U.S. patent application number 11/638103 was filed with the patent office on 2007-08-30 for bioabsorbable implant of hyaluronic acid derivative for treatment of osteochondral and chondral defects.
This patent application is currently assigned to Anika Therapeutics, Inc.. Invention is credited to Carol A. Joth, Khalid K. Sadozai, Charles H. Sherwood.
Application Number | 20070202084 11/638103 |
Document ID | / |
Family ID | 37891972 |
Filed Date | 2007-08-30 |
United States Patent
Application |
20070202084 |
Kind Code |
A1 |
Sadozai; Khalid K. ; et
al. |
August 30, 2007 |
Bioabsorbable implant of hyaluronic acid derivative for treatment
of osteochondral and chondral defects
Abstract
A method for treating an osteochondral defect or a chondral
defect in a subject includes implanting a composite in a site of
the osteochondral or chondral defect. The composite includes a
hyaluronic acid derivative; and at least one member of the group
consisting of a cell, a cellular growth factor and a cellular
differentiation factor, which is impregnated in, or coupled to, the
hyaluronic acid derivative. In one embodiment, carboxyl
functionalities of the hyaluronic acid derivative are each
independently derivatized to include an N-acylurea or O-acyl
isourea, or both N-acylurea and O-acyl isourea. In another
embodiment, the hyaluronic acid derivative is prepared by reacting
an uncrosslinked hyaluronic acid with a biscarbodimide in the
presence of a pH buffer in a range of between about 4 and about 8.
The composite can be used for regenerating or stimulating
regeneration of meniscal tissues in a subject in need thereof.
Inventors: |
Sadozai; Khalid K.;
(Shrewsbury, MA) ; Joth; Carol A.; (Webster,
NY) ; Sherwood; Charles H.; (Sudbury, MA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Anika Therapeutics, Inc.
Woburn
MA
|
Family ID: |
37891972 |
Appl. No.: |
11/638103 |
Filed: |
December 13, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60751414 |
Dec 14, 2005 |
|
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60751237 |
Dec 14, 2005 |
|
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60751381 |
Dec 14, 2005 |
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Current U.S.
Class: |
424/93.7 ;
514/54 |
Current CPC
Class: |
A61K 2300/00 20130101;
A61K 31/573 20130101; A61K 2300/00 20130101; C08L 5/08 20130101;
A61K 2300/00 20130101; A61K 2300/00 20130101; C08L 5/08 20130101;
A61L 27/3843 20130101; A61K 35/32 20130101; A61L 27/48 20130101;
A61L 27/227 20130101; A61L 27/48 20130101; A61L 27/56 20130101;
A61K 31/728 20130101; A61K 35/32 20130101; A61K 31/729 20130101;
A61K 31/573 20130101; A61K 45/06 20130101; A61P 19/02 20180101;
A61K 9/06 20130101; A61L 2430/06 20130101; A61K 9/0024 20130101;
A61K 35/28 20130101; A61L 27/3817 20130101; C08L 5/08 20130101;
A61K 47/36 20130101; A61K 9/0019 20130101; A61L 27/20 20130101;
A61M 5/178 20130101; A61K 38/1841 20130101; A61L 27/52 20130101;
A61L 27/3834 20130101; A61K 31/728 20130101; A61K 38/1841 20130101;
A61L 27/20 20130101; A61K 35/28 20130101; A61K 2300/00
20130101 |
Class at
Publication: |
424/093.7 ;
514/054 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 35/12 20060101 A61K035/12; A61K 31/728 20060101
A61K031/728 |
Claims
1. A method for treating an osteochondral defect or a chondral
defect in a subject, comprising implanting a composite in a site of
the osteochondral or chondral defect, the composite including: a) a
hyaluronic acid derivative, wherein carboxyl functionalities of the
hyaluronic acid derivative are each independently derivatized to
include an N-acylurea or O-acyl isourea, or both N-acylurea and
O-acyl isourea; and b) at least one member of the group consisting
of a cell, a cellular growth factor and a cellular differentiation
factor, which is impregnated in, or coupled to, the hyaluronic acid
derivative.
2. The method of claim 1, wherein the composition includes at least
one member selected from mesenchymal stem cells, fibrochondrocytes,
osteochondrocytes, chondrocytes, TGF.beta. supergene family
members, tissue growth hormones, encoding genes thereof, and
synthetic peptide analogues thereof.
3. The method of claim 2, wherein the composite includes a
cartilage chondrocyte, osteochondrocyte or mesenchymal stem
cell.
4. The method of claim 1, further including the step of stabilizing
the composite within the site of the osteochondral or chondral
defect so that the composite does not move during the regeneration
or repair of the osteochondral or chondral defect.
5. The method of claim 1, wherein at least about 1% by mole of the
carboxyl functionalities have been derivatized.
6. The method of claim 5, wherein at least about 25% by mole of the
derivatized carboxyl functionalities are O-acyl isoureas and/or
N-acylureas.
7. The method of claim 5, wherein the hyaluronic acid derivative
includes at least one crosslink represented by the following
structural formula: HA'--U--R.sub.2--U--HA' wherein: each HA' is
the same or a different hyaluronic acid molecule; each U is
independently an optionally substituted O-acyl isourea or N-acyl
urea; and each R.sub.2 is independently a substituted or
unsubstituted hydrocarbylene group optionally interrupted by one or
more heteroatoms.
8. The method of claim 1, wherein the composite has interconnected
pores of sizes that can provide molecular cuing for the impregnated
or coupled cell to migrate through, or a path for migration of the
impregnated or coupled cellular growth or differentiation
factor.
9. The method of claim 1, wherein the composite further includes a
biocompatible, biodegradable support, wherein the hyaluronic acid
derivative is at the support.
10. The method of claim 9, wherein the support includes at least
one member selected from the group consisting of crosslinked
alginates, gelatin, collagen, crosslinked collagen, collagen
derivatives, crosslinked hyaluronic acid, chitosan, chitosan
derivatives, cellulose and derivatives thereof, dextran
derivatives, polyanionic polysaccharides and derivatives thereof,
polylactic acid (PLA), polyglycolic acid (PGA), a copolymer of a
polylactic acid and a polyglycolic acid (PLGA), lactides,
glycolides, polyoxanones, polyoxalates, copolymer of
poly(bis(p-carboxyphenoxy)propane)anhydride (PCPP) and sebacic
acid, poly(l-glutamic acid), poly(d-glutamic acid), polyacrylic
acid, poly(dl-glutamic acid), poly(l-aspartic acid),
poly(d-aspartic acid), poly(dl-aspartic acid), polyethylene glycol,
copolymers of polyamino acids with polyethylene glycol,
polypeptides, polycaprolactone, poly(alkylene succinates),
poly(hydroxy butyrate) (PHB), poly(butylene diglycolate),
nylon-2/nylon-6-copolyamides, polydihydropyrans, polyphosphazenes,
poly(ortho ester), poly(cyano acrylates), polyvinylpyrrolidone and
polyvinylalcohol.
11. The method of claim 1, wherein the composition further includes
a material that enhances adherence of the composite to tissue.
12. The method of claim 11, wherein the material that enhances
adherence of the composite to tissue is a polymer selected from the
group consisting of fibrin, collagen, crosslinked collagen,
collagen derivative and a polymer that includes a peptide sequence
having arginine, glycine and aspartic acid.
13. The method of claim 1, further including the step of
fabricating the composite in the shape of the osteochondral or
chondral defect.
14. The method of claim 1, further including the steps of: forming
the composite in a sheet or film; and cutting, trimming and
contouring the sheet or film to fill the osteochondral or chondral
defect.
15. A method for regenerating or promoting regeneration of
cartilage and/or bone in an osteochondral or chondral defect in a
subject, comprising: a) forming a scaffold that includes a
hyaluronic acid derivative and a support, wherein carboxyl
functionalities of the hyaluronic acid derivative are each
independently derivatized to include an N-acylurea or O-acyl
isourea, or both N-acylurea and O-acyl isourea; b) impregnating in,
or coupling to, the scaffold at least one member of the group
consisting of a cell, and a cellular growth and differentiation
factor in the scaffold; and c) implanting the scaffold that is
impregnated or coupled with said at least one member of the group
consisting of a cell, and a cellular growth and differentiation
factor at a site of the osteochondral or chondral defect of the
subject, thereby providing a mechanism for delivery of the cell,
cellular growth factor or cellular differentiation factor to the
site of the osteochondral or chondral defect to regenerate or
promote regeneration of cartilage and bone in the osteochondral or
chondral defect.
16. The method of claim 15, wherein the cell, cellular growth
factor and cellular differentiation factor include at least one
member selected from the group consisting of mesenchymal stem
cells, fibrochondrocytes, osteochondrocytes, chondrocytes,
TGF.beta. supergene family members, and hormones that stimulate
tissue growth.
17. The method of claim 16, wherein the scaffold include a
cartilage chondrocyte, osteochondrocyte or mesenchymal stem
cell.
18. The method of claim 15, further including the step of
stabilizing the composite within the osteochondral or chondral
defect so that the composite does not move during the regeneration
or repair of the osteochondral or chondral defect.
19. The method of claim 15, wherein at least 1% by mole of the
carboxyl functionalities have been derivatized.
20. The method of claim 19, wherein at least 25% by mole of the
derivatized carboxyl functionalities are O-acyl isoureas and/or
N-acylureas.
21. The method of claim 15, wherein the hyaluronic acid derivative
includes at least one crosslink represented by the following
structural formula: HA'--U--R.sub.2--U--HA' wherein: each HA' is
the same or a different hyaluronic acid molecule; each U is
independently an optionally substituted O-acyl isourea or N-acyl
urea; and each R.sub.2 is independently a substituted or
unsubstituted hydrocarbylene group optionally interrupted by one or
more heteroatoms.
22. The method of claim 15, wherein the scaffold has interconnected
pores of sizes that can provide molecular cuing for the impregnated
or coupled cell to migrate through, or a path for migration of the
impregnated or coupled cellular growth factor or cellular
differentiation factor.
23. The method of claim 15, wherein the support is a biocompatible
and biodegradable support.
24. The method of claim 23, wherein the support includes at least
one member selected from the group consisting of crosslinked
alginates, gelatin, collagen, crosslinked collagen, collagen
derivatives, crosslinked hyaluronic acid, chitosan, chitosan
derivatives, cellulose and derivatives thereof, dextran
derivatives, polyanionic polysaccharides and derivatives thereof,
polylactic acid (PLA), polyglycolic acid (PGA), a copolymer of a
polylactic acid and a polyglycolic acid (PLGA), lactides,
glycolides, polyoxanones, polyoxalates, copolymer of
poly(bis(p-carboxyphenoxy)propane)anhydride (PCPP) and sebacic
acid, poly(l-glutamic acid), poly(d-glutamic acid), polyacrylic
acid, poly(dl-glutamic acid), poly(l-aspartic acid),
poly(d-aspartic acid), poly(dl-aspartic acid), polyethylene glycol,
copolymers of polyamino acids with polyethylene glycol,
polypeptides, polycaprolactone, poly(alkylene succinates),
poly(hydroxy butyrate) (PHB), poly(butylene diglycolate),
nylon-2/nylon-6-copolyamides, polydihydropyrans, polyphosphazenes,
poly(ortho ester), poly(cyano acrylates), polyvinylpyrrolidone and
polyvinylalcohol.
25. The method of claim 15, wherein the scaffold further includes a
material that enhances adherence of the composite to tissue.
26. The method of claim 25, wherein the material that enhances
adherence of the composite to tissue is a polymer selected from the
group consisting of fibrin, collagen, crosslinked collagen,
collagen derivative and a polymer that includes a peptide sequence
having arginine, glycine and aspartic acid.
27. The method of claim 15, further including the step of
fabricating the scaffold in the shape of the osteochondral or
chondral defect.
28. The method of claim 15, further including the steps of: forming
the scaffold in a sheet or film; and cutting, trimming and
contouring the sheet or film to fill the osteochondral or chondral
defect.
Description
INCORPORATION BY REFERENCE
[0001] This application claims the benefit of U.S. Provisional
Application Nos. 60/751,237; 60/751,381; and 60/751,414, all of
which were filed Dec. 14, 2005. The entire teachings of the
above-mentioned applications are incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] Articular cartilage is an elastic tissue that covers the
ends of bones in joints and enables the bones to move smoothly. The
repair of damaged articular cartilage represents one of the most
challenging problems in orthopedics today. When articular cartilage
is damaged, it does not heal as rapidly or effectively as other
tissues in the body. Rather, the damage tends to spread and the
bones may rub directly against each other, which results in pain,
reduced mobility and loss of joint function.
[0003] Various therapeutic approaches to stimulate regeneration of
articular cartilage and subchondral bone have been developed with
varying degrees of success. Examples of such therapeutic approaches
include osteochondral grafting and autologous chondrocyte
implantation. However, there is still a need to develop methods for
treating articular cartilage defects, such as osteochondral and
chondral defects.
SUMMARY OF THE INVENTION
[0004] The present invention generally is directed to a method for
treating an osteochondral defect or a chondral defect in a subject
by the use of a composite that includes a hyaluronic acid
derivative and at least one member of the group consisting of a
cell, a cellular growth factor and a cellular differentiation
factor.
[0005] In one embodiment, the invention is directed to a method for
treating an osteochondral defect or a chondral defect in a subject.
The method includes implanting a composite at a site of the
osteochondral or chondral defect. The composite includes a
hyaluronic acid derivative; and at least one member of the group
consisting of a cell, a cellular growth factor and a cellular
differentiation factor. The cell, cellular growth factor or
cellular differentiation factor is impregnated in, or coupled to,
the hyaluronic acid derivative. The hyaluronic acid derivative
includes carboxyl functionalities that are each independently
derivatized to include an N-acylurea or O-acyl isourea, or both
N-acylurea and O-acyl isourea.
[0006] In another embodiment, the invention is directed to a method
for regenerating or promoting regeneration of cartilage and/or bone
in an osteochondral defect or a chondral defect in a subject. The
method includes forming a scaffold that includes a hyaluronic acid
derivative and a support, wherein a portion of carboxyl
functionalities of the hyaluronic acid derivative is derivatized to
include an N-acylurea or O-acyl isourea, or both N-acylurea and
O-acyl isourea. The method also includes the steps of impregnating
in, or coupling to, the scaffold at least one member of the group
consisting of a cell, a cellular growth factor and a cellular
differentiation factor; and implanting the scaffold impregnated or
coupled with at least one member of the group consisting of a cell,
a cellular growth factor and a cellular differentiation factor at a
site of the osteochondral defect or chondral defect, thereby
providing a mechanism for the delivery of the cell, cellular growth
factor or cellular differentiation factor to the site of the
osteochondral or chondral defect to regenerate or promote
regeneration of cartilage and bone in the osteochondral defect or
chondral defect.
[0007] The natural repair of osteochondral or chondral defects can
be enhanced with a proper matrix that provides structural support
and molecular cuing to stimulate repair. For example, in the
current invention, subject's own cells, such as healthy cartilage
cells or mesenchymal stem cells, can be harvested and impregnated
in, or coupled to, the hyaluronic acid derivative or the hyaluronic
acid derivative and one or more biocompatible, biodegradable
supports. The hyaluronic acid derivative optionally together with
the biocompatible support can provide structural support and
molecular cuing for the impregnated or coupled cells to migrate,
multiply and stimulate regeneration of cartilage. Likewise, the
cellular growth and differentiation factors can be loaded into the
matrix and provide additional molecular cuing for the cells to
produce cartilage or bone tissue, or provide signals for the cells
to differentiate down the chondrogenic or osteogenic lineage. The
biocompatible, biodegradable support and/or hyaluronic acid
derivative will be absorbed by the body while the cartilage tissue
regeneration takes place. Thus, there is no need to remove the
support and hyaluronic acid derivative from the subject after the
regenerated articular cartilage restores its function, leaving no
artificial materials at the treatment site.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIGS. 1A and 1B are scanning electron microscopy (SEM)
images of a composite of the invention, including a freeze-dried
crosslinked hyaluronic acid (HA) sponges, at two different
magnifications.
[0009] FIGS. 2A and 2B are scanning electron microscopy (SEM)
images of a composite of the invention, including a freeze-dried
crosslinked hyaluronic acid (HA) sponges, at two different
magnifications.
[0010] FIG. 3 is a cross sectional view of the composite of FIGS.
2A-2B, showing interconnected structural support that can provide
cues for ingrowth of cells, cellular growth factors or cellular
differentiation factors for treating an osteochondral or chondral
defect.
DETAILED DESCRIPTION OF THE INVENTION
[0011] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings. The drawings are not
necessarily to scale, emphasis instead being placed upon
illustrating the principles of the invention.
[0012] As used herein, the term "hyaluronic acid derivative" means
hyaluronic acid derivatized in that carboxyl functionalities of the
hyaluronic acid (HA) (a portion or all) are each independently
derivatized to include an N-acylurea or O-acyl isourea, or both
N-acylurea and O-acyl isourea. As used herein, hyaluronic acid, and
any of its salts which are often referred to as "hyaluronan" (e.g.,
sodium, potassium, magnesium, calcium or ammonium salts) are
represented by the term "HA." Typically, HA comprises disaccharide
units of D-glucuronic acid (GlcUA) and N-acetyl-D-glucosamine
(GlcNAc), which are alternately linked, forming a linear
polymer.
[0013] N-acylurea and O-acyl isourea derivatives for the invention
are as shown in the bracketed fragments in the following structural
formulas (I) and (II): ##STR1##
[0014] In structural formulas (I) and (II), each R.sub.1 can be the
same or different. Each R.sub.1 is selected from the group
consisting of hydrogen; substituted or unsubstituted hydrocarbyl
groups (linear or branched, or cyclic or acyclic) optionally
interrupted by one or more heteroatoms; substituted or
unsubstituted alkoxy; substituted or unsubstituted aryloxy; and
substituted or unsubstituted aralkyloxy. Examples of substituted or
unsubstituted hydrocarbyl groups (linear or branched, or cyclic or
acyclic) optionally interrupted by one or more heteroatoms include
optionally substituted aliphatic groups (e.g., alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl and
cycloaliphaticalkyl); optionally substituted aryl groups (including
heteroaryl groups); optionally substituted aliphatic groups
interrupted by one or more heteroatoms (e.g., heterocyclyl,
cycloaliphaticalkyl and heterocyclylalkyl); and optionally
substituted, partially aromatic and partially aliphatic groups
(e.g., aralkyl and heteroaralkyl). Suitable optional substituents
are those that do not substantially interfere with the properties
of the resulting crosslinked HA composition. Suitable substituents
for carbon atoms of hydrocarbyl groups include --OH, halogens
(--Br, --Cl, --I, --F), --OR.sup.a, --O--COR.sup.a, --COR.sup.a,
--CN, --NCS, --NO.sub.2, --COOH, --SO.sub.3H, --NH.sub.2,
--NHR.sup.a, --N(R.sup.aR.sup.b), --COOR.sup.a, --CHO,
--CONH.sub.2, --CONHR.sup.a, --CON(R.sup.aR.sup.b), --NHCOR.sup.a,
--NR.sup.bCOR.sup.a, --NHCONH.sub.2, --NHCONR.sup.aH,
--NHCON(R.sup.aR.sup.b), --NR.sup.bCONH.sub.2,
--NR.sup.bCONR.sup.aH, --NR.sup.cCON(R.sup.aR.sup.b),
--C(.dbd.NH)--NH.sub.2, --C(.dbd.NH)--NHR.sup.a,
--C(.dbd.NH)--N(R.sup.aR.sup.b), --C(.dbd.NR.sup.c)--NH.sub.2,
--C(.dbd.NR.sup.c)--NHR.sup.a,
--C(.dbd.NR.sup.c)--N(R.sup.aR.sup.b), --NH--C(.dbd.NH)--NH.sub.2,
--NH--C(.dbd.NH)--NHR.sup.a, --NH--C(.dbd.NH)--N(R.sup.aR.sup.b),
--NH--C(.dbd.NR.sup.c)--NH.sub.2,
--NH--C(.dbd.NR.sup.c)--NHR.sup.a,
--NH--C(.dbd.NR.sup.c)--N(R.sup.aR.sup.b),
--NR.sup.dH--C(.dbd.NH)--NH.sub.2,
--NR.sup.d--C(.dbd.NH)--N(R.sup.aR.sup.b),
--NR.sup.d--C(.dbd.NR.sup.c)--NH.sub.2,
--NR.sup.d--C(.dbd.NR.sup.c)--NHR.sup.a,
--NR.sup.d--C(.dbd.NR.sup.c)--N(R.sup.aR.sup.b), --NHNH.sub.2,
--NHNHR.sup.a, --NHR.sup.aR.sup.b, --SO.sub.2NH.sub.2,
--SO.sub.2NHR.sup.a, --SO.sub.2NR.sup.aR.sup.b, --SH, --SR.sup.a,
--S(O)R.sup.a, and --S(O).sub.2R.sup.a. In addition, an alkyl,
alkylene, alkenyl or alkenylene group can be substituted with
substituted or unsubstituted aryl group to form, for example, an
aralkyl group such as benzyl. Similarly, aryl groups can be
substituted with a substituted or unsubstituted alkyl or alkenyl
group.
[0015] R.sup.a-R.sup.d are each independently an alkyl group, aryl
group, including heteroaryl group, non-aromatic heterocyclic group
or --N(R.sup.aR.sup.b), taken together, form a substituted or
unsubstituted non-aromatic heterocyclic group. The alkyl, aromatic
and non-aromatic heterocyclic group represented by R.sup.a-R.sup.d
and the non-aromatic heterocyclic group represented by
--N(R.sup.aR.sup.b) can optionally be substituted.
[0016] In other embodiments, R.sub.1 is an optionally substituted
aliphatic group (cyclic or acyclic, or linear or branched). More
preferably, R.sub.1 is an alkyl group, such as C1-C6 alkyl (e.g.,
methyl, ethyl, propyl, butyl, 2-propyl, tert-butyl, and the like).
Preferably, each R.sub.1 is ethyl.
[0017] Each R.sub.2 is independently a substituted or unsubstituted
linking group including one or more of hydrocarbylene groups
(cyclic or acyclic, or linear or branched) optionally interrupted
by one or more heteroatoms. Examples include optionally substituted
aliphatic groups (e.g., alkylene, alkenylene, alkynylene,
cycloalkylene, cycloalkenylene, cycloalkynylene and
cycloaliphaticalkylene); optionally substituted arylene (including
heteroaryl groups); optionally substituted aliphatic groups
interrupted by one or more heteroatoms (e.g., heterocyclylene,
cycloaliphaticalkylene and heterocyclylalkylene); and optionally
substituted, partially aromatic and partially aliphatic groups
(e.g., aralkylene and heteroaralkylene). Suitable optional
substituents are as those described above for R.sub.1.
[0018] In some embodiments, R.sub.2 includes or is interrupted by
other groups, e.g, carbonyl, amide, oxy, sulfide, disulfide, and
the like. In other embodiments, R.sub.2 is a cycloaliphatic,
arylene, heteroarylene, or heterocyclylene group. In still other
embodiments, R.sub.2 is 1,6-hexamethylene, octamethylene,
decamethylene, dodecamethylene, PEG,
--CH.sub.2CH.sub.2--S--S--CH.sub.2CH.sub.2-,
para-phenylene-S--S-para-phenylene,
meta-phenylene-S--S-meta-phenylene,
ortho-phenylene-S--S-ortho-phenylene, ortho-phenylene,
meta-phenylene or para-phenylene. More preferably, R.sub.2 is
phenylene. Preferably, R.sub.2 is para-phenylene.
[0019] In one embodiment, the wavy line connected to R.sub.2 in
structural formulas (I) and (II) represents hydrogen, substituted
or unsubstituted hydrocarbyl groups (linear or branched, or cyclic
or acyclic) optionally interrupted by one or more heteroatoms;
alkoxy; aryloxy; or aralkyloxy, as described for R.sub.1. In
another embodiment, the wavy line connected to R.sub.2 in
structural formulas (I) and (II) represents optionally substituted
N-acyl urea group or O-acyl isourea group, as shown below in
structural formulas VI-VIII.
[0020] In general, the modified HA derivative is prepared by
reacting hyaluronic acid, or a salt thereof, with a carbodiimide,
preferably a multifunctional carbodiimide, such as a
biscarbodiimide, in the absence of a nucleophile or a polyanionic
polysaccharide other than HA, to form an N-acylurea or O-acyl
isourea.
[0021] Examples of suitable carbodiimides in the invention include
a monocarbodiimide and a multifunctional carbodiimide, such as a
biscarbodiimide. The monocarbodiimide has the formula:
R.sub.3--N.dbd.C.dbd.N--R.sub.4 (III) wherein R.sub.3 and R.sub.4
are each independently as described above for R.sub.1 (e.g.,
hydrocarbyl, substituted-hydrocarbyl, alkoxy, aryloxy or
alkaryloxy). Examples of suitable monocarbodiimides include:
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC);
1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide
metho-p-toluenesulfonate (CMC);
1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide methiodide (EAC);
1,3-dicyclohexylcarbodiimide (DCC); and
1-benzyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
(BDC).
[0022] Examples of suitable biscarbodiimides may be represented by
those difunctional compounds having the formula:
R.sub.1--N.dbd.C.dbd.N--R.sub.2--N.dbd.C.dbd.N--R.sub.1 (IV). Each
R.sub.1 can be different or the same. R.sub.1 and R.sub.2 are each
independently as described above. Suitable specific examples of
biscarbodiimides include 1,6-hexamethylene bis(ethylcarbodiimide),
1,8-octamethylene bis(ethylcarbodiimide), 1,10 decamethylene
bis(ethylcarbodiimide), 1,12 dodecamethylene
bis(ethylcarbodiimide), PEG-bis(propyl(ethylcarbodiimide)),
2,2'-dithio-bis(ethyl(ethylcarbodiimde)),
1,1'-dithio-ortho-phenylene-bis(ethylcarbodiimide),
1,1'-dithio-para-phenylene-bis(ethylcarbodiimide), and
1,1'-dithio-meta-phenylene bis(ethylcarbodiimide). In a preferred
embodiment, the biscarbodiimide is
para-phenylene-bis(ethylcarbodiimide). Methods of preparing
biscarbodiimides are described, for example, in U.S. Pat. Nos.
6,013,679; 2,946,819; 3,231,610; 3,502,722; 3,644,456; 3,972,933;
4,014,935; 4,066,629; 4,085,140; 4,096,334; 4,137,386, 6,548,081,
and 6,620,927 the teachings of which are incorporated herein by
reference in their entireties.
[0023] In a preferred embodiment, the HA derivative is crosslinked.
In a more preferred embodiment, the HA derivative is at least about
1% by mole crosslinked, and the HA derivative includes at least one
crosslink, e.g., the linking group connecting through a group U at
each end to a HA' molecule, as shown in the following structural
formula: HA'--U--R.sub.2--U--HA' (V). Each HA' in the preceding
formula can be different or the same HA' molecule, e.g., the
crosslink can be an intermolecular or intramolecular crosslink.
Each U can be the same or different and is an optionally
substituted N-acyl urea or O-acyl isourea. As used herein, the term
"at least about 1% by mole crosslinked" means that HAs are
crosslinked with each other via derivatized carboxyl
functionalities of the HAs, such as O-acylisoureas or N-acylureas,
wherein the derivatized carboxyl functionalities are at least about
1% by mole of the total carboxyl functionalities of the individual
HA.
[0024] In an even more preferred embodiment, the N-acylurea or
O-acylisourea results from crosslinking with the multifunctional
carbodiimide. Alternatively, a monocarbodiimide may be employed in
combination with a multifunctional carbodiimide. Suitable examples
of monocarbodiimides and multifunctional carbodiimides are as
described above. Use of a multifunctional carbodiimide to prepare
the modified HA derivative causes crosslinking of the hyaluronic
acid. For example, use of a biscarbodiimide results in a
crosslinking between COOH groups present in the repeating
disaccharide unit of hyaluronic acid, since the biscarbodiimide is
difunctional. The COOH group may be present in the same polymer
chain, resulting in an intramolecular crosslinked product, or
present on two different polymer chains, resulting in an
intermolecular crosslinked product.
[0025] The reaction of HA with a biscarbodiimide rather than a
monocarbodiimide does not change the mechanism of reaction, but can
cause the product to be crosslinked.
[0026] The reaction of HA with a biscarbodiimide crosslinking
reagent, in the presence of an available proton, is believed to
comprise protonation in the first step. The acid anion can then
attach to the carbon atom of the cation formed, resulting in the
formation of an O-acyl isourea intermediate. The acyl group in the
intermediate can migrate from the oxygen atom to a nitrogen atom to
produce a N-acyl isourea derivative of the HA. It is believed that
the O-to-N migration can be incomplete, resulting in a product
reaction mixture that can include both the N-acyl urea and the
O-acyl isourea. Thus, a crosslink resulting from reaction of a
biscarbodiimide with the uncrosslinked HA precursor typically can
contain two O-acyl isoureas connected through R.sub.2, as
represented in the following structural formula (VI): ##STR2## or
an O-acyl isourea and an N-acyl urea connected through R.sub.2, as
represented in the following structural formula (VII): ##STR3## or
two N-acyl ureas connected through R.sub.2, as represented in the
following structural formula (VIII): ##STR4##
[0027] The mixed products can be used separately or together to
prepare the compositions according to embodiments of the
invention.
[0028] The term "hydrocarbyl," as used herein, means a monovalent
moiety obtained upon removal of a hydrogen atom from a parent
hydrocarbon. As used herein, hydrocarbylene groups are divalent
hydrocarbons. Typically, hydrocarbyl and hydrocarbylene groups
contain 1-25 carbon atoms, 1-12 carbon atoms or 1-6 carbon atoms.
Hydrocarbyl and hydrocarbylene groups can be independently
substituted or unsubstituted, cyclic or acyclic, branched or
unbranched, and saturated or unsaturated. Optionally, hydrocarbyl
and hydrocarbylene groups independently can be interrupted by one
or more hetero atoms (e.g., oxygen, sulfur and nitrogen). Examples
of hydrocarbyl groups include aliphatic and aryl groups.
Substituted hydrocarbyl and hydrocarbylene groups can independently
have more than one substituent.
[0029] The term "substituent," as used herein, means a chemical
group which replaces a hydrogen atom of a molecule. Representative
of such groups are halogen (e.g., --F, --Cl, --Br, --I), amino,
nitro, cyano, --OH, alkoxy, alkyl, alkenyl, alkynyl, aryl,
haloalkoxy, haloalkyl, haloalkenyl, haloalkynyl, alkyl amino,
haloalkyl amino, aryl amido, sulfamido, sulfate, sulfonate,
phosphate, phosphino, phosphonate, carboxylate, carboxamido, and
the like.
[0030] An "alkyl" group, as used herein, is a saturated aliphatic
group. The alkyl group can be straight chained or branched, or
cyclic or acyclic. Typically, an alkyl group has 1-25 carbon atoms.
Examples of alkyl groups include methyl, ethyl, propyl, butyl,
pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl,
tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl,
nonodecyl, eicosyl, heneicosyl, docosyl, tricosyl, tetracosyl,
pentacosyl, and the isomeric forms thereof. An alkyl group may be
substituted with one or more substituents independently selected
for each position.
[0031] An "alkylene" group, as used herein, is a saturated
aliphatic group that is bonded to two other groups each through a
single covalent bond. The alkylene group can be straight chained or
branched, or cyclic or acyclic. Typically, an alkylene group has
1-25 carbon atoms. Examples of alkylene groups include methylene,
ethylene, propylene, butylene, pentylene, hexylene, heptylene,
octylene, 1,6-hexamethylene, 1,8-octamethylene, 1,10-decamethylene,
1,12-dodecamethylene and the isomeric forms thereof. An alkylene
group may be substituted with one or more substituents
independently selected for each position.
[0032] As used herein, an "alkenyl" group is an aliphatic group
that contains a double bond. Typically, an alkenyl group has 2 to
25 carbon atoms. Examples include vinyl, allyl, butenyl, pentenyl,
hexenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl,
tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl,
octadecenyl, nonadecenyl, eicosenyl, heneicosenyl, docosenyl,
tricosenyl, tetracosenyl, pentacosenyl, and isomeric forms
thereof.
[0033] As used herein, an "alkenylene" group is an aliphatic group
that contains a double bond. Typically, an alkenylene group has 2
to 25 carbon atoms. Examples include butenylene, pentenylene,
hexenylene, octenylene, nonenylene and isomeric forms thereof.
[0034] As used herein, an "alkynyl" group is an aliphatic group
that contains a triple bond. Typically, an alkynyl group has 2 to
25 carbon atoms. Examples include vinyl, allyl, butynyl, pentynyl,
hexynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl,
tridecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl,
octadecynyl, nonadecynyl, eicosynyl, heneicosynyl, docosynyl,
tricosynyl, tetracosynyl, pentacosynyl, and isomeric forms
thereof.
[0035] As used herein, an "alkynylene" group is an aliphatic group
that contains a triple bond. Typically, an alkynylene group has 2
to 25 carbon atoms. Examples include vinylene, allylene,
butynylene, pentynylene, hexynylene, octynylene and isomeric forms
thereof.
[0036] The term "aryl" as used herein refers to an aromatic ring
(including heteroaromatic ring). Particularly, an aryl group that
includes one or more heteroatoms is herein referred to
"heteroaryl." Examples of aryl groups include phenyl, tolyl, xylyl,
naphthyl, biphenylyl, triphenylyl, and heteroaryl, such as
pyrrolyl, thienyl, furanyl, pyridinyl, oxazolyl, isooxazolyl,
thiazolyl, isothiazolyl and quinolinyl. An aryl group may be
substituted with one or more substituents independently selected
for each position.
[0037] The term "arylene" as used herein refers to an aryl ring in
a molecule that are bonded to two other groups each through a
single covalent bond from two of its ring atoms. Particularly, an
arylene group that includes one or more heteroatoms is herein
referred to "heteroarylene." Examples of arylene groups include
phenylene [--(C.sub.6H.sub.4)--], such as meta-phenylene and
para-phenylene; and heteroarylene groups, such as pyridylene
[--(C.sub.5H.sub.3N)--]; and furanylene [--(C.sub.4H.sub.2O)--]. An
arylene group may be substituted with one or more substituents
independently selected for each position.
[0038] An alkyl, alkylene, alkenyl, alkenylene group, alkynyl or
alkynylene can be optionally substituted with substituted or
unsubstituted aryl group to form, for example, an aralkyl group
(e.g. benzyl), or aralylene (e.g. --CH.sub.2--(C.sub.6H.sub.4)-- or
--CH.dbd.CH.sub.2--(C.sub.6H.sub.4)--). Similarly, aryl or arylene
groups can be optionally substituted with a substituted or
unsubstituted alkyl, alkenyl or alkynyl group.
[0039] The term "heterocyclyl" refers to a cycloalkyl group wherein
one or more ring carbon atoms are replaced with a heteroatom, e.g.,
aziridyl, azetidyl, pyrrolidyl, piperidyl, thiiranyl, thietanyl,
tetrahydrothiophenyl, tetrahydrothiopyranyl, oxiranyl, oxetanyl,
tetrahydrofuranyl, tetrahydropyranyl, morpholinyl, and the
like.
[0040] The term "heterocyclylene" refers to a cycloalkylene group
wherein one or more ring carbon atoms are replaced with a
heteroatom, e.g., 2,5-tetrahydrofuranylene.
[0041] An alkoxy group is an alkyl group connected through an
oxygen atom, e.g., methoxy, ethoxy, propoxy and the like.
[0042] An aryloxy group is an aryl group connected through an
oxygen atom, e.g., phenoxy and the like.
[0043] An aralkyloxy group is an aralkyl group connected through an
oxygen atom, e.g., benzyl oxy and the like.
[0044] In one embodiment, the modified HA derivative is at least
about 1% by mole crosslinked. The crosslinked HA gel can be
water-soluble or substantially water-insoluble.
[0045] In another embodiment, at least about 1% by mole, such as at
least about 2% by mole, at least about 5% by mole, or between about
1% by mole and about 20% by mole, of the carboxyl functionalities
of the modified hyaluronic acid are derivatized. In yet another
embodiment, at least about 25% by mole, such as between about 25%
by mole and about 75% by mole, of the derivatized functionalities
are O-acylisoureas and/or N-acylureas. In yet another embodiment,
the carboxyl functionalities of the modified hyaluronic acid are
derivatized, and the derivatized carboxyl functionalities result
from crosslinking of HAs with a multifunctional carbodiimide
described above, preferably biscarbodiimide. Conditions for such
crosslinkings are known in the art, for example, in U.S. Pat. No.
6,548,081, the entire teachings of which are incorporated herein by
reference.
[0046] The steps required to make a biocompatible HA derivative
include providing a sample of HA or a salt thereof, such as sodium
hyaluronate. HA from any of a variety of sources, including HA
extracted from animal tissues or harvested as a product of
bacterial fermentation, can be used as a starting material.
Alternatively, the HA used to make the composites of this invention
can be produced in commercial quantities by bioprocess technology,
as described, for example, in Nimrod et al., PCT Publication No. WO
86/04355.
[0047] In one example, the sample of HA or its salt is dissolved in
water to make an aqueous solution. In a particular example, the
concentration of HA in this first aqueous solution is in the range
of between about 0.1% and 5% weight/weight ("w/w"), that is, 1
mg/ml solution to 50 mg/ml solution. In another particular example,
the reactions are carried out with a range of about between about
0.4% and 0.6% weight/weight, or 4 to 6 mg of hyaluronic acid per
milliliter. The precise concentration used will vary depending on
the molecular weight of the HA. At significantly lower
concentrations, the reactions are slower and less effective. At
significantly higher HA concentrations, the end product may be
difficult to handle due to the increase in viscosity. One skilled
in the art will be able to determine, with no more than routine
experimentation, an acceptable concentration of HA to be used for a
particular embodiment. Examples of various acceptable
concentrations of HA are described in U.S. Pat. No. 5,356,883, to
Kuo et al., the teachings of which are incorporated herein by
reference in their entirety.
[0048] The pH of the HA solution is then adjusted by the addition
of a suitable acid or a suitable pH buffer known in the art, so
that the aqueous HA solution is acidic, preferably having a pH of
about between 4.0 and 8.0, such as about between 4.0 and about 6.0
or between about pH 4.75 and about pH 5.5. The pH buffer can
include any buffer agent known to one skilled in the art, e.g.,
2-(N-morpholino)ethanesulfonic acid (MES);
2,2-bis(hydroxymethyl)-2,2',2''-nitrotriethanol; succinate/succinic
acid; KH.sub.2PO.sub.4; N-tris(hydroxymethyl-2-aminoethanesulfonic
acid; triethanolamine; diethylbarbituate;
tris(hydroxymethyl)aminoethane; N-tris(hydroxy)methylglycine; and
N,N-bis(2-hydroxyethyl)glycine. The buffer agent can be employed
with an additional acid or base, e.g.,
2-(N-morpholino)ethanesulfonic acid with NaOH;
2,2-bis(hydroxymethyl)-2,2',2''-nitrotriethanol with HCl; succinate
with succinic acid; KH.sub.2PO.sub.4 with borax;
N-tris(hydroxymethyl-2-aminoethanesulfonic acid with NaOH;
triethanolamine with HCl; diethylbarbituate with HCl;
tris(hydroxymethyl)aminoethane with HCl;
N-tris(hydroxy)methylglycine with HCl; and
N,N-bis(2-hydroxyethyl)glycine with HCl. Preferably, the buffer
includes 2-(N-morpholino)ethanesulfonic acid and NaOH.
[0049] Once the pH of the aqueous HA solution has been adjusted,
the carbodiimide can be added. Generally an excess of the
stoichometric proportion of carbodiimide is advantageous to promote
the desired reaction. Preferably the molar equivalent ratio of the
carbodiimide to HA is equal to or greater than about 5%.
[0050] In one example, the pH of the aqueous HA solution is
adjusted by the addition of a suitable acid, such as an HCl
solution. Preferably, the carbodiimide is dissolved in an
appropriate water-mixable solvent and added drop-wise. In this
example, as the carbodiimide and the HA are mixed, the pH of the
solution generally increases. Films and gels with various desired
physical properties can be obtained by simply allowing the pH to
rise as the reaction proceeds. However, the reaction is monitored
by a pH meter, and HCl may be added to maintain the pH of the
reaction mixture, for example, about between 4.0 and 8.0, such as
about between 4.0 and about 6.0 or between about pH 4.75 and about
pH 5.5. The reaction is then allowed to proceed at room temperature
for about two hours. The reaction may be directed to favor the
formation of the N-acylurea derivatives by increasing the pH with a
suitable aqueous base. The progress of the reactions described
above may be followed by monitoring the pH. When the pH is
stabilized, the reactions are substantially complete.
[0051] In another example, the carbodiimide, such as
biscarbodiimide, is reacted with the HA in the presence of a
suitable pH buffer, wherein the buffer is at a pH between about 4
and about 8. Suitable examples of pH buffer agents are as described
above. Typically, the buffer agent is mixed in aqueous media, in a
concentration between about 5 mM (millimolar) and about 250 mM
(e.g., about 75 mM). Typically, the HA is mixed in aqueous media,
e.g., the pH buffer solution, in a concentration between about 1 mM
(millimolar) and about 100 mM (e.g., about 37 mM). The particular
concentration employed can vary depending on the molecular weight
of the HA.
[0052] The carbodiimide can be combined with the HA solution alone,
or more typically as a solution in a water-miscible organic
solvent, e.g., acetone, methyl ethyl ketone, dimethyformamide,
dimethyl sulfoxide, methanol, ethanol, 2-propanol, acetonitrile,
tetrahydrofuran, N-methyl pyrrolidone, and the like. When a
biscarbodiimide is utilized, typically, the solvent is acetone, and
the biscarbodiimide is at a concentration of between about 0.1
mg/mL and about 100 mg/mL. The HA and the carbodiimide, such as
carbodiimide, can be combined in any molar equivalent ratio, e.g.,
between about 1% and about 200%, typically between about 2% and
about 30%. The reaction can be carried out at a temperature range
of between about 0.degree. C. and about 60.degree. C., typically
between 25-30.degree. C.
[0053] Crosslinked HA can be formed by reacting uncrosslinked HA
with a crosslinking agent, such as a biscarbodiimide as described
above, under suitable reaction conditions by methods known in the
art, for example, U.S. Patent Application Publication Nos.
10/743,557, 5,356,883, 5,502,081, 6,013,679, 6,537,979, and
6,548,081, the entire teachings of which are incorporated herein by
reference. The uncrosslinked HA used as a precursor for the
crosslinking typically has typically an average molecular weight
range of from between about 6.times.10.sup.4 to about
8.times.10.sup.6 Daltons, or 150 to 20,000 disaccharide repeat
units. Uncrosslinked HA having lower or higher molecular weights
than these can also be used in the invention.
[0054] The reaction conditions for HA crosslinking with a
biscarbodiimide are similar to those used for HA-monocarbodiimide
coupling reactions. Advantageously, the crosslinking reactions are
carried out with (1) an increase of the HA concentration in the
reaction mixture, and/or (2) a decrease of the biscarbodiimide
concentration in the addition solution. This creates a condition
favorable to intermolecular crosslinking versus intramolecular
crosslinking.
[0055] At the conclusion of the reactions described above, the
desired HA derivative may be separated from the reaction mixtures
by conventional methods of precipitation, washing and
re-precipitation. The completeness of the reaction, the nature of
the products and the extent of chemical modification can be
determined by, for example, proton NMR, or by studying the
resistance to enzymatic hydrolysis or studying other changes in the
physical or chemical behavior of the product.
[0056] If a colored product is desired, a solution of a
biocompatible dye or stain, e.g., Coomassie.TM. Brilliant Blue
R-250, can be admixed to the reaction mixtures described above. The
resulting product will have a blue color which makes the gel, film
or sponge easy to see when it is handled during surgery and when it
is in place.
[0057] When the reaction is complete, sodium chloride is typically
added to the reaction mixture to adjust the sodium chloride
concentration to 1M. Ethanol is added to form a precipitate of
chemically-modified, HA derivative. The precipitate is separated
from the solution, washed, and dried by vacuum. The freeze dried
material can be washed with appropriate solvents to remove
contaminants of the reaction and dried and then sterilized by
ethylene oxide (EtO) sterilization or sterilization by gamma
irradiation before loading the cells and implanting them into
mammals.
[0058] To make a gel of the HA derivative, the precipitate is
re-suspended in water and stirred in a cold room. The gel of the HA
derivative is a hydrogel. The term "hydrogel" is defined herein to
mean a macromolecular network swollen in water or biological
fluids. The degree of hydration is dependent on the degree of
crosslinking.
[0059] To make a sponge, the precipitate is then re-suspended in
water, poured into a mold having a desired shape, and, preferably,
dried, such as by air-drying, freeze-drying or heat-drying. A film
may be prepared by further drying the gel. Alternatively, a film
can be formed by compressing a gel under conditions that permit
water to escape, as, for example, by compressing the gel between
two surfaces, at least one of which is porous. See, for example,
Malson et al., U.S. Pat. No. 4,772,419, the teachings of which are
incorporated herein by reference in their entirety.
[0060] The composites of the invention can include the modified HA
derivative described above without biocompatible, biodegradable
supports (e.g., polymers) other than the modified HA derivative.
Preferably, in this embodiment, the modified HA derivative is a
highly-crosslinked HA, such as at least about 75% by mole
crosslinked HA.
[0061] Alternatively, the composites of the invention can include
the modified HA derivative described above and one or more
biocompatible, biodegradable supports (e.g., polymers) other than
the modified HA derivative. In this embodiment, the modified HA is
at the support(s). Preferably, in this embodiment, the modified HA
derivative is a crosslinked HA of low degree of crosslinking, such
as less than about 20% by mole, such as about 5% by mole or about
18% by mole, or between about 1% by mole and about 10% by mole.
[0062] As used herein, a "biocompatible" support is one that has no
medically unacceptable toxic or injurious effects on biological
function. As used herein, a "biodegradable" support is one that is
capable of being decomposed by natural biological processes.
[0063] Examples of the physical form of a suitable support include:
a biocompatible, biodegradable matrix, sponge, film, sheet, thread,
tube, non-woven fabric and cord. The biodegradable support may be
formed from a material which is porous, and the pore sizes may be
large enough so that when a layer of the hyaluronic acid (HA)
derivative is spread on the support, the molecules of the HA
derivative can partially or fully penetrate into the pores of the
support to make an anchor. Examples of compositions to be used as a
suitable support include: crosslinked alginates, gelatin, collagen,
crosslinked collagen, collagen derivatives, such as, succinylated
collagen or methylated collagen, crosslinked hyaluronic acid,
chitosan, chitosan derivatives, such as,
methylpyrrolidone-chitosan, cellulose and cellulose derivatives
such as cellulose acetate or carboxymethyl cellulose, dextran
derivatives such carboxymethyl dextran, starch and derivatives of
starch such as hydroxyethyl starch, other glycosaminoglycans and
their derivatives, other polyanionic polysaccharides or their
derivatives, polylactic acid (PLA), polyglycolic acid (PGA), a
copolymer of a polylactic acid and a polyglycolic acid (PLGA),
lactides, glycolides, and other polyesters, polyoxanones and
polyoxalates, copolymer of
poly(bis(p-carboxyphenoxy)propane)anhydride (PCPP) and sebacic
acid, poly(l-glutamic acid), poly(d-glutamic acid), polyacrylic
acid, poly(dl-glutamic acid), poly(l-aspartic acid),
poly(d-aspartic acid), poly(dl-aspartic acid), polyethylene glycol,
copolymers of the above listed polyamino acids with polyethylene
glycol, polypeptides, such as, collagen-like, silk-like, and
silk-elastin-like proteins, polycaprolactone, poly(alkylene
succinates), poly(hydroxy butyrate) (PHB), poly(butylene
diglycolate), nylon-2/nylon-6-copolyamides, polydihydropyrans,
polyphosphazenes, poly(ortho ester), poly(cyano acrylates),
polyvinylpyrrolidone, polyvinylalcohol, polycasein, keratin,
myosin, and fibrin.
[0064] A sample of highly crosslinked HA, for example at least
about 75% by mole, crosslinked HA, may form a support for a sample
of modified HA which is not highly crosslinked. One example of the
highly-crosslinked HA is a thiol-containing, highly crosslinked HA
(see U.S. Pat. No. 6,620,927, the entire teachings of which are
incorporated herein by reference). The HA support can be made by,
for example, pouring a mixture of uncrosslinked HA and a
crosslinking agent, such as a biscarbodiimide having an
intramolecular disulfide bond, into a mold and freeze dried in a
desired shape.
[0065] The composite of the invention may be in a form of sponge,
film, sheet, gel, thread, tube, non-woven fabrics, cords and
meshes. In one embodiment, the composite is in the form of a
sponge. In another embodiment, the composite is in the form of a
sheet or film, preferably hydrophilic sheet or film. The
hydrophilic sheet or film can be stacked together for stitching,
for example, to correctly fit or fully fill a treatment site.
[0066] When the HA derivative is employed in combination with one
or more biocompatible, biodegradable supports as described above,
there are several ways in which the HA derivative (e.g., gel, film
or sponge) can be immobilized on the support to make the composite
device of this invention (see, for example, U.S. Pat. No.
6,548,081). For example, a layer of derivatized HA may be applied,
either by soaking or dipping or spraying or spreading or by any
other method of application, to at least one surface of a support
to form a composite. A suitable support may be a matrix, sponge,
film, sheet, gel, thread, tube, non-woven fabrics, cords and
meshes, which may be porous. If the surface of the support is
porous, the HA derivative will soak into the pores at the support
surface. For example, porous beads may be soaked in the hyaluronic
acid derivative for a sufficient period of time to allow the
hyaluronic acid derivative to be absorbed and adsorbed by the pores
of the beads. The composite is then dried under conditions that
permit the escape of water from the composite.
[0067] In another embodiment, a composite sponge or film having
hyaluronic acid derivative on both sides of the support is prepared
by pouring the water-insoluble gel of derivatized HA prepared
according to the procedure described above, into a first mold
having the desired shape and depth, and spreading the gel in the
first mold to form a first gel layer of even thickness. A suitable
support may be a matrix, sponge, film, or particles such as beads
made from another biocompatible material, for example collagen or
gelatin. The support is spread on top of the evenly-spread first
gel layer of derivatized HA. A second mold of the same size, shape
and depth, is placed on the top of the support. Gel is poured into
the second mold, and spread to form a second gel layer of even
thickness in the second mold. In this manner, the polymer used as
supporting matrix is sandwiched between the two layers of
derivatized HA gel which are molded to the support. The composite
is freeze-dried. The freeze-dried composite may be cut into
specimens of the desired shape and size.
[0068] The composite of the invention can optionally include a
material that enhances adherence of the composite to tissue.
Materials that are suitable for enhancing adherence of the
composite to tissue include fibrin, collagen, crosslinked collagen,
and collagen derivatives, and any other polymers that include a
peptide sequence having arginine (R), glycine (G), and aspartic
acid (D), such as a peptide sequence consisting of arginine (R),
glycine (G), and aspartic acid (D).
[0069] In a preferred embodiment, the composite of the invention is
an implantable composite. More preferably, the implantable
composite has interconnected pores of sizes that can provide
molecular cuing for the impregnated or coupled cells, cellular
growth factors or cellular differentiation factors to migrate (or
move) through. The interconnected pores of sizes can also provide
molecular cuing for cells of a subject that are surrounding the
osteochondral or chondral defect of the subject (e.g., cartilage,
bone or synovium) to migrate (or move) through. In a specific
example, the implantable composite is freeze-dried and has
interconnected pores of sizes that can provide molecular cuing as
described above.
[0070] These composites can provide structural support and
molecular cuing to stimulate regeneration of cartilage and/or bone,
or repair of osteochondral or chondral defects by providing a
mechanism for the delivery of, for example, cells to a site of
osteochondral or chondral defects, or a site in need of
regeneration of cartilage and/or bone. For example, the composite
of the invention can provide the cells, cellular growth factors or
cellular differentiation factors that are impregnated in, or
coupled to, the composite to regenerate articular cartilage and/or
bone in the osteochondral or chondral defect, or repair the
osteochondral or chondral defect. The biocompatible, biodegradable
support and the hyaluronic acid derivative will be absorbed by the
body (e.g., by the regenerated tissues), while the repair or
regeneration takes place. After the biodegradation of the
composite, for example, the regenerated articular cartilage can
have physical characteristics of natural articular cartilage and
assume its normal function.
[0071] The rate of biodegradation of derivatized HA (e.g., the rate
of release of derivatized HA) can be controlled, in part, by the
degree of crosslinking of HA, and the quantity of the crosslinked
HA loaded on the support. The residence time of unmodified HA in
the human body is generally less than a week. However, when HA is
derivatized, the residence time can be appreciably increased. In
general, an increase in the degree of crosslinking results in an
increase in the time of residence. By controlling the degree of
crosslinking, a crosslinked HA of desired residence time can be
prepared. In some embodiments, the derivatized HA selected for a
particular use may have a biodegradation rate which is faster than
the biodegradation rate of the support. The support, in fact, can
be itself made of a sample of crosslinked HA having a slower rate
of biodegradation than that of the derivatized HA loaded on the
support.
[0072] The rate at which the gel, film or sponge of the HA
derivative degrades and diffuses also depends on the insolubility,
the density, and the degree of crosslinking of the modified HA in
the composite. Just as gels, films and sponges which have a high
degree of crosslinking are slow to degrade, modified HA which is
more insoluble, or which has a higher degree of crosslinking, will
degrade at a slower rate. Preferably, the density of modified HA in
the film or sponge will be in the range of from about 0.1
mg/cm.sup.2 to about 100 mg/cm.sup.2. Those skilled in the art will
know, or will be able to ascertain with no more than routine
experimentation, the appropriate combination of insolubility,
density and crosslinking that will yield a gel, film or sponge
having the desired rate of degradation for a given situation.
[0073] The rate at which the cellular growth or differentiation
factor in the composites of the invention is released can also be
controlled by varying the physical characteristics of the
composite, such as porosity and interconnectivity, or varying the
HA crosslink density. Typically, the retention of the cellular
growth or differentiation factor will range from about 1 day to 6
months, and preferably in the 2-4 week range.
[0074] A composite of the invention includes at least one member of
the group consisting of a cell, a cellular growth factor and a
cellular differentiation factor, which is impregnated in, or
coupled to, the HA derivative, or the composite that includes the
HA derivative, and biocompatible and biodegradable support.
Suitable examples of the cell, cellular growth factor and cellular
differentiation factor include mesenchymal stem cells from various
tissue sources (e.g., cartilage, periosteum, synovium, bone marrow,
fat, etc.), fibrochondrocytes, osteochondrocytes, chondrocytes,
TGF.beta. supergene family members, such as BMPs, IGF, PDGF, GDFs,
CDMPs and GFG, and tissue growth hormones. In addition, genes
encoding for these proteins, as well as synthetic peptide analogues
of these proteins can be contemplated. In a preferred embodiment,
the composite include at least one member selected from the group
consisting of cartilage chondrocytes, osteochondrocytes and
mesenchymal stem cells. In another preferred embodiment, the
composite includes both a cell, and a cellular growth or
differentiation factor. The cellular growth or differentiation
factor can provide molecular cuing for the cell to produce
cartilage or bone tissue, or provide signals for the cell to
differentiate down the chondrogenic or osteogenic lineage.
[0075] Typically, the cells, cellular growth factor and/or cellular
differentiation factors are harvested from various sources and
impregnated in, or coupled to, the HA derivative or the HA
derivative and biocompatible support, by methods known in the art.
For example, autologous cells (e.g., chondrocytes or marrow-derived
pluripotent stem cells) can be harvested, optionally expanded in
culture, and loaded onto a composite of the invention by
conventional cell seeding methods. The cell-loaded composite may be
further cultured prior to implantation. Additionally, cells,
cellular growth factors and cellular differentiation factors can be
loaded prior to implantation by various means. These cells or
factors can be loaded before (e.g., suspension, covalent linking,
etc) or after (e.g., soak-loading, surface immobilization, etc.)
manufacture of the composite.
[0076] In one embodiment, the invention provides a method for
treating an osteochondral defect or a chondral defect in a subject
employing the composite described above. The method includes
implanting the composite in a site of the osteochondral or chondral
defect. Typically, the implanting can be done by surgical
insertion.
[0077] The implanted composition is located at a treatment site for
an extended period of time (e.g., a time period of at least days, a
week, a month, two months, six months, a year or longer than two
years).
[0078] As used herein, the term "treating" refers to resulting in a
beneficial clinical outcome of or exerting a positive influence on,
the condition being treated with the composite of the invention
compared with the absence of treatment. For example, the term
"treating" an osteochondral defect or a chondral defect includes
repair the osteochondral or chondral defect, and regenerating or
promoting regeneration of cartilage and/or bone in an articular
defect. As used herein, a "treatment site" is the site in a subject
that is in need of treatment for an osteochondral defect or a
chondral defect. The treatment site also includes the site in need
of regeneration of cartilage and/or bone in a subject.
[0079] As used herein a subject is a mammal, preferably a human,
but can also be an animal in need of veterinary treatment, such as
a companion animal (e.g., dogs, cats, and the like), a farm animal
(e.g., cows, sheep, pigs, horses, and the like) or a laboratory
animal (e.g., rats, mice, guinea pigs, and the like).
[0080] The implantable composite of the invention can provide, for
example, scaffolds that have tear strength and tear propagation
resistance and can be surgically sutured or anchored to stabilize
them within the osteochondral or chondral defect so that they do
not move during the healing and tissue regeneration. Once sutured
or anchored in place, these scaffolds will provide a matrix into
which the impregnated or coupled cells, or surrounding cells, for
example, from the cartilage, bone and synovium, can begin to
migrate and/or multiply, or the impregnated or coupled cellular
growth or differentiation factors can exert local activity. The HA
derivative in the composite can influence on cell infiltration, the
formation and degradation of a fibrin matrix, swelling of the
matrix, phagocytosis and vascularisation. As the repair or
regeneration of articular cartilage or bone takes place, the
composite will be absorbed by the body and the regenerated
cartilage or bone will restore its function, reduce pain, and
possibly retard or suspend the degenerative process caused with an
osteochondral or chondral defect.
EXEMPLIFICATION
Example 1
[0081] This example illustrates an embodiment of the invention in
which a biscarbodiimide, p-phenylene-bis(ethylcarbodiimide), and HA
are reacted at a molar equivalent ratio of 16.0%.
[0082] A solution of HA (5.4 mg/ml; 200-ml; 2.69 mequiv) was
reacted with a solution of p-phenylene-bis(ethylcarbodiimide) (1
mg/ml in acetone; 46.1-ml; 0.215 mmol; 0.43 mequiv) according to a
procedure described in U.S. Pat. Nos. 5,356,883, 5,502,081 and
6,013,679, the teachings of which are incorporated herein by
reference in their entirety. The precipitate of the crosslinked HA
was separated from the solution, washed, and resuspended in saline.
The suspension was stirred for 2 days in a cold room to form a
water-insoluble gel of about 4 mg/ml concentration. Chloroform
equal to 1/2 of the volume of the aqueous solution was added to the
solution and contents were vigorously stirred for seven days in the
cold room. The reaction mixture was then centrifuged at 4.degree.
C. and 43 k rpm for one hour to remove chloroform. The aqueous/gel
layer was aseptically collected and the concentration of sodium
chloride in the collected aqueous/gel was adjusted to 1M. The
mixture was stirred for 15 minutes under aseptic conditions.
Ethanol equal to 3 volumes of the solution was added to precipitate
the crosslinked HA and the precipitate was collected, squeezed to
remove ethanol, and shredded into small pieces under aseptic
conditions. The precipitate was re-dissolved in injection grade
water to reconstitute a gel of desired concentration.
Example 2
[0083] This example illustrates an embodiment of the invention in
which a biscarbodiimide, p-phenylene-bis(ethylcarbodiimide), and HA
are reacted at a molar equivalent ratio of 8.0%.
[0084] A solution of HA (5.4 mg/ml; 200-ml; 2.69 mequiv) was
reacted with a solution of p-phenylene-bis(ethylcarbodiimide) (1
mg/ml in acetone; 23.0-ml; 0.108 mmol; 0.216 mequiv) according to a
procedure described in U.S. Pat. Nos. 5,356,883, 5,502,081 and
6,013,679, the teachings of which are incorporated herein by
reference in their entirety. The precipitate of the crosslinked HA
was separated from the solution, washed, and resuspended in saline.
The suspension was stirred for 2 days in a cold room to form a
water-insoluble gel of about 4 mg/ml concentration. Chloroform
equal to 1/2 of the volume of the aqueous solution was added to the
solution and contents were vigorously stirred for seven days in the
cold room. The reaction mixture was then centrifuged at 4.degree.
C. and 43 k rpm for one hour to remove chloroform. The aqueous/gel
layer was aseptically collected and the concentration of sodium
chloride in the collected aqueous/gel was adjusted to 1M. The
mixture was stirred for 15 minutes under aseptic conditions.
Ethanol equal to 3 volumes of the solution was added to precipitate
the crosslinked HA and the precipitate was collected, squeezed to
remove ethanol, and shredded into small pieces under aseptic
conditions. The precipitate was re-dissolved in injection grade
water to reconstitute a gel of desired concentration.
Example 3
[0085] This example illustrates an embodiment of the invention in
which a biscarbodiimide, p-phenylene-bis(ethylcarbodiimide), and HA
are reacted at a molar equivalent ratio of 8.0% in MES buffer.
[0086] A solution of HA (15.0 mg/ml; 133.3-ml; 4.99 mequiv) in MES
buffer (pH 5.5) was reacted with a solution of
p-phenylene-bis(ethylcarbodiimide) (15 mg/ml in acetone; 2.8-ml;
0.2 mmol; 0.4 mequiv) according to a procedure described in U.S.
Patent Application 2005/0136122 A1. The reaction mixture was
thoroughly mixed (mixing with either a glass rod or an overhead
mechanical stirrer, e.g., for about 1 minute, results in a white
paste from the clear reaction mixture), and the mixture was allowed
to stand at room temperature for about 96 hours. Sodium chloride
(6.5 g, to make the mixture 5% by weight of sodium chloride) was
mixed into the resulting gel, which was allowed to stand for 1
hour. The crosslinked HA gel was precipitated by addition into
about 1.2 L of vigorously stirred ethanol. The precipitate was
collected and dried under reduced pressure yielding the crosslinked
hyaluronic acid. The dry crosslinked HA precipitate was milled. The
powder was packed in a Tyvek.RTM./Mylar.RTM. pouch, sealed and
sterilized by ethylene oxide. The precipitate was re-dissolved in
injection grade water to reconstitute a gel of desired
concentration.
Example 4
[0087] Example 4 describes the preparation of Sponge 1 shown in
FIGS. 1A and 1B, an embodiment of the invention which is a
composite including crosslinked HA derivative only. To make Sponge
1, a gel of crosslinked HA prepared according to the procedure
described in Example 1, was poured into an 8 cm.times.8 cm mold
under aseptic conditions. The mold containing the crosslinked HA
gel was frozen at -45.degree. C. and then freeze-dried under
aseptic conditions for 24 hours under vacuum of less then 10
millimeters. The freeze-dried sponge was cut under aseptic
conditions into 4 cm.times.4 cm pieces. These sponges were put in
sterile pouches and sealed to keep them sterile.
Example 5
[0088] Example 5 describes the preparation of Sponge 2 shown in
FIGS. 2A and 2B, an embodiment of the invention which is a
composite including crosslinked HA derivative only. To make Sponge
2, a gel of crosslinked HA prepared according to the procedure
described in Example 2 and 3, was poured into an 8 cm.times.8 cm
mold under aseptic conditions. The mold containing the crosslinked
HA gel was frozen at -45.degree. C. and then freeze-dried under
aseptic conditions for 24 hours under vacuum of less then 10
millimeters. The freeze-dried sponge was cut under aseptic
conditions into 4 cm.times.4 cm pieces. These sponges were put in
sterile pouches and sealed to keep them sterile.
Example 6
[0089] This example describes the preparation of an embodiment of
the invention, Sponge 3, a composite having HA derivative on both
sides of a support made of collagen. The HA derivative has at least
about 1% crosslinking, and was prepared according to the following
procedure.
[0090] A solution of hyaluronic acid (MWt. 2.35.times.10.sup.6
Daltons, 1922 ml, 6 mg/ml, pH 4.75, 28.76 mmoles) in saline was
crosslinked using a solution of cross-linker
p-phenylene-bis(ethylcarbodiimide) in acetone (1 mg/ml, 246 ml,
1.15 mmoles). The crosslinked HA was precipitated, separated from
the solution and washed with ethanol.
[0091] A weighed portion of the precipitate was dissolved in
sterile water to form crosslinked HA gel of about 7.7 mg/ml
concentration.
[0092] Non-sterile collagen sponge was cut in to square pieces of
desired dimensions. Crosslinked HA gel (7.7 mg/ml, 24 ml), prepared
according to the procedure described above was poured in to the
lower chamber of a 12 cm.times.8 cm mold and spread into a layer of
even thickness. A 14 cm.times.10 cm piece of collagen sponge was
placed on the top of the spread gel and it was covered with another
layer of crosslinked HA gel (7.7 mg/ml, 24 ml). The collagen sponge
was allowed to soak in the gel for 1 hour under aseptic condition
in a refrigerator. The mold containing the composite was frozen at
-46.degree. C. and then freeze-dried for 24 hours under vacuum of
less then 10 millimeters. The sides of the freeze-dried composite
were trimmed to make a sponge, 11.5 cm.times.7.5 cm. This larger
piece of sponge was then cut in to four 5.5 cm.times.3.5 cm pieces.
Each piece was individually packed in a Tyvek.RTM./Mylar.RTM.
pouch, sealed and sterilized by ethylene oxide (EtO).
Examples 7-9 (Prophetic)
[0093] In each of these examples, reagents are used in the amounts
indicated in Table 1. Uncrosslinked HA (2.0 g) is dissolved in
133.4 mL of MES buffer at the pH 5.5 and combined with a 15 mg/mL
acetone solution of p-phenylene-bis(ethylcarbodiimide) (PBCDI),
resulting in the specified molar equivalent ratio (MER %) and mol %
between PBCDI:HA. The reaction mixture is then thoroughly mixed
(mixing with either a glass rod or an overhead mechanical stirrer,
e.g., for about 1 minute, can result in a white paste from the
clear reaction mixture), and the mixture is poured in to the molds
designed in any desired shape, allowed to stand at room temperature
for about 72 hours. The mold containing the crosslinked HA gel is
frozen at -45.degree. C. and then freeze-dried for 24 hours under
vacuum of less then 10 millimeters. The freeze dried material is
soaked and washed with organic solvent to remove the undesired
contaminants of the reaction. The solvent is removed and the
scaffold was dried under vacuum. The dried scaffold is sealed in
Tyvek/Mylar pouch and sterilized by EtO (ethylene oxide).
TABLE-US-00001 TABLE 1 Details for synthesizing crosslinked HA in
Examples 7-9 PBCDI (15 mg/mL) Hyaluronic acid (15 mg/mL) MER
Example mL mg mmol mequiv mL g mmol mequiv % Mol % 7 17.8 267 1.25
2.5 133.3 2.0 5.0 5.0 50 25 8 26.7 400 1.87 3.74 133.3 2.0 5.0 5.0
75 37.4 9 35.6 534 2.5 5.0 133.3 2.0 5.0 5.0 100 50
Example 10 (Prophetic)
[0094] Sponges 1 and 2 of Examples 4 and 5, the composite made by
method of Example 6 and sponges that can be made by the methods
described in Examples 7-9, respectively, can provide scaffolds
adapted for the loading and ingrowth of cells, such as cartilage
chondrocytes or osteochondrocytes, or loading of cellular growth or
differentiation factors. FIG. 3 shows a cross sectional SEM image
of Sponge 2, showing interconnected pores that can provide cues for
the loaded cells to move or migrate, and multiply, or for the
loaded cellular growth or differentiation factors to move or
migrate, and exert local activity, to thereby treat osteochondral
or chondral defects.
EQUIVALENTS
[0095] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *