Note: Descriptions are shown in the official language in which they were submitted.
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 '"
-- 2
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
3 --
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
-- 5
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~
7 --
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
-- 10 --
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
-- 13 --
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~
-- 14
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
-- 15 --
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
- 17 -
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~
-- 18 --
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
-- 19 --
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
-- 20 --
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
-- 21 --
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
-- 22 --
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
-- 23
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
-- 25 --
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
-- 27 --
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
-- 28 --
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
-- 29 --
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
-- 31 --
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~
- 33 -
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
- 34 -
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~
- 35 -
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
- 36 -
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
- 37 -
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
38 --
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
-- 39 --
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
- 40 -
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
- 41 -
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
- 43 -
~-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)>
EXAMPLE 3
~ 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. ~.
EXAMPLE 4
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~ ~
- 44 -
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.
EXAMPLE 6
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
-- 45 --
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
:
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