U.S. patent application number 12/808737 was filed with the patent office on 2010-12-09 for crosslinked-hyaluronic acid in emulsion.
This patent application is currently assigned to Novozymes BioPharma DK A/S. Invention is credited to Birgitte Moelholm Malle.
Application Number | 20100311963 12/808737 |
Document ID | / |
Family ID | 40429993 |
Filed Date | 2010-12-09 |
United States Patent
Application |
20100311963 |
Kind Code |
A1 |
Malle; Birgitte Moelholm |
December 9, 2010 |
CROSSLINKED-HYALURONIC ACID IN EMULSION
Abstract
The present invention relates to methods of producing
crosslinked hyaluronic acid microbeads, as well as the produced
microbeads, said method comprising the steps of: (a) mixing an
aqueous alkaline solution comprising hyaluronic acid, or a salt
thereof, with a solution comprising a crosslinking agent; (b)
forming microdroplets having a desired size from the mixed solution
of step (a) in an organic or oil phase to form a water in organic
or water in oil (W/O) emulsion; (c) continuously stirring the W/O
emulsion, whereby the reaction of hyaluronic acid with
divinylsulfone takes place to provide crosslinked hyaluronic acid
microbeads; and (d) purifying the crosslinked hyaluronic acid
microbeads.
Inventors: |
Malle; Birgitte Moelholm;
(Birkeroed, DK) |
Correspondence
Address: |
NOVOZYMES NORTH AMERICA, INC.
500 FIFTH AVENUE, SUITE 1600
NEW YORK
NY
10110
US
|
Assignee: |
Novozymes BioPharma DK A/S
Bagsvaerd
DK
|
Family ID: |
40429993 |
Appl. No.: |
12/808737 |
Filed: |
December 19, 2008 |
PCT Filed: |
December 19, 2008 |
PCT NO: |
PCT/EP2008/068050 |
371 Date: |
June 17, 2010 |
Current U.S.
Class: |
536/53 |
Current CPC
Class: |
A61K 8/025 20130101;
A61P 35/00 20180101; C08J 3/26 20130101; C08L 5/08 20130101; A61P
17/14 20180101; A61K 8/375 20130101; C08B 37/0072 20130101; Y10T
428/2982 20150115; A61K 8/608 20130101; C08J 3/07 20130101; A61P
9/00 20180101; A61P 27/02 20180101; A61P 17/16 20180101; A61P 19/02
20180101; A61K 8/735 20130101; A61K 8/894 20130101; A61K 8/37
20130101; A61P 17/02 20180101; A61Q 19/08 20130101; C08J 2305/08
20130101 |
Class at
Publication: |
536/53 |
International
Class: |
C08B 37/08 20060101
C08B037/08 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 19, 2007 |
EP |
07150176.1 |
Claims
1-42. (canceled)
43. A method of producing crosslinked hyaluronic acid microbeads,
said method comprising the steps of: (a) mixing an aqueous alkaline
solution comprising hyaluronic acid or a salt thereof, with a
solution comprising a crosslinking agent; (b) forming microdroplets
having a desired size from the mixed solution of step (a) in an
organic or oil phase to form a water in organic or water in oil
(W/O) emulsion; (c) continuously stirring the W/O emulsion, whereby
the reaction of hyaluronic acid with divinylsulfone takes place to
provide crosslinked hyaluronic acid microbeads; and (d) purifying
the crosslinked hyaluronic acid microbeads.
44. The method of claim 43, wherein the hyaluronic acid or salt
thereof is recombinantly produced in a Bacillus host cell.
45. The method of claim 43, wherein the hyaluronic acid or salt
thereof has a number average molecular weight of between 100 and
3,000 kDa.
46. The method of claim 43, wherein the alkaline solution comprises
dissolved hyaluronic acid or a salt thereof, in a concentration of
between 0.1%-40% (w/v).
47. The method of claim 43, wherein the alkaline solution comprises
dissolved sodium hydroxide in a concentration of between 0.001 -2.0
M.
48. The method of claim 43, wherein the crosslinking agent is
divinylsulfone (DVS).
49. The method of claim 48, wherein DVS is comprised in the mixed
solution of step (a) in a weight ratio of between 1:1 and 100:1 of
HA/DVS (dry weight).
50. The method of claim 43, wherein the reaction of hyaluronic acid
with divinylsulfone takes place at a temperature in the range of
5-100.degree. C.
51. The method of claim 43, wherein the stirring in step (c) is
continued for a period of between 1-180 minutes.
52. The method of claim 43, wherein the microdroplets of step (b)
have an average diameter in the range of about 1 nanometer to 1
millimeter.
53. The method of claim 43, wherein the purifying step comprises
dialyzing the crosslinked microbeads against de-ionized water using
a dialysis membrane that allows free diffusion of molecules having
a size less than 13,000 daltons.
54. The method of claim 43, wherein the purifying step comprises
neutralizing the pH of the crosslinked microbeads with a buffer or
an acid.
55. The method of claim 54, wherein the buffer comprises a buffer
with a pH in the range of 2.0-8.0.
56. The method of claim 54, wherein the buffer comprises a buffer
with a pH, which results in that the crosslinked microbeads have a
pH between 5.0 and 7.5 after the purifying step.
57. The method of claim 54, wherein the buffer comprises a
phosphate buffer and/or a saline buffer.
58. The method of claim 43, wherein the crosslinked microbeads are
washed at least once with water, and/or a phosphate and/or saline
buffer with a pH in the range of 2.0-8.0.
59. The method of claim 43, wherein a preservative is added as a
component to the crosslinked microbeads either before or after the
crosslinking reaction.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods of producing
crosslinked hyaluronic acid microbeads, as well as the produced
microbeads, said method comprising the steps of:
[0002] (a) mixing an aqueous alkaline solution comprising
hyaluronic acid, or a salt thereof, with a solution comprising a
crosslinking agent;
[0003] (b) forming microdroplets having a desired size from the
mixed solution of step (a) in an organic or oil phase to form a
water in organic or water in oil (W/O) emulsion;
[0004] (c) continuously stirring the W/O emulsion, whereby the
reaction of hyaluronic acid with divinylsulfone takes place to
provide crosslinked hyaluronic acid microbeads; and
[0005] (d) purifying the crosslinked hyaluronic acid
microbeads.
BACKGROUND OF THE INVENTION
[0006] The present invention relates to a process for the
preparation of modified hyaluronic acid (HA), in particular
crosslinked HA in emulsion, for use in biomedical and
pharmaceutical applications.
[0007] Hyaluronic acid (HA) is a natural and linear carbohydrate
polymer belonging to the class of the non-sulfated
glycosaminoglycans. It is composed of beta-1,3-N-acetyl glucosamine
and beta-1,4-glucuronic acid repeating disaccharide units with a
molecular weight (MW) up to 6 MDa. HA is present in hyaline
cartilage, synovial joint fluid, and skin tissue, both dermis and
epidermis. HA may be extracted from natural tissues including the
connective tissue of vertebrates, from the human umbilical cord and
from cocks' combs. However, it is preferred today to prepare it by
microbiological methods to minimize the potential risk of
transferring infectious agents, and to increase product uniformity,
quality and availability (WO 03/0175902, Novozymes).
[0008] Numerous roles of HA in the body have been identified. It
plays an important role in the biological organism, as a mechanical
support for the cells of many tissues, such as the skin, tendons,
muscles and cartilage. HA is involved in key biological processes,
such as the moistening of tissues, and lubrication. It is also
suspected of having a role in numerous physiological functions,
such as adhesion, development, cell motility, cancer, angiogenesis,
and wound healing. Due to the unique physical and biological
properties of HA (including viscoelasticity, biocompatibility,
biodegradability), HA is employed in a wide range of current and
developing applications within ophthalmology, rheumatology, drug
delivery, wound healing and tissue engineering. The use of HA in
some of these applications is limited by the fact that HA is
soluble in water at room temperature, i.e. about 20.degree. C., it
is rapidly degraded by hyaluronidase in the body, and it is
difficult to process into biomaterials. Crosslinking of HA has
therefore been introduced in order to improve the physical and
mechanical properties of HA and its in vivo residence time.
[0009] U.S. Pat. No. 4,582,865 (Biomatrix Inc.) describes the
preparation of crosslinked gels of HA, alone or mixed with other
hydrophilic polymers, using divinyl sulfone (DVS) as the
crosslinking agent. The preparation of a crosslinked HA or salt
thereof using a polyfunctional epoxy compound is disclosed in EP 0
161 887 B1. Other bi- or poly-functional reagents that have been
employed to crosslink HA through covalent linkages include
formaldehyde (U.S. Pat. No. 4,713,448, Biomatrix Inc.),
polyaziridine (WO 03/089476 A1, Genzyme Corp.), L-aminoacids or
L-aminoesters (WO 2004/067575, Biosphere S.P.A.). Carbodiimides
have also been reported for the crosslinking of HA (U.S. Pat. No.
5,017,229, Genzyme Corp.; U.S. Pat. No. 6,013,679, Anika Research,
Inc). Total or partial crosslinked esters of HA with an aliphatic
alcohol, and salts of such partial esters with inorganic or organic
bases, are disclosed in U.S. Pat. No. 4,957,744. Crosslinking of HA
chains with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride ("EDAC") and adipic acid dihydrazide in a
water/acetone mixture was disclosed in U.S. 2006/0040892
(University of North Texas). WO 2006/56204 (Novozymes A/S) also
discloses methods for the preparation of crosslinked gels of HA
using divinyl sulfone (DVS) as the crosslinking agent.
[0010] WO 2008/100044 was published in the priority year of the
present application and describes a method of preparing hyaluronic
hydrogel nanoparticles by crosslinking hyaluronic acid, the method
comprising mixing i) an oil phase containing a surfactant dissolved
therein with ii) a water phase, containing hyaluronic acid and a
water-soluble crosslinker dissolved in an aqueous basic solution
where divinylsulfone is not mentioned, so as to a form a w/o
emulsion, and crosslinking the hyaluronic acid in the w/o emulsion,
the oil phase comprising dodecane, heptane or
cetylethylhexanoate.
[0011] EP 0 830 416 (equivalent of U.S. Pat. No. 6,214,331)
describes the preparation of a crosslinked water-soluble polymer
particle preparation wherein the particles are less than 212 .mu.m
in diameter and wherein at least 80% of the particles are
spherical, obtainable by adding an aqueous polymer solution,
comprising a water-soluble polymer selected from hyaluronic acid,
chondroitin sulfate, dermatan sulfate, keratan sulfate, celluloses,
chitin, chitosan, agarose, carrageenans, curdlan, dextrans,
emulsan, gellan, xanthans, poly(ethyleneoxide), poly(vinyl
alcohol), poly(N-vinyl pyrrolidone), proteins, glycoproteins,
peptidoglycans, proteoglycans, lipopolysaccharides, or combinations
thereof, and an aqueous medium, to an oil base containing a water
in oil emulsifying agent, agitating the mixture to form an emulsion
containing polymer droplets, and crosslinking the polymer droplets
in situ by a crosslinking agent resulting in the formation of
crosslinked polymer particles. For the production of hyaluronic
acid microspheres the crosslinking agent is added directly to an
emulsion of aqueous hyaluronic acid in toluene. The crosslinking
agent is first deactivated by adjusting the pH of the aqueous
solution to pH 11 and then activated by lowering the pH to 7 to 8.
It is preferred to use toluene, o-xylene or isooctane as oil phase.
The weight ratio of aqueous phase to oil phase is about 1 to 1.
[0012] Nurettin Sahiner and Xinqiao Jai (Turk J Chem, 32 (2008),
397-409) describe the preparation of hyaluronic acid based
submicron hydrogel particles using isooctane as oil phase. For
preparing the emulsion 0.54 ml of aqueous hyaluronic acid solution
was added to 15 ml of isooctane, resulting in a weight ratio of
aqueous phase to oil phase is higher then 10 to 1.
SUMMARY OF THE INVENTION
[0013] It is clear from the above, that several crosslinking agents
suitable for preparing crosslinked HA-gels are known. However, it
is still of commercial interest to provide new formulations of HA
that are particularly suitable for various application. For
instance, new methods of providing gel-like crosslinked HA
microbeads of desired size, with cross-sections ranging from nano-
to micrometers, are of commercial interest. Such microbeads of
crosslinked HA may be used for any number of applications, such as,
as delivery vehicles for pharmaceutical drugs, as bioactives in
themselves, as constituents in compositions and in a whole range of
biomedical applications.
[0014] Accordingly, in a first aspect the invention provides a
method of producing crosslinked hyaluronic acid microbeads, said
method comprising the steps of:
[0015] (a) mixing an aqueous alkaline solution comprising
hyaluronic acid, or a salt thereof, with a solution comprising a
crosslinking agent;
[0016] (b) forming microdroplets having a desired size from the
mixed solution of step (a) in an organic or oil phase to form a
water in organic or water in oil (W/O) emulsion;
[0017] (c) continuously stirring the W/O emulsion, whereby the
reaction of hyaluronic acid with divinylsulfone takes place to
provide crosslinked hyaluronic acid microbeads; and
[0018] (d) purifying the crosslinked hyaluronic acid
microbeads.
[0019] In a second aspect, the invention relates to a microbead
comprising hyaluronic acid, or salt thereof, crosslinked with
divinylsulfone; preferably made by the method of the first
aspect.
[0020] In a third aspect, the invention relates to a composition
comprising a microbead as defined in the second aspect, and an
active ingredient, preferably the active ingredient is a
pharmacologically active agent.
[0021] A fourth aspect of the invention relates to a pharmaceutical
composition comprising an effective amount of a microbead as
defined in the second aspect, together with a pharmaceutically
acceptable carrier, excipient or diluent.
[0022] A fifth aspect relates to a pharmaceutical composition
comprising an effective amount of a microbead as defined in the
second aspect as a vehicle, together with a pharmacologically
active agent, preferably encapsulated as a dispersion or solution
in the microbead.
[0023] In a sixth aspect, the invention relates to a sanitary,
medical or surgical article comprising a microbead as defined in
the second aspect or a composition as defined in any of the third,
fourth, or fifth aspects, preferably the article is a diaper, a
sanitary towel, a surgical sponge, a wound healing sponge, or a
part comprised in a band aid or other wound dressing material.
[0024] An important aspect relates to a medicament capsule or
microcapsule comprising a microbead as defined in the second aspect
or a composition as defined in any of the third, fourth, or fifth
aspects.
[0025] A number of aspects relate to uses of a microbead as defined
in the second aspect or a composition as defined in any of the
third, fourth, or fifth aspects, for the manufacture of a
medicament for the treatment of osteoarthritis, cancer, the
manufacture of a medicament for an ophtalmological treatment, the
manufacture of a medicament for the treatment of a wound, the
manufacture of a medicament for angiogenesis, the manufacture of a
medicament for the treatment of hair loss or baldness, the
manufacture of a moisturizer, the manufacture of dermal fillers,
drug delivery systems/vehicles or tissue augmentation devices.
BRIEF DESCRIPTION OF DRAWINGS
[0026] FIG. 1 shows a photomicrograph of isolated crosslinked
hyaluronic acid gel particles showing the spherical profile of the
particle with a diameter 22 micrometer.
[0027] FIG. 2 shows a photomicrograph of the emulsion including a
pH indicator as coloring agent. The picture shows the contribution
of small particles (1-5 micrometer) surrounded by the larger
particles.
[0028] FIG. 3 shows a photomicrograph of isolated crosslinked
hyaluronic acid gel particles (80 x) showing gel particles of
maximum 1 mm in diameter.
[0029] FIG. 4 shows a photomicrograph of isolated microbead
particles showing the spherical profile of the particles with a
diameter of various sizes, in the range of 500-1000 micrometer.
[0030] FIG. 5 shows a photomicrograph of particles after washing
procedure.
DEFINITIONS
[0031] The term "hyaluronic acid" is used in literature to mean
acidic polysaccharides with different molecular weights constituted
by residues of D-glucuronic and N-acetyl-D-glucosamine acids, which
occur naturally in cell surfaces, in the basic extracellular
substances of the connective tissue of vertebrates, in the synovial
fluid of the joints, in the endobulbar fluid of the eye, in human
umbilical cord tissue and in cocks' combs.
[0032] The term "hyaluronic acid" is in fact usually used as
meaning a whole series of polysaccharides with alternating residues
of D-glucuronic and N-acetyl-D-glucosamine acids with varying
molecular weights or even the degraded fractions of the same, and
it would therefore seem more correct to use the plural term of
"hyaluronic acids". The singular term will, however, be used all
the same in this description; in addition, the abbreviation "HA"
will frequently be used in place of this collective term.
[0033] "Hyaluronic acid" is defined herein as an unsulphated
glycosaminoglycan composed of repeating disaccharide units of
N-acetylglucosamine (GIcNAc) and glucuronic acid (GlcUA) linked
together by alternating beta-1,4 and beta-1,3 glycosidic bonds.
Hyaluronic acid is also known as hyaluronan, hyaluronate, or HA.
The terms hyaluronan and hyaluronic acid are used interchangeably
herein.
[0034] Rooster combs are a significant commercial source for
hyaluronan. Microorganisms are an alternative source. U.S. Pat. No.
4,801,539 discloses a fermentation method for preparing hyaluronic
acid involving a strain of Streptococcus zooepidemicus with
reported yields of about 3.6 g of hyaluronic acid per liter.
European Patent No. EP0694616 discloses fermentation processes
using an improved strain of Streptococcus zooepidemicus with
reported yields of about 3.5 g of hyaluronic acid per liter. As
disclosed in WO 03/054163 (Novozymes), which is incorporated herein
in its entirety, hyaluronic acid or salts thereof may be
recombinantly produced, e.g., in a Gram-positive Bacillus host.
[0035] Hyaluronan synthases have been described from vertebrates,
bacterial pathogens, and algal viruses (DeAngelis, P. L., 1999,
Cell. Mol. Life Sci. 56: 670-682). WO 99/23227 discloses a Group I
hyaluronate synthase from Streptococcus equisimilis. WO 99/51265
and WO 00/27437 describe a Group II hyaluronate synthase from
Pasturella multocida. Ferretti et al. discloses the hyaluronan
synthase operon of Streptococcus pyogenes, which is composed of
three genes, hasA, hasB, and hasC, that encode hyaluronate
synthase, UDP glucose dehydrogenase, and UDP-glucose
pyrophosphorylase, respectively (Proc. Natl. Acad. Sci. USA. 98,
4658-4663, 2001). WO 99/51265 describes a nucleic acid segment
having a coding region for a Streptococcus equisimilis hyaluronan
synthase.
[0036] Since the hyaluronan of a recombinant Bacillus cell is
expressed directly to the culture medium, a simple process may be
used to isolate the hyaluronan from the culture medium. First, the
Bacillus cells and cellular debris are physically removed from the
culture medium. The culture medium may be diluted first, if
desired, to reduce the viscosity of the medium. Many methods are
known to those skilled in the art for removing cells from culture
medium, such as centrifugation or microfiltration. If desired, the
remaining supernatant may then be filtered, such as by
ultrafiltration, to concentrate and remove small molecule
contaminants from the hyaluronan. Following removal of the cells
and cellular debris, a simple precipitation of the hyaluronan from
the medium is performed by known mechanisms. Salt, alcohol, or
combinations of salt and alcohol may be used to precipitate the
hyaluronan from the filtrate. Once reduced to a precipitate, the
hyaluronan can be easily isolated from the solution by physical
means. The hyaluronan may be dried or concentrated from the
filtrate solution by using evaporative techniques known to the art,
such as lyophilization or spraydrying.
[0037] The term "microbead" is used herein interchangeably with
microdrop, microdroplet, microparticle, microsphere, nanobead,
nanodrop, nanodroplet, nanoparticle, nanosphere etc. A typical
microbead is approximately spherical and has an number average
cross-section or diameter in the range of between 1 nanometer to 1
millimeter. Though, usually the microbeads of the present invention
will be made with a desired size in a much more narrow range, i.e.,
they will be fairly uniform. The microbeads preferably have a
diameter in the range of about 100 -1,000 nanometer; or in the
range of 1,000 nanometer to 1,000 micrometer. The size-distribution
of the microbeads will be low and the polydispersibility
narrow.
Host Cells
[0038] A preferred embodiment relates to the method of the first
aspect, wherein the hyaluronic acid or salt thereof is
recombinantly produced, preferably by a Gram-positive bacterium or
host cell, more preferably by a bacterium of the genus
Bacillus.
[0039] The host cell may be any Bacillus cell suitable for
recombinant production of hyaluronic acid. The Bacillus host cell
may be a wild-type Bacillus cell or a mutant thereof. Bacillus
cells useful in the practice of the present invention include, but
are not limited to, Bacillus agaraderhens, Bacillus alkalophilus,
Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,
Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus
lautus, Bacillus lentus, Bacillus licheniformis, Bacillus
megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus
subtilis, and Bacillus thuringiensis cells. Mutant Bacillus
subtilis cells particularly adapted for recombinant expression are
described in WO 98/22598. Non-encapsulating Bacillus cells are
particularly useful in the present invention.
[0040] In a preferred embodiment, the Bacillus host cell is a
Bacillus amyloliquefaciens, Bacillus clausii, Bacillus lentus,
Bacillus licheniformis, Bacillus stearothermophilus or Bacillus
subtilis cell. In a more preferred embodiment, the Bacillus cell is
a Bacillus amyloliquefaciens cell. In another more preferred
embodiment, the Bacillus cell is a Bacillus clausii cell. In
another more preferred embodiment, the Bacillus cell is a Bacillus
lentus cell. In another more preferred embodiment, the Bacillus
cell is a Bacillus licheniformis cell. In another more preferred
embodiment, the Bacillus cell is a Bacillus subtilis cell. In a
most preferred embodiment, the Bacillus host cell is Bacillus
subtilis A164.DELTA.5 (see U.S. Pat. No. 5,891,701) or Bacillus
subtilis 168.DELTA.4.
Molecular Weight
[0041] The content of hyaluronic acid may be determined according
to the modified carbazole method (Bitter and Muir, 1962, Anal
Biochem. 4: 330-334). Moreover, the number average molecular weight
of the hyaluronic acid may be determined using standard methods in
the art, such as those described by Ueno et al., 1988, Chem. Pharm.
Bull. 36, 4971-4975; Wyatt, 1993, Anal. Chim. Acta 272: 1-40; and
Wyatt Technologies, 1999, "Light Scattering University DAWN Course
Manual" and "DAWN EOS Manual" Wyatt Technology Corporation, Santa
Barbara, Calif.
[0042] In a preferred embodiment, the hyaluronic acid, or salt
thereof, of the present invention has a molecular weight of about
10,000 to about 10,000,000 Da. In a more preferred embodiment it
has a molecular weight of about 25,000 to about 5,000,000 Da. In a
most preferred embodiment, the hyaluronic acid has a molecular
weight of about 50,000 to about 3,000,000 Da.
[0043] In a preferred embodiment, the hyaluronic acid or salt
thereof has a molecular weight in the range of between 300,000 and
3,000,000; preferably in the range of between 400,000 and
2,500,000; more preferably in the range of between 500,000 and
2,000,000; and most preferably in the range of between 600,000 and
1,800,000.
[0044] In yet another preferred embodiment, the hyaluronic acid or
salt thereof has a low number average molecular weight in the range
of between 10,000 and 800,000 Da; preferably in the range of
between 20,000 and 600,000 Da; more preferably in the range of
between 30,000 and 500,000 Da; even more preferably in the range of
between 40,000 and 400,000 Da; and most preferably in the range of
between 50,000 and 300,000 Da.
Salts and Crosslinked HA
[0045] A preferred embodiment relates to a method of the first
aspect, which comprises an inorganic salt of hyaluronic acid,
preferably sodium hyaluronate, potassium hyaluronate, ammonium
hyaluronate, calcium hyaluronate, magnesium hyaluronate, zinc
hyaluronate, or cobalt hyaluronate.
Other Ingredients
[0046] In a preferred embodiment, the product produced by the
method of the invention may also comprise other ingredients,
preferably one or more active ingredient, preferably one or more
pharmacologically active substance, and also preferably a
water-soluble excipient, such as lactose or a non-biologically
derived sugar.
[0047] Non-limiting examples of an active ingredient or the one or
more pharmacologically active substance(s) which may be used in the
present invention include vitamin(s), anti-inflammatory drugs,
antibiotics, bacteriostatics, general anaesthetic drugs, such as,
lidocaine, morphine etc. as well as protein and/or peptide drugs,
such as, human growth hormone, bovine growth hormone, porcine
growth hormone, growth hormone releasing hormone/peptide,
granulocyte-colony stimulating factor, granulocyte
macrophage-colony stimulating factor, macrophage-colony stimulating
factor, erythropoietin, bone morphogenic protein, interferon or
derivative thereof, insulin or derivative thereof, atriopeptin-Ill,
monoclonal antibody, tumor necrosis factor, macrophage activating
factor, interleukin, tumor degenerating factor, insulin-like growth
factor, epidermal growth factor, tissue plasminogen activator,
factor IIV, factor IIIV, and urokinase.
[0048] A water-soluble excipient may be included for the purpose of
stabilizing the active ingredient(s), such excipient may include a
protein, e.g., albumin or gelatin; an amino acid, such as glycine,
alanine, glutamic acid, arginine, lysine and a salt thereof;
carbohydrate such as glucose, lactose, xylose, galactose, fructose,
maltose, saccharose, dextran, mannitol, sorbitol, trehalose and
chondroitin sulphate; an inorganic salt such as phosphate; a
surfactant such as TWEEN.RTM. (ICI), poly ethylene glycol, and a
mixture thereof. The excipient or stabilizer may be used in an
amount ranging from 0.001 to 99% by weight of the product.
[0049] Several aspects of the invention relate to various
compositions and pharmaceuticals comprising, among other
constituents, an effective amount of the crosslinked HA product,
and an active ingredient, preferably the active ingredient is a
pharmacologically active agent; a pharmaceutically acceptable
carrier, excipient or diluent, preferably a water-soluble
excipient, and most preferably lactose.
[0050] In addition, aspects of the invention relate to articles
comprising a product as defined in the first aspect or a
composition as defined in the aspects and embodiments above, e.g.,
a sanitary article, a medical or surgical article. In a final
aspect the invention relates to a medicament capsule or
microcapsule comprising a product as defined in the first aspect or
a composition as defined in other aspects and embodiments of the
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0051] The first aspect of the invention relates to a method of
producing crosslinked hyaluronic acid microbeads, said method
comprising the steps of:
[0052] (a) mixing an aqueous alkaline solution comprising
hyaluronic acid, or a salt thereof, with a solution comprising a
crosslinking agent;
[0053] (b) forming microdroplets having a desired size from the
mixed solution of step (a) in an organic or oil phase to form a
water in organic or water in oil (W/O) emulsion;
[0054] (c) continuously stirring the W/O emulsion, whereby the
reaction of hyaluronic acid with divinylsulfone takes place to
provide crosslinked hyaluronic acid microbeads; and
[0055] (d) purifying the crosslinked hyaluronic acid
microbeads.
[0056] It has previously been described how to produce hyaluronic
acid recombinantly in a Bacillus host cell, see WO 2003/054163,
Novozymes NS, which is incorporated herein in its entirety.
[0057] Accordingly, in a preferred embodiment, the invention
relates to the method of the first aspect, wherein the hyaluronic
acid, or salt thereof, is recombinantly produced in a Bacillus host
cell.
[0058] Various molecular weight fractions of hyaluronic acid have
been described as advantageous for specific purposes.
[0059] A preferred embodiment of the invention relates to a method
of the first aspect, wherein the hyaluronic acid, or salt thereof,
has an number average molecular weight of between 100 and 3,000
kDa, preferably between 500 and 2,000 kDa, and most preferably
between 700 and 1,800 kDa.
[0060] The initical concentration of hyaluronic acid, or a salt
thereof, in the method of the invention, influences the properties
of the resulting crosslinked microbeads. Therefore, a preferred
embodiment of the invention relates to a method of the first
aspect, wherein the alkaline solution comprises dissolved
hyaluronic acid, or salt thereof, in a concentration of between
0.1%-40% (w/v).
[0061] The pH value during the crosslinking reaction also
influences the outcome, so in a preferred embodiment the invention
relates to a method of the first aspect, wherein the alkaline
solution comprises dissolved sodium hydroxide in a concentration of
between 0.001-2.0 M.
[0062] It is also noteworthy that the concentration of the
crosslinking agent has a profound impact on the resulting
microbeads.
[0063] Consequently, a preferred embodiment of the invention
relates to a method of the first aspect, wherein the crosslinking
agent is divinylsulfone (DVS); preferably DVS is comprised in the
mixed solution of step (a) in a weight ratio of between 1:1 and
100:1 of HA/DVS (dry weight), preferably between 2:1 and 50:1 of
HA/DVS (dry weight).
[0064] Other crosslinking agents are also envisioned as being
suitable for the methods of the instant invention, such as,
crosslinking agents based on bisepoxide crosslinking technology:
GDE=glycerol diglycidyl ether and BDE: 1,4-butanediol diglycidyl
ether.
[0065] Crosslinking agents suitable for the methods of the instant
invention are for example poly functional (>=2) OH-reactive
compounds. Examples for suitable crosslinking agents are
divinylsulfone (DVS) or crosslinking agents based on bisepoxide
crosslinking technology, for example GDE=glycerol diglycidyl ether
or BDE: 1,4-butanediol diglycidyl ether. The crosslinking agent is
preferably selected from divinylsulfone, glycerol diglycidyl ether
or 1,4-butanediol diglycidyl ether. The most preferred crosslinking
agent of the invention is divinylsulfone which is preferably used
in the weight ratio mentioned above.
[0066] The inventors found that an initial period of stirring
during and/or immediately after mixing the solution comprising the
crosslinking agent and the HA-solution was desirable to achieve
satisfactory gelling.
[0067] Accordingly, a preferred embodiment of the invention relates
to a method of the first aspect, wherein the reaction of hyaluronic
acid with divinylsulfone takes place at a temperature in the range
of 5.degree. C.-100.degree. C., preferably in the range of
15.degree. C.-50.degree. C., more preferably in the range of
20.degree. C.-30.degree. C.
[0068] In another preferred embodiment of the method of the first
aspect, the stirring in step (c) is continued for a period of
between 1-180 minutes.
[0069] The present inventors determined that a heating step was
beneficial after mixing the solutions.
[0070] Accordingly, a preferred embodiment of the invention relates
to a method of the first aspect, wherein the mixed solution is
heated to a temperature in the range of 20.degree. C.-100.degree.
C., preferably in the range of 25.degree. C.-80.degree. C., more
preferably in the range of 30.degree. C.-60.degree. C., and most
preferably in the range of 35.degree. C.-55.degree. C., and wherein
the temperature is maintained in this range for a period of at
least 5 minutes, preferably at least 10 minutes, 20 minutes, 30,
40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or
most preferably at least 180 minutes after mixing the solutions;
preferably without stirring.
[0071] It is advantageous to leave the reaction mixture at room
temperature for a brief period after the crosslinking reaction has
taken place, but still with continuous stirring.
[0072] In a preferred embodiment of the method of the first aspect,
the reaction mixture is maintained after the reaction has taken
place for a period of at least 5 minutes, preferably at least 10
minutes, 20 minutes, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120,
130, 140, 150, 160, 170, or most preferably at least 180 minutes,
at a temperature in the range of 0.degree. C.-40.degree. C.,
preferably in the range of 10.degree. C.-30.degree. C.
[0073] It might by advantageous when the microdroplets of step (b)
have a number average diameter in the range of from about 1
nanometre to 1 millimetre. The maximum of the particle size
distribution of the microdroplets of step (b) is preferably in the
range of from 0.1 to 100 pm, more preferably from 0.5 to 10 .mu.m
and most preferably from 1 to 2 .mu.m. The size of the droplets can
be adjusted by the choice of emulsifier used and the intensity of
stirring. The combination of emulsifier used and intensity of
stirring necessary to obtain droplets with the desired size can be
determined by simple test series. The size of the droplets or
microbeads can be determined with an Accusizer (Accusizer 780
Optical Particle Sizer, PSS NICOMP, Santa Barbara, Calif., USA with
Accusizer 780AD CW788-Nicomp software, V 1.68 (2000)).
[0074] In a preferred aspect, the invention relates to methods of
the first aspect, wherein the microdroplets of step (b) have a
number average diameter in the range of about 1 nanometer to 1
millimeter. It is also preferred that the crosslinked microbead of
the second aspect has a number average diameter in the range of
about 1 nanometer to 1 millimeter.
[0075] It might be advantageous to obtain a dispersion in step (c)
that comprises almost none unreacted crosslinking agent. Preferably
the dispersion more preferably the microbeads comprise less than 10
ppm by weight (wppm), more preferably less than 5 wppm. The
concentration of free crosslinking agent in the dispersion
especially needs to be low if the dispersion is directly used in
pharmaceutical or biomedical application/device compositions
because the unreacted crosslinking agent might be a toxicological
threat. It is therefore preferred to last the reaction of step (c)
till a dispersion is obtained comprising the unreacted crosslinking
agent in the concentration mentioned above.
[0076] Compounds from at least one of the following groups can be
employed as nonionic emulsifiers or surfactants: addition products
of from 2 to 100 mol of ethylene oxide and/or 0 to 5 mol of
propylene oxide on linear fatty alcohols having 8 to 22 C atoms, on
fatty acids having 12 to 22 C atoms and on alkylphenols having 8 to
15 C atoms in the alkyl group, C12/18-fatty acid mono- and diesters
of addition products of from 1 to 100 mol of ethylene oxide on
glycerol, glycerol mono- and diesters and sorbitan mono- and
diesters of saturated and unsaturated fatty acids having 6 to 22
carbon atoms and ethylene oxide addition products thereof, alkyl
mono- and oligoglycosides having 8 to 22 carbon atoms in the alkyl
radical and ethylene oxide addition products thereof, addition
products of from 2 to 200 mol of ethylene oxide on castor oil
and/or hydrogenated castor oil, partial esters based on linear,
branched, unsaturated or saturated C6-C22-fatty acids, ricinoleic
acid and 12-hydroxystearic acid and glycerol, polyglycerol,
pentaerythritol, dipentaerythritol, sugar alcohols (e.g. sorbitol),
alkyl glucosides (e.g. methyl glucoside, butyl glucoside, lauryl
glucoside) and polyglucosides (e.g. cellulose), mono-, di- and
trialkyl phosphates and mono-, di- and/or tri-PEG-alkyl phosphates
and salts thereof, polysiloxane/polyether copolymers (Dimethicone
Copolyols), such as e.g. PEG/PPG-20/6 Dimethicone, PEG/PPG-20/20
Dimethicone, Bis-PEG/PPG-20/20 Dimethicone, PEG-12 or PEG-14
Dimethicone, PEG/PPG-14/4 or 4/12 or 20/20 or 18/18 or 17/18 or
15/15, polysiloxane/polyalkyl polyether copolymers and
corresponding derivatives, such as e.g. Lauryl or Cetyl Dimethicone
Copolyols, in particular Cetyl PEG/PPG-10/1 Dimethicone (ABIL.RTM.
EM 90 (Evonik Degussa)), mixed esters of pentaerythritol, fatty
acids, citric acid and fatty alcohol according to DE 11 65 574
and/or mixed esters of fatty acids having 6 to 22 carbon atoms,
methylglucose and polyols, such as e.g. glycerol or polyglycerol,
citric acid esters, such as e.g. Glyceryl Stearate Citrate,
Glyceryl Oleate Citrate and Dilauryl Citrate.
[0077] Preferred emulsifiers used in the present invention are
selected from those having a HLB-value of from 3 to 9, preferably 4
to 6 and more preferably about 5. Preferred emulsifiers are
selected from polyglyceryl-4-diisostearat/polyhydroxysterat/sebacat
(ISOLAN.RTM. GPS), PEG/PPG-10/1 dimethicone, (ABIL.RTM. EM 90),
Polyglyceryl-4 Isostearate (ISOLAN.RTM. GI 34), Polyglyceryl-3
Oleate (ISOLAN.RTM. GO 33), Methylglucose Isostearate (ISOLAN.RTM.
IS), Diisostearoyl Polyglyceryl-3 Dimer Dilinoleate (ISOLAN.RTM.
PDI), Glyceryl Oleate (TEGIN.RTM. O V), Sorbitan Laurate (TEGO.RTM.
SML), Sorbitan Oleate (TEGO.RTM. SMO V) and Sorbitan Stearate
(TEGO.RTM. SMS). These preferred emulsifiers are available from
Evonik Goldschmidt GmbH.
[0078] Anionic emulsifiers or surfactants can contain groups which
confer solubility in water, such as e.g. a carboxylate, sulphate,
sulphonate or phosphate group and a lipophilic radical. Anionic
surfactants which are tolerated by skin are known in large numbers
to the person skilled in the art and are commercially obtainable.
In this context these can be alkyl sulphates or alkyl phosphates in
the form of their alkali metal, ammonium or alkanolammonium salts,
alkyl ether-sulphates, alkyl ether-carboxylates, acyl sarcosinates
and sulphosuccinates and acyl glutamates in the form of their
alkali metal or ammonium salts.
[0079] Cationic emulsifiers and surfactants can also be added.
Quaternary ammonium compounds, in particular those provided with at
least one linear and/or branched, saturated or unsaturated alkyl
chain having 8 to 22 C atoms, can be employed in particular as
such, thus, for example, alkyltrimethylammonium halides, such as
e.g. cetyltrimethylammonium chloride or bromide or
behenyltrimethylammonium chloride, but also dialkyldimethylammonium
halides, such as e.g. distearyldimethylammonium chloride.
[0080] Monoalkylamidoquats, such as e.g.
palmitamidopropyltrimethylammonium chloride, or corresponding
dialkylamidoquats can furthermore be employed. Readily
biodegradable quaternary ester compounds, which can be quaternized
fatty acid esters based on mono-, di- or triethanolamine, can
furthermore be employed. Alkylguanidinium salts can furthermore be
admixed as cationic emulsifiers.
[0081] Typical examples of mild surfactants, i.e. surfactants which
are particularly tolerated by skin, are fatty alcohol polyglycol
ether-sulphates, monoglyceride sulphates, mono- and/or dialkyl
sulphosuccinates, fatty acid isethionates, fatty acid sarcosinates,
fatty acid taurides, fatty acid glutamates, ether-carboxylic acids,
alkyl oligoglucosides, fatty acid glucamides, alkylamidobetaines
and/or protein-fatty acid condensates, the latter for example based
on wheat proteins.
[0082] It is furthermore possible to employ amphoteric surfactants,
such as e.g. betaines, amphoacetates or amphopropionates, thus e.g.
substances such as the N-alkyl-N, N-dimethylammonium glycinates,
for example coco-alkyldimethylammonium glycinate,
N-acylaminopropyl-N,N-dimethylammonium glycinates, for example
coco-acylamimopropyldimethylammonium glycinate, and
2-alkyl-3-carboxymethyl-3-hydroxyethylimidazolines having in each
case 8 to 18 C atoms in the alkyl or acyl group, and
coco-acylaminoethylhydroxyethylcarboxymethyl glycinate.
[0083] Of the ampholytic surfactants, those surface-active
compounds which contain, apart from a C8/18-alkyl or -acyl group,
at least one free amino group and at least one -COOH or -SO3H group
in the molecule and are capable of formation of inner salts can be
employed. Examples of suitable ampholytic surfactants are
N-alkylglycines, N-alkylpropionic acids, N-alkylaminobutyric acids,
N-alkyliminodipropionic acids,
N-hydroxyethyl-N-alkylamidopropylglycines, N-alkyltaurines,
N-alkylsarcosines, 2-alkylaminopropionic acids and alkylaminoacetic
acids having in each case about 8 to 18 C atoms in the alkyl group.
Further examples of ampholytic surfactants are
N-coco-alkylaminopropionate, coco-acylaminoethylaminopropionate and
012/18-acrylsarcosine.
[0084] Preferred emulsifiers or surfactants used for formulating
the composition are identical to those used in the production of
the microbeads.
[0085] Many types of buffers or acids, as are well known to the
skilled person, have been envisioned as suitable for the swelling
and neutralizing of the crosslinked microbeads of the invention. In
a preferred embodiment the buffer comprises a buffer with a pH
value in the range of 2.0-8.0, preferably in the range of
5.0-7.5.
[0086] Optimally, a suitable buffer is chosen with a pH value,
which results in that the crosslinked microbeads have a pH value as
close to neutral as possible. In a preferred embodiment, the buffer
comprises a buffer with a pH value, which results in that the
crosslinked microbeads have a pH value between 5.0 and 7.5.
[0087] It is preferred that the buffer in the method of the first
aspect comprises a phosphate buffer and/or a saline buffer.
[0088] It is also preferred that the crosslinked microbeads are
washed at least once with water, water and an acid, water and a
phosphate buffer, water and a saline buffer, or water and a
phosphate buffer and a saline buffer, with a pH value in the range
of of 2.0-8.0, preferably in the range of 5.0-7.5.
[0089] The purifying step may comprise any separation technique
known in the art, e.g. filtration, decantation, centrifugation and
so on. It might be advantageous to combine one or more purifying
steps with one or more neutralizing steps.
[0090] A preferred embodiment of the first aspect relates to the
method, wherein the purifying step comprises dialyzing the
crosslinked microbeads against de-ionized water using a dialysis
membrane that allows free diffusion of molecules having a size less
than 13,000 Daltons.
[0091] It is preferred to use standard emollients used in cosmetic
or personal care formulations as oil phase. Such standard
emollients are not hydrocarbons or aromatic hydrocarbons,
especially not toluene, o-xylene, dodecane, heptane, isooctane or
cetylethylhexanoate. Preferred emollients used in the present
invention are selected from mono- or diesters of linear and/or
branched mono- and/or dicarboxylic acids having 2 to 44 C atoms
with linear and/or branched saturated or unsaturated alcohols
having 1 to 22 C atoms, the esterification products of aliphatic
difunctional alcohols having 2 to 36 C atoms with monofunctional
aliphatic carboxylic acids having 1 to 22 C atoms, long-chain aryl
acid esters, such as e.g. esters of benzoic acid with linear and/or
branched C6-C22-alcohols, or also benzoic acid isostearyl ester,
benzoic acid butyloctyl ester or benzoic acid octyldodecyl ester,
carbonates, preferably linear C6-C22-fatty alcohol carbonates,
Guerbet carbonates, e.g. dicaprylyl carbonate, diethylhexyl
carbonate, longer-chain triglycerides, i.e. triple esters of
glycerol with three acid molecules, at least one of which is
longer-chain, triglycerides based on C6-C10-fatty acids, linear or
branched fatty alcohols, such as oleyl alcohol or octyldodecanol,
and fatty alcohol ethers, such as dialykl ether e. g. dicaprylyl
ether, silicone oils and waxes, e.g. polydimethylsiloxanes,
cyclomethylsiloxanes, and aryl- or alkyl- or alkoxy-substituted
polymethylsiloxanes or cyclomethylsiloxanes, Guerbet alcohols based
on fatty alcohols having 6 to 18, preferably 8 to 10 carbon atoms,
esters of linear C6-C22 fatty acids with linear C6-C22-fatty
alcohols, esters of branched C6-C13-carboxylic acids with linear
C6-C22-fatty alcohols, esters of linear C6-C22-fatty acids with
branched C8-C18-alcohols, in particular 2-ethylhexanol or
isononanol, esters of branched C6-C13-carboxylic acids with
branched alcohols, in particular 2-ethylhexanol or isononanol,
esters of linear and/or branched fatty acids with polyhydric
alcohols (such as e.g. propylene glycol, dimer diol or trimer
triol) and/or Guerbet alcohols, liquid mono-/di-/triglyceride
mixtures based on C6-C18-fatty acids, esters of C6-C22-fatty
alcohols and/or Guerbet alcohols with aromatic carboxylic acids,
plant oils, branched primary alcohols, substituted cyclohexanes,
ring-opening products of epoxidized fatty acid esters with polyols
and/or silicone oils or a mixture of two or more of these
compounds. The emollient used is preferably not miscible with water
without phase separation.
[0092] Monoesters which are suitable as emollients and oil
components are e.g. the methyl esters and isopropyl esters of fatty
acids having 12 to 22 C atoms, such as e.g. methyl laurate, methyl
stearate, methyl oleate, methyl erucate, isopropyl myristate,
isopropyl palmitate, isopropyl stearate, isopropyl oleate. Other
suitable monoesters are e.g. n-butyl stearate, n-hexyl laurate,
n-decyl oleate, isooctyl stearate, isononyl palmitate, isononyl
isononanoate, 2-ethylhexyl laurate, 2-ethylhexyl palmitate,
2-ethylhexyl stearate, 2-hexyldecyl stearate, 2-octyldodecyl
palmitate, oleyl oleate, oleyl erucate, erucyl oleate and esters
which are obtainable from technical-grade aliphatic alcohol cuts
and technical-grade aliphatic carboxylic acid mixtures, e.g. esters
of unsaturated fatty alcohols having 12 to 22 C atoms and saturated
and unsaturated fatty acids having 12 to 22 C atoms, such as are
accessible from animal and plant fats. However, naturally occurring
monoester and wax ester mixtures such as are present e.g. in jojoba
oil or in sperm oil are also suitable. Suitable dicarboxylic acid
esters are e.g. di-n-butyl adipate, di-n-butyl sebacate,
di-(2-ethylhexyl) adipate, di-(2-hexyldecyl) succinate,
di-isotridecyl azelate. Suitable diol esters are e.g. ethylene
glycol dioleate, ethylene glycol di-isotridecanoate, propylene
glycol di-(2-ethylhexanoate), butanediol di-isostearate, butanediol
di-caprylate/caprate and neopentyl glycol di-caprylate.
[0093] There may be mentioned here by way of example fatty acid
triglycerides; as such, for example, natural plant oils, e.g. olive
oil, sunflower oil, soya oil, groundnut oil, rapeseed oil, almond
oil, sesame oil, avocado oil, castor oil, cacao butter, palm oil,
but also the liquid contents of coconut oil or of palm kernel oil,
as well as animal oils, such as e.g. shark-fish liver oil, cod
liver oil, whale oil, beef tallow and butter-fat, waxes, such as
beeswax, carnauba palm wax, spermaceti, lanolin and neat's foot
oil, the liquid contents of beef tallow or also synthetic
triglycerides of caprylic/capric acid mixtures, triglycerides from
technical-grade oleic acid, triglycerides with isostearic acid, or
from palmitic acid/oleic acid mixtures, can be employed as
emollients (oil phase).
[0094] In another preferred embodiment of the first aspect, the
organic or oil-phase comprises mineral oil or TEGOSOFT.RTM. M.
[0095] Preferably, the emulsifier is chosen from polyoxyethylene
sorbitan fatty acid esters, sucrose fatty acid esters, sorbitan
fatty acid esters, polysorbates, polyvinyl alcohol, polyvinyl
pyrrolidone, gelatin, lecithin, poly-oxyethylene castor oil
derivatives, tocopherol, tocopheryl polyethylene glycol succinate,
tocopherol palmitate and tocopherol acetate,
polyoxyethylene-polyoxypropylene co-polymers, or their
mixtures.
[0096] The microbeads of the invention give access to the
compositions of the invention comprising these microbeads. The
compositions of the invention may comprise at least one additional
component chosen from the group of emollients, emulsifiers and
surfactants, thickeners/viscosity regulators/stabilizers, UV light
protection filters, antioxidants, hydrotropic agents (or polyols),
solids and fillers, film-forming agents, insect repellents,
preservatives, conditioning agents, perfumes, dyestuffs, biogenic
active compounds, moisturizers and solvents. The additional
components might be inside and/or outside the microbeads.
Preferably the additional ingredients are present in the
composition of the invention outside or within the microbeads.
[0097] In a preferred embodiment, the composition of the invention
can be an emulsion, a suspension, a solution, a cream, an ointment,
a paste, a gel, an oil, a powder, an aerosol, a stick or a
spray.
[0098] The microbeads or the compositions of the invention may be
used as a transdermal drug delivery system/vehicle. When applied
topically the microbeads congregate in wrinkles and folds of the
skin (results not shown).
EXAMPLES
Example 1
Preparation of DVS Crosslinked Microparticles in Emulsion
[0099] This example illustrates the preparation of DVS-crosslinked
microparticles.
[0100] Sodium hyaluronate (HA, 580 kDa, 1.90 g) was dissolved in
aqueous NaOH (0.2 M, 37.5 ml) by vigorous stirring at room
temperature for 3 hours until a homogenous solution was obtained.
Sodium chloride (0.29 g) was added and mixed shortly.
[0101] Mineral oil (10.0 g) and ABIL.RTM. EM 90 surfactant (Cetyl
PEG/PPG-10/1 Dimethicone, 1.0 g) were mixed by stirring.
[0102] Divinylsulfone (DVS, 320 microliter) was added to the
aqueous alkaline HA-solution and mixed for 1 min. to obtain a
homogeneous distribution in the aq. phase. The water phase was then
added within 2 minutes to the oil phase with mechanical stirring at
low speed. An emulsion was formed immediately and stirring was
continued for 30 minutes at room temperature. The emulsion was left
over night at room temperature.
[0103] The emulsion was neutralized to pH 7.0 by addition of aq.
HCI (4 M, approx. 2.0 ml) and stirred for approx. 40 min.
Example 2
Preparation of DVS Crosslinked Microparticles in Emulsion
Neutralized with use of pH Indicator
[0104] This example illustrates the preparation of DVS-crosslinked
microparticles with neutralization using a pH indicator.
[0105] Sodium hyaluronate (HA, 580 kDa, 1.88 g) was dissolved in
aqueous NaOH (0.2 M, 37.5 ml) by vigorous stirring at room
temperature for 2 hours until a homogenous solution was obtained.
Bromothymol blue pH indicator (equivalent range pH 6.6-6.8) was
added (15 drops, blue color in solution). Sodium chloride (0.25 g)
was added and mixed shortly.
[0106] Mineral oil (10.0 g) and ABIL.RTM. EM 90 surfactant (Cetyl
PEG/PPG-10/1 Dimethicone, 1.0 g) were mixed by stirring.
[0107] Divinylsulfone (DVS, 320 microliter) was added to the
aqueous alkaline HA-solution and mixed very vigorously for 30 to 60
seconds to obtain a homogeneous distribution in the aq. phase. The
water phase was then added within 30 sec. to the oil phase with
mechanical stirring at 400 RPM. An emulsion was formed immediately
and stirring was continued for 30min. at room temperature.
Neutralization was performed by addition of aq. HCI (4 M, 1.6 ml)
and the emulsion was left at room temperature with magnetic
stirring for 4 hours. The pH indicator present in the gel particles
changed color to green. pH in the emulsion was measured by pH stick
to 3-4. The emulsion was left in fridge over night. The pH
indicator present in the gel particles had changed to yellow.
Example 3
Phase Separation of Emulsion, Swelling and Isolation of
Microparticles
[0108] This example illustrates the breakage of the W/O emulsion
followed by phase separation and dialysis. The crosslinked HA
microparticles were separated from the W/O emulsion by organic
solvent extraction.
[0109] The W/O emulsion (5 g) and a mixture of n-butanol/chloroform
(1/1 v%, 4.5 ml) was mixed vigorously by whirl mixing in a test
tube at room temperature. Extra mQ-water (20 ml) was added to
obtain phase separation. The test tube was centrifuged and three
phases were obtained with the bottom phase being the organic phase,
middle phase of gel particles and upper phase of clear aqueous
solution. The top and bottom phases were discarded and the middle
phase of gel particles was transferred into a dialysis tube (MWCO
12-14,000, Diameter 29 mm, Vol/Length 6.4 ml/cm).
[0110] The sample was dialyzed overnight at room temperature in
MilliQ.RTM.-water. The dialysate was changed two more times and
left overnight. The resulting gel was thick and viscous and had
swelled to a volume of approximately 50 ml, which correlated to
0.004 g HA/cm.sub.3.
Example 4
Preparation of DVS Crosslinked Microparticles in Emulsion and
Separation of Microparticles
[0111] This example illustrates the preparation of DVS-crosslinked
HA microparticles.
[0112] Sodium hyaluronate (HA, 580 kDa, 1.89 g) was dissolved in
aqueous NaOH (0.2 M, 37.5 ml). Sodium chloride (0.25 g) was added
and the solution was stirred by magnetic stirring for 1 hour at
room temperature until a homogeneous solution was obtained.
[0113] TEGOSOFT.RTM. M (10.0 g) oil and ABIL.RTM. EM 90 surfactant
(Cetyl PEG/PPG-10/1 Dimethicone, 1.0 g) were mixed by stirring.
[0114] Divinylsulfone (DVS, 320 microliter) was added to the
aqueous alkaline HA-solution and mixed for 1 min. to obtain a
homogenoues distribution in the aq. phase. The water phase was then
added within 2 min. to the oil phase with mechanical stirring (300
RPM). An emulsion was formed immediately and stirring was continued
for 30 min. at room temperature.
[0115] The emulsion was neutralized by addition of stociometric
amounts of HCI (4 M, 1.8 ml) and stirred for approx. 40 min. The
emulsion was broken by addition of a n-butanol/chloroform mixture
(1:1 v%, 90 ml) and extra MilliQ.RTM.-water (100 ml) followed by
magnetic stirring. The upper phase was separated in a volume of
approx. 175 ml. The organic phase was mixed with mQ-water (30 ml)
for a final washing. The combined water/gel phase (205 ml) were
transferred to a dialysis tube (MWCO 12-14,000, Diameter 29 mm,
Vol/Length 6.4 ml/cm) and dialysed against MilliQ.RTM.-water
overnight at room temperature. The conductivity were decreased to
0.67 micro-Sievert/cm after subsequent change of water (3 times)
and dialysis overnight (2 nights). The microparticles were assessed
by microscopy (DIC 200.times.), see FIG. 1; the cross-section of
one microparticle is indicated and labelled "21,587.92 nm".
Example 5
Phase Separation of Emulsion and Isolation of Microparticles
[0116] This example illustrates the breakage of the W/O emulsion
and isolation of the gel microparticles.
[0117] The gel microparticles were separated from the W/O-emulsion
by organic extractions. Examples of organic solvents which were
used for this extraction were mixtures of butanol/chloroform in
volume ratios (v%) of 75:20 to 20.80, respectively. The weight
ratio (w%) of W/O emulsion to organic solvent was approximately
1:1.
[0118] Separation in small scale: The W/O emulsion (5 g) was
weighed in centrifuge tubes (50 ml). A mixture of butanol/
chloroform was prepared (1:1 v%) and from this mixture 4.5 ml was
added (corresponds to 5 g) to the test tube. The test tube was
carefully mixed to secure that all emulsion was dissolved. The test
tube was mixed by Whirl mixing and left at room temperature for
phase separation. Phase separation with water phase on top and
organic phase at bottom with a white emulsion phase in between was
often observed. Addition of more water and organic phases improved
separation. The water phase was separated by decanting and further
purified or characterized.
Example 6
Preparation of Water-in-Oil Emulsions
[0119] This example illustrates a composition in which the HA
microparticles were formed.
[0120] A hot/cold procedure can be used with incorporation of a
cold water phase B into a hot oil phase, which will shorten the
time of manufacture. A non-limiting example of formulation could be
as follows:
Phase A:
[0121] 2.0% ABIL.RTM. EM 90 (cetyl PEG/PPG-10/1 dimethicone) [0122]
20.0% Mineral oil (or TEGOSOFT.RTM. M)
Phase B:
[0122] [0123] 0.5% Sodiumchloride [0124] 3.8% Hyaluronic acid
[0125] 0.2 M NaOH (aq) up to 100%
Phase C:
[0126] Approx. 0.6% Divinylsulfone
Preparation:
[0127] 1. Mix phase A at room temperature. [0128] 2. Phase B:
Solubilize hyaluronic acid (Hyacare.RTM.) in aq. NaOH by stirring;
then add NaCl and stir. [0129] 3. Add DVS to phase B and stir for 1
min. [0130] 4. Add phase B slowly to phase A with stirring. [0131]
5. Homogenise or stir for a short time and leave to react. [0132]
6. Stirring and swelling. [0133] 7. Continue stirring below
30.degree. C. [0134] 8. Neutralize.
Example 7
Preparation and Separation of DVS Cross-Linked Microparticles
[0135] Sodium hyaluronate (HA, 580 kDa, 1.88 g) was dissolved in
aqueous NaOH (0.2 M, 37.5 mL). Sodium chloride (0.25 g) was added
and the solution was stirred by magnetic stirring for 1 hour at
room temperature until a homogeneous solution was obtained. The
oil: TEGOSOFT.RTM. M (10.0 g) and surfactant: ABIL.RTM. EM 90
(Cetyl PEG/PPG-10/1 Dimethicone, 1.0 g) was mixed by stirring.
Divinylsulfone (DVS, 320 microliter) was added to the aqueous
alkaline HA-solution and mixed for 1 min to obtain a homogenoues
distribution in the aq. phase. The water phase was then added
within 2 min to the oil phase with mechanical stirring (300 RPM).
An emulsion was formed immediately and stirring was continued for
30 min at room temperature.
[0136] The emulsion was neutralized by addition of stociometric
amounts of HCI (4 M, 1.8 mL) and stirred for approx. 40 min. The
emulsion was transferred to a separation funnel, and broken by
addition of a n-butanol/chloroform mixture (1:1 v%, 90 mL) and
extra millliQ.TM.-water (100 mL) followed by vigorous shaking. The
upper phase was separated in a volume of approx. 175 mL. The
organic phase was washed with millliQ.TM.-water (100 mL). The
combined water/gel phase was transferred to a dialysis tube (MWCO
12-14,000, Diameter 29 mm, Vol/Length 6.4 mL/cm) and dialysed
against millliQ.TM.-water overnight at room temperature. The
conductivity was decreased to 10 micro-Sievert/cm after subsequent
change of water (3 times) and dialysis overnight (2 nights). The
microparticles were assessed by microscopy (FIG. 4).
Example 8
Washing Procedure to Purify Microparticles
[0137] This example illustrates the final isolation and
purification of the microparticles.
[0138] 100 mL particles previously isolated were re-suspended in a
Na.sub.2HPO.sub.4/NaH.sub.2PO.sub.4 buffer (0.15 M, 400 mL), and
stirred slowly for 1/2 hour. The suspension stood at 5.degree. C.
for 2 hours and solidified oil droplets were removed. The solution
was then filtered through a mesh and washed further with 2.times.50
mL buffer. Particles were allowed to drip-dry, before
characterization (FIG. 5).
Example 9
Investigation of Rheological Properties of Microparticles
[0139] This example illustrates performance of rheological studies
on particles. A particle sample is analyzed on an Anton Paar
rheometer (Anton Paar GmbH, Graz, Austria, Physica MCR 301,
Software: Rheoplus), by use of a 50 mm 2.degree. cone/plate
geometry. First the linear range of the visco-elastic properties G'
(Storage modulus) and G'' (Loss modulus) of the material is
determined by an amplitude sweep with variable strain, .gamma..
Secondary a Frequency sweep is made, and based on values of the
visco-elastic values, G' and G'', tan .delta. can be calculated as
a value for week/strong gel behaviors.
Example 10
Investigation of Syringeability Experiments on Texture Analyzer
[0140] This example illustrates performance of an investigation of
force applied to inject at a certain speed, as a function of the
homogeneity of the sample. A particle sample is transferred to a
syringe applied with a needle, either 27G.times.1/2 '',
30G.times.1/2 '', and is set in a sample rig, in a texture analyzer
(Stable Micro Systems, Surrey, UK, TA.XT Plus, SoftWare: Texture
Component 32). The test is performed with an injection speed at
12.5 mm/min., over a given distance.
* * * * *