U.S. patent application number 10/310629 was filed with the patent office on 2003-05-22 for sodium hyaluronate microspheres.
This patent application is currently assigned to Clear Solutions Biotech, Inc.. Invention is credited to Dehazya, Philip, Lu, Cheng.
Application Number | 20030096734 10/310629 |
Document ID | / |
Family ID | 24792994 |
Filed Date | 2003-05-22 |
United States Patent
Application |
20030096734 |
Kind Code |
A1 |
Dehazya, Philip ; et
al. |
May 22, 2003 |
Sodium hyaluronate microspheres
Abstract
The present invention relates to microspheres comprising
hyaluronan derivatized with a bifunctional crosslinker to form
microspheres. Methods of making such microspheres, comprising
mixing hyaluronic acid and a dihydrazide with a crosslinker in an
aqueous solution, adding a solvent and an emulsifying agent to form
an emulsion, and lowering the pH of the emulsion to allow
intramolecular and intermolecular crosslinking to occur, are also
disclosed. The invention also provides for pharmaceutical or
cosmetic formulations based on the microspheres described herein,
further containing one or more active or cosmetic agents, and
methods of using such formulations.
Inventors: |
Dehazya, Philip; (Westbury,
NY) ; Lu, Cheng; (Livingston, NJ) |
Correspondence
Address: |
DARBY & DARBY P.C.
Post Office Box 5257
New York
NY
10150-5257
US
|
Assignee: |
Clear Solutions Biotech,
Inc.
|
Family ID: |
24792994 |
Appl. No.: |
10/310629 |
Filed: |
December 5, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10310629 |
Dec 5, 2002 |
|
|
|
09695445 |
Oct 24, 2000 |
|
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Current U.S.
Class: |
514/1 ; 514/54;
536/53 |
Current CPC
Class: |
A61K 9/5036
20130101 |
Class at
Publication: |
514/1 ; 514/54;
536/53 |
International
Class: |
A61K 031/00; C08B
037/00; A61K 031/728 |
Claims
We claim:
1. A microsphere comprising hyaluronan functionalized with a
crosslinker at glucuronic acid sites of the hyaluronan, wherein the
derivitized hyaluronan is crosslinked intramolecularly and
intermolecularly.
2. The microsphere of claim 1, wherein the crosslinker is a
dihydrazide having the formula: H.sub.2N--NH--CO-A-CO--NH--NH.sub.2
wherein A is a substituted hydrocarbyl, unsubstituted hydrocarbyl,
substituted heterocarbyl or unsubstituted heterocarbyl moiety, said
moiety having one to twenty carbons or heteroatoms.
3. The microsphere of claim 2, wherein A is a heterocarbyl having
heteroatoms selected from the group consisting of nitrogen, oxygen,
and sulfur.
4. The microsphere of claim 2, wherein the carboxyl groups of the
glucuronic acid residues have been activated with a
carbodiimide.
5. The microsphere of claim 4, wherein the carbodiimide is
1-ethyl-dimethylaminopropyl carbodiimide.
6. The microsphere of claim 1, wherein the microsphere is formed by
mixing hyaluronan and a dihydrazide in an aqueous solution, adding
a substantially non-water miscible liquid and an emulsifying agent
to form a water in oil type-emulsion, and lowering the pH of the
emulsion.
7. The microsphere of claim 1, further comprising a component that
is incorporated into the microsphere.
8. A method of making a functionalized hyaluronic acid microsphere
comprising mixing hyaluronic acid and a dihydrazide with a
crosslinking activator in an aqueous solution, adding a
substantially non-water miscible liquid and an emulsifying agent to
form an oil in water-type emulsion, and lowering the pH of the
emulsion to allow intramolecular and intermolecular crosslinking to
occur.
9. The method of claim 8, wherein the pH of the emulsion is lowered
to the range from about pH 7 to about pH 4.
10. The method of claim 8, further comprising dehydrating the
microspheres after they have formed.
11. The method of claim 8, wherein the crosslinking activator is a
carbodiimide.
12. The method of claim 8, wherein at least one molar equivalent of
a dihydrazide is added per molar equivalent of glucuronic acid
groups on the hyaluronic acid.
13. The method of claim 8, wherein at least one molar equivalent of
a carbodiimide is added per molar equivalent of glucuronic acid
groups on the hyaluronic acid.
14. The method of claim 8, wherein the dihydrazide has the formula:
H.sub.2N--NH--CO-A-CO--NH--NH.sub.2 wherein A is a substituted
hydrocarbyl, unsubstituted hydrocarbyl, substituted heterocarbyl or
unsubstituted heterocarbyl moiety, said moiety having one to twenty
carbons or heteroatoms.
15. The method of claim 8, wherein A is a substituted heterocarbyl
or an unsubstituted heterocarbyl having heteroatoms selected from
the group consisting of nitrogen, oxygen, or sulfur.
16. A pharmaceutical or cosmetic formulation comprising a
pharmacologically effective amount of the microsphere of claim 7
and an acceptable carrier, excipient, or diluent.
17. A method of administering microspheres to a human or animal
comprising administering a pharmacologically effective amount of
the pharmaceutical or cosmetic formulation of claim 16.
Description
[0001] This is a continuation of application Ser. No. 09/695,445,
filed Oct. 24, 2000. The prior application is hereby incorporated
herein by reference, in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to microspheres comprised of
dihydrazide derivatives of sodium hyaluronate and a method for
preparing such microspheres.
BACKGROUND OF THE INVENTION
[0003] Encapsulated pharmacological and cosmetic agents have
several advantages over non-encapsulated agents. The
bioavailability of the encapsulated agent can be improved, the
active agent can be protected from degradation in a finished
formulation, and delayed or slow, sustained release of the active
agent is possible if the agent is encapsulated.
[0004] Active pharmacological or cosmetic agents can be
encapsulated by incorporation into microspheres made of
biocompatible, biodegradable natural polymers. A microsphere is a
substantially spherical particle with a diameter in the .mu.m
range. Biocompatible, biodegradable natural polymers suitable for
use in microspheres include collagen, fibrin, fibronectin, albumin,
gelatin, starch, and hyaluronic acid.
[0005] Hyaluronic acid (HA) is a viscoelastic biopolymer composed
of repeating disaccharide units of N-acetyl-D-glucosamine (GlcNAc)
and D-glucuronic acid (GlcUA). Sodium hyaluronate is the
predominate form of hyaluronic acid at physiological pH. Sodium
hyaluronate and hyaluronic acid are collectively referred to as
hyaluronan. Hyaluronan molecules have differing molecular weights
due to the fact that the number of repeating disaccharide units in
each molecule is variable. Sodium hyaluronate occurs naturally in
cellular surfaces, in the basic extracellular substances of the
connective tissues of vertebrates, in the synovial fluid of joints,
in the vitreous humor of the eye, and in the tissue of umbilical
cord. Sodium hyaluronate acts as a regulator of viscosity, tissue
hydration, lubrication, and repair, and is involved in cell
mobility, cell differentiation, wound healing, and cancer
metastasis. Hyaluronan solutions and cross-linked hyaluronan gels
can be used as drug delivery systems (U.S. Pat. No. 5,128,326). A
drug can be dispersed in a hyaluronan solution, and a cross-linked
hyaluronan gel can serve as a macromolecular cage in which a drug
substance can be dispersed. In this manner, a hyaluronan gel or
solution can serve as a vehicle that allows for the slow release of
a drug that is incorporated into the gel or solution.
[0006] A large number of sodium hyaluronate derivatives have been
synthesized by esterification of the carboxyl group of the
D-glucuronic acid moiety of the sodium hyaluronate. (U.S. Pat. No.
4,851,521; Goei, L., et al., Pharmaceutical Research 6(9) S94
(1989); Ghezzo, E., et al., International Journal of Pharmaceutics
87: 21-29 (1992)). Ester derivatives of sodium hyaluronate have
been used to form microspheres (Kyyronen, K., et al., International
Journal of Pharmacetuics 80: 161-169 (1992)); Ghezzo, E., et al.,
International Journal of Pharmaceutics 87: 21-29 (1992);
Richardson, J. L., et al., International Journal of Pharmaceutics
115: 9-15 (1995); Benedetti, L., et al., Biotechnology and
Bioengineering 53: 232-237 (1997); U.S. Pat. No. 5,690,954). In
addition, cross-linked esters of hyaluronic acid have been used to
form microspheres, which can be incorporated into a bioabsorbable
matrix to form wound implant materials (U.S. Pat. No.
5,766,631).
[0007] Sodium hyaluronate can also be derivitized by covalent
attachment of hydrazides at carboxyl groups of glucuronic acid
moieties (Pouyani, T. & Prestwich, G. D., Bioconjugate
Chemistry 5: 339-347 (1994); Vercruysse, K. P., et al.,
Bioconjugate Chemistry 8: 686-694 (1997); U.S. Pat. No. 5,652,347,
U.S. Pat. No. 5,616,568). Hyaluronate functionalized with hydrazide
has a pendant hydrazide group that allows for subsequent coupling
and crosslinking reactions (Pouyani, T. & Prestwich, G. D.,
Bioconjugate Chemistry 5: 339-347 (1994); Vercruysse, K. P., et
al., Bioconjugate Chemistry 8: 686-694 (1997); U.S. Pat. No.
5,652,347, U.S. Pat. No. 5,616,568).
[0008] Various techniques have been used to produce microspheres
made of sodium hyaluronate derivatives. A spray drying process has
been used to prepare microspheres composed of sodium hyaluronate
esters (Kyyronen, K., et al., International Journal of
Pharmaceutics 80: 161-169 (1992)). An emulsion and solvent
extraction procedure has been used to prepare microspheres composed
of water-insoluble sodium hyaluronate esters (Ghezzo, E., et
al.,International Journal of Pharmaceutics 87: 21-29(1992)). In
this approach, an emulsion was prepared in which the internal phase
was a 6% w/v hyaluronate ester solution in dimethylsulphoxide
(DMSO) containing the agents to be encapsulated, and the external
phase consisted of mineral oil and 0.5% w/v of a surfactant. The
inner phase was added to the outer phase with continuous stirring.
Extraction with ethyl acetate proceeded until microspheres were
formed. The microspheres were washed extensively with n-hexane and
dried under a vacuum. Complete separation of the residual solvents
could not be achieved with this emulsion/solvent extraction method,
however, and a relevant percentage of liquid was retained within
the microspheres. The presence of DMSO, ethyl acetate, and n-hexane
in a composition that is to be administered to humans or animals is
undesirable.
[0009] Attempts have been made to use a rapid expansion of
supercritical solutions (RESS) process and a supercritical
antisolvent process (SAS) to prepare microspheres composed of
sodium hyaluronate benzylic esters (Bendetti, L., et al.,
Biotechnology and Bioengineering 53: 232-237(1997)). In the RESS
process, a supercritical fluid is used to solubilize a nonvolatile
solute. The resulting solution is highly compressible and a sharp
decrease in the solvent density, which can be obtained by a
relatively small change in pressure, leads to a large decrease in
the solubility of the solute. Solute nucleation, triggered by a
sudden pressure decrease, is performed in media in which high
supersaturation ratios are uniformly reached, which can lead to the
formation of microparticles.
[0010] The SAS process is performed by first dissolving a solid of
interest in an organic liquid (DMSO). Then, the supercritical fluid
(CO.sub.2), which is not able to dissolve the solid but is
completely miscible with the liquid, is added to the solution in
order to precipitate the solute. The SAS continuous process is
performed in the critical region of the CO.sub.2-DMSO system. The
SAS batch process involves a low pressure gradient value and a
uniform distribution of the antisolvent in the liquid.
[0011] The RESS process could not be used to prepare hyaluronic
acid benzylic ester microspheres because the solubility of the
hyaluronic acid ester in CO.sub.2 was too low (Bendetti, L., et
al., Biotechnology and Bioengineering 53: 232-237(1997)). Using the
SAS continuous process, appreciable amounts of solute were
produced, but the particles formed were not regular in shape and
morphology, and agglomerate structures were obtained. When the SAS
process was carried out in a batch mode, microspheres of the sodium
hyaluronate ester were obtained that had an average diameter of 0.4
.mu.m and a narrow particle size distribution.
[0012] To date, the art has not provided a hydrophilic hyaluronic
acid microsphere, e.g., that may be useful for delivery of a
pharmaceutical or cosmetic.
SUMMARY OF THE INVENTION
[0013] The present invention relates to microspheres comprising
hyaluronan functionalized with a homobifunctional crosslinker at
glucuronic acid sites of the hyaluronan, wherein the derivitized
hyaluronan is crosslinked intramolecularly and intermolecularly in
the form of a microsphere.
[0014] In another aspect, the invention relates to a method of
making a functionalized hyaluronic acid microsphere comprising
mixing hyaluronic acid and a dihydrazide with a crosslinker in an
aqueous solution, adding a solvent and an emulsifying agent to form
an emulsion, and lowering the pH of the emulsion to allow
intramolecular and intermolecular crosslinking to occur.
[0015] In yet another aspect, the invention relates to a
pharmaceutical or cosmetic formulation comprising a
pharmacologically effective amount of said microspheres and an
acceptable carrier, excipient, or diluent.
[0016] In a further aspect, the invention relates to a method of
administering microspheres to a human or animal comprising
administering a pharmacologically effective amount of said
pharmaceutical or cosmetic formulation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1A shows a photograph of microspheres comprised of
dihydrazide derivatized hyaluronate. The photograph was taken using
a Zeiss microscope fitted with a Polaroid camera, type 347 film,
with 25.times. magnification and normal illumination.
[0018] FIG. 1B shows a photograph of microspheres comprised of
dihydrazide derivatized hyaluronate. The photograph was taken using
a Zeiss microscope fitted with a Polaroid camera, type 347 film,
with 25.times. magnification and phase contrast illumination.
[0019] FIG. 2A shows a photograph of desiccated FITC-labeled
microspheres, prepared as described in Example 2 (see below). The
photograph was taken using a Zeiss microscope fitted with a
Polaroid camera, type 347 film, with 25.times. magnification and
phase contrast illumination.
[0020] FIG. 2B shows a photograph of FITC-labeled microspheres in
water. FITC-labeled microspheres were prepared as described in
Example 2 (see below), and thereafter resuspended in water for
several minutes. The photograph, taken using identical conditions
to those described in FIG. 2A, shows the change in size of the
microspheres, and confirms their resistance to dissolution after
crosslinking
[0021] FIG. 3A depicts the size distribution of microspheres
suspended in 100 ml isopropyl alcohol, analyzed as described in
Example 3 (see below).
[0022] FIG. 3B depicts the size distribution of microspheres
recovered from isopropyl alcohol and resuspended in distilled water
for 7 min prior to size distribution analysis.
[0023] FIG. 4 shows a photograph of microspheres prepared from
silicone oil. See Example 4. The photograph was taken using a Zeiss
microscope fitted with a Polaroid camera, with 100.times.
magnification.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention relates to microspheres comprised of
hyaluronan derivitized with homobifunctional crosslinking groups.
The homobifunctional crosslinking groups allow functionalized
hyaluronan to be crosslinked and to serve as an intermediate for
attachment of bio-effecting agents, drugs, peptides, fluorocarbons,
oxygen-carrying agents, and other molecules of biological
interest.
[0025] Hyaluronan possesses a number of characteristics that make
its use as a drug carrier advantageous: it is biocompatible,
non-immunogenic, subject to natural degradation by enzymes in the
body, and possesses a number of functional groups such as OH, COOH
and CH.sub.2OH that are amenable to covalent modification.
Hyaluronan, however, is known to be unstable and undergoes
degradation below about pH 2 and above about pH 9. The mild
reaction conditions used in the invention avoid this degradation.
Moreover, the modified products show improved resistance to pH
extremes.
[0026] Definitions
[0027] The term "hyaluronan" is commonly used to describe a series
of naturally occurring, water soluble polysaccharides with anywhere
from about 100 to about 10,000 alternating disaccharides of
D-glucuronic acid and N-acetyl-D-glucosamine, and to describe
degraded fractions of the same. The polymer is hydrophilic and
highly viscous in aqueous solution at relatively low solute
concentrations. Hyaluronic acid often occurs naturally as the
sodium salt, sodium hyaluronate. Hyaluronic acid/sodium hyaluronate
preparations are often referred to, and are referred to herein, as
"hyaluronan" or "HA". Although the plural form "hyaluronans" may
seem more appropriate, the discussion herein shall continue to use
the singular form to refer to hyaluronan in its various forms,
including its molecular fractions. Hyaluronan can be obtained from,
e.g., Sigma, Genzyme, Lifecore, and Kraeber GMBH. Methods of
preparing commercially available hyaluronic acid and salts thereof
are well known in the art.
[0028] The term "hydrocarbyl" as used herein means the monovalent
moiety obtained upon removal of a hydrogen atom from a parent
hydrocarbon, or the divalent moitey obtained upon removal of two
hydrogen atoms. Non-limiting examples of monovalent hydrocarbyls
include alkyl, aryl, alkylaryl and arylalkyl
[0029] The term "substituted hydrocarbyl" as used herein means the
hydrocarbyl moiety as previously defined wherein one or more
hydrogen atoms have been replaced with a chemical group which does
not adversely affect the desired preparation of the product
derivative. Representative of such groups are amino-,
phosphino-,quaternary nitrogen (ammonium), quaternary phosphorous
(phosphonium), hydroxyl, amide, alkoxy, mercapto, nitro, alkyl,
halo, sulfone, sulfoxide, phosphate, phosphite, carboxylate,
carbamate groups and the like.
[0030] As used herein, the term "about" or "approximately" means
within 25%, preferably 15%, more preferably 5%, and most preferably
1% of the given value. Alternatively, the term "about" means the
standard deviation or variance for a given value, if available.
[0031] The term "microsphere" is intended to mean a substantially
spherical particle with a diameter of about 1 .mu.m to about 500
.mu.m. Microspheres need not be uniform in size. Microspheres maybe
solid or hollow and may encapsulate a pharmacologically active
substance, a cosmetic agent, a biopolymer, or a growth factor. A
microsphere may contain any substance, such as, but not limited to,
pharmacologically active agents, polynucleotides, polypeptides, and
cosmetic agents. Preferably, the substance is lipophilic when
making microspheres using an oil-in-water emulsion; and hydrophilic
when making microspheres using a water-in-oil technique.
[0032] The terms "pharmacologically active agent" or "cosmetic
agent" as used herein refer to any chemical material, compound, or
composition suitable for administration to humans and animals which
provides any desired pharmacological or cosmetic effect.
[0033] The term "effective amount" of pharmacologically active
agent or cosmetic agent refers to a nontoxic but sufficient amount
of a compound to provide the desired effect and performance at a
reasonable benefit/risk ratio attending any medical treatment.
[0034] The term "crosslinking agent" means any composition of
matter that facilitates a cross linking reaction, i.e., that makes
a crosslinking reaction occur more rapidly or efficiently. The term
"crosslinker" means a molecule with two reactive groups, which can
join to separate molecules or regions of a molecule by forming
covalent bonds with two different functional groups on the
molecule. As used herein, the term "bifunctional molecule" refers
to a molecule with two reactive groups. The bifunctional molecule
may be homobifunctional or heterobifunctional. Homobifunctional
molecules have at least two reactive functional groups, which are
the same. Heterobifunctional molecules have at least two reactive
functional groups, which are different.
[0035] The term "pK.sub.a" is used to express the extent of
dissociation or the strength of weak acids, so that, for example,
the pK.sub.a of the protonated amino group of amino acids is in the
range of about 12-13, in contrast to the pK.sub.a of the protonated
amino groups of the dihydrazides useful herein, which is less than
about 7.
[0036] The term "therapeutic drugs" is intended to include those
defined in the Federal Food, Drug and Cosmetic Act. The United
States Pharmacopeia (USP) and the National Formulary (NF) are the
recognized standards for potency and purity for most common drug
products.
[0037] The term "biocompatible" means non-toxic or non-damaging to
human and non-human tissue.
[0038] The term "growth factor" means any composition of matter
that specifically stimulates target cells to proliferate,
differentiate, or alter their function or phenotype.
[0039] The term "biopolymer" means a molecule in which naturally
occurring monomers, such as, for example, sugars or amino acids,
are linked by covalent chemical bonds.
[0040] Preparation of HA Microspheres
[0041] In the present invention, microspheres are formed from
hyaluronan derivatives. The HA crosslinking process involves the
following steps: hyaluronate is mixed with a homobifunctional
crosslinker; the pH is lowered to allow functionalization and
crosslinking of HA to occur; and the pH of the suspension is raised
after the crosslinking reaction is considered complete. In a
preferred embodiment, an activating agent is also present to
further facilitate the crosslinking reaction. An emulsifier is
added to the HA solution before, during, or after,
functionalization of HA. In a preferred embodiment, an emulsifier
is added to the HA solution prior to lowering the pH. Once HA
crosslinking has occured, and microspheres have been formed, the
microspheres are dehydrated, dried and, optionally, sonicated. Each
aspect of this process will be described more fully below.
[0042] Functionalization and crosslinking. The hyaluronate is
functionalized by covalent attachment of homobifunctional
crosslinking groups, such as pendant hydrazido groups. The
hyaluronan is preferably functionalized at carboxyl groups of
glucuronic acid moieties. Multiple crosslinking groups maybe
introduced into a hyaluronan polysaccharide. For example, the
functionalization of hyaluronan with dihydrazide may be represented
as Scheme 1 below: 1
[0043] Crosslinkers include a reactive group for conjugation to a
substituent; in the present case, for reaction with a carbonyl
functional group on hyaluronic acid, and more particularly with a
glucuronic acid functional group. As used herein, the term
"reactive group" refers to a functional group on the crosslinker
that reacts with a functional group on HA. The term "functional
group" retains its standard meaning in organic chemistry.
Carboxylic acids can be activated in the presence of carbodiimides,
such as EDC, allowing for interaction with various nucleophilic
reactive groups including primary and secondary amines. Alkylation
of carboxylic acids to form stable esters can be achieved by
interaction with sulfur or nitrogen mustards or crosslinkers
containing either an alkyl or aryl aziridine moiety.
[0044] Dihydrazides useful in the invention can be represented by
the following formula:
H.sub.2N--NH--CO-A-CO--NH--NH.sub.2
[0045] The symbol "A" in the dihydrazide formula maybe considered
to be a spacer group whose purpose is to allow one hydrazide to
react with the hyaluronan carboxylate while leaving the second
hydrazido group available for further chemical modification. "A"
can be a hydrocarbyl, heterocarbyl, substituted hydrocarbyl
substituted heterocarbyl, and the like. Suitable hydrocarbyls
include alkyl, aryl, alkylaryl or arylalkyl and suitable
heterohydrocarbyls also include oxygen, sulfur and/or nitrogen
atoms, in addition to carbon atoms. An alkyl maybe branched or
unbranched and may contain one to 20 carbons, or other carbon-sized
atoms, preferably 2 to 10, more preferably, 4 to 8 carbons or
carbon-sized heteroatoms, such as oxygen, sulfur or nitrogen. The
alkyl maybe fully saturated or may contain one or more multiple
bonds. The carbon atoms of the alkyl may be continuous or separated
by one or more functional groups such as an oxygen atom, a keto
group, an amino group, an oxycarbonyl group, and the like. The
alkyl may be substituted with one or more aryl groups. The alkyl
may, in whole or in part, be in form of rings such as cyclopentyl,
cyclohexyl, and the like. The non-cyclic or cyclic groups described
above may be hydrocarbyl or may include heteroatoms such as oxygen,
sulfur, or nitrogen and maybe further substituted with inorganic,
alkyl or aryl groups, including halo, hydroxy, amino, carbonyl,
etc. Any of the alkyl groups described above may have double or
triple bond(s). Moreover, any of the carbon atoms of the alkyl
group may be separated from each other or from the dihydrazide
moiety with one or more groups such as carbonyl, oxycarbonyl,
amino, and also oxygen and sulfur atoms singly or in a
configuration such as --S--S--, --O--CH.sub.2--CH.sub.2--O--,
S--S--CH.sub.2--CH.sub.2-- and NH(CH.sub.2).sub.nNH--. Aryl
substituents are typically substituted or unsubstituted phenyl, but
may also be any other aryl group such as pyrrolyl, furanyl,
thiophenyl, pyridyl, thiazoyl, etc. The aryl group maybe further
substituted by an inorganic, alkyl or other aryl group including
halo, hydroxy, amino, thioether, oxyether, nitro, carbonyl, etc.
The alkylaryl or arylalkyl groups maybe a combination of alkyl and
aryl groups as described above. These groups may be further
substituted as described above. Generally, to obtain dihydrazides,
two hydroxy groups of a dicarboxylic acid are substituted with
NH.sub.2NH.sub.2 yielding the dihydrazide.
[0046] Aliphatic dihydrazides can have the formula:
NH.sub.2NHCO(CH.sub.2).sub.nCONHNH.sub.2
[0047] wherein n'=1 to 18. Aliphatic dihydrazides useful in the
invention include, for example, succinic (butandioic) (n'=2),
adipic (hexanedioic) (n'=4) and suberic (octanedioic) (n'=6),
oxalic (ethanedioic) (n'=0), malonic (propanedioic) (n'=1),
glutaric (pentanedioic) (n'=3), pimelic (heptanedioic) (n'=5),
azelaic (nonanedioic) (n'=7), sebacic (decanedioic) (n'=8),
dodecanedioic, (n'=10), brassylic (tridecanedioic), (n'=11), etc.
up to n'=20. Other dicarboxylic acids include, for example, maleic
(HO.sub.2CCH.dbd.CHCO.sub.2H), fumaric (HO.sub.2CCH.dbd.CHCO.sub.2-
H) and aromatic dicarboxylic acids. Aromatic dihydrazides include
terephthalic acid C.sub.6H.sub.4(COOH).sub.2. Some preferred
dihydrazides are at least partially soluble in water and include
succinic, adipic and suberic dihydrazides; also pimelic, sebacic,
tridecane dioic, maleic, fumaric, isophthalic; as well as malonic,
glutaric, and azelaic dodecanedioic dihydrazide. Another preferred
dihydrazide is terephtalate dihydrazide (Lancaster, Pelham, N.H.),
which requires an organic solvent for its solvation, such as, but
not limited to, DMSO. Most preferred because of their commercial
availability, are adipic and suberic dihydrazides, and also
preferred are phthalic dihydrazide and dihydrazides containing oxa,
thio, amino, disulfide (--CH.sub.2--S--S--CH.sub.2--),
--S(CH.sub.2).sub.2S--, --O(CH.sub.2).sub.nO-- or
--NH(CH.sub.2).sub.nNH--(n=2 to 4) groups. The preferred
dihydrazides are also weak bases or weak acids having a pK.sub.a
for the protonated form, less than about 8, preferably in the range
of 1 to 7, and most preferably 2 to 6.
[0048] The carbodiimides useful in the invention may be represented
by formula:
R.sup.1--N.dbd.C.dbd.N--R.sup.2
[0049] wherein R.sup.1 and R.sup.2 are each independently selected
from the group consisting of hydrogen, hydrocarbyl of 1-25 carbon
atoms, and including substituted-hydrocarbyl, alkoxy, aryloxy,
alkaryloxy, and the like. The carbodiimides used in the invention
are well known compounds, as represented by the formula given
above. Carbodiimides having the above formula are preferred where
R.sup.1 and/or R.sup.2 represent more specifically alkyl,
cycloalkyl, aryl or substituted forms thereof. Most preferred are
carbodiimides which are at least partly water soluble at ambient
temperature and up to 80.degree. C. Representative of a preferred
class of monofunctional carbodiimides of the above formula are:
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI);
N,N'-dicyclohexylcarbodiimide;
N-allyl-N'-(.beta.-hydroxyethyl)carbodiimi- de;
N-.alpha.-dimethylaminopropyl)-N'tert-butylcarbodiimide;
N-(.alpha.-dimethyl-aminopropyl)-N'-(.beta.-bromoallyl)carbodiimide;
1-(3-dimethylaminopropyl)-3-(6-benzoylaminohexyl)-carbodiimide;
cyclohexyl-.beta.-(N-methylmorpholino)ethyl-carbodiimide-p-toluene-sulfon-
ate (CMC); and the like.
[0050] The functionalization of hyaluronan with dihydrazides is
preferably carried out under mild conditions including a pH of
about 2to about 8. A particularly preferred pH range is from about
pH 3 to about 5. The hyaluronate is dissolved in water, which may
also contain water-miscible solvents such as dimethylformamide,
dimethylsulfoxide, and hydrocarbyl alcohols, diols, or glycerols.
In one embodiment, at least one molar equivalent of dihydrazide per
molar equivalent carboxyl hyaluronate is added. For maximum
percentage functionalization, a large molar excess of the
dihydrazide (e.g., 10-100 fold) dissolved in water or an
aqueous-organic mixture is added and the pH of the reaction mixture
is adjusted by the addition of dilute acid, e.g., HCl. A sufficient
molar excess (e.g., 2 to 100 fold) of carbodiimide reagent
dissolved in water, in any aqueous-organic mixture, or
finely-divided in solid form, is then added to the reaction
mixture. The hyaluronate and dihydrazide should preferably be mixed
together before addition of the carbodiimide. An increase in pH
maybe observed after addition of the carbodiimide and additional
dilute HCl or other acid maybe added to adjust the pH.
Alternatively, a suitable buffer, such as Bis-Tris, may be
included. In such cases, a more concentrated acid solution may be
required to lower the pH of the solution (or emulsion, if
emulsifier has been added (see below)). The reaction is allowed to
proceed at a temperature of about 0.degree. C. to about 100.degree.
C. (e.g.,just above freezing, 0.degree. C., to just below boiling
(100.degree. C.)), preferably at or near room temperature for
purposes of convenience. The time of the reaction is from about 0.5
to about 48 hours, preferably about one to about five hours.
Polymerization is a function of the chosen reactants and their
concentrations, pH, temperature, and all other variables that
affect chemical reaction rates. In the case where an emulsifier has
been added, agitation of the emulsion also affect the
polymerization rate. Periodically (e.g., once every 10 minutes
during the first hour; thereafter every hour) the pH is measured,
using either pH test strips (e.g., from EM Science) or a pH meter
with an electrode, and adjusted, if necessary, by small additions
of acid to maintain the pH within the chosen range. The
crosslinking reaction may optionally be stopped by, e.g.,
increasing the pH to within the range of about 7 to about 9, for
example by adding ammonium hydroxide. After the crosslinking
reaction is complete, the pH may be adjusted to an approximately
neutral range.
[0051] Formation of Microspheres. An emulsion technique is used to
form microspheres from functionalized hyaluronate. An emulsifier,
such as, but not limited to, Span 60 (obtained from Aldrich Co.)
can be added to the HA solution before, during, or after the
derivatization reaction. In a preferred embodiment, an emulsifier
is added to an aqueous solution of HA, a hydrazide, and a
carbodiimide, prior to lowering pH.
[0052] Preferably, the emulsifier is first dissolved in a solvent.
In one embodiment, the emulsifier concentration in the organic
solvent ranges from about 0.01-10%, or, more preferably, from 1-2%
(weight/volume); and about an equal volume of the solvent
containing emulsifier is added to the HA solution. However, for
each specific set of chosen reagents and experimental conditions
the appropriate relative amounts of emulsifier, solvent, and HA
solution may vary, but can be optimized by one skilled in the art.
Also, suitable solvents and emulsifier concentrations maybe chosen
based upon, e.g., toxicity and or emulsifier characteristics.
Preferably, the solvent is a non-water-miscible liquid. Preferred,
although non-limiting, non-water-miscible liquids include toluene
(e.g, from Aldrich Co.), silicone oil (Dow Corning 200/0.65 cts),
mineral oils of suitable viscosity, and vegetable oils such as
olive oil. A solvent such as silicone oil may result in
microspheres less prone to aggregation. In addition, hydroxypropyl
methylcellulose has been identified as an additive allowing
microspheres to be recovered after drying with minimal
agglomeration.
[0053] The emulsifier is advantageously added slowly to a HA
solution stirred using, e.g., a direct drive mixer (obtained from
Cole-Parmer) with a metal propeller, or any other suitable mixer.
The stirring rate may range from about 60 rpm to about 2000 rpm.
Preferably, a stirring rate of about 800 rpm is used. For purposes
of convenience, the temperature of the HA solution is preferably at
or near room temperature, but other temperatures ranging from about
0.degree. C. to about 100.degree. C. (e.g., just above freezing,
0.degree. C., to just below boiling (100.degree. C.)), may be
employed if so desired. After the addition of the organic phase, an
emulsion is effected. The formed globules can be observed using,
e.g., a microscope at about 40.times. magnification. If the
emulsifier is added prior to HA derivatization, the pH of the
emulsion is brought to the chosen pH range (see above), and
maintained by adding small amounts of dilute acid, to allow both
intramolecular and intermolecular crosslinking to occur within and
between derivatized hyaluronate molecules until the crosslinking
reaction is considered complete.
[0054] Thereafter, the suspension may be dehydrated by the addition
of a liquid, preferably an alcohol. The alcohol such as, but not
limited to, isopropanol (obtained from Aldrich Co.). The suspension
is separated into two layers and the organic solvent layer is
removed. Isopropanol (or another dehydrating alcohol) is added to
the solution, and, after mixing, the isopropanol is removed. If
desired, additional isopropanol may be added, and the solution
stirred overnight. Depending, for instance, on the choice of oil or
solvent, some aggregation of microspheres may occur, as shown in
the Examples below. In some cases, aggregation can be reversed to
some degree by sonicating the suspension for a suitable length of
time, e.g., about 45 minutes. As shown in the Examples below, the
appearance as well as the aggregation characteristics of a
microspheres preparation maybe influenced by the choice of solvent.
Once a phase change is effected, microspheres will settle from the
vessel when stirring is terminated. The microspheres may be
collected by sedimentation under gravity, centrifugation, or
filtration, and, optionally, dried at an elevated temperature.
[0055] Use of HA Microspheres
[0056] Microspheres may contain any substance that can be dissolved
in an oil phase when making microspheres using an oil in water
emulsion, or dissolved in the water phase when using a water in oil
technique. In the case of the microspheres of the invention,
substances to be incorporated have to be present prior to lowering
the pH to initiate HA polymerization. However, once the
microspheres have been formed, they may also be useful as carriers
of substances that will adhere or be attached to pendant hydrazide
groups on the spheres. Thus, the microspheres of this invention can
be used as carriers for a wide variety of releasable biologically
active substances having curative or therapeutic value for human or
non-human animals. Included among biologically active materials
that are suitable for incorporation into the microspheres of the
invention are therapeutic drugs, e.g., anti-inflammatory agents,
anti-pyretic agents, steroidal and non-steroidal drugs for
anti-inflammatory use, hormones, growth factors, contraceptive
agents, antivirals, antibacterials, antifungals, analgesics,
hypnotics, sedatives, tranquilizers, anti-convulsants, muscle
relaxants, local anesthetics, antispasmodics, antiulcer drugs,
peptidic agonists, sympathiomimetic agents, cardiovascular agents,
antitumor agents, oligonucleotides and their analogues, etc.
Examples of preferred active substances are all anti-inflammatory
compositions, including, but not limited to, ibuprofen, naproxen,
ketoprofen and indomethacin. Other preferred biologically active
substances are peptides that are naturally occurring, non-naturally
occurring or synthetic polypeptides or their isosteres, such as
small peptide hormones or hormone analogues and protease
inhibitors. Also preferred are spermicides, antibacterials,
antivirals, antifungals and antiproliferatives such as
fluorodeoxyuracil and adriamycin. These substances are all known in
the art.
[0057] The actual preferred amounts of active compound in a
specified case will vary according to the specific compound being
utilized, the particular compositions formulated, the mode of
application, and the particular situs and organism being treated.
Dosages for a given host can be determined using conventional
considerations, e.g. by customary comparison of the differential
activities of the subject compounds and of a known agent, e.g., by
means of an appropriate convention pharmacological protocol.
[0058] The pendant hydrazido group of the derivitized hyaluronan of
the microspheres may be used for the coupling of compounds to
hyaluronan. For example, drugs may be covalently attached through
the intermediacy of hydrolytically and/or enzymatically labile
bonds, which allows for the preparation of controlled release
formulations. Such labile linkages include ethers, imidates,
thioimidates, esters, amides, thioethers, thioesters, thioamides,
carbamates, ethers, disulfides, hydrazides, hydrazones, oxime
ethers, oxime esters and amines. Carboxylate-containing chemicals
such as the anti-inflammatory drugs ibuprofen or
hydrocortisone-hemisuccinate can be converted to the corresponding
N-hydroxysuccinimide (NHS) active esters and can further react with
a primary amino group of the dihydrazides. Non-covalent entrapment
of a pharmacologically active agent in the microspheres is also
possible. Electrostatic or hydrophobic interactions can facilitate
retention of a pharmaceutically active agent in the microspheres.
For example, the hydrazido of the invention can non-covalently
interact, e.g., with carboxylic acid-containing steroids and their
analogs, and anti-inflammatory drugs such as Ibuprofen
(2-(4-iso-butylphenyl) propionic acid). The protonated hydrazido
group can form salts with a wide variety of anionic materials such
as proteins, heparin or dermatan sulfates, oligonucleotides,
phosphate esters, and the like.
[0059] The microspheres of the invention maybe directly labeled for
in vivo imaging purposes, such as CAT, PET, and MRI scanning.
Labels for use in the invention include colloidal gold, colored
latex beads, magnetic beads, fluorescent labels (e.g., fluorescene
isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR),
rhodamine, free or chelated lanthanide series salts, especially
Eu.sup.3+, to name a few fluorophores), chemiluminescent molecules,
radioisotopes (.sup.125I, .sup.32P, .sup.35S, chelated Tc, etc.) or
magnetic resonance imaging labels.
[0060] Materials that have been incorporated into the microspheres
can be subject to sustained release by chemical, enzymatic and
physical erosion of the microsphere and/or the covalent
hyaluronate-drug linkage over a period of time, providing improved
therapeutic benefits of the compounds. Sustained release is
particularly useful with anti-inflammatories, anti-infectives,
sperimicidal and anti-tumor agents.
[0061] The microspheres of this invention can incorporated into a
formulation for the purpose of administration to humans and
animals. Suitable formulations include, but are not limited to,
solutions, suspensions, emulsions, creams, ointments, powders,
liniments, salves, aerosols, etc., which are, if desired,
sterilized or mixed with auxiliary agents, e.g., preservatives,
stabilizers, wetting agents, buffers, or salts for influencing
osmotic pressure, etc. The microspheres of this invention can be
employed in admixture with conventional excipients, i.e.,
pharmaceutically acceptable organic or inorganic carrier substances
suitable for parenteral or topical application which do not
deleteriously react with the active compounds. Suitable
pharmaceutically acceptable carriers include, but are not limited
to, water, salt solutions, alcohols, gum arabic, vegetable oils,
benzyl alcohols, polyethylene glycols, gelatine, carbohydrates such
as lactose, amylose, or starch, magnesium stearate, talc, silicic
acid, viscous paraffin, perfume oil, fatty acid monoglycerides and
diglycerides, pentaerythritol fatty acid esters, hydroxymethyl
cellulose, and polyvinyl pyrrolidone, merely to name a few. The
pharmaceutical preparations can be sterilized, and if desired,
mixed with auxiliary agents, e.g., lubricants, preservatives,
stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic pressure, buffers, coloring, flavoring and/or aromatic
substances and the like, which do not deleteriously react with the
active compounds. The pharmaceutical preparations can also be
combined where desired with other active agents, e.g.,
vitamins.
[0062] Hyaluronic acid is a useful moisturizing and lubricating
agent in skin creams, shampoos, and a variety of cosmetics. For
cosmetic applications, additional biocompatible or biologically
inert materials maybe incorporated into the hyaluronan microspheres
of the invention. Additional cosmetic materials include humectants,
i.e., substances having affinity for water such as glycerine,
propylene glycol or isopropanolpropylene glycol; organic or
inorganic salts such as quaternary ammonium compounds and zinc
salts; enzymes; peptides; alcohols such as benzyl alcohol or lower
aliphatic alcohols; polymer latices; fillers such as silica and
talc; oils such as mineral oil, castor oil and petrolatum; wetting
or dispersing agents or surfactants such as block copolymers of
ethylene oxide and propylene oxide to reduce adherence to skin;
dyes; fragrances; pigments; antisolar or UV absorbing agents such
as actinoquinol, anthranilates, cinnamates, benzyl and homomenthyl
salicylate; para-aminobenzoic acid and its ester derivatives; zinc
oxide and titanium dioxide; topical medicaments such as
methylsalicylate, nicotinates, capsaicin and menthol; antiacne
medicaments such as benzoyl peroxide, resorcinol and retinoic acid;
topical antibacterials such as silver sulfadiazine, tetracycline
and cefazolin; skin hydrating agents such as sodium pyrrolidine
carboxylic acid; and other compounds such as fatty acids having
about 2 to about 24 carbon atoms, which change the rheological
properties of the modified hyaluronan. As is apparent from this
list of biocompatible materials, the microspheres of the invention
may be used for cosmetic treatments and dressings.
[0063] The following non-limiting examples further illustrate the
invention.
EXAMPLES
Example 1
Preparation of HA Microspheres
[0064] One opening of a 500 ml organic reaction kettle (with
interchangeable covers and four openings) was fitted with a
safe-lab stirrer bearing. Six-hundred mg (1.44 mmol) of sodium
hyaluronate (HA), 378 milligrams (2.16 mmol) of adipic dihydrazide
(ADH), and 100 ml of water were placed in the kettle. The HA and
ADH were completely dissolved in the water, resulting in the
formation of a viscous liquid with a pH of 6 to 7. Two-hundred
seventy-eight mg (1.44 mmol) of 1-ethyl-dimethylamino-propyl
carbodiimide (EDCI) in 10 ml of water was added to the kettle. The
mixture was stirred for five minutes.
[0065] One-hundred ml of toluene solution containing 1.5% w/v Span
60 was added to the kettle and an emulsion was formed by vigorous
stirring at 800-1000 rpm. After the emulsion was formed, the pH of
the suspension was decreased to between 4 and 5 using 1N HCl. The
crosslinking reaction was continued for six hours. The pH was then
raised to between 7 and 9 using 10% (w/v) ammonium hydroxide.
[0066] One-hundred ml of the dehydrating agent isopropanol was
added to the suspension and the mixture was stirred for
approximately 10 minutes. The suspension was separated into two
layers. The supernatant contained toluene, which was removed. An
additional 100 ml of isopropanol was added to the suspension. The
isopropanol was decanted and an additional 150 ml of isopropanol
was added and the suspension was stirred at 500 rpm overnight.
[0067] The microspheres were examined with an optical microscope
(FIGS. 1A, 1B). Some microspheres adhered together to form large
particles. The large microsphere particles were sonicated in the
isopropanol suspension for 45 minutes to break the large particles
into individual microspheres. The suspension was filtered to
recover the microspheres and the microspheres were dried at
37.degree. C.
Example 2
Preparation of FITC-labeled HA microspheres
[0068] Microspheres containing FITC-labeled bovine serum albumin
(BSA) were prepared for studies of release of this marker protein
from the spheres under various conditions. Essentially,
experimental conditions were similar to those described in Example
1, except for the addition of FITC-labeled BSA to the HA solution,
at a concentration of 10 mg/100 ml HA solution. After dissolution
and addition of all materials, polymerization was effected as
described. The microspheres were thereafter washed by repeated
sedimentation and resuspension in isopropyl alcohol. The labeled
microspheres were recovered by sedimentation and allowed to dry in
an oven for 24 h at 37.degree. C.
Example 3
Determination of Microsphere Size Distribution
[0069] Particle size distribution was determined using a Malvern
Mastersizer S instrument, operated at ambient temperature, after
adjusting the particle suspension obscurescence to approximately
10%.
Example 4
The Effect of pH on the Crosslinking Rate
[0070] The effect of pH on the crosslinking rate of sodium
hyaluronate (HA) with adipic dihydrazide (ADH) in the presence of
1-ethyl-dimethylaminopropyl carbodiimide (EDCI) was determined at
room temperature. The results of the experiment are illustrated in
Table 1. Sodium hyaluronate and adipic dihydrazide (in the amount
shown in Table 1) were dissolved in water, and EDCI (in the amount
shown in Table 1) was added to the mixture. The molar ratio of HA
to ADH to EDCI was approximately 1 to 1.5 to 1, respectively. The
pH was adjusted to either 10, 9, 8, 7, 6, 5, 4, or 3 with dilute
NaOH or HCl. Crosslinking reactions were continued for 1, 12, and
24 hours and at each time point the viscosity of the solutions were
noted. A viscous liquid indicated that crosslinking had not
occurred, whereas an unmovable, solid gel indicated extensive
crosslinking. At pH 10, crosslinking did not occur even after 24
hours, and the viscosity of the solution remained unchanged. At pH
7, 8, and 9 the viscosity of the solutions increased from 1 to 12
to 24 hours, but the solutions were still rather liquid after 24
hours. At pH 4, 5, and 6 the viscosity of the solutions also
increased from 1 to 12 to 24 hours, but the solutions were more
viscous at each time point than were the solutions at pH 7, 8, and
9. At pH 3, the solution became an unmovable gel after only seven
minutes.
1TABLE 1 The Effect of Time and pH on the Crosslinking Rate HA
H.sub.2O ADH EDCI (mg) (g) (mg) (mg) pH 1 hr 12 hr 24 hr 17.8
2.9985 12.0 8.1 10 -- -- -- 18.1 2.9985 12.5 8.5 9 * ** *** 17.8
3.0280 11.6 8.6 8 * ** *** 17.9 3.0374 11.8 8.3 7 * ** *** 18.1
2.9958 13.2 8.4 6 ** *** **** 18.0 2.9923 13.3 8.5 5 ** *** ****
17.9 3.0018 11.8 8.4 4 ** *** **** 18.3 2.9871 12.6 8.6 3 solid gel
solid gel solid gel
[0071] The viscosity of the solution is indicated by the number of
asterisks, with more asterisks indicating a more viscous
solution.
Example 4
Polymerization of Microspheres Containing FITC-BSA in Silicone
Fluid Dow 200 using ADH and Cosmetic Grade HA.
[0072] Three-hundred mg HA and 0.42 g Bis-Tris was dissolved in 100
ml water with slow stirring, using flat impeller. Three-hundred mg
ADH was added and dissolved. About 1 mg FITC-BS was added and
mixed. Twenty mg hydroxypropyl methylcellulose (HPMC, Sigma) was
added to 15 ml water with heating. The cloudy suspension was added
to the HA reaction mix. The remainder of the suspension was
recovered by adding reaction mixture to the vial-mixture and add
remainder to vessel. Thereafter, pH was adjusted to about 4.0 with
1N HCl (pH strips, about 30 drops). Three-hundred mg EDCI was
dissolved in 10 ml water, added immediately to the reaction
mixture, and mixed for about 2 min.
[0073] The mixture was then emulsified at approximately 600 rpm
with 100 ml Silicone Dow Corning 200 fluid, containing 1% (w/w)
Span 80, and allowed to polymerize for 5 h at room temperature.
After 2 hours, pH was adjusted to about 5 with 1N HCl (about 10
drops). After 5 hours, pH was adjusted to about 9-10 with 1N NaOH
(about 30 drops). The remainder of the emulsion was stirred, with
moderate reduction in speed, for about 30 min. The emulsion was
thereafter decanted into 4.times.50 ml centrifuge tubes, and
centrifuged at 1500 rpm for 20 min. Three layers were obtained:
upper (oil), middle (emulsion) and minimal lower (aqueous). The
upper oil layer was decanted, the middle emulsion layer was scooped
out, and the aqueous layer was re-centrifuged at room temperature.
The 1.sup.st cut emulsion layer and the 2.sup.nd cut emulsion layer
(creamiest portion--small amount) was added to IPA as follows. The
emulsion layer was added, scoopula wise, to 250 ml IPA 25 ml water,
adjusted to about pH 7 by the addition of 1N NaOH, and stirred at
about 600 rpm. The pH was checked, and adjusted to about 7 with 1N
HCl. The emulsion was stirred for about 30 min at 700 rpm, and
thereafter left overnight without stirring. The alcohol suspension
was centrifuged at 1500 rpm for about 20 min at room temperature,
and the tubed drained well. About half of the sedimented material
was suspended in several milliliters of IPA, and the other half in
several milliliters of water.
[0074] The water suspension got thick and sticky and contained
spheres (See FIG. 4). The alcohol suspension remained slightly
granular and contained spheres. The suspensions were dried at about
37.degree. C. in separate watch glasses. The IPA dried suspension
(about 3 hrs) was flakey but could be re-suspended in water to give
microspheres with large size variation with lots of debris. There
was not a lot of aggregation. Spheres dried from aqueous suspension
and resuspended in water did not reassume the morphology expected.
Swelled material resembled gel fragments of an irregular shape and
were somewhat amorphous. HPMC was used because it had been
identified as an additive that would allow microspheres to be
recovered after drying with minimal agglomeration. However, as this
experiment shows, what was more important was whether the
microspheres were dried from aqueous or alcohol suspensions.
Example 5
Effect of Various Parameters on Microsphere Preparation
[0075] In this Example, hydrophilic hyaluronate sodium microspheres
were prepared from the HA-ADH-EDCI crosslinking reaction by
inversion emulsion polymerization. Table 2 lists the various
parameters applied for each preparation, as well as the weight and
appearance of the microspheres formed.
2TABLE 2 Preparations of sodium hyaluronate microspheres Albumin
Preparation No. 1 2 3 4 5 6 7 Water Phase: HA (mg) 600 600 600 600
600 600 600 ADH (mg) 376 377 377 366 377 377 377 Water (ml) 100 100
100 100 100 100 100 EDCI (mg) 277 277 277 346 346 346 246 Initial
PH 6-7 (Buffer) 6-7 6-7 6-7 6-7 6-7 6-7 HA:ADH:EDCI, 1:1.5:1
1:1.5:1 1:1:5:1 1:1.5:1.25 1:1.5:1.25 1:1.5:1.25 1:1.5:1.25 (mmol
ratio) Oil Phase: Span 60 (g) 1.5 1.5 1.5 1.5 1.5 1.5 1.5 Toluene
(ml) 100 100 100 100 100 100 100 Stirring rate (rpm) 800 800 800
700 600 600 600 emul. 1 hr Reaction time (h) 4 6 6 6 4 4 4 Final pH
6-7 .fwdarw. 6-7 3 .fwdarw. 10 6-7 9-10 6-7 3 .fwdarw. 7 3 .fwdarw.
4 (10% NH.sub.4OH) Dehydrating 2 .times. 150 2 .times. 100 2
.times. 150 2 .times. 150 2 .times. 150 250, 150 250, 150 agent;
IPA (ml) Particles & Sphere & gel & beads & Beads
& Beads & Beads & Microspheres & shape Aggregated
beads Particles particles particles Aggregated particles beads
beads weight (g) 0.2860 0.7734 0.6667 0.6119 0.7149 -- 0.6548
[0076] The present invention thus relates to microspheres
comprising hyaluronan functionalized with a homobifunctional
crosslinker at glucuronic acid sites and methods of making such
microspheres. The derivatized hyaluronan microspheres have useful
pharmacological and cosmetic applications as delivery vehicles for
active pharmacological and cosmetic agents.
[0077] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0078] It is further to be understood that all numerical values are
approximate, and provided for description.
[0079] All patents, patent applications, publications, and other
materials cited herein are hereby incorporated herein reference in
their entireties. In case of conflicting terminology, the present
disclosure controls.
* * * * *