U.S. patent application number 13/667581 was filed with the patent office on 2013-09-19 for hyaluronic acid-collagen matrices for dermal filling and volumizing applications.
This patent application is currently assigned to ALLERGAN, INC.. The applicant listed for this patent is ALLERGAN, INC.. Invention is credited to Nicholas J. Manesis, Jacob Pollock, Xiaojie Yu.
Application Number | 20130244943 13/667581 |
Document ID | / |
Family ID | 49158184 |
Filed Date | 2013-09-19 |
United States Patent
Application |
20130244943 |
Kind Code |
A1 |
Yu; Xiaojie ; et
al. |
September 19, 2013 |
HYALURONIC ACID-COLLAGEN MATRICES FOR DERMAL FILLING AND VOLUMIZING
APPLICATIONS
Abstract
Hydrogels comprising a macromolecular matrix and water may be
used for aesthetic fillers, for example, dermal fillers. The
macromolecular matrix may include a crosslinked combination of
hyaluronic acid and collagen.
Inventors: |
Yu; Xiaojie; (Irvine,
CA) ; Manesis; Nicholas J.; (Summerland, CA) ;
Pollock; Jacob; (Charleston, WV) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALLERGAN, INC. |
Irvine |
CA |
US |
|
|
Assignee: |
ALLERGAN, INC.
Irvine
CA
|
Family ID: |
49158184 |
Appl. No.: |
13/667581 |
Filed: |
November 2, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13605565 |
Sep 6, 2012 |
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13667581 |
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13603213 |
Sep 4, 2012 |
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13605565 |
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61555970 |
Nov 4, 2011 |
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61531533 |
Sep 6, 2011 |
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61531533 |
Sep 6, 2011 |
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Current U.S.
Class: |
514/17.2 |
Current CPC
Class: |
A61K 8/042 20130101;
A61K 2800/91 20130101; A61L 2430/34 20130101; A61Q 19/08 20130101;
A61L 27/52 20130101; A61L 27/26 20130101; A61L 27/26 20130101; A61L
27/26 20130101; A61L 27/3695 20130101; C08L 89/06 20130101; C08L
5/08 20130101; A61K 8/735 20130101; A61L 27/24 20130101; A61L
2400/06 20130101 |
Class at
Publication: |
514/17.2 |
International
Class: |
A61L 27/24 20060101
A61L027/24; A61L 27/52 20060101 A61L027/52 |
Claims
1. A soft tissue aesthetic product comprising: a filler comprising
a hydrogel having a form suitable for injecting into mammalian
tissue; and a label comprising instructions to inject the filler
into the tissue; wherein the hydrogel comprises water, and a
crosslinked macromolecular matrix comprising: a hyaluronic acid
component; and a collagen component; wherein the hyaluronic acid
component is crosslinked to the collagen component by a
crosslinking component; and wherein the crosslinking component
comprises a plurality of crosslink units, wherein at least a
portion of the crosslink units comprise an ester bond or an amide
bond.
2. The product of claim 1, wherein the collagen component comprises
collagen type I or collagen type III.
3. The product of claim 1, wherein crosslinked macromolecular
matrix has a weight ratio of the hyaluronic acid component to the
collagen component of about 1 to about 3.
4. The product of claim 1, wherein the hydrogel is in the form of a
dermal filler.
5. The product of claim 1, wherein the hydrogel is in the form of a
lip filler.
6. A method of improving an aesthetic quality of soft tissue of a
human being comprising: injecting a hydrogel composition into a
soft tissue of the human being to thereby improve the aesthetic
quality of the soft tissue; wherein the hydrogel composition
comprises water, and a crosslinked macromolecular matrix
comprising: a hyaluronic acid component; and a collagen component;
wherein the hyaluronic acid component is crosslinked to the
collagen component by a crosslinking component; and wherein the
crosslinking component comprises a plurality of crosslink units,
wherein at least a portion of the crosslink units comprise an ester
bond or an amide bond.
7. The method of claim 6, wherein the collagen component comprises
collagen type I or collagen type III.
8. The method of claim 6, wherein the hydrogel is in the form of a
dermal filler.
9. The method of claim 6, wherein the hydrogel is in the form of a
lip filler.
10. A dermal filler comprising: a hydrogel having a form suitable
for injecting into mammalian tissue; wherein the hydrogel comprises
water, and a crosslinked macromolecular matrix comprising: a
hyaluronic acid component; and a collagen component; wherein the
hyaluronic acid component is crosslinked to the collagen component
by a crosslinking component; and wherein the crosslinking component
comprises a plurality of crosslink units, wherein at least a
portion of the crosslink units comprise an ester bond or an amide
bond.
11. The dermal filler of claim 10, wherein the collagen component
comprises collagen type I or collagen type III.
12. The dermal filler of claim 10, wherein crosslinked
macromolecular matrix has a weight ratio of the hyaluronic acid
component to the collagen component of about 1 to about 3.
13. The dermal filler of claim 10 wherein the crosslinked
macromolecular matrix has a weight ratio of the hyaluronic acid
component to the collagen component of about 1 to about 7.
Description
[0001] This application claims priority to U.S. Provisional Patent
Application No. 61/555,970, filed Nov. 4, 2011, and also is a
continuation-in-part of U.S. patent application Ser. No.
13/605,565, filed on Sep. 6, 2012, which claims priority to U.S.
Provisional Patent Application No. 61/531,533, filed on Sep. 6,
2011, and which is a continuation-in-part of U.S. patent
application Ser. No. 13/603,213, filed Sep. 4, 2012, which claims
priority to U.S. Provisional Patent Application No. 61/531,533,
filed Sep. 6, 2011, the entire disclosure of each of these
applications being incorporated herein by this specific
reference.
[0002] This application generally relates to biocompatible,
implantable compositions and more specifically relates to
hyaluronic acid-collagen based compositions useful as dermal
fillers.
SUMMARY
[0003] The present invention generally relates to a soft tissue
aesthetic product. In one aspect, the product comprises a filler
comprising a hydrogel having a form suitable for injecting into
human tissue; and a label comprising instructions to inject the
filler into the human tissue; wherein the hydrogel comprises water,
and a crosslinked macromolecular matrix described herein.
[0004] Some embodiments include a method of improving an aesthetic
quality of soft tissue of a human being comprising: injecting a
hydrogel composition into a soft tissue of the human being to
thereby improve the aesthetic quality of the soft tissue; wherein
the hydrogel composition comprises water, and a crosslinked
macromolecular matrix described herein.
[0005] Some embodiments include a method of generating tissue
comprising contacting a tissue with a hydrogel composition to
generate an additional amount of the tissue, wherein the hydrogel
composition comprises water and a crosslinked macromolecular matrix
described herein.
[0006] Some crosslinked molecular matrices may comprise a
hyaluronic acid component; and a collagen component; wherein the
hyaluronic acid component is crosslinked to the collagen component
by a crosslinking component; and wherein the crosslinking component
comprises a plurality of crosslink units, wherein at least a
portion of the crosslink units comprise an ester bond or an amide
bond.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] Some aspects of the present disclosure may be more clearly
understood with reference to the appended drawings of which:
[0008] FIG. 1A is a plot of frequency sweep and FIG. 1B is a plot
of strain sweep for a hydrogel in accordance with this
disclosure.
[0009] FIG. 2 is an extrusion profile through a 30 G needle for a
hydrogel from Example 4.
[0010] FIGS. 3A-3C show respectively, micrographs (at 5.times.
magnification) of (A) tissue adjacent to an implanted control
composition of commercial crosslinked hyaluronic acid gel, (B)
tissue adjacent to an implanted composition of Example 3, and (C)
tissue adjacent to an implanted composition from Example 4.
DETAILED DESCRIPTION
[0011] Some embodiments include a method of improving an aesthetic
quality of soft tissue of a human. Such a method may comprise
injecting a hydrogel composition, or a composition comprising a
hydrogel, into a soft tissue of the human being to thereby improve
the aesthetic quality of the soft tissue.
[0012] Some embodiments may include a soft tissue aesthetic product
comprising: a filler comprising a hydrogel having a form suitable
for injecting into human tissue and a label comprising instructions
to inject the filler into the human tissue.
[0013] A filler comprising a hydrogel or a hydrogel composition may
be any kind of filler that is suitable for injecting into human
tissue to improve an aesthetic quality of a soft tissue, such as a
dermal filler, a breast augmentation or reconstruction filler, a
lip filler, hand rejuvenation, or the like. When injected, a
hydrogel may stimulate tissue in-growth and generation after being
injected into the soft tissue. In some embodiments, a hydrogel may
stimulate collagenesis after being injected into the soft
tissue.
[0014] Injecting a hydrogel comprising a crosslinked macromolecular
matrix comprising a hyaluronic acid component that is crosslinked
to a collagen component may provide improved aesthetic quality for
an extended duration, as compared to injecting an identical
hydrogel except that hyaluronic acid component and the collagen
component are not crosslinked.
[0015] Some embodiments include a method of generating tissue
comprising contacting a tissue with a hydrogel composition to
generate an additional amount of the tissue. This method may be
used to generate tissue both ex vivo and in vivo. In some
embodiments, contact between a tissue and a hydrogel may be ex
vivo. In some embodiments, contact between a tissue and a hydrogel
may be in vivo. Tissue types that may be generated include, but are
not limited to, adipose tissue, muscle tissue, tendon tissue,
cardiovascular tissue, neural tissue, bone tissue, and the
like.
[0016] Some embodiments include a packaged product comprising a
syringe loaded with a hydrogel and a needle. A syringe may be
fitted with a needle of any size that is appropriate for injecting
the hydrogel into the soft tissue of interest, such as a needle
with about a #25, about a #30, or a larger gauge.
[0017] A filler comprising a hydrogel may be suitable for injection
if it can be injected into the soft tissue of interest without
unreasonable difficulty, and includes fillers that can be dispensed
from cannulas having gauge as low as about #30 or about #25 under
normal manual pressure with a smooth extrusion plateau.
[0018] Injection of a hydrogel may provide a soft tissue
augmentation that mimics the natural components of the skin. A
hydrogel may be injected intradermally or subcutaneously to augment
soft tissue and to repair or correct congenital anomalies, acquired
defects, or cosmetic defects. Examples of such conditions include
congenital anomalies such as hemifacial microsomia, malar and
zygomatic hypoplasia, unilateral mammary hypoplasia, pectus
excavatum, pectoralis agenesis (Poland's anomaly), and
velopharyngeal incompetence secondary to cleft palate repair or
submucous cleft palate (as a retropharyngeal implant); acquired
defects (post traumatic, post surgical, or post infectious) such as
depressed scars, subcutaneous atrophy (e.g., secondary to discoid
lupis erythematosis), keratotic lesions, enopthalmos in the
unucleated eye (also superior sulcus syndrome), acne pitting of the
face, linear scleroderma with subcutaneous atrophy, saddle-nose
deformity, Romberg's disease, and unilateral vocal cord paralysis;
and cosmetic defects such as glabellar frown lines, deep nasolabial
creases, circum-oral geographical wrinkles, sunken cheeks, and
mammary hypoplasia.
[0019] Crosslinked hyaluronic acid, collagen, and crosslinked
collagen, such as those used in dermal fillers, do not promote
cellular infiltration and tissue in-growth. Similarly, collagen
simply blended into hyaluronic acid hydrogels does not promote
tissue integration or de novo tissue generation. However, some
hydrogels described herein do promote cellular migration into the
hydrogels and tissue formation within the gels when implanted in
vivo.
[0020] A hydrogel may comprise water and a crosslinked
macromolecular matrix. Typically, a crosslinked molecular matrix
may comprise a hyaluronic acid component and a collagen component,
wherein the hyaluronic acid component is crosslinked to the
collagen component by a crosslinking component. A crosslinking
component may comprise a plurality of crosslink units, wherein at
least a portion of the crosslink units comprise an ester bond or an
amide bond.
[0021] A crosslinked macromolecular matrix for a hydrogel may be
synthesized by coupling a hyaluronic acid with a collagen using a
coupling agent, such as a carbodiimide. In these hydrogels,
hyaluronic acid may serve as a biocompatible water-binding
component, providing bulk and isovolumetric degradation.
Additionally, collagen may impart cell adhesion and signaling
domains to promote cell attachment, migration, and other cell
functions such as extra-cellular matrix deposition. The biopolymers
form homogeneous hydrogels with tunable composition, swelling, and
mechanical properties. Compositions can be made to be injectable
for minimally invasive implantation through syringe and needle.
[0022] Hyaluronic acid is a non-sulfated glycosaminoglycan that
enhances water retention and resists hydrostatic stresses. It is
non-immunogenic and can be chemically modified in numerous
fashions. Hyaluronic acid may be anionic at pH ranges around or
above the pKa of its carboxylic acid groups.
##STR00001##
[0023] Collagen is a protein that forms fibrils and sheets that
bear tensile loads. Collagen also has specific integrin-binding
sites for cell adhesion and is known to promote cell attachment,
migration, and proliferation. Collagen may be positively charged
because of its high content of basic amino acid residues such as
arginine, lysine, and hydroxylysine.
[0024] Because hyaluronic acid may be anionic and collagen may be
cationic, the two macromolecules may form polyionic complexes in
aqueous solution. A polyionic complex may be significantly less
soluble in water than either hyaluronic acid or collagen, and thus
may precipitate out of aqueous solution when the two macromolecules
are together in a mixture.
[0025] Under certain conditions, a hyaluronic acid and a collagen
may be combined in an aqueous liquid in which both components are
soluble. A hyaluronic acid and a collagen may then be crosslinked
while both are dissolved in an aqueous solution to form a hydrogel.
Reaction conditions such as the concentration of hyaluronic acid,
the concentration of collagen, the pH of the solution, and salt
concentration may be adjusted to help to prevent polyionic complex
formation between anionic hyaluronic acid and cationic collagen.
They may also help to prevent collagen microfibril formation.
[0026] Some embodiments include a method of crosslinking hyaluronic
acid and collagen. This method generally comprises a dissolution
step which results in an aqueous pre-reaction solution. In a
dissolution step, hyaluronic acid and collagen are dissolved in an
aqueous solution that has a low pH and/or a salt to form an aqueous
pre-reaction solution.
[0027] A hyaluronic acid-collagen crosslinking method further
comprises an activation step. In an activation step, an aqueous
pre-reaction solution is modified at least by adding a water
soluble coupling agent and/or by increasing the pH of the solution.
If needed, a salt may also be added to keep the hyaluronic acid and
collagen in solution at the higher pH. Thus, a crosslinking
reaction mixture comprises hyaluronic acid and collagen dissolved
or dispersed in an aqueous medium, a water soluble coupling agent,
and a salt, and has a higher pH than the aqueous pre-reaction
solution from which it was derived. The crosslinking reaction
mixture is allowed to react to thereby crosslink the hyaluronic
acid and the collagen.
[0028] In some embodiments, the pH of the aqueous pre-reaction
solution may be increased and a substantial amount of fiber
formation may be allowed to occur in the solution before adding the
water soluble coupling agent. In some embodiments, the water
soluble coupling agent may be added to the aqueous pre-reaction
solution before substantially any fiber formation occurs.
[0029] A crosslinking reaction mixture can react to form a
crosslinked macromolecular matrix. Since reaction occurs in an
aqueous solution, a crosslinked macromolecular matrix may be
dispersed in an aqueous liquid in hydrogel form as it is formed by
a crosslinking reaction. A crosslinked macromolecular matrix may be
kept in hydrogel form because, in many instances, a crosslinked
macromolecular matrix may be used in hydrogel form.
[0030] In some embodiments, an aqueous pre-reaction solution or a
crosslinking reaction mixture may further comprise about 10% to
about 90% of an organic solvent in which hyaluronic acid has poor
solubility, such as ethanol, methanol, isopropanol, or the
like.
[0031] After a crosslinking reaction has occurred, the crosslinked
macromolecular matrix may be particulated or homogenized through a
mesh. This may help to form an injectable slurry or hydrogel. A
mesh used for particulating a crosslinked macromolecular matrix may
have any suitable pore size depending upon the size of particles
desired. In some embodiments, the mesh may have a pore size of
about 10 microns to about 100 microns, about 50 microns to about 70
microns, or about 60 microns.
[0032] A hydrogel comprising a crosslinked molecular matrix may be
treated by dialysis for sterilization or other purposes. Dialysis
may be carried out by placing a semipermeable membrane between the
hydrogel and another liquid so as to allow the hydrogel and the
liquid to exchange molecules or salts that can pass between the
membrane.
[0033] A dialysis membrane may have a molecular weight cutoff that
may vary. For example, the cutoff may be about 5,000 daltons to
about 100,0000 daltons, about 10,000 daltons to about 30,000
daltons, or about 20,000 daltons.
[0034] The dialysis may be carried out against a buffer solution,
or the liquid on the other side of the membrane from the hydrogel
may be a buffer solution. In some embodiments, the buffer solution
may be a sterile phosphate buffer solution that may comprise
phosphate buffer, potassium chloride, and/or sodium chloride. A
sterile phosphate buffer solution may be substantially isosmotic
with respect to human physiological fluid. Thus, when dialysis is
complete, the liquid component of a hydrogel may be substantially
isosmotic with respect to human physiological fluid.
[0035] In some embodiments, a crosslinked macromolecular complex
may further comprise an aqueous liquid. For example, the
crosslinked macromolecular complex may absorb the aqueous liquid so
that a hydrogel is formed. An aqueous liquid may comprise water
with a salt dissolved in it, such as a phosphate buffer, sodium
chloride, potassium chloride, etc. In some embodiments, an aqueous
liquid may comprise water, sodium chloride at a concentration of
about 100 mM to about 200 mM, potassium chloride at a concentration
of about 2 mM to about 3 mM, and phosphate buffer at a
concentration of about 5 mM to about 15 mM, wherein the pH of the
liquid is about 7 to about 8.
[0036] A hydrogel may be used in a soft tissue aesthetic product. A
soft tissue aesthetic product may comprise: an aesthetic device
having a form suitable for injecting or implanting into human
tissue; and a label comprising instructions to inject or implant
the aesthetic component into human tissue; wherein the aesthetic
device comprises a crosslinked macromolecular matrix described
herein. Some products may comprise the crosslinked macromolecular
matrix in hydrogel form.
[0037] Some embodiments include a method of improving an aesthetic
quality of an anatomic feature of a human being comprising:
injecting or implanting an aesthetic device into a tissue of the
human being to thereby improve the aesthetic quality of the
anatomic feature; wherein the aesthetic device comprises a
crosslinked macromolecular matrix comprising described herein. In
some embodiments, the crosslinked macromolecular matrix used in the
product may be in hydrogel form.
[0038] In some embodiments, a hydrogel of a crosslinked
macromolecular complex may have a storage modulus of about 1 Pa to
about 10,000 Pa, about 50 Pa to 10,000 Pa, about 500 Pa to about
1000 Pa, about 556 Pa, about 560 Pa, about 850 Pa, about 852 Pa, or
any value in a range bounded by, or between, any of these
values.
[0039] In some embodiments, a hydrogel of a crosslinked
macromolecular complex may have a loss modulus of about 1 Pa to
about 500 Pa, about 10 Pa to 200 Pa, about 100 Pa to about 200 Pa,
about 20 Pa, about 131 Pa, about 152 Pa, or any value in a range
bounded by, or between, any of these values.
[0040] In some embodiments, a hydrogel of a crosslinked
macromolecular complex may have an average extrusion force of about
20 N to 30 N, or about 25 N, when the hydrogel is forced through a
30 G needle syringe by moving the plunger of a 1 mL syringe
containing the hydrogel at a rate of 100 mm/min for about 11 mm,
and measuring the average force from about 4 mm to about 10 mm.
[0041] A crosslinked macromolecular complex may have tunable
swelling properties based on reaction conditions and hydrogel
dilution. In some embodiments, a crosslinked macromolecular complex
may have a swelling ratio of about 1 to about 7. A swelling ratio
is the ratio of the weight of the crosslinked macromolecular
complex when saturated with water to the weight of the crosslinked
macromolecular complex without any water. More specifically, the
swelling ratio is the ratio of the mass of the gel which has been
allowed to fully swell to the mass of the gel at its initial
concentration.
[0042] In a crosslinking reaction, the molecular weight of a
hyaluronic acid may vary. In some embodiments, a hyaluronic acid
may have a molecular weight of about 500,000 daltons to about
10,000,000 daltons, about 1,000,000 daltons to about 5,000,000
daltons, or about 1,000,000 daltons to about 3,000,000 daltons.
When the crosslinking reaction occurs, the resulting crosslinked
macromolecular product may have a hyaluronic acid component derived
from the hyaluronic acid in the crosslinking reaction. Thus, the
ranges recited above may also apply to the molecular weight of a
hyaluronic acid component, e.g. about 500,000 daltons to about
10,000,000 daltons, about 1,000,000 daltons to about 5,000,000
daltons, or about 1,000,000 daltons to about 3,000,000 daltons. The
term "molecular weight" is applied in this situation to a portion
of the matrix even though the hyaluronic acid component may not
actually be a separate molecule due to the crosslinking.
[0043] The concentration of hyaluronic acid in an aqueous
pre-reaction solution or a crosslinking reaction mixture may vary.
In some embodiments, hyaluronic acid is present at about 3 mg/mL to
about 100 mg/mL, about 6 mg/mL to about 24 mg/mL, about 1 mg/mL to
about 30 mg/mL, about 1.7 mg/mL, about 3 mg/mL, about 6 mg/mL,
about 12 mg/mL, about 16 mg/mL, or about 24 mg/mL
[0044] Any type of collagen may be used in the methods and
compositions described herein. In some embodiments, collagen type
I, collagen type III, collagen type IV, collagen type VI, or a
combination thereof, may be used. In some embodiments, a collagen
or a collagen component comprises collagen type I or collagen type
III. In some embodiments, the collagen component comprises collagen
type V.
[0045] A collagen may be derived from cell culture, animal tissue,
or recombinant means, and may be derived from human, porcine, or
bovine sources. Some embodiments comprise collagen derived from
human fibroblast culture. Some embodiments comprise collagen that
has been denatured to gelatin. The source and/or collagen
extraction/processing conditions can alter the way in which
collagen macromolecules bundle together to form supramolecular
structures. These higher order structures can have effects on the
gel physical properties (stiffness, viscosity) and may also have an
effect on the reactivity of the collagen to crosslinking
reagents.
[0046] Collagen concentration in an aqueous pre-reaction solution
or a crosslinking reaction mixture may vary. In some embodiments,
collagen may be present at a concentration of about 1 mg/mL to
about 40 mg/mL, about 1 mg/mL to about 15 mg/mL, about 3 mg/mL to
about 12 mg/mL, about 1.7 mg/mL, about 3 mg/mL, about 6 mg/mL,
about 8 mg/mL, or about 12 mg/mL. The collagen concentration has an
effect on the physical properties of the gel (stiffness,
viscosity). In general, higher collagen concentrations lead to a
higher elastic modulus.
[0047] In some embodiments, the weight ratio of hyaluronic acid to
collagen in a aqueous pre-reaction solution or a aqueous
pre-reaction solution or a crosslinking reaction mixture (e.g. [wt
hyaluronic acid]/[wt collagen]) may be about 0.5 to about 3, about
1 to about 3, about 1 to about 2, about 1, or about 2. When the
crosslinking reaction occurs, the resulting crosslinked
macromolecular product may have a collagen component derived from
the collagen in the crosslinking reaction. Thus, the resulting
crosslinked macromolecular matrix may have a weight ratio of
hyaluronic acid component to collagen component that corresponds to
the weight ratio in the crosslinking reaction, e.g. about 0.5 to
about 3, about 1 to about 3, about 1 to about 2, about 1, or about
2.
[0048] In other embodiments of the invention, the compositions have
an HA to collagen ratio of between about 0.5 to 1 and about 7 to
1.
[0049] A salt may help to screen the negative charges of hyaluronic
acid from positive charges of collagen, and may thus prevent
precipitation of a polyionic ion complex from solution. However,
high concentrations of salt may reduce the solubility of some
components in solution. Thus, in some embodiments, the salt
concentration of an aqueous pre-reaction solution or a crosslinking
reaction mixture may be high enough to screen the charges so that
the polyionic ion complex is not formed, but also low enough so
that the components of the mixture remain in solution. For example,
the total salt concentration of some aqueous pre-reaction solutions
or crosslinking reaction mixtures may be about 10 mM to about 1 M,
for example, between about 5 mM to about 0.5 M, for example,
between about 2 mM to about 0.2 M.
[0050] Some salts in an aqueous pre-reaction solution or a
crosslinking reaction mixture may be non-coordinating buffers. Any
non-coordinating buffer may be used that is capable of buffering
the mixture and does not coordinate with metal atoms or ions in the
collagen. In some embodiments, the buffer does not react with the
crosslinking reagents (carbodiimide and additive). For example,
acetate or phosphate buffers may not be used in these embodiments.
Examples of suitable non-coordinating buffers may include, but are
not limited to, 2-(N-morpholino)ethanesulfonic acid
(MES),3-(N-morpholino)propanesulfonic acid
(MOPS),4-(2-hydroxyethyl)-1-piperazinyl)ethanesulfonic acid
(HEPES), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid
(HEPPS), N-cyclohexyl-2-aminoethanesulfonic acid (CHES),
N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), etc.
##STR00002##
[0051] The concentration of a non-coordinating buffer may vary. For
example, some aqueous pre-reaction solutions or crosslinking
reaction mixtures may have a buffer concentration in a range of
about 10 mM to about 1 M, about 10 mM to about 500 mM, about 20 mM
to about 100 mM, or about 25 mM to about 250 mM. Some aqueous
pre-reaction solutions or crosslinking reaction mixtures comprise
MES at a concentration of about 20 mM to about 200 mM, about 20 mM
to about 100 mM, about 100 mM, or about 180 mM.
[0052] Non-buffering salts may also be included in an aqueous
pre-reaction solution or a crosslinking reaction mixture as an
alternative to, or in addition, to buffering salts. Some examples
may include sodium chloride, potassium chloride, potassium bromide,
sodium bromide, lithium chloride, lithium bromide, sodium iodide,
and potassium iodide The concentration of a non-buffering salt may
vary. For example, some mixtures may have a non-buffering salt
concentration in a range of about 10 mM to about 1 mM, about 30 mM
to about 500 mM, or about 50 mM to about 300 mM. In some
embodiments, sodium chloride may be present at a concentration in a
range of about 0.5% w/v to about 2% about 0.9% w/v, about 1.6% w/v,
about 20 mM to about 1 mM, about 40 mM to about 500 mM, about 50 to
300 mM, about 80 mM to about 330 mM, about 150 mM, or about 270
mM.
[0053] The pH of an aqueous pre-reaction solution may be lower than
the pH of a crosslinking reaction mixture. If the salt content of
the aqueous pre-reaction solution is low, the pH may be lower to
enhance solubility of the hyaluronic acid and the collagen. If the
salt content is higher, the pH may be higher in the aqueous
pre-reaction solution. In some embodiments, the pH of the aqueous
pre-reaction mixture is about 1 to about 8, about 3 to about 8,
about 4 to about 6, about 4.7 to about 7.4, or about 5.4. For low
salt concentrations, the pH may be about 1 to about 4 or about 1 to
about 3.
[0054] In some embodiments, pH may be adjusted to neutral to allow
collagen gelation or fiber formation before adding a coupling
agent.
[0055] In some embodiments, the pH may be adjusted to neutral
immediately prior to, around the time of, or after adding a
coupling agent, such that collagen gelation is reduced or does not
substantially occur.
[0056] Any water-soluble coupling agent may be used that can
crosslink hyaluronic acid to collagen. Some non-limiting examples
of a coupling agent include carbodiimides such as
N,N'-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide
(DIC), or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).
Carbodiimide coupling agents may facilitate ester or amide bond
formation without becoming part of the linkage. However, other
coupling agents that become part of the crosslinking group may be
used. The concentration of a coupling agent may vary. In some
embodiments, a coupling agent may be present at about 2 mM to about
150 mM, about 2 mM to about 50 mM, about 20 mM to about 100 mM, or
about 50 mM. In some embodiments, the coupling agent is EDC that is
present at a concentration of about 20 mM to about 100 mM, about 2
mM to about 50 mM, or about 50 mM.
##STR00003##
[0057] As a result of a crosslinking reaction, a crosslinked
macromolecular matrix may comprise a crosslinking component that
crosslinks or covalently connects the hyaluronic acid component to
the collagen component. A crosslink component comprises a plurality
of crosslink units, or individual covalent bonding links, between
the hyaluronic acid component and the collagen component. At least
a portion of the crosslink units comprise an ester bond or an amide
bond. In some embodiments, at least a portion of the crosslink
units may be --CON-- or --CO.sub.2--, where the N is a nitrogen
from an amino acid residue.
[0058] An activating agent may be used to increase the ratio of
amide bonds compared to ester bonds formed in the crosslinked
product. In some embodiments, an activating agent may be a triazole
such as hydroxybenzotriazole (HOBT) or 1-hydroxy-7-azabenzotriazole
(HOAT); a fluorinated phenol such as pentafluorophenol; a
succinimide such as N-hydroxysuccinimide (NHS) or
N-hydroxysulfosuccinimide (sulfoNHS), and the like.
##STR00004##
[0059] The concentration of an activating agent may vary. In some
embodiments, the activating agent may have a concentration of about
2 mM to about 200 mM, about 2 mM to about 50 mM, about 20 mM to
about 100 mM, or about 50 mM. In some embodiments, the activating
agent may be NHS or sulfoNHS is at a concentration of about 2 mM to
about 50 mM. In some embodiments, the activating agent may be
N-hydroxysulfosuccinimide, sodium salt, at a concentration of about
20 mM to about 100 mM, or about 50 Mm.
[0060] In some embodiments, a crosslinking reaction mixture may
comprise a carbodiimide coupling agent and an activating agent. In
some embodiments, the coupling agent is EDC and the activating
agent is NHS or sulfoNHS. In some embodiments EDC is present at a
concentration of about 2 mM to about 50 mM and NHS or sulfoNHS is
present at about 2 mM to about 50 mM.
[0061] In some embodiments, a crosslinking reaction mixture may
comprise hyaluronic acid at a concentration of about 1.7 mg/mL,
collagen at a concentration of about 1.7 mg/mL,
2-(N-morpholino)ethanesulfonic acid at a concentration of about 100
mM, sodium chloride at a concentration of about 0.9 wt % or about
150 mM, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a
concentration of about 50 mM, and N-hydroxysulfosuccinimide sodium
salt at a concentration of about 50 Mm, wherein the solution has a
pH of about 5.4.
[0062] In some embodiments, a crosslinking reaction mixture may
comprise hyaluronic acid at a concentration of about 6 mg/mL,
collagen at a concentration of about 6 mg/mL,
2-(N-morpholino)ethanesulfonic acid at a concentration of about 180
mM, sodium chloride at a concentration of about 1.6 wt % or about
270 mM, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a
concentration of about 50 mM, and N-hydroxysulfosuccinimide sodium
salt at a concentration of about 50 mM, wherein the solution has a
pH of about 5.4.
[0063] In some embodiments, a crosslinking reaction mixture may
comprise hyaluronic acid at a concentration of about 16 mg/mL of,
collagen at a concentration of about 8 mg/mL,
2-(N-morpholino)ethanesulfonic acid at a concentration of about 100
mM, sodium chloride at a concentration of about 0.9 wt % or about
150 mM, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a
concentration of about 50 mM, and N-hydroxysulfosuccinimide sodium
salt at a concentration of about 50 mM, wherein the solution has a
pH of between about 4.5 and 5.5, for example, about 5.2.
[0064] In some embodiments, a crosslinking reaction mixture may
comprise hyaluronic acid at a concentration of about 12 mg/mL,
collagen at a concentration of about 12 mg/mL,
2-(N-morpholino)ethanesulfonic acid at a concentration of about 100
mM, sodium chloride at a concentration of about 0.9 wt % or about
150 mM, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a
concentration of about 50 mM, and N-hydroxysulfosuccinimide sodium
salt at a concentration of about 50 mM.
[0065] In some embodiments, a crosslinking reaction mixture may
comprise hyaluronic acid at a concentration of about 3 mg/mL,
collagen at a concentration of about 3 mg/mL,
2-(N-morpholino)ethanesulfonic acid at a concentration of about 100
mM, sodium chloride at a concentration of about 0.9 wt % or about
150 mM, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a
concentration of about 50 mM, and N-hydroxysulfosuccinimide sodium
salt at a concentration of about 50 mM, wherein the solution has a
pH of about 5.4.
[0066] In some embodiments, a crosslinking reaction mixture may
comprise hyaluronic acid at a concentration of about 12 mg/mL,
collagen at a concentration of about 6 mg/mL,
2-(N-morpholino)ethanesulfonic acid at a concentration of about 100
mM, sodium chloride at a concentration of about 0.9 wt % or about
150 mM, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a
concentration of about 50 mM, and N-hydroxysulfosuccinimide sodium
salt at a concentration of about 50 mM, wherein the solution has a
pH of about 5.4.
[0067] In some embodiments, a crosslinking reaction mixture may
comprise hyaluronic acid at a concentration of about 24 mg/mL,
collagen at a concentration of about 12 mg/mL,
2-(N-morpholino)ethanesulfonic acid at a concentration of about 100
mM, sodium chloride at a concentration of about 0.9 wt % or about
150 mM, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a
concentration of about 50 mM, and N-hydroxysulfosuccinimide sodium
salt at a concentration of about 50 mM, wherein the solution has a
pH of about 5.4.
[0068] In some embodiments, a crosslinking reaction mixture may
comprise hyaluronic acid at a concentration of about 1 mg/mL to
about 20 mg/mL, collagen at a concentration of about 1 mg/mL to
about 15 mg/mL, 2-(N-morpholino)ethanesulfonic acid at a
concentration of about 20 mM to about 200 mM, sodium chloride at a
concentration of about 0.5 wt % to about 2 wt % or about 80 mM to
about 330 mM, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at a
concentration of about 20 mM to about 100 mM, and
N-hydroxysulfosuccinimide sodium salt at a concentration of about
20 mM to about 100 mM, wherein the solution has a pH of about 4 to
about 6.
Example 1
Method of Making an Injectable Composition
[0069] Solutions of hyaluronic acid (HA) and collagen were produced
by dissolving 15 mg of 2.0 MDa hyaluronic acid in 5 mL of human
collagen(III) solution at 3 mg/mL in 0.01 N hydrochloric acid
(Fibrogen). The hyaluronic acid/collagen solution was then
lyophilized at -50.degree. C. and 0.02 Torr. The resulting sponges
were soaked in 20 mL of ethanol:water mixture at ratios varying
from 1:2 to 5:1 with 50 mM of
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and 50 mM of
N-hydroxysulfosuccinimide sodium salt for 24 hrs. The crosslinked
gels were then washed in 70% isopropanol/30% water for
sterilization followed by five washes in sterile phosphate buffer
for purification.
Example 2
Method of Making an Injectable Composition
[0070] A solution of HA at 3.4 mg/mL was created by dissolving 34
mg of 2 MDa HA in 10 mL of 100 mM MES buffer with 0.9 wt % NaCl, pH
4.7. Upon full hydration and dissolution of the HA, this solution
was mixed with 10 mL of 3.4 mg/mL human collagen(III) solution in
100 mM HCl. The pH of the resulting HA/collagen(III) solution was
adjusted to 5.4 with 10 mM NaOH solution. EDC (192 mg) and 217 mg
of sulfoNHS (50 mM each) were added to the HA/collagen(III)
solution and mixed thoroughly. The crosslinking reaction proceeded
for 18 hrs before the gel was particulated through a 100 micron
pore-sized mesh.
Example 3
Method of Making an Injectable Composition
[0071] Rat tail collagen(I) in 0.01 N hydrochloric acid
(Invitrogen) was concentrated from 5 mg/mL to 8 mg/mL using a
centrifugal filtration device with 20 kDa molecular weight cutoff.
HA (160 mg, 2 MDa) was added to 10 mL of the collagen solution and
allowed to hydrate for 60 minutes. The solution was then
homogenized by passing from syringe to syringe through a leur-leur
connector. NaCl (93 mg) and 201 mg of MES were added to the
solution and mixed. EDC (98 mg) and 111 mg of sulfoNHS were added
to the solution and quickly mixed. Finally, 200 .mu.L of 1 N NaOH
was added to the solution which was mixed by syringe-to-syringe
passing. The reaction solution was transferred to a glass vial and
centrifuged for 5 min at 4000 RPM to remove air bubbles. The gel
was then particulated through a 60 micron pore-sized mesh.
Following sizing, the gel was sterilized by dialysis through a 20
kDa molecular-weight cut-off cellulose ester membrane against 70%
isopropanol/30% water for 3 hrs at 4.degree. C. Dialysis was then
continued against sterile phosphate buffer for 48 hrs at 4.degree.
C. with three changes of buffer. The gel was then dispensed into
syringes under aseptic conditions.
Example 4
Method of Making an Injectable Composition
[0072] Rat tail collagen(I) in 0.01 N hydrochloric acid
(Invitrogen) was concentrated from 5 mg/mL to 12 mg/mL using a
centrifugal filtration device with 20 kDa molecular weight cutoff.
HA (120 mg, 2 MDa) was added to 10 mL of the collagen solution and
allowed to hydrate for 60 minutes. The solution was then
homogenized by passing from syringe to syringe through a leur-leur
connector. NaCl (93 mg) and 201 mg of MES were added to the
solution and mixed. EDC (98 mg) and 111 mg of sulfoNHS were added
to the solution and quickly mixed. Finally, 200 .mu.L of 1 N NaOH
was added to the solution which was mixed by syringe-to-syringe
passing. The reaction solution was transferred to a glass vial and
centrifuged for 5 min at 4000 RPM to remove air bubbles. The gel
was then particulated through a 60 micron pore-sized mesh.
Following sizing, the gel was sterilized by dialysis through a 20
kDa molecular-weight cut-off cellulose ester membrane against 70%
isopropanol/30% water for 3 hrs at 4.degree. C. Dialysis was then
continued against sterile phosphate buffer for 48 hrs at 4.degree.
C. with three changes of buffer. The gel was then dispensed into
syringes under aseptic conditions.
Example 5
Method of Making an Injectable Composition
[0073] Rat tail collagen(I) in 0.01 N hydrochloric acid
(Invitrogen) was concentrated from 5 mg/mL to 12 mg/mL using a
centrifugal filtration device with 20 kDa molecular weight cutoff.
HA (120 mg, 2 MDa) was added to 10 mL of the collagen solution and
allowed to hydrate for 60 minutes. The solution was then
homogenized by passing from syringe to syringe through a leur-leur
connector. NaCl (93 mg), 201 mg of MES, and 200 .mu.L of 1 N NaOH
were added to the solution, mixed, and given 45 minutes for
collagen polymerization. EDC (98 mg) and 111 mg of sulfoNHS were
then added and the final solution was mixed by syringe-to-syringe
passing. The reaction solution was transferred to a glass vial and
centrifuged for 5 min at 4000 RPM to remove air bubbles. The gel
was then particulated through a 60 micron pore-sized mesh.
Following sizing, the gel was sterilized by dialysis through a 20
kDa molecular-weight cut-off cellulose ester membrane against 70%
isopropanol/30% water for 3 hrs at 4.degree. C. Dialysis was then
continued against sterile phosphate buffer for 48 hrs at 4.degree.
C. with three changes of buffer. The gel was then dispensed into
syringes under aseptic conditions.
Example 6
Rheology Characterization of the Compositions
[0074] Oscillatory parallel plate rheology was used to characterize
the mechanical properties of gels using an Anton Paar MCR 301. A
plate diameter of 25 mm was used at a gap height of 1 mm. A
frequency sweep from 0.1 to 10 Hz at a fixed strain of 2% with
logarithmic increase in frequency was applied followed by a strain
sweep between 0.1% and 300% at a fixed frequency of 5 Hz with
logarithmic increase in strain. The storage modulus (G') and loss
modulus (G'') were determined from frequency sweep measurements at
5 Hz.
[0075] The gel from Example 4 had a storage modulus (G') of 556 Pa
and loss modulus (G'') of 131 Pa. The frequency sweep (A) and
strain sweep (B) are shown in FIG. 1.
Example 7
Extrusion Test
[0076] In order to determine the force required to extrude the
gels, they were ejected from 1 mL BD syringes through 30G needles
using an Instron 5564 with Bluehill 2 software. The plunger was
pushed at a rate of 100 mm/min for 11.35 mm and the extrusion
profile was recorded.
[0077] The extrusion profile through a 30 G needle for gel from
Example 4 is shown in FIG. 2. The gel had an average extrusion
force of 25 N from 4 through 10 mm.
Example 8
Method of Making Dermal Fillers
[0078] Hyaluronic acid, 2 MDa molecular weight, was dissolved in
human collagen(I) solution in 0.01 N hydrochloric acid (Advanced
BioMatrix). Sodium chloride was added at 0.9 wt % and
2-(N-morpholino)ethanesulfonic acid was added at 100 mM to the
solution and mixed. The hyaluronic acid was allowed to hydrate for
1 hr and the solution was homogenized by syringe-to-syringe mixing.
The pH of the solution was adjusted to 5.4 by addition of 1 N
sodium hydroxide. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (50
mM) and N-hydroxysulfosuccinimide sodium salt (50 mM) were added to
the hyaluronic acid/collagen solution and quickly mixed by
syringe-to-syringe transfer. The solution was transferred to a
glass vial and centrifuged for 5 min at 4000 RPM to remove air
bubbles. The resulting gel was allowed to react for 16 hrs at
4.degree. C. The gel was then particulated through a 100 micron
pore-sized mesh. Following sizing, the gel was sterilized by
dialysis through a 20 kDa molecular-weight cut-off cellulose ester
membrane against 70% isopropanol/30% water for 3 hrs at 4.degree.
C. Dialysis was then continued against sterile phosphate buffer, pH
7.4, for 48 hrs at 4.degree. C. with four changes of buffer. The
gel was then dispensed into syringes under aseptic conditions.
[0079] This procedure was used to produce hydrogels with varying
concentrations of hyaluronic acid and collagen. When required,
human collagen(I) in 0.01 N hydrochloric acid was concentrated from
3 mg/mL to the desired reaction concentration in 20 kDa
molecular-weight cut-off centrifugal filtration devices. A 50 mL
sample of each gel was synthesized, sterilized by exposure to 70%
isopropanol, and purified by dialysis against phosphate buffer, pH
7.4. The gels synthesized are described in Table 2 along with their
rheological properties.
TABLE-US-00001 TABLE 2 Hyaluronic acid-human collagen(I) hydrogel
synthesis concentrations and rheological properties Sample [HA]
[Col(I)] G' G'' ID (mg/mL) (mg/mL) (Pa) (Pa) A 3 3 199 24.6 B 12 6
1260 154 C 16 8 2450 288 D 12 12 3160 420 E 24 12 5440 433 F 12 3
1110 52.2 G 16 3 1490 60.6 H 20 3 1770 49.5
Example 9
Biopolymer Concentration of the Dermal Fillers
[0080] In order to determine the biopolymer concentration in gels,
the weight of the hydrated gel was compared to that of dried gel. A
2 mL sample of gel was weighed and dried by flash-freezing in
liquid nitrogen followed by lyophilization at -50.degree. C. and
0.02 Torr. A solution of the appropriate buffer was also weighed
and dried in the same fashion to account for salt content of the
gel. The total solids content of the gel was calculated by dividing
the dry weight by the wet volume, assuming 1 g/mL density for the
wet gel, to give a value in mg/mL. The salt solids content was then
subtracted from this value to determine the biopolymer
concentration in the gel.
TABLE-US-00002 TABLE 3 Final concentrations of hyaluronic
acid-human collagen(I) hydrogels Final Sample [Col(I)]
concentration ID [HA] (mg/mL) (mg/mL) (mg/mL) A 3 3 5.3 B 12 6 16.3
C 16 8 19.4 D 12 12 22.6 E 24 12 31.6
Example 10
Swelling Ratios
[0081] Swelling ratios relative to initial water content were
determined for gels by increase in weight when equilibrated with
phosphate buffer. For each gel, approximately 1 mL was injected
into a 15 mL Falcon tube and weighed, followed by addition of 10 mL
of phosphate buffered saline, pH 7.4. The gels were thoroughly
mixed with the buffer and vortexed for 30 seconds. The gels were
then allowed to equilibrate in the buffer for 48 hrs at 4.degree.
C. After this time, the suspensions were centrifuged at 4000 RPM in
a swinging bucket rotor for 5 minutes. The supernatant buffer was
then decanted and the weight of the swollen gel was measured. The
swelling ratio was determined by dividing the final weight of the
swollen gel by the weight of the initial gel.
TABLE-US-00003 TABLE 4 Swelling ratios of hyaluronic acid-human
collagen(I) hydrogels Sample [HA] [Col(I)] Swelling ID (mg/mL)
(mg/mL) ratio A 3 3 0.96 B 12 6 1.67 C 16 8 1.69 D 12 12 1.49 E 24
12 1.65
Example 11
HA/Collagen for Facial Defects of Check
[0082] This example illustrates the use of compositions and methods
disclosed herein for a facial disorder.
[0083] A 58-year-old woman presented with a lean face. She felt her
face looked old, sad and bitter because of the less fullness of her
cheek contour. Pre-operative evaluation of the person includes
routine history and physical examination in addition to thorough
informed consent disclosing all relevant risks and benefits of the
procedure. The physician evaluating the individual determines that
she is a candidate for administration of the dermal filler
compositions and methods disclosed herein.
[0084] A composition of the invention, such as described in EXAMPLE
4, is provided in a 20 mL syringe. One-holed blunt infiltration
cannulas (3 mm inner diameter) are used to place about 15 mL of the
composition in the syringe subcutaneously and under superficial
musculoaponeurotix system into the left and right checks.
[0085] The individual is monitored for approximately 7 days. The
physician evaluates the treatment area and determines that the
treatment was successful. The woman's cheeks are fuller than prior
to treatment, Both the woman and her physician are satisfied with
the results of the procedure because she looks younger than she did
when she came in for treatment.
Example 12
Treatment of Facial Defects of Eyelids
[0086] This example illustrates the use of compositions and methods
disclosed herein for a treatment of eyelid defects.
[0087] A 37-year-old woman presented with fine wrinkles around her
eyes and she reports that her eyes made her look old and angry.
Pre-operative evaluation of the person includes routine history and
physical examination in addition to thorough informed consent
disclosing all relevant risks and benefits of the procedure. The
physician evaluating the individual determines that she is a
candidate for administration of the dermal filler compositions and
methods disclosed herein.
[0088] A composition, such as made as described in Example 5, is
provided in a 20 mL syringe. About 2.5 mL of the composition is
injected with a fine needle subcutaneously in the skin beneath the
wrinkles into the regions adjacent the eyes.
[0089] The individual is monitored for approximately 7 days. The
physician evaluates the eye of the patient and determines that the
treatment was successful. Both the woman and her physician are
satisfied with the results of the procedure because her eyes appear
refreshed and the skin appears rejuvenated. Approximately one year
after the procedure, the woman indicates that her quality of life
has improved.
Example 13
Treatment of Acne Scars
[0090] This example illustrates the use of compositions and methods
disclosed herein for treatment of acne scars.
[0091] A 25-year-old man presents with moderate acne scarring on
his jaw line including depressions and pitting. He reports that he
is dissatisfied with his appearance and feels he is socially
inhibited due to his perception of his appearance. Pre-operative
evaluation of the person includes routine history and physical
examination in addition to thorough informed consent disclosing all
relevant risks and benefits of the procedure. The physician
evaluating the individual determines that he is a candidate for
administration of the dermal filler compositions and methods
disclosed herein.
[0092] A composition, such as that made as described in Example 12,
is provided in 10 mL syringes. The physician injects a small amount
of the composition below the skin in each depressed or pitted area
of the patient's jawline to raise the area to match the surrounding
skin.
[0093] The individual returns for a follow up visit with the
physician in 14 days. The physician evaluates the patient and
determines that the treatment was successful. The man reports he is
satisfied with the results of the procedure because his skin is
more smooth in appearance and the acne scarring is substantially
less visible. Approximately six months after the procedure, the man
returns for a follow up treatment. He reports to the physician that
his quality of life has greatly improved since the procedure and he
is no longer shy about his appearance.
[0094] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as molecular weight,
reaction conditions, and so forth used in the specification and
claims are to be understood as being modified in all instances by
the term "about." Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the specification and
attached claims are approximations that may vary depending upon the
desired properties sought to be obtained. At the very least, and
not as an attempt to limit the application of the doctrine of
equivalents to the scope of the claims, each numerical parameter
should at least be construed in light of the number of reported
significant digits and by applying ordinary rounding
techniques.
[0095] The terms "a," "an," "the," and similar referents used in
the context of describing the invention (especially in the context
of the following claims) are to be construed to cover both the
singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. All methods described herein can
be performed in any suitable order unless otherwise indicated
herein or otherwise clearly contradicted by context. The use of any
and all examples, or exemplary language (e.g., "such as") provided
herein is intended merely to better illuminate the invention and
does not pose a limitation on the scope of any claim. No language
in the specification should be construed as indicating any
non-claimed element essential to the practice of the invention.
[0096] Groupings of alternative elements or embodiments disclosed
herein are not to be construed as limitations. Each group member
may be referred to and claimed individually or in any combination
with other members of the group or other elements found herein. It
is anticipated that one or more members of a group may be included
in, or deleted from, a group for reasons of convenience and/or
patentability. When any such inclusion or deletion occurs, the
specification is deemed to contain the group as modified thus
fulfilling the written description of all Markush groups used in
the appended claims.
[0097] Certain embodiments are described herein, including the best
mode known to the inventors for carrying out the invention. Of
course, variations on these described embodiments will become
apparent to those of ordinary skill in the art upon reading the
foregoing description. The inventor expects skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than specifically described
herein. Accordingly, the claims include all modifications and
equivalents of the subject matter recited in the claims as
permitted by applicable law. Moreover, any combination of the
above-described elements in all possible variations thereof is
contemplated unless otherwise indicated herein or otherwise clearly
contradicted by context.
[0098] In closing, it is to be understood that the embodiments
disclosed herein are illustrative of the principles of the claims.
Other modifications that may be employed are within the scope of
the claims. Thus, by way of example, but not of limitation,
alternative embodiments may be utilized in accordance with the
teachings herein. Accordingly, the claims are not limited to
embodiments precisely as shown and described.
* * * * *