U.S. patent application number 12/648960 was filed with the patent office on 2010-04-22 for cross-linked collagen and uses thereof.
This patent application is currently assigned to ALLERGAN, INC.. Invention is credited to Gregory S. Dapper, Kenneth C. Olson, Jacqueline A. Schroeder.
Application Number | 20100099623 12/648960 |
Document ID | / |
Family ID | 40039895 |
Filed Date | 2010-04-22 |
United States Patent
Application |
20100099623 |
Kind Code |
A1 |
Schroeder; Jacqueline A. ;
et al. |
April 22, 2010 |
Cross-Linked Collagen and Uses Thereof
Abstract
The present invention discloses collagen cross-linked in a micro
to non-fibrillar form and at a high concentration. The cross-linked
collagen gel has improved volume stability or persistence than
collagen cross-linked at a neutral pH. Also disclosed are methods
for preparing the inventive cross-linked collagen and using such
for augmenting soft tissues in mammals.
Inventors: |
Schroeder; Jacqueline A.;
(Boulder Creek, CA) ; Dapper; Gregory S.; (Newark,
CA) ; Olson; Kenneth C.; (Foster City, CA) |
Correspondence
Address: |
ALLERGAN, INC.
2525 DUPONT DRIVE, T2-7H
IRVINE
CA
92612-1599
US
|
Assignee: |
ALLERGAN, INC.
Irvine
CA
|
Family ID: |
40039895 |
Appl. No.: |
12/648960 |
Filed: |
December 29, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12125337 |
May 22, 2008 |
|
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12648960 |
|
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60939664 |
May 23, 2007 |
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Current U.S.
Class: |
514/17.2 ;
530/356 |
Current CPC
Class: |
C07K 14/78 20130101;
A61P 17/00 20180101 |
Class at
Publication: |
514/12 ;
530/356 |
International
Class: |
A61K 8/65 20060101
A61K008/65; C07K 14/78 20060101 C07K014/78; A61P 43/00 20060101
A61P043/00 |
Claims
1. Cross-linked collagen prepared according to a method comprising
the steps of: obtaining micro to non-fibrillar collagen; treating
the micro to non-fibrillar collagen with a cross-linking agent; and
isolating cross-linked collagen.
2. The cross-linked collagen of claim 1, wherein the number of free
hydroxy lysine and lysine residues per 1000 amino acid residues in
the cross-linked collagen is in the range of 22-32.
3. The cross-linked collagen of claim 2, wherein the number of free
hydroxy lysine and lysine residues per 1000 amino acid residues in
the cross-linked collagen is in the range of 24-29.
4. The cross-linked collagen of claim 1, wherein the cross-linked
collagen is in a gel but not fibrous state.
5. The cross-linked collagen of claim 4, wherein the cross-linked
collagen locks water in the gel and does not disperse like a
fibrous collagen suspension.
6. The cross-linked collagen of claim 4, wherein the cross-linked
collagen in the gel state maintains its shape in vivo better than
cross-linked collagen in a fibrous state.
7. The cross-linked collagen of claim 1, wherein the fibers of the
cross-linked collagen are smaller than those of fibrous collagen
cross-linked at a neutral pH.
8. The cross-linked collagen of claim 1 wherein the micro to
non-fibrillar collagen is obtained by incubating fibrillar collagen
in a suspension or solution of pH 2-5 or pH 9-12.
9. The cross-linked collagen of claim 8, wherein the micro to
non-fibrillar collagen is obtained by incubating the fibrillar
collagen in a suspension or solution of pH 4.2-5.0.
10. The cross-linked collagen of claim 1, wherein the concentration
of the micro to non-fibrillar collagen is in the range of 3-150
mg/mL.
11. The cross-linked collagen of claim 10, wherein the
concentration of the micro to non-fibrillar collagen is in the
range of 38-52 mg/mL.
12. The cross-linked collagen of claim 1, wherein the treating step
includes treating the micro to non-fibrillar collagen with the
cross-linking agent at pH 2-5 or pH 9-12, followed by treating the
micro to non-fibrillar collagen with the cross-linking agent at pH
6-8.
13. The cross-linked collagen of claim 1, wherein the cross-linked
collagen derives from type I, II, III, IV or V collagen, or a
combination thereof.
14. The cross-linked collagen of claim 1, wherein the cross-linked
collagen derives from telo-containing collagen, atelo-collagen or
derivatized collagen, or a combination thereof.
15. The cross-linked collagen of claim 1, wherein the cross-linking
agent is capable of forming covalent bonds between amino acid
residues in the micro to non-fibrillar collagen.
16. The cross-linked collagen of claim 1, wherein the cross-linking
agent is selected from the group consisting of carbodiimides,
polyaldehydes, polysulfones, activated PEGs, epoxides, imidazoles
and diisocyanates.
17. The cross-linked collagen of claim 10, wherein the
cross-linking agent is glutaraldehyde.
18. A dermal filler composition comprising: a cross-linked collagen
prepared according to a method comprising the steps of obtaining
micro to non-fibrillar collagen, treating the micro to
non-fibrillar collagen with a cross-linking agent and isolating
cross-linked collagen; and a local anesthetic agent admixed with
the cross-linked collagen.
19. The composition of claim 18, wherein the local anesthetic agent
is lidocaine.
20. Cross-linked collagen comprising: micro to non-fibrillar
collagen; and a cross-linking agent, wherein the micro to
non-fibrillar collagen is cross-linked by the cross-linking
agent.
21. The cross-linked collagen of claim 20, wherein the cross-linked
collagen derives from type I, II, III, IV or V collagen, or a
combination thereof.
22. The cross-linked collagen of claim 20, wherein the cross-linked
collagen derives from telo-containing collagen, atelo-collagen or
derivatized collagen, or a combination thereof.
23. The cross-linked collagen of claim 20, wherein the
cross-linking agent is capable of forming covalent bonds between
amino acid residues in the micro to non-fibrillar collagen.
24. The cross-linked collagen of claim 20, wherein the
cross-linking agent is selected from the group consisting of
carbodiimides, polyaldehydes, polysulfones, activated PEGs,
epoxides, imidazoles and diisocyanates.
25. The cross-linked collagen of claim 20, wherein the
cross-linking agent is glutaraldehyde.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/125,337, filed on May 22, 2008 which claims the benefit
of 35 U.S.C. .sctn.120 to U.S. Provisional Patent Application No.
60/939,664 filed on May 23, 2007, the disclosure of each of which
is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates in general to body treating
compositions and methods. More specifically, the invention provides
a cross-linked collagen implant of improved volume stability
("persistence") for augmenting soft tissue in mammals.
BACKGROUND OF THE INVENTION
[0003] Collagen has been used as a pharmaceutical carrier, as a
surgical prosthesis (sutures and wound dressings), and as an
implant material. In many instances, the collagen is cross-linked
with chemical agents, radiation, or other means to improve its
mechanical properties, decrease its immunogenicity, and/or increase
its resistance to resorption. For example, U.S. Pat. No. 4,424,208
describes a collagen composition including cross-linked collagen
and reconstituted collagen fibers having enhanced persistence.
While these materials are remarkably effective, they shrink in
volume after implantation due primarily to absorption of their
fluid component by the body, although the shrinkage (syneresis) is
less than non-cross-linked collagen. Since a constant volume or
persistence is desirable, an additional injection or injections of
supplemental implant material is required. It would thus be
advantageous to provide collagen compositions having enhanced
persistence after being introduced in vivo to a soft tissue
treatment site.
SUMMARY OF THE INVENTION
[0004] The present invention relates to collagen cross-linked in a
micro to non-fibrillar form and at a high concentration for
augmenting soft tissue in mammals. The cross-linked collagen of the
present invention has improved volume stability or persistence
compared with collagen cross-linked at a neutral pH.
[0005] In one aspect, the invention features a method for preparing
cross-linked collagen. The method involves the steps of obtaining
micro to non-fibrillar collagen, treating the micro to
non-fibrillar collagen with a cross-linking agent, and isolating
cross-linked collagen. The micro to non-fibrillar collagen may be
obtained by incubating fibrillar collagen in a suspension or
solution of pH 2-5 or pH 9-12. In a preferred embodiment, the pH of
the suspension or solution is between 4.2 and 5.0. The
concentration of the micro to non-fibrillar collagen at the time of
cross-linking is in the range of 3-150 mg/mL, and preferably, 38-52
mg/mL. Typically, the treating step includes treating the micro to
non-fibrillar collagen with the cross-linking agent at pH 2-5 or pH
9-12, followed by treating the micro to non-fibrillar collagen with
the cross-linking agent at pH 6-8 to encourage completion of the
cross-linking. The method of the invention optionally may further
include a step of admixing a local anesthetic agent (e.g.,
lidocaine) with the cross-linked collagen. Cross-linked collagen so
prepared is within the invention.
[0006] Also provided in the present invention is cross-linked
collagen comprising micro to non-fibrillar collagen and a
cross-linking agent. The micro to non-fibrillar collagen is
cross-linked by the cross-linking agent. Such cross-linked collagen
may be prepared according to the method described above.
[0007] The cross-linked collagen may derive from type I, II, III,
IV, or V collagen, or a combination thereof. It may also derive
from telo-containing collagen, atelo-collagen, or derivatized
collagen, or a combination thereof.
[0008] Preferably, the cross-linking agent is capable of forming
covalent bonds between amino acid residues in the micro to
non-fibrillar collagen. Examples of suitable cross-linking agents
include, but are not limited to, carbodiimides, polyaldehydes,
polysulfones, activated PEGs, epoxides, imidazoles, and
diisocyanates. In one embodiment, the cross-linking agent is
glutaraldehyde.
[0009] In some embodiments of the cross-linked type I collagen, the
number of free hydroxy lysine and lysine residues per 1000 amino
acid residues is in the range of 22-32, and more typically, in the
range of 24-29.
[0010] The cross-linked collagen of this invention is in a gel but
not fibrous state. It locks water in the gel and does not disperse
like a fibrous collagen suspension. The cross-linked collagen in a
gel state maintains its shape in vivo better than cross-linked
collagen in a fibrous state. The fibers of the cross-linked
collagen are smaller than those of fibrous collagen cross-linked at
a neutral pH.
[0011] The invention further provides a composition containing the
cross-linked collagen of the invention and a local anesthetic agent
such as lidocaine. The local anesthetic agent is admixed with the
cross-linked collagen. In addition, the invention provides a
packaged product containing a syringe fitted with a needle, wherein
the syringe is loaded with the cross-linked collagen of the
invention.
[0012] In another aspect, the invention features a method for
filling voids and defects and increasing tissue volume in a mammal.
The method involves administering to a mammal the cross-linked
collagen of the invention. Preferably, the cross-linked collagen is
administered by intradermal or subcutaneous injection.
[0013] As used herein, "micro to non-fibrillar collagen" refers to
collagen with a diameter of 5-70 nm; "fibrillar collagen" refers to
collagen with a diameter of >70 nm; "fibrous collagen" refers to
fibrillar collagen and larger fibers. "Telo-containing collagen"
refers to collagen with intact telo peptide; "atelo-collagen"
refers to collagen wherein the telo portions are removed partially
or totally; "derivatized collagen" refers to chemically modified
collagen. Examples of derivatized collagen include, but are not
limited to, deamidated, methylated, succinylated, and
phosphorylated collagen. Cross-linked collagen in a "gel state"
contains micro to non-fibrillar collagen with a fiber diameter
range of 5-70 nm; cross-linked collagen in a "fibrous state"
contains fibrillar collagen with a fiber diameter of greater than
70 nm.
[0014] As used herein, "free hydroxyl-lysine and lysine" in
collagen refers to unmodified hydroxyl-lysine and lysine; "neutral
pH" refers to pH 6-8.
[0015] The present invention provides a cross-linked collagen
filler for augmenting and filling soft tissue defects and voids
with a material that plumps and bulks the soft tissue. The
cross-linking collagen of the invention is particularly useful for
deep dermal correction and sculpting. The superior shape retention
makes it ideal for areas that are hard to correct and where a
biocompatible bolus can provide mechanical strength to the
body.
[0016] The above-mentioned and other features of this invention and
the manner of obtaining and using them will become more apparent,
and will be best understood, by reference to the following
description, taken in conjunction with the accompanying drawings.
The drawings depict only typical embodiments of the invention and
do not therefore limit its scope.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 is a flow chart illustrating representative
conventional and inventive processes for preparation of
cross-linked collagen.
[0018] FIG. 2 shows the persistence of the inventive cross-linked
collagen relative to the conventional cross-linked collagen.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention is based, at least in part, on the
unexpected discovery that collagen cross-linked in a micro to
non-fibrillar state and preferably at a high concentration has
improved persistence compared to collagen cross-linked at a neutral
pH.
[0020] More specifically, fibrillar collagen is fairly porous and
allows the transport of cells and large molecules. By cross-linking
smaller fiber (microfibrillar) collagen, more rigidity within the
network was created to further increase the diffusion hindrance
(Rosenblatt and Shenoy, 1993, Chain Rigidity and Diffusional
Release in Biopolymer Gels, Proceed Intern Symp Control Rel Bioact
Mater 20, Controlled Release Society, Inc.). While fibrillar
collagen matrices are capable of moderating the diffusion of large
molecules, smaller fiber and non-fibrillar collagen can modulate
the diffusion of smaller molecules (Rosenblatt et al., 1989, The
Effect of Collagen Fiber Size Distribution on the Release Rate of
Protein from Collagen Matrices by Diffusion, J Controlled Release
9:195-203). Cross-linked micro-fibrillar collagen has a tighter
network or mesh, which creates a collagen matrix more resistant or
persistent to biological degradation. In the present invention, as
described in the Example below, type I collagen matrices were
modified to reduce the penetration of cells and proteins into the
matrices.
[0021] The present invention is particularly directed to the
filling of voids and defects and increasing tissue volume in
mammals with injectable cross-linked Type I/III collagen implants.
Accordingly, the invention provides a method for preparing
cross-linked collagen by cross-linking collagen in a micro to
non-fibrillar state and preferably at a high concentration.
[0022] The cross-linked collagen of the present invention primarily
derives from mammalian source materials, such as bovine or porcine
corium, although human placenta material, collagen produced from
human fibroblast cell culture, or recombinantly produced collagen
expressed from a cell line may also be used. The donor need not be
genetically similar to the host into which the material is
ultimately implanted.
[0023] Referring to FIG. 1, in a conventional process, purified,
type I, pepsin digested human collagen from Allergan Medical
Biomaterials (48490 Milmont Drive, Fremont, Calif. 94538) is
reconstituted from a solution by neutralizing the solution at
reduced temperatures and ionic strengths hypotonic relative to
physiological conditions. The pH of the solution is raised to a
level at which the collagen in solution reaggregates into fibrils.
The reconstituted fibrous collagen is cross-linked with a
cross-linking agent at a neutral pH. The cross-linked collagen is
then harvested by centrifugation, formulated/homogenized, smoothed,
and screened.
[0024] In contrast, the process of the present invention involves
concentrating the collagen suspension by centrifugation. The pellet
is homogenized, and the pH is adjusted to a non-neutral level prior
to cross-linking. The collagen concentration at the time of
cross-linking is usually in the range of 3-150 mg/mL, more
typically 30-60 mg/mL, 35-55 mg/mL, or 38-52 mg/mL. The pH is
adjusted to a non-neutral level appropriate for the cross-linking
agent to be used. For example, the pH may be adjusted to about 4.5
for cross-linking by glutaraldehyde or to about 10 for
cross-linking by divinylsulfone. A dilute solution of HCl or the
like is typically added to adjust the pH to a desired acidic level,
while NaOH or the like is used for adjusting the pH to a desired
alkaline level.
[0025] The cross-linking begins at pH 2-5 or pH 9-12, more
preferably at pH 2-3, 2-4, 3-4, 3-5, or 4-5, or pH 9-10, 9-11,
10-11, 10-12, or 11-12. At these pH ranges, the collagen fiber is
unraveled to a micro to non-fibrillar state. More lysine groups are
exposed and available for cross-linking. In addition, a high
collagen concentration increases the reaction rate. The collagen is
stabilized while being cross-linked into small fiber structures.
Preferably, after the initial cross-linking, the pH is further
adjusted to a neutral pH, e.g., pH 6-8, using 0.5 M phosphate, pH
11.2, or the like, to encourage complete cross-linking. The pH can
then be adjusted back to neutral without causing the cross-linked
collagen gel to collapse or to spontaneously form fibers.
[0026] Usually a cross-linking agent is polyfunctional, and more
usually bifunctional. The cross-linking conditions of the present
invention are such as to produce covalently cross-linked collagen
that has improved persistence relative to an implant of a
comparable formulation prepared according to the conventional
process. When this degree of cross-linking has been reached, the
cross-linking reaction is optionally quenched by adding a quenching
agent. The quenching agent forms a water soluble adduct with the
cross-linking agent. The concentration of the collagen in the
suspension at the time of cross-linking, the concentration of the
cross-linking agent, and the duration of the cross-linking reaction
are important process conditions for obtaining the kind and degree
of cross-linking that provides a product having enhanced
persistence.
[0027] The collagen can be cross-linked by any of a number of
conventional chemical cross-linking agents, including, but not
limited to, glutaraldehyde, divinylsulfone, epoxides,
carbodiimides, imidazole, N-hydroxy-succinimide (NHS), thiol
derivatized polyethylene glycol (PEG), and the like.
[0028] Aldehydes are preferred cross-linking agents. Examples of
aldehydes that may be used to cross-link collagen are formaldehyde,
acetaldehyde, glyoxal pyruvic aldehyde, and dialdehyde starch.
Glutaraldehyde is particularly preferred. Compounds that have
functional groups that react with the functional groups of the
cross-linking agent (e.g., aldehyde group) to form water soluble
adducts may be used to quench the cross-linking reaction. Quenching
agents that have free amino groups such as amino acids are
preferred. Glycine is particularly preferred. The concentration of
glutaraldehyde in the reaction mixture is typically about 0.001% to
about 0.05% by weight. The glutaraldehyde reacts with hydroxy
lysine and lysine residues of the collagen fibers, thereby reducing
the number of free hydroxy lysine and lysine in the collagen. At
the glutaraldehyde concentrations mentioned above, the number of
free hydroxy lysine and lysine residues per 1000 amino acid
residues after cross-linking is about 22-32, more typically about
24-29. Hydroxy lysine and lysine content may be measured by
reducing the cross-linked collagen with borohydride and hydrolyzing
the reduced material under vacuum in 5.7 N HCl for 24 hours at
100.degree. C. Amino acid analysis may be performed with available
analyzers (e.g., a Durrum Model D-500 analyzer) and the hydroxy
lysine and lysine residues quantified by comparing the hydroxy
lysine and lysine/alanine ratio to those observed in
non-cross-linked controls.
[0029] The duration of the cross-linking reaction is usually in the
range of one-half hour to about one week. The reaction is normally
carried out at about 10.degree. C. to about 35.degree. C. The
quenching agent is added in at least stoichiometric proportions
relative to the cross-linking agent.
[0030] A particularly preferred cross-linking protocol is about 38
to about 52 mg/mL collagen concentration, pH about 4.2 to about
5.0, and about 0.01% by weight glutaraldehyde for about 16 hours at
approximately 22.degree. C.
[0031] After the cross-linking reaction has been terminated, the
cross-linked collagen product may be washed with an aqueous buffer
solution to remove unreacted aldehyde, aldehyde polymers, and, if
quenching was employed, unreacted quenching agent, and
aldehyde-quenching agent adducts. A sodium phosphate-sodium
chloride buffer solution, pH 6.9 to 7.4, is preferred. The washed
product may be concentrated, such as by filtration or
centrifugation, to a suitable protein concentration range,
typically about 20 to about 65 mg/mL, more usually about 25 to
about 40 mg/mL. Protein concentration may be adjusted to this range
by addition of buffer or further concentration, as the case may be.
The washed product has a free aldehyde content below about 20
ppm.
[0032] Formulation of the cross-linked collagen typically involves
adjusting the ionic strength to isotonicity (i.e., about 0.15 to
about 0.2) and adding a local anesthetic, such as lidocaine, to a
concentration of about 0.3% by weight to reduce local pain upon
injection. A particularly preferred cross-linking product has a
collagen concentration of 30.0-37.0 mg/mL, a lidocaine
concentration of 2.7-3.3 mg/mL, and a pH of 7.0-7.6. The
cross-linked product is further homogenized, smoothed by
microfluidization, and screened by forcing the collagen fibers
through a screen of defined pore size.
[0033] The cross-linked collagen is then loaded into syringes
fitted with a #25 gauge or larger gauge needle for injection. In
the case of formulations used for dermal augmentation, the term
"injectable" means that the formulation can be dispensed from
syringes having a gauge as low as #25 under normal manual pressure
without substantial spiking.
[0034] The above described steps for preparing the inventive
cross-linked collagen are preferably carried out in sterile
conditions using sterile materials.
[0035] The cross-linked collagen of the present invention may be
injected intradermally or subcutaneously to augment soft tissue, to
repair or correct congenital anomalies, acquired defects or
cosmetic defects. Examples of such conditions are 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, enophthalmos 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.
[0036] In particular, the invention provides a soft tissue
augmentation injectable that fills the space with a durable strong
biocompatible bulking agent. Compared to the conventional
cross-linked collagen, the collagen fiber of the present invention
is reduced in size and forms a network that takes up more space.
The inventive cross-linked collagen is elastic and resilient. It
keeps its shape over time and resists breakdown and cellular
infiltration.
[0037] The following example is intended to illustrate, but not to
limit, the scope of the invention. While such example is typical of
those that might be used, other procedures known to those skilled
in the art may alternatively be utilized. Indeed, those of ordinary
skill in the art can readily envision and produce further
embodiments, based on the teachings herein, without undue
experimentation.
Example
Preparation of Inventive Cross-Linked Collagen
[0038] Purified, type I, pepsin digested human collagen from
Allergan Medical Biomaterials (48490 Milmont Drive, Fremont, Calif.
94538) was precipitated by raising the pH to 7.0-7.6 and then
centrifuging at 17000.times.g for 5-7 minutes. The supernatant was
aseptically decanted from the centrifuge bottle, and the collagen
pellet aseptically suctioned into a homogenization vessel. The
precipitated collagen was aseptically homogenized. The protein
concentration was 91.6 mg/mL.
[0039] 0.05 M HCl buffer and sterile filtered WFI (water for
injection) were mixed with the homogenate. The protein
concentration was 44.6 mg/mL; the pH was 4.8.
[0040] 3000 ppm glutaraldehyde buffer was mixed with the acid
homogenate. The mixture was allowed to incubate for 1.5 hours and
remixed, further incubated for 23 hours and remixed, and incubated
again for 72 hours and remixed. The protein concentration was 37.1
mg/mL; the pH was 4.6.
[0041] The cross-linked homogenate was mixed with 0.5 M sodium
phosphate buffer, pH 11.2, followed by 0.04 M sodium phosphate/2.6
M sodium chloride/60 mg/mL lidocaine buffer. The homogenate was
allowed to incubate for 24 hours and remixed. The protein
concentration was 31.9 mg/mL. The formulated homogenate was then
passed through a microfluidizer and screened.
Comparison of Conventional and Inventive Cross-Linked Collagen
[0042] The conventional cross-linked collagen was obtained by
precipitating and cross-linking purified, type I, pepsin digested
human collagen from Allergan Medical Biomaterials (48490 Milmont
Drive, Fremont, Calif. 94538) at about 3 mg/mL and a neutral pH.
The conventional cross-linked collagen was harvested by
centrifugation, and then homogenized, formulated, smoothed using
microfluidization and screened. The inventive cross-linked collagen
was obtained by precipitating purified, type I, pepsin digested
human collagen from Inamed Biomaterials (48490 Milmont Drive,
Fremont, Calif. 94538) at about 3 mg/mL at a neutral pH and
harvesting the collagen by centrifugation. The pH was reduced to
4.4-4.8, and the collagen was cross-linked. The inventive
cross-linked collagen was then homogenized, formulated, smoothed
using microfluidization, and screened.
[0043] The biocompatibility of the inventive cross-linked collagen
was tested and compared to that of the conventional cross-linked
collagen. Safety was assessed through a cytotoxicity study and
multiple rabbit subcutaneous implantation studies. The data
demonstrates that the inventive cross-linked collagen, like the
conventional cross-linked collagen, was biocompatible.
[0044] More specifically, the cytotoxicity study was performed
using the ISO Elution Method. The inventive cross-linked collagen
implants caused no cell lysis or toxicity (Table I).
TABLE-US-00001 TABLE I Cytotoxicity Study of the Conventional and
Inventive Cross-linked Collagen Implants Percent Cells Confluent
Percent without Intrascyto- Percent Monolayer Rounding plasmic
Granules Lysis Grade Reactivity Conventional 0 0 0 0 0 None
Inventive 0 0 0 0 0 None
[0045] The rabbit subcutaneous implantation assay was used to
compare tissue responses to the conventional and inventive
cross-linked collagen implants at several different time-points.
The tissue response to the inventive implant was similar to that
seen with the conventional implant (Table II). The microscopic
scores ranging from non-irritant to slight irritant are within the
range of acceptable variability and considered to be
satisfactory.
TABLE-US-00002 TABLE II Rabbit Implantation Evaluation Time Points
Conventional Inventive 1 week Non-irritant Non-irritant 4 weeks
Non-irritant Slight irritant 9 weeks Not tested Not tested 12 weeks
Non-irritant Slight irritant
[0046] A rat subcutaneous implantation study was performed to
compare the persistence of the inventive cross-linked collagen
implant relative to the conventional cross-linked collagen implant.
As part of the rat implantation study, a macroscopic evaluation of
the implant site was performed. There was no capsule formation or
adverse reaction for either implant for all time points studied
(Table III).
TABLE-US-00003 TABLE III Rat Subcutaneous Macroscopic Evaluation
Time Points Conventional Inventive 4 weeks No capsule formation No
capsule formation or adverse reaction or adverse reaction 9 weeks
No capsule formation No capsule formation or adverse reaction or
adverse reaction 13 weeks No capsule formation No capsule formation
or adverse reaction or adverse reaction 24 weeks No capsule
formation No capsule formation or adverse reaction or adverse
reaction
[0047] To assess effectiveness, the persistence of the inventive
relative to the conventional cross-linked collagen was evaluated
using wet weight recovery in conjunction with shape retention in
the rat subcutaneous model (McPherson et al., 1988, Development and
Biochemical Characterization of Injectable Collagen, J Dermatol
Surg Oncol 14, Suppl 1). Shape retention is considered to be good
measurement of the collagen implant's ability to maintain wrinkle
correction. If the implant cannot maintain its shape, it may not
correct a wrinkle effectively.
[0048] The data from the rat subcutaneous studies, summarized in
FIG. 2, indicates that the inventive implant, on average, had
greater wet weight recovery and maintained its height better than
the conventional implant after 24 weeks of implantation.
[0049] All patents and articles cited herein are incorporated by
reference in their entirety.
* * * * *