U.S. patent application number 10/552881 was filed with the patent office on 2007-02-01 for cross-linked polysaccharide composition.
Invention is credited to Geoffrey Kenneth Heber, Simone Charlotte Vonwiller.
Application Number | 20070026070 10/552881 |
Document ID | / |
Family ID | 31500871 |
Filed Date | 2007-02-01 |
United States Patent
Application |
20070026070 |
Kind Code |
A1 |
Vonwiller; Simone Charlotte ;
et al. |
February 1, 2007 |
Cross-linked polysaccharide composition
Abstract
The present invention provides a process for making cross-linked
polysaccharide gels under basic conditions. More particularly, the
present invention provides a process for forming cross-linked
hyaluronic acid gels under basic conditions. The resulting gels
possess improved degradation characteristics, and are useful in a
variety of medical and cosmetic applications.
Inventors: |
Vonwiller; Simone Charlotte;
(Lane Cove, AU) ; Heber; Geoffrey Kenneth;
(Annandale, AU) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
2040 MAIN STREET
FOURTEENTH FLOOR
IRVINE
CA
92614
US
|
Family ID: |
31500871 |
Appl. No.: |
10/552881 |
Filed: |
April 16, 2004 |
PCT Filed: |
April 16, 2004 |
PCT NO: |
PCT/AU04/00509 |
371 Date: |
July 17, 2006 |
Current U.S.
Class: |
424/486 ;
424/488; 525/54.2 |
Current CPC
Class: |
A61P 17/02 20180101;
A61K 31/738 20130101; A61P 37/06 20180101; C08B 37/0072 20130101;
A61K 47/36 20130101; A61P 43/00 20180101; A61P 19/02 20180101 |
Class at
Publication: |
424/486 ;
424/488; 525/054.2 |
International
Class: |
A61K 9/14 20060101
A61K009/14; C08G 63/91 20060101 C08G063/91; C08G 63/48 20060101
C08G063/48 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 17, 2003 |
AU |
2003901834 |
Claims
1-35. (canceled)
36. A process for producing a cross-linked polysaccharide gel
comprising: (a) contacting a polysaccharide mixed in an alkaline
medium with a bifunctional or polyfunctional epoxide to provide an
essentially epoxy cross-linked polysaccharide wherein the epoxide
is substantially linked to the polysaccharide by ether bonds; (b)
drying the epoxy cross-linked polysaccharide without substantially
removing epoxide from the alkaline medium to form a cross-linked
polysaccharide matrix; (c) optionally washing the cross-linked
polysaccharide matrix with a water miscible solvent; and (d)
neutralising the cross-linked polysaccharide matrix with an acidic
medium to form a cross-linked polysaccharide gel.
37. The process according to claim 36 wherein the polysaccharide is
hyaluronic acid, pectin, xanthan or alginic acid.
38. The process according to claim 36 wherein the polysaccharide is
an anionic derivative of carboxymethyl cellulose, carboxymethyl
dextran, hyaluronic acid or carboxymethyl starch.
39. The process according to claim 38 wherein the polysaccharide is
hyaluronic acid.
40. The process according to claim 36 wherein the epoxide is
1,4-butanediol diglycidyl ether, 1,2-ethanediol diglycidyl ether or
an epoxy-substituted pentaerythritol.
41. The process according to claim 40 wherein the epoxide is
1,4-butanediol diglycidyl ether.
42. The process according to claim 36 wherein the alkaline medium
has a pH in the range of about 9 to 12.
43. The process according to claim 42 wherein the alkaline medium
comprises between 1 and 5 wt/vol percent polysaccharide and between
0.05 and 0.5 wt/vol percent epoxide.
44. The process according to claim 43 wherein the epoxide contacts
the polysaccharide at a temperature of at least about 45.degree.
C.
45. The process according to claim 44 wherein the polysaccharide
matrix is dried under vacuum at a temperature of at least about
35.degree. C.
46. The process according to claim 36 wherein steps (a) to (c) are
performed under alkaline conditions.
47. The process according to claim 46 wherein the optional washing
step (c) further comprises washing the cross-linked polysaccharide
matrix with acetone.
48. The process according to claim 47 wherein the neutralisation
step (d) further comprises freeze drying the cross-linked
polysaccharide gel and reconstituting the gel.
49. The process according to claim 48 wherein the freeze dried
cross-linked polysaccharide gel is reconstituted in phosphate
buffered saline.
50. The process according to claim 49 further comprising combining
the polysaccharide with a biologically active substance.
51. A cross-linked polysaccharide gel substantially resistant to
hyaluronidase degradation prepared by the process according to
claim 36.
52. The gel according to claim 51 wherein the gel releases less
than about 75 percent uronic acid under hyaluronidase
treatment.
53. The gel according to claim 51 wherein the gel releases no more
than about 70 percent uronic acid under hyaluronidase
treatment.
54. The gel according to claim 51 wherein the gel releases no more
than about 65 percent uronic acid under hyaluronidase
treatment.
55. The gel according to claim 51 wherein the gel releases less
than about 75 percent uronic acid after being extruded or expelled
from a 32 gauge needle.
56. The gel according to claim 51 wherein the gel releases no more
that about 70 percent uronic acid after being extruded or expelled
from a 30 gauge needle.
57. The gel according to claim 51 further comprising a biologically
active substance.
58. The gel according to claim 57 wherein the biologically active
substance is a hormone, cytokine, vaccine, cell, tissue augmenting
substance, or mixture thereof.
59. The gel according to claim 58 wherein the tissue augmenting
substance is collagen, starch, dextranomer, polylactide,
poly-beta-hydroxybutyrate, or copolymers thereof.
60. The gel according to claim 57 wherein the biologically active
substance is an alkaloid, peptide, phenothiazine, benzodiazepine,
thioxanthene, hormone, vitamin, anticonvulsant, antipsychotic,
antiemetic, anesthetic, hypnotic, anorexigenic, tranquilizer,
muscle relaxant, coronary vasodilator, antineoplastic, antibiotic,
antibacterial, antiviral, antimalarial, carbonic anhydrase
inhibitor, nonsteroid antiinflammatory agent, vasoconstrictor,
cholinergic agonist, cholinergic antagonist, adrenergic agonist,
adrenergic antagonist narcotic antagonist or combination
thereof.
61. A pharmaceutical composition comprising: a cross-linked
polysaccharide gel substantially resistant to hyaluronidase
degradation prepared by the process according to claim 36; a
biologically active substance; and a pharmaceutically acceptable
carrier.
62. The pharmaceutical composition according to claim 61 wherein
the preparation is in the form of a pill, tablet, capsule,
suppository, spray, cream ointment or sticking plaster.
63. A method of treating or preventing a disorder or condition
selected from the group consisting of tissue augmentation,
arthritis, tissue adhesions, immunogenicity, diseases of the
mucosa, dermatological conditions, ophthalmological conditions,
hormonal conditions, joint lubrication conditions and cosmetic
conditions, in a subject in need thereof, comprising administering
a therapeutically effective amount of a gel substantially resistant
to hyaluronidase degradation prepared by the process according to
claim 36.
64. The method according to claim 63 wherein the gel further
comprises a biologically active substance, and a pharmaceutically
acceptable carrier.
65. The method according to claim 63, wherein the administration to
the subject is by injection.
66. The method according to claim 63, wherein the administration to
the subject is by topical application.
Description
TECHNICAL FIELD
[0001] The present invention relates to cross-linked polysaccharide
compositions, processes for preparing the compositions, and uses of
the compositions in cosmetic, medical and pharmaceutical
applications.
BACKGROUND ART
[0002] Hyaluronic acid (HA) is a member of a class of polymers
known as glycosaminoglycans. HA is a long chain linear
polysaccharide and is usually present as a sodium salt having the
molecular formula (C.sub.14H.sub.20NNa.sub.11).sub.n where n may
vary according to the source of the HA and the method of isolating
the HA. Molecular weights of HA of up to 14.times.10.sup.6 have
been reported.
[0003] HA and its salts may be isolated from many sources including
the human umbilical cord, rooster combs and nearly all connective
matrices of vertebrate organisms. HA is also a capsular component
of bacteria such as streptococci and may therefore be obtained by
fermentation methods such as reported in U.S. Pat. No. 5,411,874
(Fermentech Ltd).
[0004] HA is non-immunogenic and therefore has great potential in
medicine. Because of its visco-elastic properties, HA having a high
molecular weight (over 1 million) has been found to be particularly
useful in a variety of clinical fields, including wound treatment,
ophthalmic surgery, orthopedic surgery and drug delivery. HA is
also potentially useful in a variety of non-medical fields,
including cosmetic applications.
[0005] However, one drawback to administering HA to humans is that
HA is degraded by enzymes such as hyaluronidase and free radicals
found in the human body. Furthermore, HA is soluble in water at
room temperature, which may also make it less suited to certain
applications.
[0006] Various attempts have been made to prepare more stable forms
of HA, in particular, by cross-linking the HA molecules. For
example, hydroxyl groups have been cross-linked via an ether
linkage and carboxyl groups via an ester linkage. HA has been
cross-linked at pH levels less than 9 at which ester bonds form via
carboxyl groups, and at pH levels greater than 9 at which ether
bonds form via hydroxyl groups. The present inventors have found
that ether bonds may be beneficial because these bonds are more
resistant to physiological degradation.
[0007] A number of documents report a variety of methods of
cross-linking HA gels. For example, U.S. Pat. No. 4,582,865
(Biomatrix Inc) reports cross-linked gels of HA formed by
cross-linking HA (either by itself or mixed with other hydrophilic
polymers) using divinyl sulfone as the cross-linking agent.
[0008] U.S. Pat. No. 5,827,937 (Agerup) reports polysaccharide gel
compositions prepared by forming an aqueous solution of the
polysaccharide, initiating cross-linking in the presence of a
polyfunctional cross-linking agent, sterically hindering the
cross-linking reaction from being terminated before gelation occurs
(e.g. by diluting the solution) and then reintroducing sterically
unhindered conditions (e.g. by evaporating the solution) so as to
continue the cross-linking such that a viscoelastic gel is formed.
The cross-linking in this method may be performed under alkaline or
acidic conditions.
[0009] WO 00/46253 (Fermentech Ltd) reports cross-linking HA with
other polymers by two different types of cross-linking bonds. The
formation of different types of bonds is achieved by cross-linking
via different functional groups. For example, one type of bond may
be formed by cross-linking via hydroxyl groups, and a different
functional bond may be formed by cross-linking via carboxyl
groups.
[0010] WO 87/07898 reports reacting a polysaccharide with a
polyfunctional epoxide, removing excess epoxide and employing a
drying operation to cross-link the polysaccharide into a film,
powdered material or similar dry product.
[0011] U.S. Pat. No. 4,963,666 (Pharmacia) reports a process in
which a polysaccharide is monosubstituted with a cross-linking
agent at low concentration under alkaline conditions to form ether
linkages. The mixture is washed to pH 5.5 inducing some ester
linkages and then, in one example, concentrated by slow evaporation
to complete cross-linking with ester linkages. In another example,
the pH is increased by the addition of ammonia, and then slowly
evaporated to complete the cross-linking with primarily ether
linkages and some ester linkages.
[0012] Although attempts have been made to improve the properties
of cross-linked HA, it would be beneficial to provide cross-linked
HA gels having improved degradation characteristics when
administered to a patient.
DISCLOSURE OF INVENTION
[0013] In one embodiment, the present invention provides a process
for producing a cross-linked polysaccharide gel. First, a
polysaccharide mixed with an alkaline medium is contacted with a
bifunctional or polyfunctional epoxide to form an essentially epoxy
cross-linked polysaccharide in which the epoxide is linked to the
polysaccharide substantially by ether bonds. The epoxy cross-linked
polysaccharide is then dried without removing the epoxide from the
alkaline medium. The resulting dried cross-linked polysaccharide
matrix may then be washed in a suitable water miscible solvent, and
treated with an acidic medium to form a cross-linked polysaccharide
gel.
[0014] A variety of polysaccharide starting materials may be used
in embodiments of the present invention. Suitable polysaccharides
include HA, pectin, xanthan or alginic acid, as well as anionic
derivatives of carboxymethyl cellulose, carboxymethyl dextran or
carboxymethyl starch. HA may be a particularly suitable starting
material. Suitable epoxides for use as the cross-linking agent
include 1,4-butanediol ether, 1,2-ethanediol diglycidyl ether
and/or epoxy-substituted pentaerythritol. It will be appreciated,
however, that other epoxides may also be suitable for the present
invention.
[0015] In another embodiment, the present invention provides a
cross-linked polysaccharide gel prepared by the process reported
herein. The gel may have improved degradation characteristics when
administered to a patient.
[0016] In yet another embodiment, the present invention provides a
biocompatible gel including HA cross-linked substantially by ether
bonds with 1,4-butanediolglycidyl ether that is sufficiently
cross-linked to resist to degradation.
[0017] As used herein, the phrase "sufficiently cross-linked to
resist degradation" means that the gel is relatively stable to
hyaluronidase attack under physiological conditions over prolonged
periods or can tolerate extrusion or being expelled from a small
gauge needle. In one embodiment, the present inventors have been
able to produce biocompatible gels which release less than 75
percent uronic acid when 0.4 ml of the gel having a concentration
of 15 mg/ml is combined with 0.5 mg hyaluronidase and 3 ml
phosphate buffered saline, and stored at a temperature of at least
37.degree. C. for two days. Uronic acid release may be measured by
the UV absorbance techniques reported in the Examples. In certain
embodiments, the gels may release no more that 70 percent uronic
acid, more particularly no more that 65 percent uronic acid under
the foregoing conditions.
[0018] In a first aspect, the present invention provides a process
for producing a cross-linked polysaccharide gel comprising: [0019]
(a) contacting a polysaccharide mixed in an alkaline medium with a
bifunctional or polyfunctional epoxide to provide an essentially
epoxy cross-linked polysaccharide wherein the epoxide is
substantially linked to the polysaccharide by ether bonds; [0020]
(b) drying the epoxy cross-linked polysaccharide without
substantially removing epoxide from the alkaline medium to form a
cross-linked polysaccharide matrix; [0021] (c) optionally washing
the cross-linked polysaccharide matrix with a water miscible
solvent; and [0022] (d) neutralising the cross-linked
polysaccharide matrix with an acidic medium to form a cross-linked
polysaccharide gel.
[0023] In a second aspect, the present invention provides a
cross-linked polysaccharide gel substantially resistant to
hyaluronidase degradation prepared by the process according to the
first aspect of the present invention.
[0024] In a third aspect, the present invention provides a
biocompatible gel comprising hyaluronic acid cross-linked
substantially by ether bonds with 1,4-butanediol diglycidyl ether
such that the gel is sufficiently cross-linked to substantially
resist degradation.
[0025] In a fourth aspect, the present invention provides a
pharmaceutical composition comprising a cross-linked polysaccharide
gel according to the second aspect of the present invention; a
biologically active substance; and a pharmaceutically acceptable
carrier.
[0026] In a fifth aspect, the present invention provides a
pharmaceutical composition comprising a biocompatible gel according
to the third aspect of the present invention; a biologically active
substance; and a pharmaceutically acceptable carrier.
[0027] In a sixth aspect, the present invention provides a method
of treating or preventing a disorder in a subject in need thereof,
comprising administering a therapeutically effective amount of a
gel according to the fourth aspect of the present invention.
[0028] In a seventh aspect, the present invention provides a method
of treating or preventing a disorder in a subject in need thereof,
comprising administering a therapeutically effective amount of a
pharmaceutical composition according to the fifth aspect of the
present invention.
[0029] In a eighth aspect, the present invention provides use of a
gel according to the third aspect of the present invention in the
manufacture of a medicament for treating or preventing a disorder
in a subject in need thereof.
[0030] In a ninth aspect, the present invention provides use of a
pharmaceutical composition according to the fourth aspect of the
present invention in the manufacture of a medicament for treating
or preventing a disorder in a subject in need thereof.
[0031] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising", will be understood to imply the inclusion of a
stated element, integer or step, or group of elements, integers or
steps, but not the exclusion of any other element, integer or step,
or group of elements, integers or steps.
[0032] Any discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is solely for the purpose of providing a context for
the present invention. It is not to be taken as an admission that
any or all of these matters form part of the prior art base or were
common general knowledge in the field relevant to the present
invention as it existed in Australia before the priority date of
each claim of this application.
[0033] In order that the present invention may be more clearly
understood, preferred embodiments will be described with reference
to the following drawings and examples.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 shows the titration curve of hyaluronidase on a
hyaluronic acid substrate. as reported in the Examples.
[0035] FIG. 2 shows a comparison of uronic acid (UA) release
between samples A and B as reported in the Examples.
[0036] FIG. 3 shows the UV absorption of UA in gels after 1 day as
reported in the Examples.
[0037] FIG. 4 shows the UV absorption of UA at 530 nm at one, two
and twelve days as reported in the Examples.
[0038] FIG. 5 shows the UV absorption of UA at 530 nm, after two
days incubation as reported in the Examples.
[0039] FIG. 6 shows a comparison between various gels as reported
in the Examples.
MODE(S) FOR CARRYING OUT THE INVENTION
[0040] In one embodiment, the present invention provides a process
for making a polysaccharide cross-linked gel. The process generally
includes the steps of:
[0041] (a) forming an epoxy cross-linked polysaccharide by
contacting a polysaccharide starting material with a bifunctional
or polyfunctional epoxide in an alkaline medium to form an
essentially cross-linked polysaccharide in which the epoxide is
substantially linked to the polysaccharide by ether bonds;
[0042] (b) drying the epoxy cross-linked polysaccharide without
substantially removing epoxide from the alkaline medium;
[0043] (c) optionally washing the dried epoxy cross-linked
polysaccharide with a water miscible solvent to form a cross-linked
polysaccharide matrix; and
[0044] (d) neutralising the epoxy cross-linked polysaccharide with
an acidic medium to form a cross-linked polysaccharide gel.
[0045] Advantageously, it has been determined that when the epoxide
cross-linked polysaccharide gel is formed in the foregoing manner,
the gel has improved resistance to degradation when compared to
conventional cross-linked polysaccharide gels.
[0046] The polysaccharide starting material may be selected from a
wide range of suitable naturally-occurring carboxylate-containing
polysaccharides, including HA, pectin, xanthan, or alginic acid, as
well as anionic derivatives of neutral polysaccharides such as
carboxymethyl cellulose, carboxymethyl dextran or carboxymethyl
starch.
[0047] In one embodiment, HA is used as the polysaccharide starting
material. HA may be extracted from a number of sources, for
example, cocks' combs. In certain embodiments, it may be desirable
to use hyaluronic acids constituting molecular fractions of the
integral acids obtained directly by extraction of organic materials
with a wide range of molecular weights. These fractions may be
obtained by various conventional procedures, including hydrolysis,
oxidation, enzymatic chemical agents or physical procedures such as
mechanical or irradiation procedures. Separation and purification
of the molecular fractions obtained may be accomplished by
molecular filtration. An example of a suitable purified HA fraction
is the "noninflammatory-NIF-NaHA sodium hyaluronate", reported by
Balazs in the pamphlet "Healon"--A guide to its use in Ophthalmic
Surgery--D. Miller & R. Stegmann, eds. John Wiley & Sons
N.Y. 81983: p. 5.
[0048] Other suitable HA starting materials include "Hyalastine"
brand and "Hyalectin" brand HA. The fraction Hyalastine has an
average molecular weight of about 50,000 to 100,000 while the
fraction Hyalectin has an average molecular weight of about 500,000
to 730,000. A combined fraction of these two fractions has also
been isolated and characterized as having an average molecular
weight of between about 250,000 and about 350,000. This combined
fraction may be obtained with a yield of 80% of the total
hyaluronic acid available in the particular starting material,
while the fraction Hyalectin may be obtained with a yield of 30%
and the fraction Hyalastine with a yield of 50% of the starting HA.
The preparation of these fractions is reported in European patent
publication No. 0138572A3. Other suitable HA starting materials
include the fibrous and powdered HA materials reported in the
Examples below.
[0049] The polysaccharide may be cross-linked by a variety of
suitable polyfunctional cross-linking epoxides, including bi- or
polyfunctional epoxides, such as lower aliphatic epoxides or their
corresponding epihalohydrins. Specific examples of suitable
epoxides include 1,4-butanediol diglycidyl ether (BDDE),
1,2-ethanediol diglycidyl ether, epoxy-substituted pentaerythritol
(e.g. SHELL 162) and epihalohydrins thereof. In one embodiment, the
poly-functional cross-linking agent includes 1,4-butanediol
diglycidyl ether.
[0050] The polysaccharide starting material may be combined with
the cross-linking agent in an alkaline medium. In one embodiment,
between about 1 and about 5 w/v percent, more particularly about 4
w/v percent, polysaccharide may be added to the alkaline medium.
The alkaline medium may be formed with sodium hydroxide or other
suitable basic materials. The concentration of sodium hydroxide or
other basic material may be between about 0.1 and about 1 w/v
percent, more particularly about 1% of the total mixture. The
cross-linking agent may be added to the alkaline mixture to produce
a cross-linking agent concentration between about 0.05 and about
0.5%, more particularly about 0.1%. The alkaline medium may have a
pH between about 9 and 12, more particularly, about 9.
[0051] The resulting alkaline mixture may be incubated under
conditions that promote cross-linking of the polysaccharide with
the epoxide. For example, the mixture may be incubated in a water
bath at about 45.degree. C. for about 2 hours. HA cross-linked
under these conditions will substantially include ether bonds which
are generally more resistant to physiological degradation than
ester bonds formed under acidic conditions.
[0052] After incubation, the cross-linked mixture may be dried by
conventional methods to form a polysaccharide matrix. For example
the cross-linked mixture may be dried by stirring the mixture
vigorously and removing the water under high vacuum for about 1.5
hours at between about 35.degree. C. and 45.degree. C. After
drying, the polysaccharide matrix may be washed with a water
miscible solvent, for example an isopropyl alcohol/water
co-solvent, for several hours. Finally, the washed matrix may be
neutralised with an acidic medium to form a cross-linked
polysaccharide gel. For example, the matrix may be treated with a
solution of 1-2 percent acetic acid in water to form the
cross-linked polysaccharide gel. Optionally, the cross-linked
polysaccharide gel may be further treated with a phosphate buffered
saline mixture to affect the viscosity of the gel.
[0053] As further reported in the Examples below, the
polysaccharide gel formed by the foregoing method is sufficiently
cross-linked to resist degradation when administered to a patient.
Because of the improved degradation characteristics of the gel, the
resulting cross-linked polysaccharide gel may be used for a variety
of applications. In one embodiment, the cross-linked polysaccharide
gel may be used for augmenting tissue, treating arthritis, treating
tissue adhesions, and for use in coating mammalian cells to reduce
immunogenicity. In another embodiment, the cross-linked
polysaccharide gel may be used in cosmetic applications, corrective
implants, hormone replacement therapy, hormone treatment,
contraception, joint lubrication, and ocular surgery.
[0054] Advantageously, the cross-linked polysaccharide gel remains
substantially resistant to degradation following extrusion through
a narrow gauge needle. Extrusion through a needle may break gels
into smaller particles if the gels are not resistant to shear
stress. In particular, the cross-linked polysaccharide gels of
embodiments of the present invention are resistant to degradation
following extrusion through a small gauge needle such as a 27, 30
or 32 gauge needle. Thus, these gels are particularly suitable for
injection into tissue or skin without substantial loss of the
structural integrity of the solution or gel.
[0055] In an alternate embodiment, the cross-linked polysaccharide
gel may be combined with a biologically active substance for
administration to a patient. Suitable biologically active
substances for use with the present invention include hormones,
cytokines, vaccines, cells, tissue augmenting substances, or
mixtures thereof. Examples of suitable tissue augmenting substances
include collagen, starch, dextranomer, polylactide,
poly-beta-hydroxybutyrate, and/or copolymers thereof.
[0056] Additional examples of biologically active substances are
reported in U.S. Pat. No. 5,676,964, which is incorporated herein
by reference for the purpose of describing suitable biologically
active substances, methods of preparing cross-linked polysaccharide
gels including these substances and methods of administering the
biologically active substances.
[0057] Suitable biologically active substances may include various
alkaloids, peptides, phenothiazines, benzodiazepines,
thioxanthenes, hormones, vitamins, anticonvulsants, antipsychotics,
antiemetics, anesthetics, hypnotics, anorexigenics, tranquilizers,
muscle relaxants, coronary vasodilators, antineoplastics,
antibiotics, antibacterials, antivirals, antimalarials, carbonic
anhydrase inhibitors, nonsteroid antiinflammatory agents,
vasoconstrictors, cholinergic agonists, cholinergic antagonists,
adrenergic agonists, adrenergic antagonists, narcotic
antagonists.
[0058] The biologically active substance may be combined with
suitable cross-linked polysaccharide gels of the present invention
by physical mixing of the biologically active substance with the
polysaccharide starting material. The biologically active substance
may be combined in solid form, for example as a freeze-dried powder
or solutions.
[0059] The use of the cross-linked polysaccharide gel as a vehicle
for biologically active substances may be particularly useful in
ophthalmology, where particular compatibility between the
cross-linked polysaccharide gels and the corneal epithelium exists.
When biologically active substances are administered in the form of
concentrated solutions with elastic-viscous characteristics or in
solid form on the corneal epithelium, homogenous and stable films
are formed that are transparent and adhering, and that provide
prolonged bioavailability of the biologically active substance. The
cross-linked polysaccharide gel vehicles of embodiments of the
present invention may also be suitable for treatment of diseases of
the mucosa (e.g. diseases of the mount) and dermatological
treatments.
[0060] In certain embodiments, the foregoing biologically active
gels may be formed into pharmaceutical preparations for oral,
rectal, parenteral, subcutaneous, local or intradermal use.
Suitable pharmaceutical preparations may be in solid or semisolid
form, for example pills, tablets, gelatinous capsules, capsules,
suppositories or soft gelatin capsules. For parenteral and
subcutaneous uses, pharmaceutical preparations intended or
intramuscular or intradermal uses or infusions or intravenous
injections may be used, and may therefore be presented as solutions
of the active compounds or as freeze-dried powders of the active
compounds to be mixed with one or more pharmaceutically acceptable
excipients or diluents. Additionally, pharmaceutical preparations
in the form of topical preparations may be suitable, for example
nasal sprays, creams and ointments for topical use or sticking
plasters specially prepared for intradermal administration.
[0061] The preparations may be administered to humans or animals.
In one embodiment, the cross-linked polysaccharide gel may contain
between about 0.01% and 10% of biologically active substance for
solutions, sprays, ointments and creams, and between about 15% and
50% of biologically active substance for the solid form
preparations.
[0062] In the context of the present invention, the term "alkaline
medium" includes, but is not limited to a hydroxide salt dissolved
in water, preferably sodium hydroxide.
[0063] In the context of the present invention, the term "acidic
medium" includes, but is not limited to an organic or inorganic
acid dissolved in water, preferably acetic acid.
EXAMPLES
Synthesis of Cross-Linked Gels
[0064] Separate samples of fibrous [Javenech HTL (MW 1.6-1.33 MD)]
and powder hyaluronic acid [Fluka from Streptococcus equi (MW 1.69
MD)] (0.5 g) were each dissolved in 1% NaOH (12.5 ml) with vigorous
stirring over a period of 1 hour. 1,4-butanediol diglycidyl ether
(BDDE)(12.5 .mu.l) was added with vigorous stirring for 5 minutes
and then the resulting solution was incubated without stirring in a
water bath at 45.degree. C. for 2 hours. At the end of the
incubation period the mixture was removed from the bath, stirred
vigorously for 1 minute and then water was removed under high
vacuum for 1.5 hours at 35-40.degree. C. The resulting transparent
polysaccharide matrices were washed with an isopropyl alcohol and
water mixture (IPA/H.sub.2O) (6:4, 25 ml) for 22 hours, and then
the IPA/H.sub.2O mixture was replaced two more times every 22 hours
(i.e. for a total wash time of 66 hours). The IPA/H.sub.2O mixture
was removed, and then 1.3 percent acetic acid in water (25 ml) was
added with stirring. After 35 minutes, both samples had produced
fully swollen gels with the "fibrous" gel ("Sample A") being
noticeably more viscous than the "powdered" gel ("Sample B").
[0065] The gels were then subjected to a series of washes with IPA
(50 ml), IPA/H.sub.2O (6:4, 25 ml), IPA/H.sub.2O (8:2, 100 ml), and
then IPA (50 ). The resulting opaque rubbery materials were then
freeze dried to give opaque hard sheets. The sheets were then
reconstituted in freshly prepared phosphate buffered saline over 24
hours at concentrations of 15 and 20 mg/ml for use in the following
Examples. Sample A was pushed under pressure through a 500 .mu.m
mesh while Sample B was pushed under pressure though a 300 .mu.m
mesh. The samples were used over a 3-month period and did not
degrade during storage.
Carbazole Assay
[0066] The reaction of uronic acids with carbazole is a
satisfactory method to estimate the quantity of uronic acids in
different compounds. The procedure reported in Bitter and Muir [T.
Bitter and H. M. Muir, Anal. Biochem. 4, 330-334 (1962)] was
followed to establish a standard titration curve.
[0067] Reagents: [0068] A: 0.025 M sodium tetraborate 10 H.sub.20
in sulfuric acid 98%; [0069] B: 0.125% carbazole in absolute
ethanol (stable 12 weeks at 4.degree. C. in the dark); [0070] C: 11
glucuronolactone solutions of 0, 1, 5, 10, 15, 20, 25, 30, 40, 50,
75 and 100 .mu.g/ml in deionized water saturated with benzoic acid
(stable for 6 months at 4.degree. C.).
[0071] Reagent A (5 ml) was placed in a tube and cooled to
-70.degree. C. Solution C (1 ml) was then added. The tube was
sealed and allowed to warm to room temperature. The tube was then
shaken and heated for 10 minutes in a vigorously boiling water
bath. The tube was then cooled to room temperature. Aliquots (0.2
ml) of reagent B were then added. The tube was again shaken and
heated for 15 minutes. After returning to room temperature, the UV
absorption was measured at 530 nm. FIG. 1 shows a titration curve
of the UV absorption values as a function of the concentration of
glucuronolactone.
Resistance to Hyaluronidase of Samples A and B
[0072] To determine the concentration of uronic acid (UA) released
by hyaluronidase from Samples A and B the procedure reported in X.
B. Zhao, J. E. Fraser, C. Alexander, C. Lockett, B. J. White, J.
Mat. Science, Materials in Medicine 13, 11-16 (2002) was followed
with some modification as reported below.
[0073] One ml of each gel at various concentrations (Sample A at 20
mg/ml, Sample A at 15 mg/ml. Sample B at 20 mg/ml and Sample B at
15 mg/ml) was suspended in 6 ml of phosphate buffered saline
(pH=7.4) containing 1 mg of hyaluronidase (containing 1010 U) and
incubated at 37.degree. C. After 5 days, 0.5 ml of each Sample was
diluted in 2 ml of isopropanol. The remaining gel, which was not
destroyed by the enzyme, was precipitated and removed by
centrifugation over 30 minutes. The supernatant liquids containing
the uronic acid were then heated in a vigorously boiling bath of
water for 30 minutes to denature the enzyme, and centrifuged again
for 30 minutes to eliminate the enzyme. The volume of each tube was
adjusted to 3.5 ml. The concentration of UA released by
hyaluronidase was determined from the titration curve shown in FIG.
1 by measuring UV absorption at 530 nm. FIG. 2 shows a comparison
of the different UV values.
[0074] Lower concentrations of UA were observed in gels containing
a lower concentration of biopolymer (e.g. Sample A at 15 mg/ml
compared to Sample A at 20 mg/ml). Also Sample A was significantly
less degraded than Sample B at concentrations of both 15 and 20
mg/ml.
[0075] The concentration of UA (in .mu.g/ml of gel solution) after
5 days of incubation was determined from the titration curve (FIG.
1). A dilution factor of 7 (i.e. 3.5/0.5) was taken into account as
the 0.5 ml sample was diluted to a volume of 3.5 ml for analysis.
[0076] [UA]: concentration of UA in the gel supernatant; [0077]
[UA.sub.dil]: concentration of UA deduced from the titration curve;
[0078] y=0.0172[UA.sub.dil]+0.0215; [0079]
[UA.sub.dil]=(y-0.0215)/0.0172 where y=maximum absorption value at
530 nm; [0080]
[UA]=[UA.sub.dil].times.7=[(y-0.0215)/0.0172].times.7; [0081]
Sample A, 20 mg: y=0.439, [UA]=170 .mu.g/ml [0082] Sample A, 15 mg:
y=0.3515, [UA]=134 .mu.g/ml [0083] Sample B, 20 mg: y=0,559,
[UA]=219 .mu.g/ml [0084] Sample B, 15 mg: y=0.539, [UA]=211
.mu.g/ml Comparison of Sample A with Commercially Available Gels
Restylane.TM. and Perlane.TM.
[0085] A comparison between Sample A and Restylane.TM. gel [Q-Med
AB, Uppsala, Sweden] and Perlane.TM. gel [Q-Med AB, Uppsala,
Sweden] was performed as reported below.
[0086] Samples (0.4 ml) of each gel were suspended in 3 ml of
phosphate buffered saline (pH=7.4) containing 0.5 mg of
hyaluronidase (505 U) and incubated at 37.degree. C. The tested
gels were Restylane.TM. gel at a concentration of 20 and 15 mg/ml,
Perlane.TM. gel at a concentration of 20 and 15 mg/ml and Sample A
at a concentration of 20 and 15 mg/ml. After 1 day, 0.25 ml of each
gel was diluted in 2 ml of isopropanol. The residual gel, which was
not destroyed by the enzyme, was precipitated and removed by
centrifugation over 30 minutes. Each tube of gel was then heated in
a vigorously boiling bath of water for 30 minutes to denature the
enzyme, and centrifuged again for 30 minutes to eliminate the
enzyme. The volume of each tube was adjusted to 2 ml. The
concentration of UA released by hyaluronidase was determined from
the titration curve by measuring UV absorption at 530 nm. The UV
absorbance curve at day 1 for each gel is shown in FIG. 3.
[0087] For both the 15 mg/ml and 20 mg/ml series, Sample A
exhibited improved degradation (i.e. lower concentration of UA
released), when compared to Perlane.TM. and Restylane.TM. gels.
Indeed Sample A at a concentration of 20 mg/ml degraded less than
Perlane.TM. gel at a concentration of 15 mg/ml.
Effect of Needle Size on Gel Deterioration
[0088] To determine the effect of needle size on degradation of the
gels, the procedure reported above was repeated on Restylane.TM.
gel expelled or extruded through a 32 G needle, Perlane.TM. gel
expelled or extruded through a 30 G needle, and Sample A (500
.mu.m) extruded through a 32 G needle and a 30 G needle. Gel
concentration was fixed at 15 mg/ml.
[0089] Initially, a trial experiment was run in order to establish
when the maximum level of degradation of the gels was obtained in
the procedure conditions (0. 15 g/l of hyaluronidase).
[0090] The values obtained after two days were slightly higher than
those obtained after one day. Consequently, a third set of
measurements was taken after twelve days, in which the release of
UA was very low compared to the first 48 hours, when the gels were
mostly degraded (see FIG. 4). From this, it was determined that a
two-day incubation period was sufficient to establish a comparison
between the UA release (ie. degradation) of the different gels.
FIG. 5, shows the UV absorption at 530 nm after two days for each
experiment.
[0091] UV maxima and UA concentrations are listed in Table 1.
TABLE-US-00001 TABLE 1 Maximum absorption (at 530 nm) (UA] in
.mu.g/ml* Sample A 1.111 511 Sample A, needle 30G 1.1193 549 Sample
A, needle 32G 1.24 571 Restylane .TM. 1.482 683 Restylane .TM.,
needle 32G 1.617 746 Perlane .TM. 1.302 600 Perlane .TM., needle
30G 1.466 676 *dilution factor: 2/0.25 = 8
[0092] Table 1 indicates that the degradation level generally
increased with the decrease of the needle size. As shown in FIG. 6,
even when Sample A was extruded through a 32 G needle, the UA
concentration remained below the values observed for both
Perlane.TM. gel and Restylane.TM. gel without extrusion, thus
indicating the improved degradation characteristics of Sample
A.
Evaluation of the Degree of Degradation of the Gels
[0093] Initially, a maximum degradation level of each gel was
established. UA extraction was performed by refluxing the gel
solutions in the presence of hyaluronidase for 1 hour. Acidic
treatment (see carbazole assay procedure) was applied to the 0.25
ml sample without centrifugation. Before analysis, the solution
volume was adjusted to 2 ml.
[0094] The UA concentrations obtained from the UV spectra and the
titration curve are presented in Table 2. TABLE-US-00002 TABLE 2
Maximum absorption (at 530 nm) [UA].sub.max in .mu.g/ml* Sample A
1.6979 784 Restylane .TM. 1.6985 784 Perlane .TM. 1.6826 111
*dilution factor: 2/0.25 = 8
[0095] The similarity in the calculated concentrations indicates
that a maximum degradation level had been reached under the
reported conditions.
[0096] Next, results from Table 1 and Table 2 provided the basis to
calculate the percentage of UA released in the experimental
conditions listed below, relative to the maximum UA release that
can be expected to measure for each gels: [0097] %
UA.=[UA]/[UA].sub.max.times.100; [0098] Gels: 0.4 ml at 15 mg/ml;
[0099] Hyaluronidase: 0.5 mg;
[0100] Solvent: PBS, 3 ml; TABLE-US-00003 TABLE 3 [UA] in .mu.g/ml*
% UA Sample A 511 65 Sample A, needle 30G 549 70 Sample A, needle
32G 571 73 Restylane .TM. 683 87 Restylane .TM., needle 32G 746 95
Perlane .TM. 600 77 Perlane .TM., needle 30G 676 87
[0101] The hyaluronidase resistance studies showed that Sample A,
formed according to an embodiment of the present invention,
exhibited lower degradation than the two commercially available
cross-linked polysaccharide gels. It should be noted that the assay
method was based on a method generally used to test dense hard gels
rather than soft flowing gels, which employed a high concentration
of enzyme. Consequently, all gels exhibited significant degradation
after 2 days. Nevertheless, the results indicate that cross-linked
polysaccharide gels formed according to embodiments of the present
invention have improved degradation characteristics over
commercially available gels.
[0102] It will be appreciated by persons skilled in the art that
numerous variations and/or modifications may be made to the
invention as shown in the specific embodiments without departing
from the spirit or scope of the invention as broadly described. The
present embodiments are, therefore, to be considered in all
respects as illustrative and not restrictive.
* * * * *