound poss~ssing both a high a:f~inity or growth factor and a low solubility can be used to remai~ at the site of an injury and to absorb at l~ast some portion of the ~;
:~ : growth factors rel~ased by the injured tissùe, thereby reducing ~:
25 the probability of over-stimulation of the wound healing ;~
process, as is ohs~rved in restenosis following angioplasty.
In view of both the beneficial and pathological properties of growth factors involved in wound repair, applicants have thus identified a n~d for compositions which 30 regulate the concentration and/or difPusion of growth factors in the area of a wound so a t~ optimize the wound~ h~aling process. Accordingly, the present invention provides low solubility polyanionic saccharide derivatives having a high .--~ W~ 93/09790 P~/V592/Og7~4 ` ~ 212~ f nega~ive charge density for affectirlg the growth of living tissue in marnmals. Al~;o provided are composition~; c:omprising an af~tive agent comprising low solubility polyanionic sact::haride deri~Jative and a physiologically acceptable carrier for the 5 sac:charide derivative.
The saccharid~ derivative pref erably ha~; a body tempera~ure solubility of lass than about ~5 grams per 100 ml of water . Acc:ording to certain pref erred ~mbodimen~;, the saccharide derivatives have sub~;tantially no solub~ lity in water 10 at body telaplerature. The term l'body temperature" a~ us~d herein rei~ rs to the range of body temperatures expected for a li~ing mammal, including the lowered body t~mperatures us~d in variou ~-~ surgical tech~iques arld îhe elevated body temperatur~s en::ountered in physiological responses ~:o infection,. Unless 15 o~herwise indicat~d~ solubility refers to ss~lubility in di tilled water.
The compositions of the pr~sent inventiorl of f er number of advantages o~rer prior art compo:;itions du~, at least part, to the low ~olubility of the active ingredient in body 20 tissues and flslid., The low solubility of the present saccharide derivatives is advantageous in ~rollnd healing methods which provide for administration directly to the site of a wound. The compo~;ition~; remain substantially at the administered loc:ation ~Eor an extended period of time. When combined with growth 2 5 f actors, th~ sa ::charide derivatives of the presen~ invention :
facilita~e controlled release of the growth factors at the wourld site, thereby regulating and greatly enhanc:ing the wound healing procs~ss . In the absence of a growth f actor, the pr~erlt compositions can, by virtue of their affinity for growth 30 ~actors, reduce the local concerltration and/or di~fusion of growth factors produ-~ed by cells at the wolmd site as well as grow~h f actors present in the blood stream . By r~duc:ing the diffusion of growth fac:tors, the compositions are c:apable of W093/0~7~ 2~ 57 PCT/~S92/~754 preventing or substantially reducing over-stimulation of the wound healing response, thereby avoiding the pathological growth of c211~ that results in such conditions as restenosis following angiopla~ty, vein graft intimal hyperplasia, and native vessel 5 atheroscl~rosis.
The present invention also provides method~ for the preparati~n of ben~ficial wound healing compositlons~ ~hese compositions compri~e r~l~tively insoluble solid forms of highly anionir polysaccharides. The method aspects comprise reacting a 10 saccharida with an anionic derivatiæing agent to g~nerate a polyanionic derivative of the saccharide, ~ollowed by salt formation of the l~rgely insoluble produc~. AlternatiYelyl :
accharides are reacted with a suitable coupling agent to ~:
generate a sparsely soluble polymer or copolymer of that 15 saccharide, followed by a react:ion with an anionic derivatizing agent. According to certain embodiments, these d~rivatized saccharides are then combined with one or more growt~ factors.
The compo~itions provided by these methods of prPparation havs - :
th~ advantageous properties of very low solubility and high 20 growth factor affinity.
The present invention also provides wound healiny methods. According to ~hese methods, the present low solubility, polyanionic saccharide derivatives are applied to the area to be treated. Such methods are adaptab~e for use in 25 the prevention of restenosis, promotion of a~giogen~sis, treatment of transplanted tissue or organs and treatment of : .
damaged or transplanted bo~e or cartilage.
rhe ability of the prese~t compounds and compositions to regulate the wound healing process offers possible life-30 saving benefits to patients who have undergone procedures su~h :-as percutaneous transluminal angioplasty (hereinafter l'PCTA~').
It has been observed that up to 43% of patients whc undergo PCTA
are afflicted by restenosis and the recurrent arterial blockag~

-- W093/09790 2 ~ 2 ~ ~ ~ 7 PCT/~S92/097~

that it causes. Thus, the long-term effectiveness of treatments for arteriosclerosis, such as angioplasty, have been substantially limited by ~he reoccurrence of restenosis. It is believed that the present compositions will substantial7y reduce 5 or eliminate restenosis and thereby have a major influence on th~ morbidity an~ mort~ y ra~e ~or pati~nts which have und~rgone angioplasty, v~in graft bypa~s operations and similar procedures. In addition, i~ is ~xpected t~at victims of cardiac, cerebral or peripheral ischemic disease will greatly 10 benefit from use of the compositions of the present invention.
In particular, patients wh~ suffer from in~arcted myocard~al tissue re~uire the establishment of new collateral blood ~-~ capillaries and vessels to supply ~lood to the infarcted tis ue.
The presen~ compositions may include growth factors t~ promote 15 angiogenes~s at the si~e of the infarcted tissue. These examples represent just a few o~E the possible life-saving benefits offer~d by the composition and methods of the present invention.

~ri ~ :
FIG. 1 ~A and B) is a schematic representation of (A) the chemical structure of ~ and ~ cyclodextrin monomer; and (B) of the three-dimensional shape o~ these cyclodextrin monomers~ ~
FIG. 2 is a schematic representation of the chemical structur~ o~ sucrose with the sites of anionic substituent groups indicated~
FIG. 3 shows the affinity of beta-cyclodextrin tetradecasulfate polymer for basic fibroblast growth factor.
FIG. 4 shows polyacrylamide gel electrophoresis of ~:
basic fibroblast growth factor and Chrondosarcoma-derived growth factor purified by cyclodex~rin copper biaffinity - chromatography. Lane 1 hows th~ protein profile of the protein ~, 1, 212~7 PC~US 9 ~ I ~ 9 7 5 ~4 03 ~e~'d PCTlPTo 1 ~ ~EC 1993 markers (phosphorylase b, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor~ beta lactoglobulin, and 1YSQZYme)~ Lanes 2 and 3 show the 18,000 molecular weight polypeptide bands of ba~ic f ibroblast growth 5 factor and Chon~rosarcoma derived growth factor, respectivel5 FIG . 5 compares the af f inikies of heparin and beta cyclod~xtrin tetrad~casulfate polymer for Chrondosarcoma derived growth f actor r _tailedl D~criptio~ Of th~ In~v~tion T~e invention is directed to comp.ounds ~ compositions and methods for aff~cting th~ growth of living tissue in mammals. Ths nc)vel compounds of the present invention are derivatized cyclodextrin pol~ners having low solubility ir 15 distilled water at body temperature and a high negative charg~
d~nsity. The present~ mpositions comprise a low ~;olubility, polyanionic sac~ haride derivati~e having a relatively high d~nsity o~ anionic substituents an* a carrier for such derivative "
2 0 One important aspect of the present compounds and compositior1s is the strong a~finity of such material for prol:einic growth factors. Although applicants do not intend to n~cessarily be bound by or limited to any particular theory, il;
. is thought that the density of the anionic groups on the 2 5 saccharid~ ompourlds of the present invention is importarlt in providing the high affinity of these compc~unds for tissue and ~rowth ~actors. Applicants have discovered khat the af~ ity of th~ present compounds and compositions for growth factors combined with th~ low solubility of the pre5ent sacc:haride 30 derivatives provides th~ ability to regulate and control the conc:entration o~ growth factors in the area of a wound. In addition, the present c:ompounds and compositions provide ac:tive agerlts in the fonn of th~ present d~rivatized sacc:harldes which !~llF3S~lTUTE SHEET

WO 93/097g0 2 1 2 4 ~ 5 7 PC~/US92/09754 g tend to adhere to living ~is~;ue. ~s a result, such compositions and compoull s have the hiyhly desirable ability to provide active wound healing agen~; at the site of an injury for extended periods of time.
The invention is also directed to methods f or preparing these composition~3 and to methods f or treating a variety o~ wounds resul~:ing ~rom accidents or surgical procedures. As the term is u~ed herein, "wound healing" refers to the repair or reconstruction of cellular tissue. The wound 10 may be the result of accident, such as injury or burns. The wounds treatable by the present compositions and methods also include wounds resulting from surgical pr oc~durçs of any ty -~ from minor intrusive procedures, such as catheterizatiorl or angioplasty resulting in woundi.ng of va~;cular or organ surf aces, 15 to major sursical procedures, such as bypass or organ transplant operations. Included in this c~onc:ept o~ wound healing is th~
r~pair of injured or fragmented bon~ or car~ilag~ and the p3~0motion of the establishment of bone grafts or implants.
I q~ T~:E C:ONPOfiITION~
Applicants have found that compositions comprisin~ a~
an active agent polyanionic sa charide derivatives having a high negative charge density and low solubility can be useful wound healing materials~ EspPcially preferred are polyanionic cyclodextrin polymers.
As used herein, the term "polyanionic saccharid~ :
derîvative" refers broadly to saccharide based compounds having 1.3 or more anionic substituents per sugar unit. The term sugar unit as used herein refers to an elementary monosaccharide building block which may, for example, be a hexose or pento~e~
30 ~xemplary monosaccharides are glucose, fructose, amylo~e, etc.
It is contemplated tha~ all compounds which include a basic sac~haride structure, as well as homologues, analogues and isomers of such compounds, are within the scope of the term W093/09790 2 1 2 4 8 5 7 PCT/US92/nYS4 9'saccharide" as used herein. The sac~haride compou~ds hereof may comprise, for example, di~accharide , trisaccharides, ~etrasaccharides, oligosaccharides, polysaccharides and polymers of such saccharides. The term "oligosa ::charide" refers to 5 saccharides of from about 5 to about 10 sugar unit having molecular weigh~s, when unsubstituted, from abo-at 650 to about 1300. The l:erm "poly~accharide'~ refers to saecharide~
comprising greater tharl about lQ sugar units per molecule.
Polysacchari~es are understood to be sac ::harides having many 10 sugar units possessing a variety of structures and vari :3~as ~:ubstituent groups . The term polymer as used her~ain ref ers to struc~ures o~ repeated and similar saccharide compounds, based c~n monomers which are linked together to ~orm the pc)lymer.
Applican~s have found tha~ the relationship between 15 the structure of the derivatized saccharide and the level of :~
negative charge density can influence the effectiveness of the present compounds, compositions ,and methods. For sxample, the anionic substituents are preferably present in the molecule to an exten~ of from, o~ average, ~bout l.0 t~ about 4 substituents 20 per sugar unit. Especially preferred ~ompounds are those based on saccharides having on average at lea~t ~bout 1.4 anionic substituents per sugar unit. For saccharide compound~ comprised o~ n sugar units and R substituents, it is preferred that the anionic substituents on the derivatized sa~charide corre~pond 25 substantially to about ~he followin~: :
If n - 2 to 3; average anionic R per n unit - or > 3.5 n = 4 to 5; average anionic R per n unit - or ~ 2.0 n = ~ 6; average anionic R per n unit = or > 1.4.
While applicants contemplate that the anionic 30 substituents of the present invention may be selected from a large group of known and available anionic substituents, it i~
generally preferred that the anionic substituen s be selected from the group consisting of sulfate, carboxylate, phosphate, W0~3/0~790 2 1 2 4 ~ ~ 7 PCT/US~2/09~54 sulfonate, and combinations of two or more of these. Preferred compositions are ba~ed on ~accharides having 6 or more sugar - units and from about 2 to about 3 subctituents per ~ugar unit, wherein the sub~ti~uents comprise su~fa~e, sulfonate and/~r .5 phosphate substituen~.
The saccharide d~ivatives of the pre~ent invention have a low solubility in di tilled water at b~dy temperature.
As the term is us~d har~in, 'llow snlubility" refers to solubility of much less than abou~ 15 grams per 100 ml of water.
10 It refers to the ability of the present saccharide compounds to remain localized in a solid state for a substantial length of time in an aqueous medium such as physiological or distilled water. According to certain preferred embodiments, the saccharide deri~atives have substantially no solubility in 15 distilled water at body temperature~.That is, it is preferred that the solubility of th~ saccharid~ derivative is much less th~n about 1 gram per 100 ml of clistilled watar, and even more pre~erably lecs than about 1 milligram per 100 ml~ 5uch insolubility is achievad, for example, by utilizing saccharide ~0 compo~itions comprising polymer alggregates or dispersions of substantially solid polymer parti.cles. While it i~ contemplated ~that various particle sizes and shapes may be utilized, it is pre~erred that the particles have an average particle size ra~ging from about a millimicron to about 1000 micron~ in 25 diameter. E~pres~ed in tPrms of molecular weight, the polymers comprising the polymer have, on average, a molecular weight of abQut one billion or greater. The high molecular w~ight of the preferxed polymers is due to the presence of many millions a~
sugar units within any of the discrete undissolved entitie~.
30 Alternatively, in other embodimen~s of the inventi~n, particl~s haviny the desired insolubility are produced ~y forming a salt comprising an a~io~ic saccharid~ in combination or associated with a polyva~ent cationic constituent.

WQ93J097gO 212 4 ~ ~ 7 PCT/US92/OQ?~
~ 12 -While the compositions of the present invention may be produced from ~he soluble ~accharides as starting materials, as indicated abo~e, it is al~o possible to ~mploy as starting mat~rials a sparsaly soluble, quasi solid or solid sacoharide~, 5 such as cellulos~ or ~tarch. Utilization of these saccharide sources preferably comprises chemically or enzym'atically degrading th~ solid saccharide, followed by providing the subs~ituent groups in accordance with thi~ invention.
A~ Cyclodextri~ ~erivati~
Especially preferred according to the pre~ent invention are compositions containîng a cyclodex~rin derivative~
Cyclodextrin~ are saccharide compounds con~aining at leas~ six ~lucopyranose units forming a ring or ~oroid shaped m~lecule, which therefore has no end gr~ups~ ~lthough cyclodextrins with ~-5 Up . tD twelve glucopyrano~e units are known, only ~he fir~t three homologs have been studied exten~ively. These compounds have the simple~ well-defined chemical structure shown in FIG~ l(A~.
The~common designations of the lower molecular weight ~ and ~ cyclodex~rins are used throughout this specification and will 20 refer to the chemical ~tructure shown in FIG. l(A) wherein n =
6, 7, or 8 glucopyranose units, respectively. The ini~ial discovery of the cyclodextrins as degradation products of star~h was made at about the turn of the century, and Schardinger showed that these compounds could be prepared by the action Qf ~`
25 Bacillus macerans amylase upon starch. In older litexature, the compounds are often referred to as Schardinger dextrins. They are also some~imes called cycloamyloses.
Topographically, the ~yclodextrins may be represe~ted as a torus, as shown in FIG. l(B~, the upper rim of which is 30 lined with primary -CH20H groups, and the lower rim with secondary hydroxyl groups. Coaxially aligned with the torus is a channel-like cavity of about 5, 6 or 7.5 ~.U. diameter for the , and ~-cyclodextrins~ respectively. These cavities make .. WO 93~0~790 2 ~ 2 ~ ~ S 7 PCI/U!~92/~9754 the cyclodextrins capable of forming inclusion compounds with hydrophobic guest molecul~s of suitable c3 iameters .
- The composiltion~; of the pres nt invention preferably includ~ polyaniorlic cyclodextrin derivative~;. In general, th 5 ~erms '~d~rivatize~ D, " "Cl:~ deriYative" and the like refer o chemically modi~ied CD~ orm~d by r~action of the primary or sec:nndary hydroxyl grouE:~s attac:hed to carbons 2, 3 or 6 of the CD
mole;:ule withollt disturbing the ~ ( 1~4 ) hemiacetal linkages . A
r~view of such preparations i5 giYen in "Tetrahedron Report 10 Number 147, S~nthesi.s of Chemically Modified Cyalodextrins, " A.
P., Cr~ft and R. A. Bartsch, T~rahedron 39~9) :1417 1474 (1983~, incorporated herein by rç~ference in tha background thereinafter referred to as "Tetrahedron Report No . 147 " ) .
The CD derivatives are preferably derivatized 15 cyclodextrin monomers, dimers, trimers; polymars or mixtures of these. In general, the cyclodextrin derivatives of the present invention are c:omprised of or for:med ~rom derivatized cyclodextrin monomeric units congisting of at least six glucopyranose units having ~ 4 ) hemiac~tal linkages . The 20 preferred derivatized cyclodex~rin monomers o~ th~ pr~sent inverltion ge~erally have the formula (I):

R n WO 93/097gO 2 1 2 ~ ~ 5 7 P~/US92/0"75~

wherein at least two of said R groups per monomeric unit are anionic substituents and t~e rem~inder of said R groups, when prssellt, are nonaniorlic: groups selected from well known and available sub~titu~nt groups. The remaining, nonanionic R
5 groups may be, f s: r exa~nple, H, alkyl, aryl, ester, ether, thioester, thioe~her a~ad -COOHo 13xemplary alkyl groups in ::luds methyl, ekhyl, propyl and butyl. The remainin~ nonanionic R
groups may be hydrophilic, hy~lrophobic or a combiraation thereoP, depending upon ~he particular requirements of the desired 10 composition ., However, it is g~nerally pref erred that the remaining nonionic R substituent~; be hydrophobic in order to mi3:limize the solubility o:l~ the compounds.
For CD monomers having the structure of Formula I
wherein n i5 from about ~ to about ~, it is preferr@d that the 15 compound have sn average at least abi:)u~ 9 anionic ~ substituents per monomer unit, more pref erably at leas~ about 1~ anionic R
substi~uen~s per monomer, and even more pref erably at least about 14 anionic R substituents pler monomer. In general it is pref 2rred that the anis: nic substi tuents be relatively evenly 2 0 dis~ributed on the monomer molecule, and accordingly compound~;
having the structure of Fonnula I wherein n is from about 6 to about ~ prafarably have from about 1 to about 3 anionic R
substituents per n uni~, more preferably from abou~ 1. 3 to about 2 . 5 anionic R substituents per n UIlit and even more preferably 25 fre~m about 1. 4 to about 2 . 2 anionic ~ substituents per n unitO
SuGh s~rllctures are believed to provide the high negative charge density founcl to be therapeutically berleficial, with the high~st :~
charge density mole~ules providing ~xce~lent result 4 ~'~
The polyaniorlic cyclodextrin monomers of the type 3 0 described abo~e are important components of the pref erred c:ompositions o~ the present invention. The monorneric units may be present in the composition in the ~orm of, f or example , insoluble polymeric or c~-polymeric strllctures or as insoluble W~ g3/û9790 2 1 2 ~ 7 PCr/7J~g2/09754 precipitated salts of derivatized cyclodextrin monomer, dimer or trimer .. Such salts may be f ormed by methods which comprise derivatizing the CD wi1:h anionic substituent and then complexing or associa~ing the derivatized CD with an appropriate polyvaler;t 5 cation to form an insoluble derivatized C~ salt. In alternative and preferred embodimen~s, the basic monomeric structure identif ied above ::ompri~3~s the repeatirlg unit of novel insoluble polymeric cyclodextrin~, a~; d~scribed more fully hereinafter.
1. Cy~lodextri~ PolyJne Acccsrding to important and pre~erred embodiments, the presellt compositions comprise derivatized cyclodextrin poly3ners.
The pres~nt polymers have a structure corre~porlding tv polymers formed from darivatized cyclodex~rirl monomers of the type illustrated above. In view o~ the presen~ disclosure, it will 15 be appreciated that polymeric materials having such struc:ture may :~e forxned by a variety of methods. For example, derivatized cyclodextrin polymers may be produced by polylTlerizing and~or cros~-linking one or more darival:ized cyclodextrins monomers, dimers, trimers, etc . with polymerizing agents, e . g .
20 epic::hlorhydrin, dii50cynanates, diepoxides and silanes using procedures known in the art to form a cyclodextrin polymer.
(Insoluble Cyclodextrin Polymer Beads t Chem. Abstr. No. 222444m, 102: ~4 j Zsads:~n and Fenyvesi ~ lst~ Int. Symp. on Cyclodextrins, J. Szejtli, ed., D. Reidel Publishing CoO, Bo~;ton, pp. 327-336;
25 Fe~yvesi et al., 1979, Ann. Uni~r. Budapest, SeGtion Chim. 15:13 22 ; and Wiedenhof et al ., 1969 , Die Starke 21 : 119-12~ ) . Thes~
polymerizing age~ts are capable of reaoting with the primary and s~condary hydroxy groups on carbons Ç, 2, and 3. Alternatively and preferably, the derivatized cyclodextrin polymers may be 30 produced by first polymerizing arld/c:r cross-linking one or more un~erivatized cyrlodextrin monomers, dimers, trimers, etc.
( ey ., ::yclodextrins having the structure of Fig 1 ) and then deri~ratizing the resulting polymer with anionic substituents.

WO 93/09790 2 ~ 2 1 8 5 7 P~/US92/1`~754 Underivatized cyclodextrin polymer is available from American Maize Produc~s Co., Hammond, IN in the form c~f an epichlc)rhydrin linked polymer of ,B~cyc:lodextrin. Underivatized commercially available polymers may be deri~atiz~d to produce the desirad 5 form of ~erivati~ed cyclodextrin polymer. The derivatized cyclodextrin polymers may also be ~orm~d by r~acting mixtures of derivatized monomers and underivatized monomers, or by copolymeri2ing and/or cro~slinking deri~atized cyclodextrin polylT ers and lmderivatized cyclodextrin pol~nars . For all 10 preparation procedures, it is preferr~d that the polym~rization method employ~d result in a solid polymer produc:t oP suf f icient pt~rosity to allow diPfusion pene~ration of molecules betweer~ the external sc: lvent and a subs~antial portion of the internal anionic monomer sit~s.
The solubility of the present cr~ polymers will depend~
inter alia, on tha mole ::ular weigh~ and size of the polymer .
The presen~ derivatized CD polymer~; are of large molecular weight so as to remain sub~;tantially in the solid state. They are solid particulates of generally about 1 to 300 micron size.
The derivatized cyclodext~in polymer of the present in~ention may be available in a variety of physical ~orms, and all such forms are within the scope of the present invention.
Suitable forms include beads, fiber~, resins or films. Many such polymers have the ability to swell in water. The 25 characteristlcs of the pol~meric product, chemical composition~
swelling and particle size distribution are controlled,at least in pa~t, by varying the conditions of preparation.
The cyclodextrin polymer deriYative prPferably comprises a polyanionic derivative of an alpha-, beta-, or 30 ga~ma-cyclodextrin polymer. In pr~ferr~d embodiments the anionic sub~tituents are selected from the group consis~ing of sulfate, sulfonate, phosphate and combinations of two or mor~ of the foregoingO Although it is possible that other anioniG

.--. W093/09790 2 1 2 ~ 7 PCT/~S92/09754 groups such as nitxate might possess some therapeutic capacity, the sulfate, sulfonate and phosphate derivatives are expected to - possess the highest th~rapeutiG potentia~. In preferred embodiments, at least abou~ lQ molar percent of khe anionic 5 substituents, and even more pre~erably at least about 50 molar percent, are sul~ate groups. Highly preferred are alpha , beta-, and gamma-cyclodextrin polym~rs containing about 10-16 sulfate groups per cyclodextrin monomer, with beta~cyclodextrin tetradecasulfate polymer being especially preferred.
B. I~soluble B~lt P~cipitat~
The present compositions may include derivatized insoluble saccharide salt precipitates, and preferably ~:
derivatized insoluble oligos~ccharide salt precipitates. As the term is used herein, "salt precipitate" mPans a polyanionic 15 saccharide deriva~ive which has be~n associated or complexed with a suitabl~, non-toxic, physiologically acceptable cakion to produ~e a salt which is substantially insoluble at body t~mperature. Suitable polyvalent cations which may be used to produce an insoluble salt precipitate of the present invention 20 include Mg, Al, Ca, La, Ce, Pb, and Ba. The cations herein ~:
list~d are presented generally in order of decreasing solubility, although this order may be different for saccharides of different types and degr~es of anionic substitution. While all such deri~atized insoluble saccharide salt precipi~ates are 25 believed to be operable within the scope of-the present : inve~tion~ the derivatized oligosaccharides are preferred. Such oligo~accharides typically have unsubstitut2d mol~cular weight~
ranging from about 65Q to about 1300. Oligosaccharides are usually obtained by procedures of degradation of starches or -:
30 cellulos~ which result in oligosaccharide fragments in a broad ~
range of sizes. Cycl~dextrins are generally obtained from ..
starches in the presence of specific enzymes that favor the -:
formation of the cyclic saccharide structures. According to WO 93/09791~ 2 1 2 1 ~ ~ 7 PCI/US92/0~75~

certain embodiments, th~ cyclodextrin salt precipitates are obtained by reacting th~ desired cyclodextrin monomer or monomers with agents that will produce the desired anionically substituted product and sub~eg[uently exchanging the cations 5 whic:h were intrc)duced by th~ synthesis f or cations of ~he desired polyvalent type. Thi~ latter step will result in precipitation of t~e insoluble :;accharide salt prec:ipitate~
The Al, Ca and E~a ~ s of c~ and ~y~CD sulfate ar~
pref erred f or use in 1~ e composition~; of the present invention, 10 with Al ,B--CD sulfate sal~s being preferred in certain embodiment~;~ As with the sac:charide derivativ!s general:Ly, various degrees of sulfation p~r glucose unit can be employe~l.
It is generally preferred~ however, tha t the derivatized _ .
c:yclodextrin salts have an average of at leas~ abou~ 1. 3 sulfate 15 groups per sugar unit, and even more preferably about two sulfate groups per sugAr unit. EspeGially preferred is ,~-CI)-TûS
which ha~ an average s:~f abou~ two sulf ate groups per glucos~
ur CO ~?olyzln~ o~ic Di~ac~:harid~ Deri~r~tiv~
Sucralfate (C:arafate~, Marian i~errill Dow, Kansas City, M0) is a complex salt of su::rose sulfate and aluminum hydroxide. Its structure is shown in Fig. 2. Sucralfate is an ~-d~glucopyranoside, ~-d-fructofuranosyl-,octakis~hydrogen sulfate3 aluminum complex. Sucralfate is used to treat ulcers 25 and was developed during studies of sulfated polysaccharide~
that bind pepsins but lack anti-ulcer efficacy~ The sulfation of sucrose and its conjugation with a basic aluminum sal~
re~ulted in a pepsin-inhibiting molecule suitable for treatment of ulcers. Denis M. MGCarthy, Sucralfate, 325:14 New Eng. J.
30 ~ed.~ 1017-1025 (1991).
Applica~ts have found that sucralfate and oth~r polyiQnic derivatives of sucrose have some properties in common with the derivatized cy~lodaxtrins of the present invention and WO 93/0979~ 2 1~ ~ ~ ;5 7 pcr/lJs92/o9754 may provide similar solubili~y and affinity for growth factors.
I~ is believ~d that sulona~e or phosph~te derivatives of sucrose combined with polyvalen~ ca~ions such as P~g, Al, Ca, La, Ce, Pb or Ba m~y result in c:ompositions of low solubility whic:h 5 can be ::om~ined with growth fac~ors to faci.litate therapeutic delivery of these growth factox~; to the site of a wound. ûral administration of sus::ralfate has been described to have therapeutic use~ulnesR in the treatment of stoma ::h ulcers .
Accordin~ to the present invention, sucralf ate and other salts lO of sucrose octasulfate may be used to deliver growth fac:tor protein~i to tissu~s or bone in need o~ repair, by prior complexing with grc)wth factors, and delivering ~he complex ~:
physically to the si~e of repair .
The frequent and/or high dosage use of aluminum salts 15 is well known to have certain heal~h risks as~ociated with it.
Alumimlm uptake iE; known or suspec:~ed to b~ associated wi th a number of diseases. See, for example, the extensive discussion~
in ~he books ALUMINUM AND HEA1TH, A CR~TICAI, ~VIE~ (~illel and :-~
Gi~elman, Ed.), Mark Decker, Publisher, 1989 and ALUMINI~ IN
20 RENAL FAILURE, Mark E. de Broi ancl Jack W. Coburn, Klewer, Publisher, 1990. .
Aluminum is kn~wn to produce abnormalities in bone metabolism, such as osteodys~rophy, osteomalacia, impaired mineralization, etc. The introduction of aluminum into the 25 blood ~tream, such as can occur in dialysis, can b~ par~icu~arly harmful. The following are but a few examples of reparts c~ncerning the harmful effect~ of aluminum: A. M. Pierides et al., Kidney Int.l Vol. 18, 115-124, 1984; H. A. Ellis ~t al., J.
Clin. Path. 32, 832, 1979. In addition to the toxic effects of 30 Aluminum when introduced into the blood stream, oral administration of aluminu~ salts can also produce a variety of harmful effects including osteomalacia and osteitis; see, e.g. O
S ~, P . Andredi , J . M . Bergstein et al ., N . Engl . J . Med ., Vol . 3 10 , WO 93/09791) 2 1 2 4 8 5 7 PCr/U~;92/~`7~4 1079 , 1984 , K.A. Carmichael~ M.D., ~a~ lon et al ., Am. J. of Med., Vol. 76, 1137, 1984.
Particulaxly prominent amorlg aluminum' s toxic effects are neur~ abnormalitîes, particularly Alzh~imer' s disease, in S which aluminum is sllspec~ed to play an important role, although by a mechanism not yet unders~ood ~ See, f or example, D . P~ .
Crapper P~cLachlan, B . J . Farnell, Aluminum in ~euronal Deg~nerati~n, in Metal Iorls in Neurolo~ a nd Psychiatry, pp~ 69-87 , 1985 , Alan R. Liss Inc: ~; D. P . Perl , P . F . Good" Uptake of 10 ~luminllm into Csntral Nervolls System Along Nasal-01~3factory Palthways, The Lancet, M y 2, P. 10~8, 1987; J.D. Birchall, J.S.
Chappell, ~luminum, Chemical Physîo~ogy, and Alzheimer~s Disease, The Lancet, October, P. 100~, I98~.
Given the possible toxi.city of aluminum the non-~5 aluminum salt forms of the highly sulfated polysaccharides are preferable over the aluminum salt:s forms in some and perhaps all therapeutic applic:ations. In particular, the polymeric e~odim~nts whic:h do not require salt pr~cipitat~ formation, are particularly pref erred .
2 0 Se~eral specif ic embodiments of the compositions of the present invention are particularly useful for oral administration ill the healing of stomac:h ulcers~ In particular, ~he non-aluminum salt-containing forms of sucrose o::taslllfate, and most pref erably the polymeric solid f orm of hi~hly sulf ated 25 c:yclodextrin are e~pec:ially advantages:us because of the absence of aluminum and its ~ide eff~cts.
Do The Form of th~ CoD~po~itio~8 In ~ri2w of the disclosure contained herein, those skilled in the ark will alppreciate that the present wound 3~ hPaling compositions are capable of having a beneficial effect in a variety of applications. It is therefore contemplated th~t the compositions of this invention may take numerous and varied f orms, depending upon the particular circumstan e of each ~ WO 93/~9790 2 1 2 ~ ~ 5 7 -P~r/US92~09754 application. For example, the derivatized saccharide may be incorporatad into a ~olid pill or may in the form of a liguid dispersion or suspension. In g~neral, theref~re, the compositions of the present invention pref erably c:omprise a 5 derivatized sa~eharide and a suitable, non-toxic, physiols: gically acceptable carrier f or the saccharide . As the tarm is used herein, carri~r r~f ers broadly to material~ which facilitate administr~ation or U;~ of the present compositions fox wound healing. A variety of non-toxic physiologically 10 accep1:able t--arriers may be used in fo:rmirlg these compositions, and it is g~nerally pre~erred that th~e c :>mpo~:itions b~ of physiologic salinity.
For some applications involving would healing in the broades~ sense, it is desirable ~o have available a physic:ally 15 applicable or implantable predetermined solid form of material corltaining the therapeutically active material of the invention.
Accordingly, it is ::ontemplated that the compositions of this invention may be incorporated in solid f orms such as rods, ne~dles, or sheets. They may thus be introduced at or near the 20 cites of ti~sue damage or sites of implantation, or~ applied exterrla~ly as wound dr~ssings, etc. In such embodiments, the compositions and compounds of the present invention are preferably combined with a solid carrier whieh itselP is bio-ac ::eptable, or the compositions comprise suitably shaped polymer .
25 or cc> polymer of the present saccharide derivatives. For manyapplications, it is pref erred that the compo~itions of the present invention are prepared in the form of an a~ueous dispersion, suspension or paste which can be direc:tly applied to the site of a wound. To prepar~ these compositions, a 3 0 polyanionic: saccharide derivative, such as polyanionic cyclodextrin polymer, carl be used as synthesized in solid form ~:
~fter suitable purif ication, dilution and addition c: f other components, if desirable, includirlg a f luid carrier, such as W0 93~09790 2 1 2 ~ 8 5 7 PCI/US92/~)~754 saline water. This will b2 the case when the procluct, saccharide salt, saccharide polymer or the saccharide c:o-polymer h~s been synthesized ~uch a8 to produce a particle form of precipitate, di~;perE;ion or suspension~. After synth~sis, the 5 solid derivative may al~;o be dried, milled, or modif ied to a desired particle size or ~olid ~orm. The particle size can be optimi2ed fc)r the intended th6~rap~utic use of the composition.
In some pref erred embodimerlts the solid particle~; range in size from about 1 micron ~o abou~ 600 mic:rons, with from about 200~
10 600 microns being even more pre~erred. Particles ranging from about 1 to abou~: 30 microns offer the best di~persion of growth f actor and f ~st reac~ivity O For a given weight quantity of particles de~ivered to the biological environment, a smaller particle size assures exposure of greater particle surface area 15 allowing greater diffusion of pro~ainic active ingredients into or out of the administered ~olid~ Particles ranging from about 30 to about lOo microns c~f~er ~air dispersion of gro~rth factor~
m~dium reactivity and a longer per:iod of delivery of growth factor . Particles E: ossessing a size in excess of 100 mic:rons 2 0 will have low reactivity, .~ut provide the longest delîvery tim~ ~:
f or growth f actors . In certain pref erred embodimen~s, these large partic:les (>100 micron) will be used to absorb, rather than deliver growth factors in vivo. ~.' In preferred embodiments 9 the carrier is an aqueous 25: medium and the compositions are prepared in the form of an aqueous suspension of solid particulate saccharide derivakive.
The amount of the derivatized saccharide preferably ranges from about 1 to 30% by wei~ht of the composition, and even more preferably from about 5 to about 15% by weight.
~. Biologically Active Pxot~
In certain embodiments, the compositions and compounds include and/or are combined with biologically active proteins. ;:
According to preferred embodiments, the biologically active .- ~ WO 93~09790 2 1 ~ 7 P~/US92/0975 protein exhibits a specific affinity for heparin, and" more specifically, is h~parin-binding growth factor, i . e~, a class of growth fartors ~ many of which are mitc3genic for endothelial cells. An example of ~uch a growth factor is basic fibroblast 5 growth factor~ Generally it will be the heparin-binding growth factor pro~eins, commonly r~f~rred to as HBGF's, whlch may be cQmbined with the saccharide derivatives of the pres~nt invention. Some of these are listed in Table I.
To de~ermine whether a protein is suitable for the 10 therap~utic compositions of the pr~se~t invention, one can det~rmine wh~ther it has a specific affinity for hepari~. A
HB~F protein is one that remains substantially bound to heparin ;~
~e.g., using a derivatized column) even in the presence o~ an aqu~ous medium having a salt concentration of substantially 15 gr~a~er than about 0.6 molar strength of NaCll Generally~ the term substantially bound refers t:o at least about 80% of such bound protein remaining attacAed under such conditions.
TABLE I
PROTEIN FA TORS
Svmbol Name Reference Interleukin 1) Henderson & Pettipher, 1988, Biochem. Pharmacol. 37:4171;
En~o et al., 1988, ~BRC
~36:1007, Hopkins et alO, 1988, Clin~ Exp. Immunol. 72:422 --30 IL 2 (Interleukin-2) Weil-Hillm~n et al., 1988, J.
Biol. Response Mod. 7:4~4; Gemlo et al., 1988, Cancer Res.
48:586~ ;~
35 IFN~ (Interferon ~ Pitha et al., 1988, J. Im~unol.
. 141:3611; Mangini et al., 1988, Blood 72:1S53 IFN~ (Interferon ~) Blanchard & Djeu. 1988, J.
I~munol. 141:4067; Cleveland et al., 1988, J. Immunol. 141:3823 WO 93/0~790 2 1 2 ~ 8 ~ 7 PCr/V~i92/0~
~ ~4 --TNF~ (Tumor necrosi~i Pl te et al., 1988, Ann. NY
factor ~ Acad3 Sci, 532 :149; Hopkins &
Pqeager, l 9 8 8, C l in . Exp .
Immunol. 73: 88; Gxanger et al ., l988, J. ~3iol. Response Med.
7:~38 EGF (Epidermal Carpenter and Coh~n~ 1979, A~
grow~h factor) Re~r. BiochemO 48 :193-2î6 FGF (Fibrobla~;t' Folkman and Klagsbrun, 1~87, growth ~ac~or, Sf~ien~e 235: 4~2-447 aciclic: and basic) IC;F-l ~Insulin-like Blundell and ~lumbel, 1~80, growth fac:tor Nature 287: 78l-787; Schoenle et l) ~ al ~, l9~2 ~ Nature 296 : 252-255 2û ~GF 2 (Insulin lik~ B.lundell and Humbel growth ~actor-PDGF (Platelet Ross et al ., l9B6 , Cell 46 : 155 derived growth 1159 ; Richardson et al ., 1988 , fac:t;:~r) Cell 53: 309 3l9 TG~ ~ Trans~Eorming Derynck, l988, Cell 54: 593-5g5 growth f actor-: 3 0 TGF-~ (Transforming Cheifetz e~ al., l987 , Cell growth factor- 4~: 409-416 ~) , It is known that the complexing ::apabili~i~s of h~pa~in tc~ward growth factor proteins are paralleled by its complexing capabilitîes for certain c:ationic dye structures, such as azure~ methylene blue and others. Other û glyc~saminoglycan sac;::harides are known not to func:tion similarly . Thus 5uc:h dyes have b-Qen uged f or many years in ~ ~
histology as specific: stains for the presence o~ heparin like ~-polysac~ haridPs; and metachromasia, i . e. the spectral shift resulting from heparin binding on the dye has been used to 45 identify actiYe heparin-like compounds having the capabilil:y o~

WC~I 93/09790 2 1 2 4 ,~ ~ 7 P~/USg2/~9754 modulating angiogenesis. Su h dye complexiny of the active protein alco is similarly resi~;tant to sal~ conc:entration as is the complexing l:o heparin., In relation to thi invention it has been discovered 5 that such dye complexing, serving as a model for proteinic growth factor compl~xing, can usefully serve as an indicator for the de ired activity of the compositions of the imrention.
Thus, the proteir~ic growth fa ::tsr compl~xing ability of the precipitates, polymers, or co-polymer~; of the compositions of 10 the prasent invention may be determined using dye cvmplexing assays .
In the course of prac:ticing heparin hinding separa~ion or chromatography fox the separation o~ pro~einic factors it has ,~
been customary and accepted that Idesorption of the complexed 15 growth factor requires the added step and invalvement of contacting with a v~ry strong sal k solution . The present ::
invention make~; u e of the important discovery and recognition that release o~` protein compl~xed ~o the saccharide herein sp~c:if ied does not require the added step of contacting with 20 high concentration e~ ectrolyte. 1i7hile such operation would be needed f or an immediate large scale desorptiorl proce s as may be desired for a separation technology, the relatively ~r~ry low external concentration of desorbed factor is maintained by an equil ibrium process involving the complexed phase Oll the solid 25 and the low biologically required solute phase in the physiological surrounding liquid. This is a basic: discovery and recc)gnition allowirlg the use of our comE: ositions as delivery agents for biomedical purposes.
Genarally, to prepare the growth factor containing 30 compositions~ der~ivatized ~accharide is c:ontacted with a solution containing a growth f act:or or combination of growth factors. The cyclodext~in derivative is thereafter separated from the conkact fluid, resulting in an enrichment o:f the growth WO 93/09790 2 1 2 1 ,~ ~ ~ Pcr/us9~/pq 75~
~ 26 factor on the cyclodextrin derivative, and a corresponding removal of the grow~h fa::tor from the fluid. The c:ontacting solution may contain a ~:ingle pre~eparat~d, pre ::oncentrated growth factor purified from tiE;sue or bodily fluids or growth 5 factor obtained from reCoD~inant DNA methods. Alternatively the contact solu~ion may compri~;e viable tis~;ue or organ materials (hereinafter organic source~;) which contairl a variety of growth f actQrs . Wheal combi~ed with ti;sue or organ material c:ontaining gro6~h f actors, the saccharide derivatives of the present :~
10 invention may act as extractants of these growth factors. When oryanic sollrceE; are used as the source fvr growth factors, it is prefarred that the organic source used for the contac:ting solution have a volume greater than about 10 to about 100 times the volume of the tissue to be treated by the cc~mbined 15 derivative and yrowth factor(s~
After contacting the partially or wholly complexed ~3ac:charide derivative, thP solid phase, can be easily separated ~rom the f luid phase that was the source of protein to be complexed. It is preferable that the source of growth factc~r -~
20 contains the protein as a dissolYed component in the absence of solids other than the saccharides to be complexedO However, some solids in the ~rrowt~. f actor sours:e solution, ma!,r not rleces~ar~ ly be undesirable or disturbing contaminants.
S~paration of solids, such as tissue or organ friagments from the 25 sas~charides, may be accomplished by sedimentation, suitable ~iltering, c:entrifugatiorl or other mechanical or other methods~
II,. MlST~ODB FO:2 ~HE~PElDTIC RB~LATION OF liO~ ~IEALI~NG
One aspect of the present invention relates to methc)ds for the therapeutic re~ulation, and preferal:ly in vivo 30 regulation, of ~ound healing, and par~icularly to in vivo regulation of the concentration and diffusion of protein factors. Such methods generally comprise therapeutic biodeli~rery of the present compositions and compounds to the ~ WO g3/~790 ~ ~ 2 4 8 5 7 PCrJUS9~J09754 wound site., The low solubility, i D e. the solid immobilizetl state, of the presen~ mat~rials allows the compositions and compounds to be administered directly to the site of a wound and for the active ingradien~s to remain at the site of application 5 f or an extended period o~ time.
VasGular cell proliferation and abnormal accumulation of extracellular ma~rix in the vessel wall are common ;~
pathological features observed in arteriosclerosis, hyper~ension ~nd diabetesO Such conditions are also observed following 10 ~ascular injuries, such as angioplastyO Intimal hyperplasia is thouyht to be mediated in part by a variety of growth factors, :~
such as platelet derived growth factor (PDGF), acting through re~eptors to stimulate vascular smos~h muscle cell proliferation ~and migration from the media into ~he intima. Thus, applicant~
15 have discovered methods for regulating migration and proliferation of the smooth muscle cells, thereby affeGting the degree of intimal thickening noted after va cular injury. ~he applicants haYe found that ~ cyclodextrin tetradecasulfate can i~hibit human vascular smooth muscle cell proliferation and 20 migration in vitro when stimulated with fetal calf serum, which contains potent growth factor actiYity~
It is seen, therefore, that the presence or absence of growth factors at the site or vicinity of a wound has an impact upon the healing process~ Applicants have found that the 25 present composition~ and compounds can be used t~ beneficially ~;~
regulate and control biologically active proteins, such as ..
growth factor, at the site of a wound. For example, when the ;:
present compounds and compositions are combined with growth factors prior to biodeli~ery as described herein, the 30 comp~sitions and compounds slowly release this growth factor into the immediat~ vicinity o~ the wound, thereby accelerating the wound healing process. It is contemplated that all growth ~actors known to accelerate or facilitate wound healing are WO 93~0g790 2 1 2 1l ~ 5 7 PCI/US92/Oq7~4 usa~ale in the pres~nt compositions and me~hods. Growth factors suitable for this acceleratioll of wound healing inclusle those lis~ed in Table I 1 as w~ll a~ brain endothelial cell gro~h f actsr and r@tina-derlv~d growth f actor . As describ~d above, 5 heparin binding growth ~actors c:an be used to ef f ec:t the repair o~ both soft and har~? ti~;sue. 'rhe potential uses for interferons/ interleukin~;, and tissu~ growth factors are well known in the art.
The invention also relates to methods f or the 10 therapeutic admirlistration of polyanionic saccharide d~ri~ati~res , or complexe~ ther~of, with a protein f actor , wherein the sacch~ride derivative is combined with or comprises a portiorl of a biocc)mpatible porous solid. Tha phrase, "biocompatible porc:us solidl' as used herein means a solid which 15 may be applied or admini~;tered to a mamxnal without provoking a substantial inf lammatory respon~;e or s:~her ~;ubs~antia3 l advers ef f ect . Such biocompatible porous sol ids include membranes such a~ collagen-based polymeric: m~mbrane , amniotic membra~es, and omentum membranes (re~,riewed in Cobb, 198~, Eur. ~. Clin.
20 In~estig. 18:32} 326). The polyanionic saccharide derivatives may be immobilized on such membranes .in a preferred embodiment by contacting the derivatized saccharide with electrostatic binding partners on th~ membrane. ~iocompatible porous solids may also include polymers of ethylene vinyl acetate,`
25 methylcellu~o~e, silicone rubber, polyurethane rubber, polyvinyl chloride, polymethylacrylate, polyhydroxyethylacrylate, polyethylene terephthalate, polypropylene, polytetrafluoroethylene, polyethylene, polyfluoroethylene, propylene, cellulose acetate, cellulose and polyvinyl alcohol 30 (reviewed in Hoffman, Synthetic Polymeric Biomateri~ls in Polymeric Materials and Arti~icial Or~ans, ACS Symposium Series #256/ (G. Gebelein~ ed.) 19~B). In preferred ~mbodiments, the cyclodextrin startiny materials are co~polymerized with monomers WO ~3/09790 2 1 2 ~! 8 5 7 PCr/U~i92/09754 of the biocompatible polymer material of the f inal prsduct composition, ~o as to create a porous cc)-polymer. This co~
polymer is subsaquently reacted c:hemic:ally to provide the saccharide portion with the ~nionic substituents required by 5 this invention. Cyclodextrins can be coupled with reactive groups , such as amine , amidQ , carboxylate end yroups , etc ., contained in the biocompatible polymer and then subsequently derivatized with ioni :: substitllents . ~fore preferably tlhe polysacc:haride, such as a cyclodextrin is introduced as a co-10 reagent in a monomer formulation to be polymerized to a ~olidpolymer or co-polymer 9 and the product i5 contac:ted subse~uently with suitable agents to derivatize the saccharide component to add aniollic substituents to the degree taught by this invention.
Particularly advantageous f or suc:h process and products are 15 those methods that will produce a pol~er or co polymer example of a f lat polymer product of polyamide polymer, manufactured by 3M Corpora~ion, and us~d as a bio-compatible patch or dressirlg on wounds. This biocompati3:~1e patch or dressing i~; designed to physically protect a wound from invasion of pathogens, and y~t 20 to have sufficiRnt porosity to allow passage of moiskure, air~
etc. Applicants' invention contemplates the coupling of the acti~e polyanionic poly~accharide with a carrier comprising such polymer, or, the coupling of the active anionic saocharide and a proteinic factor together with a polymeric carrier. Such 25 combination is designed expressly for applications of deliberate promotion or inhibitio~ of cellular growth processes. The HB~s bind to the immobllized, derivatized saccharide-based molecules, either incorporated into or alr~ady present in biomembranes.
Biological membranes such as omentum and amnion are well known 30 in the.art as wound dressings. Collagen based synthetic biomembranes are being used in the treatment of burns. The presence of derivatize~ saccharide of the present invention in natural membranes such as amnion and the ability of these WO93~0s790 PCT/U~92/~754 212~57 - 30 - `

derivatives to bind collagen which is used as a base for synthetic membranes will allow such biomembranes, when combined with the compositions of ~he present inven~ion~ to b~ used as ::
novel delivery vehicles for HBGFso A. R~te~0~8 Arteriosclero-~is is a disorder involving thickening and hardening of the wall portions of the larger arteries of mammals, and is l~rgely re~ponsible for coronary artery disease, ~:
aortic aneurisms and arterial dis~ases of the lower extremitiesO
10 Arteriosclerosis also plays a major r~le in cerebral vascular disease.
Angioplasty has heretofore been a widely us~d meth~d for treating arteriosclerosis. For example, percutaneous transluminal c~ronary angioplasty (herPinafter 'iPTC~') was 15 performed over 200~000 times in the Uni~ed States alone during 198~. PTCA procedures involva inserting a deflated balloon cath~ter through the skin and into the v~s~el or artery containing the stenosis. The cath~t~r is then passed through ~he lumen o~ he vessel until it reaches the stenotic region, 20 which is cha~acterized by a build up of fatt~ streak~, fibrous plaques and complicated lesions on the vessel wall, which result in a narrowing of the vessel and blood flow r~striction. In order to overcome the harmful narrowing of the artery caused by ::
the arteriosclerotic condition, the balloon is inflated, thus 25 flattening the plaque against the arterial wall and otherwise : expanding the arterial lumBn~ ~
AlthQugh PTCA has producPd excellent results and low ;:
complication rates, there has, however, b~en diffi~ulties associated with the use of this technique. In particular, the ~;
30 arterial wall being enlarged frequently experiences damage and injury during expansisn of the balloon against th~ arterial wall. While this damage itself is not believ2d to be paxticularly harmful to the health or the life of the patient, . WO 93/~979û PCI`/lJS9~/Og754 2~ 24 ~ 7 the healing response triggered by this damage can cause a reoccurrence of the arterio~;clerotiG condition. In particular, it has been observed that the smooth musc:le cells as~;ociated with the stenotic region of the artery initiate cell di~ision in 5 response ~o direct or inflammatory injury of the artery~ As the smooth mu~;cle cells prolif~rate and migrate into the internal layer o~ the artery, th~y catlse thickening of the art~rial wall.
Initially, this thickening is clue to the increased mlmber of smooth muscle cells. Subsequ~ntly, however, further thickening 10 of he arterial wall and narrowing of the lumen is due to increa~;ed srnooth mus~:le cell volume and accumulati on of ex-tracellular matrix and connecti~ve tissue. rhis thic:kenirlg of ~he cell wall and narrowing of the lumen following treatment of arteriosclerosis is ref~rred to herein as restenosis.
Although applicants do not wish to be bound by any theory or theories for the basis of restenosi~;, it ic: believed that restenosis is due in part to the presence of growth factors procluced by injured endothelium which actis~ate excessive proliferation of the smooth muscle cells which are exposed to 20 ~:he endothelial injury. Accordingly, applicants have found that the present saccharide derivatives, when substanti~lly free of growth factors prior to biodelivery, are extremely effective for preventing or at lea~t substantially reducing intima~ thickening following b~lloon angioplasty. By virtue of their affini~y for 25 gr~wth factors, such compo-eitions can provide an in vivo absorption or reduction of the local concentration and/or di~fusion of such growth factors. That is, such wound site growth factor5, whether they are produced by the cells at the ~`
wound site or are otherwise in the bloodstr~am, can be taken up 30 by the present saccharide derivatives, thereby reducing the restenoic effect of such materials on the wounded tissue.
~ ccording to the present methods, mammals, including humans, which have arterial reqions subject to angioplasty, are WO 93/09790 ~I '2 4 ~) 5 ~ PCI/US92/0~754 -- 3z ~

treated ~y administering to ~he mammal a polyanionic saccharide derivative of the pr~sent invention in an araount ef f ective ~o inhibit arterial smooth muscle cell prolif~ratiorl. It is contempla~ed that the degree of restenosis inhibition may vary 5 within the scope hereof, d~pending upon such f ac:tors as the patient being treat~d and the extent of art~3rial injury during angic:~plasty . It is generally pref erred, however, l~hat the saccharide derivative be administered in an amount ef f ective to c:ause a subs~antial reduc:tion in restenosis. A.s the term is 10 u ed herein, sub~;tantial r~duction in rest~no~is means a post treatment restenosis v2l1ue of no greater than about 50g~.
According to pref erred embodiments, the post treatment ~estsnosis value is no greater than abollk 25~6. As the term is used herein, post treatment restenosis vaIue ref ers to the 15 restenosis valu. measured at a~u-t one month after angioplasty.
The term restenosis value refers ~o the res~enosi~; rate calculated as a loss of greater than or equal to 50% of the initial gain in mirlimum lumen diameter achieved by angioplasty. ~ ~:
Thus, the present invention contempla~es a method of 2 0 inhibiting restenosis in a patien1 which comprises administering ;~
to the patient an amount of a ~;accharide based derivative ef~ectiYe t~ inhibit formation of a restenotic lesion in a patient who has undergone an~iop~asty. It is contemplated that ~:
: the saccharide derivative may be administèred before, during 25 and~or after angioplasty treatment of the stenosed artery. It i~ generally preferred that the administration comprise administering the compound locally at the wound site. In preferred embodiments, local administration comprises infusing the saccharid~ derivative directly into the injured tissue. In 30 the case of reskenosis, such step prPferably comprises infusing the compound directly into the arterial wall at the site of the angioplasty.
Applicants have surprisingly found that particularly ~ W093/0~790 2 1 2 ~ 8 ~ 7 PCT/US92fO97~

beneficial antirestenoic results are obtained for embodiments in which the step of administering the saccharide derivative also co~prises the step of dilatin~ the vessel lumen to effect angioplasty. For example, applicants have found that a 5 preferred administration step comprises infusing an aqueous suspension or dispersion of saccharide derivative directly into the arterial wall at tha site of balloon angiopla ty. This is preferably accomplish~d using a modified infusion balloon catheter having a plurality of holes in the wall oX the balloon 10 portion of the catheter. These holes are configured and ~ized to allow the balloon to be both inflated an~ to leak the inflation solu~ion through thP wall of the balloon. ~ccording t,o preferred embodiments, the balloon is inflated under relati~ely low pre~suxe conditions, such as 2 3 atmospheres.
15 Examples of por~us balloon cathelers which may b~ used t~ apply the oompositions of the pre~ent invention are made by U.S.C.I.-Bard and Schneider. Ralloons of this type are referred to as Wolinsky balloons or "Rweating balloons~" It is anticipated that a variety of infusion angioplasty balloon catheters may be -~
:20 used for application of the compcsitions of the present invention and that one skilled in the art would be readily able to determine w~ich types o~ balloon infusion catheters would be appropriate. Another technique which involves the lo al administration of the saccharide derivatives of ~he present 25 in~ention utilizes bîoabsorbable intravascular stents. The ~:;
s~cc~arides of the present invention, particularly the cyclodextrin polymer derivatives may be incorporated into a bioabsorable stent and that stent positioned a~ or near the site of tissue damage.
It will be appreciated by those skilled in the art that the particular characteristics and properties of the suspension containing the saccharide derivative may vary widely :~
depending upon numerous factors not necessarily related to the ~-WOg3/09790 2 ~ 2 ~ ~ 5 7 PCT/U~2/0~754 present invention. However, the administration step preferably comprises infusing an aqu~ous suspension or dispersion of polyanionic saccharide derivate particl~, and preferably a su~pension of sulfated b~ta-cyclodextrin polymer particles, 5 ranging in siz~ from about l to 600 microns directly into the arterial wall at ~he site o~ balloon angioplasty. Applicants believe that such parti~ instilled in~o the arterial wall will remain present at the site of applization for several days, in sufficien~ quantity to resul~ in an inhibition of restenosîs.
The aqueous suspension comprises a aqueous carrier of physiological salinity and an active saccharide deri~ative. The active saccharide derivative is preferably present in an amount r~nging from about l to about 30% by weight, and even more preferably from about 5 to about 15% by weight of the 15 composition. In preferred embodimen~s, deri~atized saccharid~s, and preferably cy~lodextrin sulfate polymer particles, are applied at about the time of angioplasty.
In some instances it ma~y be ~esirable to prevent restenosis but allow angiogenesis. To meet these requirements 20 it is preferred to use a dispersion of an Al or Ba salt of a polyanionic saccharide derivative, and even more preferably an Al or Ba salt o~ a poly ~ulfated beta-cyclodextrin. If it i5 desired to allow the normal progression of anglogenesis a~ the vascular injury site while simultaneously inhibiting restenosis, 25 it is preferred to use sucralfate, an aluminum salt of sucrose octasulfate available from the Marian Merrill Dow Comp~ny, Kansas City, M0. ~:
B. I~hibition o~ I~timal Thi~e~ing of Vei~ ~raft~
Venous segments are frequently harvested at the time 30 of surgery and used as bypass grafts t~ treat vascular occlusive disorders. Specifically, they ha~e been used in the coronary, renal, femoral and popliteal arterial circulations, by way of example. One major limitation of this form of therapy is that :~

W093/09790 2 1 2 4 ~ ~ 7 PCT/US92J097~

intimal thic~enlng occurs whlch compromises the luminal cross-sectional area and results in reduced flow. This frequently, but not exclusively occurs at the anastomosis. Applicants propose that the placement of ~-cyclodextrin tetrade~asulfate S polymeric particles in the perivascular space at th~ time of surg~ry, will substantially limit the ingrowth of smooth mu~le cells into ~he intima and will improve the long term success of these graftsO
c. a~giogQ~i8 lo Angiogene~is is the formation of new blood vessels.
Angiogenic stimuli cause the elong~ion and proliferation of endothelial cells and the generation of new blood ves~els. A
~mber o* the HBGFs are known to promote angiogenesis. The new blood vessels produced by angiogenesis resu~t in 15 neovascularization of tissue.
There are a variety of diseases associat d with deficient blood supply to tissue and organs. A deficiency of thi~ kind~ known as ischaemia, may be due to the functional constriction or actual obstruction of a blood vessel~ These 20 diseases can be grouped into cardiac, cerebr~l and peripheral ischemic diseases. Cardiac ischa~mia may result in chronic angina or acute myocardial infarction. Cerebral ischaemia m~y result in a stroke. Peripheral ischaemia may re~ult in a number of diseases including arterial embolism and frostbite. In 25 severe cases of p~ripheral ischaemia, necrosis of the tissues supplied by the occluded blood vessels necessitates amputation. ~:~
To oYQxcome ischaemia, an alternative blood supply to the affec~ed tissue must be established.
According to preferred embodiments, angiogenesis is ;~
30 promoted by first contacting a saccharide derivative of the present invention with growth factor(s) and then administering the compositi~n locally to the location of the ischemic tissue, by hypode~mic injection for example, to promote angiogenesis and W~93/Og790 1 2 ~ ~ S 7 PCT/U~92/0~754 the formation of collateral blood vessels. ~s the term is used herein, colla~eral ~lood ve~ Qls are blood vessels which are absent under normal physiological condition~ but develop in respon ~ to appropriate s~imuli~ such as th~ presence of HB&Fs.
5 It is anticipated that a~ministration of compositions whic~
include saccharide derivative and growth factor will result i.n the formation of c:ollatQral blood v~ssels and reva~cularization o~ ischemi :: tissue.
In preferred embodiments, angiogenesis is promoted by 10 methods in which the saccharide derivative comprises a highly anionic cyclodextrin derivatiye or a salt form of same~ and even more pref~rably a polysulfated polymer or copolymer of a ~yclodextrin. It is preferred ~hat the cyclodextrin derivative be combined with basic fibroblast growth factor at a 15 ~yclodextrin:basic fibrobla~t growth factor weight ratio of from about 10:1 to 100:1.
~ Ti~su~ ~d org~ r~ft~ or TE~P1a~ ~ ~:
As described above, HBGFs are known to stimulate neovascularization and endothelial cell growth. In transplan ~0 tation, the graft re~resents a wound~ and success of the :~
gxafting procedure depends critically on the rapidity of establishing an adequate blood supply to the grafted or ~ransplanted tissue. Thus 9 we envision the application of the compositions of the present invention combined with growth 25 factor(s) at the site of the gra~t to.promote the establishment of an adequate blood supply to the grafted or transplanted tissue. The growth factor-containing compositions may be coated on the surfaces to be joined, sprayed on the surfaces, or applied in the form of an aqueous suspension with or without 30 viscosity enhancers such as glycerol. Xn addition, the organ or tissue to be grafted or tran~planted may be presoaked in a tr2ating solution containing the compositions of the present invention, prior to transplantation. The compositions of the . W~93/Og790 2 1 2 ~ ~ ~ 7 PCT/US9~0g754 present invention may also be injected into the transplant site or surface of both items to be joined.
In a preferred method for pr~.paring the compositions used in treating grafted or transplan~ed ~issue and organs, the 5 saccharide derivatives of the present invention are precontActed with growth factor cont~ining organic ~ources (e.g.~ tis~u~ or organ debris, ground mattar, ~r liquid extrac~) so as to extract th~ growth factors pres~nt in these sources. In highly preferred methods, ~he organic source used for contact is about lO 10 to about 100 times greater in volume than the transplanted or grafted tissue to be ~reated by the composi~ionO A mnre direct and often more economic method will involve contacting the ~accharide derivatives of the pr~sent invention with growth ~.
factor substances created by recombinant biochemical and 15 biotechnological procedures. In this manner specific growth factor prQteins are more readily chosen for a contempla~ed therapeutic application.
E~ Bo~ Gr~fti~g a~d Tr~a~pla~tatio~
The response of bone to injuries such as fractures, 20 infe~tion and interruption of blood supply is relatively limited. In ord~r for the damaged bone tissUe to heal, dead bone must be resorbed and new bone must be formed, a process carried out in association with new blood vessels growing into the involved area. HBGFs can in~uce n~ovascularization arld ~he 25 pr~liferation of bone forming cells. It is therefore contemplated to use th present eompounds in combination with gro~th factor ~or the purposes of aiding the healing of bone fractures, the joining of implanted and host bone, and the ;~
mineralization of bone (where such is intended).
In preferred embodiments, the present saccharide derivati~es are combined with growth factors and powdPred bone substance and~or finely dispersed demineralized bone matter t~
form a paste. Suitable meth~ds for preparation of such a paste W~ 93/09790 2 1 2 ~ ~ 5 7 P~IU~92~09754 are present~3d in Repair of Major Cranlo-Orbital Defects with an Elastomer Coated ~sh and Autoger~ous Bone Paste, Mutaz B. Habal et al ., 61 : 3 , ~lastic and ~ec:or~ ruc~ive ~urgery, 394 , 396 (1978). The bone tissue used to produce the paste may be 5 obtained :~rom iliac:: crest or c:alvarium . It is pref erred t~ use autogenou~ bone for implant purposes ~and to use partially demin~ralized bone over fully d~mineralized bone pow~er.
Demineralized bone powder ob~ained from allogenic and x~nogeneic sources may be u~;ed in preparing the bone powder. To make a 10 soft paste absorbable cellulose cotton or similar rnaterial may ~-be used. Although applicarlts do no~ wish to be bound by any theory or theox ie~, it is thought tha~ the bone paste produced these methods functions as an induc:tion matrix from which new bone will form after being invaded with a network of blood ~:.
15 vessels. The paste is applied to the surfac:e~ of bone to be j oined in implant procedures or used to f ill ~ractures of contuur bone to be repaired.
F ~ Ski~ ~loe~ ~li~g One debilitating disordl~r affecting millions of people 20 including, but limited to the aged, paraplegics, trauma vi~tims, and ~ia~etics are cutanes~us nonhealing ~;kin ulcers or d~cubiti.
In many cases, inadequate blood supply to the damaged tissue prevents the deli~rery of adequate nutrients for healing. It is an~icipa~ed that the application of poly~eric beads of 25 cyclo~extrin derivatives, preabsorbed with combinations of compounds such as epidermal growth factor and basi~ fibroblast ~:
growth factor, to the ulcer directly, will lead to increased angiogenesis, improved blood supply, increased keratinocyte ingrowth, and faster ulcer closure and healing.
~ ~er~tologicæl Applic~tio~s The control of blood vessel growth is an important aspect of normal and of pathological states encountered in derma~ology. In particular, the abnormal growth o~ cellular W~ 93/09790 2 1 2 4 ~ S 7 Pt~/US92/097S4 materials and vessels accomparlies several pathological sates, psoriasis being one prominent example. In many cases excesses of growth stimulating protein f actors are involve~ .
Abnormalities of this type are often associated with imbalanc~s 5 in proteirlic growth f actors For example, in th~ c:aæe of patients with cutaneous mastocytosis, extrac:t~ fro~ involYed ~;kin had 15-fold higher lev~ls of chymotryptic aativity than extr~cts of uninvolved skin or from ::ontrol samples of patients without such def ic:iency .
lû t See Human Skin Chymotryptic Proteas~, N . M~ Schechter, J . E.
Fraki , J . C Geesin , G . S 0 Lazarus , J . Biol . Chem ., 258 , 2973-2978, 1983. The Chymase Involved Is a Heparin Bin~ing Fac:t~r ~SPe S. Sayam~, R. V. Iozzo, G. S. Lazarus, No M. Schec:hter, Human Skin Chymotrypsin-like Proteinase Chymase, J. Biol. Chem.
15 262, 6808-6815, 1987. It appear~; that the ch~notrypsin like proteases car- degrade the epidermal junc:tion and can r~sult in ~-epide:n~al-dermal separation ( Se~ Sayama et al . above~ O
Another example of a growth promo~ing factor in~ olved in dermal abnormalcies is epid~rmal plasminogen activator, which 2 0 is elevated in a variety of dermal pathologies ( See Epidermal Pl asminogen Activat4r is Abnormal in Cutaneous Lesions ' ~ P . J .
Jensen et al., J. Invest. Dermat. 9Q-777-782, 19 88).
Certain embodiments of this invention, namely highly sulfated solid dispersions or o~her physical variants of highly 25 sulfated polysaccharides, and preferably those ~omprising : cyclodex~rin structures, are particularly amenable to dermal th~rapy in those cases where excess growth of cellular components is involved. In such case the agents of the present invention can be introduced at or near the tissue involved.
30 This may be accomplished by cutaneous or sub-cutaneous injection of fine par~icle dispersion of the agent, or the implantation of solid polymer shapes suitably shaped for effertive contact, or the agent may be comprised in material such as patches, or other W093/09790 7 PcT~usg2/n~754 suitable forms of externally applied materials containing agents of the invention.
It will be under~tood that depending on the pathology and diseas~ condition, the application of the agents ~f this 5 in~ention without pre~contacting with proteinic ~rowth factor is contemplated. This will be the case in conditions as exemplified above, where it is intended to reduce any growth promoting factor or f~ctor~.
In other cas~s o~ dermal damage or disease, and in 10 certain phases of ~reatment, i~ may be desirable to use the combined proteinic factors. This would be the case in connec~ion with healing processes where angiogenesis, that is ~he establi~hment of new and added blood supplies are desired.
EXAMP~LES
Th~ following examples are provided ~o illustrate this invention~ ~owever, they are no~ to be construed as necessarily limiting the scope of the invention, which scope is determined by th~ appended claims. All amounts and proportions shown are by weight unless explicitly tated to be otherwise.
EX~MPLE 1 PREPARATION OF SULFATED BETA-CYCLODEXTRIN~POLYMER
Beta cyclodextrin polymer beads (American Maize Products) of 20-60 mesh particle size were deri~atized to ~orm a novel i ~ obilized CD polymer sulfate derivative according to the 25 present inven~ion. The composition approaches a degr~e Qf sulfation of nearly two sulfates per glucose ring of the eD
pol~mer. About 0.4 g of carefully dried polymer were reacted with about 1~7 g of 6 trimethylammonium sulfur trioxide compl~x (Aldrich3 in about 100 ml of dried dimethylformamide (DMF), with 30 mild agitation at about 62 to 72C for 3 to 4 days. The solids were washed in DMF, reacted with 30% aqueous sodium acetate for 24 hours, and washed and stored in distilled water. The sulfur content of the produ~t was a~out 14.7 wt.%. This compares . W093/0979~ 2 ~ 2 4 8 ~ ~ PCT/US92/09754 favDrably to the value of 17.5% if the polymer mass were composed 100% of ,B-cyclodextrin tetradecasulfate without cross linking components, and all glucose hydroxyl uni~s were sterically available (which cannot be expected for the polymer~.
. EXAMPLE 2 - PREPARATIO~_~F~ DERIV~TIVES~OF CYCLOD~XTRIN
(A) ~ CD-TDS~Na).
~ cyclod~xtrin ~9~ pu~e dihydra~e) was purchased from Chemalog (a division of General Dynamics Corp.)~ South 10 Plainfield, NJ.
About 5.0 grams of ~ cyclodextrin ~about 4.4 mmoles, i.e., about 92 meq) -OH) was di~solved in about ~50 ml of di~methyl-formamide (DMF). To this solu~ion was~added about 15 grams of (CH3)3N~SO3 (about 108 mmolesj in a single portion and 15 the reaction mixture was heated to about 70~C. After two hours ~:
at a~out 70~C, a gu ~ y material ~egan to precipitate. The reaction mixture was maintained at 70C with vigor~us stirring, and then cooled to room tempe~ature. The ~F layer was then decanted and discarded, and the solid residue was dissolved in 20 about 250 ml of water followed by addition of abou~ 75 ml of 30%
sodium acetate. The mixture was stirred viqorously for 4 hours and then poured into about 4000 ml of ethanol. After standing overnight r the mixture was filtered to recover ~he crystallized solids. The filter cake was washed with ethan~l (absolute~
25 followed by diethyl ether. The product was ~hen dried under vacuum over P2O5. About 10.3 grams of white powder was recovered. The product was hygroscopic.
The product was analyzed under conditions such that sorption of water was minimized. Elemental analysis gave the 30 following results: C=18.84, H=2.65~ S=17.33 (Calculated for C6H8OI~S2N~2; C=19.67, H=2.19, SQ17~49)~ ~]D22=750 (C=~63 in 0.S M
NaCl). The analysis corresponds to that expected for an average substitution of two hydroxyl groups for each glucopyranose unit, WO 93/û9790 PClr/US92~Q~754 212~857 42 ;. `

i . e. 14 hydroxyls per CD molecules . The calculated yield for such ,B-CD-TDS salt is 10. 96 grams, about 6~6 higher than the observed 10 . 3 gram~
and ~y-CD-S (Na sa lt ):
The procedur~ dQscrihed above was used f or these preparations except that about 8 6 ~noles of CH3N-S03 was used with ,B-CD, and about 117 ~oles with the ~ D.
'rhe sulfaLted ~-CD alt analyzed C=~ . 76; H=2 . 60;
S=16 . 22 . This corresponds on average to a substitution of about 10 11. 7 hydroxyl uni~s per ,B~C~ molecule.
The sulfated ~y; CD salt analyzed C=18 . 92; ~-2 . ~9;
S--14 ~ 84 . This corresponds on average to a substîtution of ~bout 1~ hydroxyl groups per ~y-CD molecule. ~
(C) ,B CD-S04 (Na salt) ~7 . ~ wtg~) S): ;
About 1. 0 gm o~ ~-cyclodextrin was dissolved into about 50 ml of DMF. TQ thi~ solution was added about 883 mg o~
~CH3N S03(7.2 equivalents). The -solution was held at about 75C
for about 12 hours, at which time no precipitate had formed.
The reaction mixture was cooled t:o room temperature. TG the 20 solutiorl was added about 200 ml of ethanol. The resulting cs:~lloidal solution was then poured into about ~00 ml of diethyl ether. A white solid fo~ned in 2 :hours. The solid was collected by ~iltration and then was dissolved in about 3 0 ml H20. This solution was stirred for 2 hours. After stirrin~ he 25 solution was poured into about 900 ml oP 2 :1 EtOH-Et20 solution.
Crystals formed s:~ver an 8 hour period. The crys~als were collec:ted and washed with E~O. The product was dried over P
under vacuum. About 1.18 gm o~ powder was recovered~ ~72 . 4%
yield~. :
Elemental analysis of the product showed C=32 . 49;
H=~ . 99; and S=7 . 06. This corresponds on average to a .~
substitution of a;bout 3 . 5 hydroxyls per ~-CD molecule. ~ .
(D) ~-CD-Propoxylate - 14 504 ~

`~' ' :'.' W0~3J09790 2 1 2 4 8 5 7 P~T~USg2~9754 ~-CD-~hydroxy~n-propyl ether~ was obtained from American Maize-Products Co. ~Ha~m~nd, IN) and the procedure d~scribed above was us~d to prepare the sulfate salt, ~-CD-~-4Pr- 14 S04)>

~ uman recombinant ba~ic fibroblast growth ~actor (bFGF3 wa~ pr~ided by Takeda Chemical Industries, Ltd. It was purified from E. coli as previously d~scribed (Kurokawa et al., lo 1~87, FEBS. Lett~rs 213:1~9-194 and Iwane et al., 1987, Biochem.
Biophys. Res, Commun. 146:470~477).
Rat ~hondrosarGoma-derived growth fac~or ~ChDGF) was i~ola~ed from the tran~plantable tumor as previously described (Shing et al., ~984, Science 223:1296-~298). About one hundred 15 ml of the crude extract prepared by collagenase digastion of the ~umor was diluted (1:1) with about 0.6 M NaCl i~ about 10 mM
Tris, pH 7 and loaded direc~ed onto a heparin-Sepharose0 colu~n (1~5 x 9 cm~ pre-equilibrated with the same buffer. The column was rin ed with about 100 ml of about 0.6 M NaCl in about 10 mM
20 Tris, pH 7. ChDG~ was subsequently eluted with about 18 ml of about ~ M NaCl in about 10 mM Tri~, pH7. ~.

BETA-CYCLODEXTRIN AFF~NITY CHROMATOGRAPHY OF FGF
The insoluble sulfated beta-cyclodextrin pol~mer 2~5: (abou~ 0.~ ml bed volume), was incubated with a~out 0.5 ml of about Q.1 M NaCl, about 10 mM Tris, about pH 7 containing about 1,000 units of human recombinant bFGF at about 4~C for abou~ 1 hour with mixin~. Subsequently, the polymer was rinsed stepwise with about 2 ml each of about 0.1, 0.6, and 2 M NaCl in about 10 30 mM Tris, pH 7. All fractions eluted from the polymer were assayed for growth factor activity.
EXAMPLE 5 ~:
GROWTH ~ r___ SSAY

W093/09790 2 1 2 ~ 8 5 7 PCT/US92/0~ ~

Growth factor activity was assessed by measuring the incorporation of [3H]thymidine into the ~NA of quiescent, ::onfluent monolayers of BALB/c mou~e 3T3 c~lls in 96~well plates. One unit of artivity was defin~d as the amount of 5 grow~h factor required ~o stimulate hal~-maximal DNA synthesis in 3T3 cells (about 10,000 cells/0.25 ml of growth medium/well).
For determination of sp~cific activities, protein concentrations of the crude ~xtract and the active ~raction elut~d from heparin-Sepharose column were determined by the method of Lowry 10 et alO (1952, ~. Biol~ Chem. 193:265-275). Protein concentration~ of the pure growth factor were e~timated by comparing the intensities of silver-stained polypeptide bands o~
~_SDS-polyacrylamide gel to those of the m~le~ular wei~ht markers.

AFFINITY OF FIB~OBL~ST GROWTH FACTOR FOR
~3=5ySLDre~5~o~Ll~3'RADECASULFATE POLYMER
Human recombinant bFGF (about ~000 units) was incubated with sulfated beta-cyclodextrin polymer. The polymer was subsequently eluted stepwise with about 0.l M, 0~6 M, and 2 ~0 M NaC1. The results are shown in Figure 3.
While most of the growt~ factor actiYity remained ~ound to the polymer at about 0.6 M NaCl, about 230 units of th~
acti~ity was recovered ~hen eluted with about 2 M NaCl. Th~se results indicate that basic fibroblast growth factor has a very 25 strong affinity for beta-cyclodextrin tetradecasulfate and is at least comparable to that of FGF for heparin. The activity peak : was analyzed by SDS polyacrylamide gel electrophoresis followed by a silver ~tain. Lane 2 in figure 4 shows the polypeptide band of basic fibroblast growth factor.
The affinities of heparin and beta-cyclodextrin tetradecasulfate for chondrosarcoma derived growth factor were also tested. Chondrosarcoma extracts which cuntained about 500 units of growth factor activity were incubated individually with WO 93/0~790 2 ~ 2 ~ 8 ~ 7 P~r/US92/09754 heparin-Sepharose0 and beta-c:yclodextrin tetradecasulfate polymer. The beads were subsequently ~lu~ed stepwise with about 0.1 M, O ~ 6 M, and about 2 M ~aCl . The r~;ults are shown in Figure 5. ~pproximately 32~6 and ~8% of tha total ac:tivity was - 5 reco~ered at ~ M NaCl with h~parin Sepharose~ and beta-c:yclod~xtrin etradeca~ulf ate polymer ~ respectiYely :
',, "

Claims (46)

What is claimed is:

1. A composition for affecting the growth of living tissue in mammals comprising a polyanionic saccharide derivative having by a body temperature solubility of less than about 15 grams/100 ml of distilled water and a physiologically acceptable carrier for the saccharide derivative.

2. The composition of claim 1 further comprising a heparin binding growth factor.

3. The composition of claim 1 wherein said saccharide derivative has on average at least about 1.4 anionic substituents per sugar unit.

4. The composition of claim 3 wherein said saccharide derivative has oil average from about 1.4 to about 4 anionic substituents per sugar unit.

5. The composition of claim 3 wherein said saccharide derivative is a compound consisting of n sugar units and R anionic substituents such that when:
n = 2 to 3; average anionic R per n unit a or > 3.5 n = 4 to 5; average anionic: R per n unit = or > 20.
n = > 6; average anionic R per n unit = or > 1.4 .

6. The composition of claim 1 wherein said saccharide derivative comprises polyanionic cyclodextrin derivative.

7. The composition of claim 6 wherein said cyclodextrin derivative comprises compounds of the formula:
wherein at least two of said R groups per monomeric unit are:
selected from the group consisting of sulfate, phosphate, sulfonate and nitrate, and the remainder of said R groups, when present, are nonanionic groups selected from the group consisting of H, alkyl, aryl, ester, ether, thioester, thioether and -COOH; and n is an integer from about 6 to about 12.

8. The composition of claim 6 wherein said cyclodextrin derivative is comprised of one or more cyclodextrin monomers having on average at least about 10 anionic substituents per monomer.

9. The composition of claim 8 wherein said monomer has on average from about 10 to about 24 anionic substituents per monomer.

10. The composition of claim 7 wherein said cyclodextrin derivative comprises cyclodextrin polymer.

11. The composition of claim 10 wherein said polymer is solid particulate dispersed or suspended in said carrier.

12. The composition of claim 6 wherein said cyclodextrin derivative comprises a salt of polyanionic alpha-, beta- or gamma-cyclodextrin.

13. The composition of Claim 12 wherein the cationic constituents of said salt are selected from the group consisting essentially of Mg, Al, Ca, La, Ce, Pb, Ba and combinations of two or more of these.

14. The composition of claim 1 wherein said derivative is substantially insoluble in water at body temperature.

15. The composition of claim 1 wherein at least a portion of said saccharide derivative is solid particulate dispersed or suspended in said carrier.

16. A method for inhibiting the pathological growth of smooth muscle cells in a tissue of a mammal comprising administering locally to said tissue a polyanionic saccharide derivative in an amount effective to inhibit said pathological growth, said derivative having a solubility at body temperature of less than about 15 grams/100 ml of water.

17. The method of claim 16 wherein said derivative is a cyclodextrin derivative.

18. The method of claim 17 wherein the anionic substituents of said cyclodextrin derivative are selected from the group consisting essentially of sulfate, sulfonate, phosphate, nitrate and combinations of two or more of these.

19. The method of claim 18 wherein said cyclodextrin derivative comprises a salt of said polyanionic cyclodextrin.

20. The method of claim 19 wherein said derivative is combined with a non-toxic pharmaceutically acceptable carrier of physiological salinity.

21. The method of claim 16 wherein said derivative comprises a sulfated derivative of beta-cyclodextrin polymer.

22. The method of claim 16 wherein said derivative comprises solid particulate suspended or dispersed in a physiologically acceptable carrier, said suspension or dispersion comprising about 1 to about 30% by weight of said derivative.

23. The method of claim 22 wherein said suspension or dispersion comprises about 5 to about 15% by weight of said derivative.

24. The method of claim 16 wherein said derivative is combined with growth factor.

25. The method of claim 16 wherein said local administration comprises infusing the saccharide derivative directly into the tissue.

26. The method of claim 16 wherein said local administration comprises infusing an aqueous suspension or dispersion of said saccharide derivative directly into the tissue using an infusion balloon catheter having a plurality of i holes in the wall of the balloon portion thereof.

27. A method for promoting angiogenesis in mammals comprising administering locally to a tissue to be treated a polyanionic saccharide derivative of a cyclodextrin combined with growth factor, said derivative being characterized by a solubility at body temperature of less than about 15 grams/100 ml of distilled water.

28. The method of claim 27 wherein said derivative is a cyclodextrin derivative.

29. The method of claim 28 wherein the anionic substituents of said cyclodextrin derivative are selected from the group consisting essentially of sulfate, sulfonate, phosphate, nitrate and combinations of two or more of these.

30. The method of claim 29 wherein said cyclodextrin derivative comprises a salt of said polyanionic cyclodextrin.

31. The method of claim 30 wherein said derivative is combined with a non-toxic pharmaceutically acceptable carrier of physiological salinity.

32. The method of claim 27 wherein said derivative comprises a sulfated derivative of beta-cyclodextrin polymer.

33. The method of claim 27 wherein said derivative comprises solid particulate suspended or dispersed in a physiologically acceptable carrier, said suspension or dispersion comprising about 1 to about 3 0% by weight of said derivative.

34. The method of claim 33 wherein said suspension or dispersion comprises about 5 to about 15% by weight of said derivative.

35. The method of claim 29 wherein said local administration comprises infusing the saccharide derivative directly into the tissue.

36,. The method of claim 27 wherein said local administration comprises infusing an aqueous suspension or dispersion of said saccharide derivative directly into the tissue using an infusion balloon catheter having a plurality of holes in the wall of the balloon portion thereof.

37. A method for treating transplanted tissue or organs in mammals comprising contacting the organ or tissue to be transplanted with a composition comprising polyanionic derivative of a cyclodextrin and growth factor, said composition being administered in an amount effective to promote acceptance of the transplanted material in the body, wherein said derivative has a solubility at body temperature of less than about 15 grams/100 ml of water.

38. The method of claim 37 wherein said contacting step comprises administering said derivative locally to the tissue or organ.

39. A method for treating damaged or transplanted bone comprising contacting bone tissue with a composition comprising a polyanionic: derivative of a cyclodextrin combined with a growth factor in an amount effective to promote the healing of said damaged or transplanted bone wherein said derivative has a solubility at body temperature of less than about 15 grams/100 ml of water.

40. The method of claim 39 wherein said composition further comprises bone tissue.

41. The method of claim 40 wherein said bone tissue comprises powdered bone.

42. The method of claim 40 wherein said bone tissue comprises finely dispersed demineralized bone.

43. The method of claim 40 wherein said composition comprises a paste.

44. The method of claim 40 wherein said bone tissue comprises autogenous bone.

45. The method of claim 40 wherein said bone tissue comprises xenogenus bone.

46. A method for preparing a compound suitable for affecting the growth of living tissue in mammals, comprising:
providing cyclodextrin polymer; and reacting said cyclodextrin polymer with an anionic derivatizing agent in the presence of dimethylformamide at a temperature of from about 62 to about 72°C for a period of about 3 to about 4 days to provide a polyanionic derivative of said cyclodextrin polymer in which the derivative is characterized by a solubility at body temperature of less than about 15 grams/100 ml of water.