U.S. patent application number 16/166713 was filed with the patent office on 2019-02-21 for composition and method of preparation of protease microparticulate slow release preparation.
The applicant listed for this patent is Hyalo Technologies, LLC. Invention is credited to Shalabh JAIN.
Application Number | 20190054157 16/166713 |
Document ID | / |
Family ID | 60326386 |
Filed Date | 2019-02-21 |
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United States Patent
Application |
20190054157 |
Kind Code |
A1 |
JAIN; Shalabh |
February 21, 2019 |
COMPOSITION AND METHOD OF PREPARATION OF PROTEASE MICROPARTICULATE
SLOW RELEASE PREPARATION
Abstract
Compositions containing microparticles loaded with one or
protease enzymes and optionally auxiliary therapeutic agents and
methods of treating conditions such as keloids therewith are
disclosed. The biodegradable polymer and the protease enzyme
therein form a controlled release matrix for extended release of
the enzyme after administration to a mammal in need thereof.
Inventors: |
JAIN; Shalabh; (Mendham,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hyalo Technologies, LLC |
Somerset |
NJ |
US |
|
|
Family ID: |
60326386 |
Appl. No.: |
16/166713 |
Filed: |
October 22, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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15157847 |
May 18, 2016 |
10137179 |
|
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16166713 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Y 304/22002 20130101;
A61K 47/32 20130101; C12Y 304/24 20130101; A61K 9/19 20130101; A61K
38/482 20130101; A61K 9/0019 20130101; A61K 38/4886 20130101; A61K
9/1647 20130101; A61K 47/34 20130101; A61K 38/4873 20130101; A61K
9/06 20130101; A61K 31/573 20130101; A61K 9/0024 20130101; A61K
9/0014 20130101; A61P 17/02 20180101; C12Y 304/21 20130101; A61K
45/06 20130101; A61K 9/5031 20130101; A61K 47/26 20130101; A61K
47/38 20130101; A61K 31/573 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 38/48 20060101
A61K038/48; A61K 47/34 20170101 A61K047/34; A61K 31/573 20060101
A61K031/573 |
Claims
1. A composition, comprising a plurality of biodegradable polymer
microparticles comprising a protease enzyme therein, the
biodegradable polymer and the protease enzyme forming a controlled
release matrix for extended release of the enzyme; wherein the
plurality of microparticles include a mixture of microparticles
containing different proteases.
2. The composition of claim 1, wherein the protease is selected
from the group consisting of collagenase, papain, elastase and
mixtures thereof.
3. The composition of claim 1, wherein the biodegradable polymer
wherein the biodegradable polymer is selected from the group
consisting of polylactic acid (PLA), polylactic co-glycolic acid
(PLGA), polyglycolic acid (PGA) polylactones, polyorthocarbonate,
polyhydroxybutyrate, polyalkylcyanoacrylates, polyanhydrides,
polyorthoesters, polyester, polyimide, polyglycolides (PGA),
polyorthoester, polyacetates, polystyrene, polycarbonates,
polysaccharides, polycaprolactone, L-polylactides, block
co-polymers of polyesters and linear or star-polyethyleneglycol,
poly-beta-hydroxybutyrate, beta-hydroxyvalerate-copolymers,
polyaminoacids, hydrophobized hyaluronic acid, dextrans, starches,
methyl methacrylate, acrylamide, bisacrylamide, albumin, cellulose,
cellulose-based polymers, chitosan, collagen, gelatin, proteins,
Polyvinyl alcohol (PVA), polyvinylpyrrolidone, polyvinylpyridine,
and ethylene glycol polymers.
4. The composition of claim 3, wherein the biodegradable polymer is
polylactic co-glycolic acid or polylactic acid.
5. The composition of claim 4, wherein the biodegradable polymer is
poly lactide-poly glycolide polymer copolymer (PLGA).
6. The composition of claim 5, wherein the PLGA has a molecular
weight of from about 7,000 to about 100,000.
7. The composition of claim 1, wherein the microparticles have a
cross-sectional diameter of from about 10 nm to about 100
.mu.m.
8. The composition of claim 7, wherein the microparticles have a
cross-sectional diameter of from about 100 nm to about 50
.mu.m.
9. The composition of claim 8, wherein the microparticles have a
cross-sectional diameter of from about 1 .mu.m to about 20
.mu.m.
10. The composition of claim 1, wherein the percent loading of the
protease in the microparticles is from about 0.1 to about 5.0.
11. The composition of claim 10, wherein the protease is
collagenase and the percent loading of the protease in the
microparticles is from about 3 to about 5%.
12. The composition of claim 10, wherein the protease is elastase
and the percent loading of the protease in the microparticles is
from about 0.1 to about 0.6%.
13. The composition of claim 10, wherein the protease is papain and
the percent loading of the protease in the microparticles is from
about 0.5 to about 0.9.
14. A composition according to claim 2, comprising a first portion
of microparticles containing collagenase, and a second portion of
microparticles containing papain or elastase.
15. A composition according to claim 2, comprising a first portion
of microparticles containing collagenase, a second portion of
microparticles containing papain and a third portion of
microparticles containing elastase.
16. The composition of claim 1, further comprising an auxiliary
therapeutic agent dissolved or dispersed within the controlled
release matrix.
17. The composition of claim 16, wherein the auxiliary therapeutic
agent is co-mingled with the microparticles in the composition.
18. The composition of claim 16, wherein the auxiliary therapeutic
agent is a steroidal or a non-steroidal inflammation reducing
agent.
19. The composition of claim 18, wherein the steroidal inflammation
reducing agent is dexamethasone.
20. A method of treating hypertrophic scars (or tissue) in a
mammal, comprising administering an effective amount of a
composition of claim 1 to a mammal in need thereof.
21. The method of claim 20, wherein said hypertrophic scar is a
keloid.
22. The method of claim 20, wherein the administering is carried
out by injecting the composition to an area requiring said
treatment or by topically applying the composition to an area
requiring said treatment.
23. The method of claim 20, wherein the composition is administered
once a week, once a month or once every two months.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/157,847 filed on May 18, 2016 the contents of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] Scar formation after a surgical procedure or injury is
unpredictable and both physicians and patients are highly concerned
with minimizing scar appearance. In spite of availability of
various in vivo and in vitro studies, limited information is
available on the exact cause of scarring. Scaring can lead to the
formation of raised nodules called keloids. Scars can also be
raised and erythematous in which case they are called hypertrophic
scars. Other manifestations of scarring include the formation of
adhesions after surgery, frozen shoulder syndrome (from adhesive
capsulitis) and acne vulgaris.
[0003] Hypertrophic scar formation is often a result of an
overproduction and excess deposition of collagen by fibroblasts
caused by an increased or prolonged activity of TGF-.beta.1. The
surface of the keloid could be smooth, but in most of the cases, it
is observed that the keloids are nodular or ridged. This is due to
the presence of thickened and hyalinized collagen fibrils, mostly
type I and III, which are randomly oriented in case of Keloids.
Collagen synthesis in keloids is 3 times greater than in
hypertrophic scars and 20 times greater than in normal skin.
Adhesive capsulitis is characterized by collagenous tissue
associated with fibroblasts and myofibroblasts.
[0004] Current treatments for hypertrophic scars and keloids
include pressure therapy, silicone based products, radiation
therapy, corticosteroid application, cryosurgery and laser surgery.
All the above listed treatment options have to be continued over an
extended course of time and are expensive and some others pose
radiation risks. These treatment modalities are not a permanent
cure, and there is a high incidence of recurrence of keloids, with
the recurrence rate being reported from 45-100%.
[0005] Most therapeutic approaches remain clinically unsatisfactory
due to lack of understanding of the physiological processes
involved in wound healing and excessive scarring. Currently no
successful clinical treatment is available to healthcare providers
and patients for conditions with excessive production of collagen.
In addition, systemic side effects are high for current treatment
options.
[0006] Though a number of the treatment strategies have been tried,
none of the current treatments are long acting in duration. Use of
protease enzymes, including collagenase, papain and elastase as
solutions has been reported previously. These systems do not work
effectively because the protease activity is self-limiting and is
effective for a few hours to 1-2 days only. This is primarily due
to the fact that protease enzymes in solution are rapidly degraded
by their own activity since all of these enzymes are proteins and
hence capable of self-degradation. In addition, once a protease
enzyme is in solution form, it is subject to chemical degradation
from reactions such as hydrolysis which also results in rapid loss
of activity. When protease solutions are injected in the body, the
protease are quickly absorbed through the blood or lymph
capillaries and removed from the area of injection. This further
reduces the amount of protease available at the site of action.
Therefore, unless the composition is injected repeatedly over a
period of several weeks, the therapy does not work. Since the
treatment of scars requires a treatment lasting several weeks or
months, there exists a need for a treatment method that does not
involve repeated applications or injections of the medicament.
SUMMARY OF THE INVENTION
[0007] This invention is based on the discovery that when injected
or topically applied protease enzymes are released at a slow rate
over a period of time, the enzyme activity is sustained over
substantially the entire release time. In accordance therewith, the
compositions of the present invention have a release rate for the
protease that is high enough to maintain an effective activity of
the protease in the application area, yet slow enough to allow
continuous presence of protease for many days or weeks.
[0008] In one aspect of the invention, there are provided
compositions which include a plurality of biodegradable polymer
microparticles containing a protease enzyme dispersed or dissolved
therein. The biodegradable polymer and the protease enzyme thus
form a controlled release matrix for extended release of the
enzyme.
[0009] In a further aspect of the invention, there is provided an
injectable or topical composition of slow release protease that
includes the protease enzymes in a matrix which comprises a
biodegradable polymer. That is, the matrix includes the polymer in
which the protease and/or active medicament is either dispersed or
dissolved. In some embodiments, the final form of this composition
is a plurality of protease-containing microparticles which can be
injected or applied at the site of need.
[0010] Each microparticle includes the matrix containing the
polymer and the protease and/or active medicament. Once injected or
applied to the body, the body fluids around the site of injection
penetrate the polymer matrix and begin to degrade the matrix. This
results in a controlled release of the entrapped protease enzyme in
solution. Since the enzyme trapped in the matrix is in solid form,
it is protected from degradation until after it is released.
Therefore, a slow release form of protease will sustain its
activity at the site of injection or application for the duration
of its release. By using a suitable biodegradable polymer, a
composition can be fabricated that allows a continuous release of
the enzymes over a period of several days, weeks or months.
[0011] In another embodiment, there are provided methods of
treating hypertrophic scars or hypertrophic tissue in a mammal,
preferably a human. The methods include administering an effective
amount of the protease-containing microparticles described herein
to a mammal in need thereof. The inventive compositions are
administered preferably via injection at the site requiring the
therapeutic effect. Alternative aspects include topical
administration of the protease-containing compositions on the
affected areas.
[0012] Another aspect of the invention includes the simultaneous
use of more than one protease enzyme that works in a synergistic
manner to dissolve mammalian scar tissue. These enzymes can be
added to the same matrix or be formulated as different matrices and
mixed prior to use into a single composition. Suitable protease
enzymes can include collagenase, elastase, papain and mixtures
thereof.
[0013] A still further aspect of the invention is the inclusion of
a suitable anti-inflammatory medicament for the purpose of
discouraging the formation of additional collagen. One example of
this medicament is dexamethasone. This medicament can be included
in the microparticle form or as pure drug along with microparticles
of the protease enzymes in the composition administered to the
mammal in need thereof.
[0014] One of the major advantages of this system is its ability to
protect the protease enzymes and their activity during the
treatment period. This is accomplished by keeping a part of the
enzyme in a solid form away from the aqueous environment while
release effective concentration of the said enzyme at and around
the site of injection or application. The solid form of enzyme
protected by the biodegradable polymer is protected from the
degrading actions of the body as well as from clearance from the
site of application by absorption into circulatory system of blood
or lymphatic vessels.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows light microscopic picture of collagenase
microparticles prepared according to Example 1 and identified as
Composition 1 in Table 1.
[0016] FIG. 2 shows light microscopic picture of elastase
microparticles prepared according to Example 3 and identified as
Composition 9 in Table 1.
[0017] FIG. 3 shows light microscopic picture of papain
microparticles prepared according to Example 3 and identified as
Composition 5 in Table 1.
[0018] FIG. 4 shows light microscopic picture of dexamethasone
microparticles prepared according to Example 4.
[0019] FIG. 5 shows protease activity of papain in solution and
microparticle forms on pig tendon tissue as set forth in Example
11. The activity is expressed as the amount of soluble protein per
gram of tendon tissue as a function of time.
[0020] FIG. 6 shows protease activity of papain in solution and
microparticle forms on pig dermal collagen as set forth in Example
12.
[0021] FIG. 7 shows protease activity of collagenase in solution
and microparticle forms on pig tendon tissue as set forth in
Example 13.
[0022] FIG. 8 shows protease activity of elastase in solution and
microparticle forms on pig tendon tissue as set forth in Example
13.
[0023] FIG. 9 shows protease activity of collagenase microparticles
alone and a combination of collagenase microparticles and papain
microparticles on pig tendon tissue as set forth in Example 14.
[0024] FIG. 10 shows protease activity of collagenase
microparticles alone and a combination of collagenase
microparticles and papain microparticles on bovine collagen tissue
as set forth in Example 14.
[0025] FIG. 11 shows protease activity of elastase microparticles
alone and in combination with other protease containing
microparticles on pig tendon tissue as set forth in Example 15.
[0026] FIG. 12 shows protease activity of elastase microparticle
alone and combination with other protease containing microparticles
on pig dermal tissue as set forth in Example 15.
[0027] FIG. 13 shows protease activity of papain microparticle
alone and combination with other protease containing microparticles
on pig tendon tissue as set forth in Example 15.
[0028] FIG. 14 shows the effect of molecular weight of
biodegradable polymer on protease activity of papain microparticles
prepared with of different molecular weights of
polylactides-polyglycolic acid polymer on bovine collagen tissue as
set forth in Example 16.
[0029] FIG. 15 shows the effect of molecular weight of
biodegradable polymer on protease activity of elastase
microparticles prepared with of different molecular weights of
polylactides-polyglycolic acid polymer on bovine collagen tissue as
set forth in Example 16.
DETAILED DESCRIPTION OF THE INVENTION
[0030] In some aspects of the invention, there are provided
compositions containing a plurality of microparticles containing
one or more protease enzymes in order to provide a sustained level
of protease activity at the site of injection or application. The
composition is based on the discovery that protease enzymes when
embedded in a biodegradable polymer matrix release the enzyme at a
controlled rate thereby overcoming the problems associated with the
current treatment with protease enzymes that rely on injection or
application of a solution of the enzymes.
[0031] Within this embodiment, the compositions include a plurality
of biodegradable polymer microparticles having a protease enzyme
therein with the biodegradable polymer and the protease enzyme
forming a controlled release matrix for extended release of the
enzyme.
[0032] For purposes of the present invention, the term "matrix"
refers to a microparticle consisting of a suitable polymer in which
one or more protease enzymes or other active medicaments are
dispersed or dissolved. A plurality of such microparticles for each
protease enzyme and anti-inflammatory drug constitute the
composition. These compositions containing protease enzymes and
anti-inflammatory agent can be combined in various ratios to obtain
the final product.
[0033] Suitable proteases which can be included in the
microparticles and controlled release matrix are generally those
capable of having a beneficial effect in the treatment of keloids
and/or related hypertrophic scar conditions. In some aspects, the
protease is collagenase, in others, the enzyme is papain, while in
still other aspects, the enzyme is elastase. The enzymes may be
obtained from commercially available sources and/or may be
recombinant in origin. For purposes of the present description,
protease and enzyme are used interchangeably.
[0034] Mixtures of the enzymes in a single controlled release
matrix system are contemplated as are compositions containing a
blend of two or more different enzyme-containing microparticles.
Stated alternatively, it is further contemplated that the
compositions described herein will contain a plurality of
microparticles include a mixture of microparticles containing
different proteases. For example, some compositions will include a
first portion of microparticles containing collagenase, and a
second portion of microparticles containing papain or elastase. In
an alternative embodiment, compositions will contain a first
portion of microparticles containing collagenase, a second portion
of microparticles containing papain and a third portion of
microparticles containing elastase. The ratio of enzyme to enzyme
in compositions containing more than enzyme can be broadly
expressed as ranging from about 99.9:0.1 to 0.1:99.9. In
alternative aspects, the ratio of the enzymes is from about 1:10 to
10:1, with amounts of about 2:1 to 1:2 also being contemplated. The
ratio of enzymes can describe either the relative amounts of each
enzyme when a combination of enzymes are included in a single
controlled release matrix or as a combination of 2 or more types of
microparticles, each containing a different enzyme. Those of
ordinary skill in the art will, of course, also recognize other
beneficial mixtures of proteases in the compositions described
herein. It is intended that all such combinations are within the
scope of the present invention.
[0035] The controlled release matrix included in the inventive
compositions includes a biodegradable polymer. While the
biodegradable polymer can be selected from a wide variety of
polymeric substances, a non-limiting list of suitable polymers
which are useful in the formation of the controlled matrix include,
without limitation, polymers such as polylactic acid (PLA),
polylactic co-glycolic acid (PLGA), polyglycolic acid (PGA)
polylactones, polyorthocarbonate, polyhydroxybutyrate,
polyalkylcyanoacrylates, polyanhydrides, polyorthoesters,
polyester, polyimide, polyglycolides (PGA), polyorthoester,
polyacetates, polystyrene, polycarbonates, polysaccharides,
polycaprolactone, L-polylactides, block co-polymers of polyesters
and linear or star-polyethyleneglycol, poly-beta-hydroxybutyrate,
beta-hydroxyvalerate-copolymers, polyaminoacids, hydrophobized
hyaluronic acid, dextrans, starches, methyl methacrylate,
acrylamide, bisacrylamide, albumin, cellulose, cellulose-based
polymers, chitosan, collagen, gelatin, proteins, polyvinyl alcohol
(PVA), polyvinylpyrrolidone, polyvinylpyridine, and ethylene glycol
polymers. In some preferred aspects of the invention, the
biodegradable polymer is a polylactic co-glycolic acid or
polylactic acid such as poly lactide-poly glycolide polymer
copolymer (PLGA). Thus, some preferred polymers used in the
invention are a copolymer of two monomers; lactic acid and glycolic
acid. These monomers are combined in a random fashion to produce a
polylactic-glycolic acid. For purposes of the present invention,
polylactic-glycolic acid and poly lactide-poly glycolide polymer
copolymer (PLGA) are used interchangeably. This polymer has been
used in various other applications and is approved by the United
States Food and Drug Administration (FDA) for use in humans.
[0036] The molecular weight for the polymers will vary somewhat
depending upon the polymer that is selected. It is contemplated
that in certain embodiments, the polymers, such as PLGA will have a
molecular weight of from about 7,000 to about 100,000. However, as
will be appreciated by those of ordinary skill, all suitable
molecular weights are contemplated for any of the polymers included
herein.
[0037] Formation of the microparticles and controlled release
matrices is shown in the examples and in commonly-assigned U.S.
patent application Ser. No. 14/600,735, the contents of which are
incorporated herein by reference. In some aspects, the resultant
microparticles have a cross-sectional diameter of from about 10 nm
to about 100 .mu.m. In other aspects, the microparticles have a
cross-sectional diameter of from about 100 nm to about 50 .mu.m,
while in still others, the microparticles have a cross-sectional
diameter of from about 1 .mu.m to about 20 .mu.m. In other
embodiments, the polymer matrix containing the polymer and the
protease enzymes is prepared as small microparticles in the size
range of less than about 1 micrometer to about 50 micrometer or
even larger, with a preferred range of size being in the range of
about 1-20 micrometers.
[0038] The mechanism of release of the proteases from polymers is
due at least in part to the erosion of the polymer in an aqueous
environment. This erosion varies with the molecular weight of the
polymer and the ratio of the lactic acid and glycolic acid
monomers. For example, higher lactic acid content for the copolymer
with respect to the glycolic acid will extend release of the
protease. In such aspects of the invention, ratios of 2:1 or 3:1
are contemplated. The duration of the protease release can also be
prolonged by selecting polymers containing esterified end groups or
end capped groups.
[0039] The amount of protease included in the microparticles can be
expressed as percent (%) loading. Generally speaking, the amount of
loading can vary depending upon the needs of the artisan, including
factors such as the protease or proteases selected for inclusion in
the final formulation, the solubility of the desired protease or
proteases, the duration and concentration of the protease delivery
in vivo, among others. Within this broad range, the percent loading
for the protease can range from about 0.1 to about 10%. In other
aspects, such as where the protease is collagenase, the percent
loading can be from about 3 to about 5%. Similarly, in other
aspects, the protease is elastase and the degree of loading is from
about 0.1. Alternatively, the protease can be papain and the degree
of loading can be from about 0.5 to about 0.9%. In alternative
embodiments, however, the microparticles can contain higher amounts
of a protease enzyme. For example, higher loadings of enzymes, e.g.
up to 50% or more than the amounts set forth above, can be achieved
by using one or a combination of techniques such micronized
protease suspensions and/or by varying the aqueous solvents, i.e.
altering pH by 1-2 units, used to prepare the loaded
microparticles. Such variations and different loading amounts of
protease enzymes are therefore included in the scope of this
invention.
[0040] In further embodiments of the invention, the compositions
may include an auxiliary therapeutic agent. The agent may be within
the biodegradable polymer matrix, that is part of the microparticle
matrix or added to the composition separately. The auxiliary
therapeutic agent can be any therapeutic substance or medicament
which enhances treatment with the protease. For example, the
auxiliary therapeutic agent can be a steroidal or a non-steroidal
inflammation reducing agent such as dexamethasone, corticosteroids
such as prednisone, methylprednisolone, etc. and other agents well
known to those of ordinary skill. Other examples of auxiliary
therapeutic agents include, for example, anti-neoplastic agents
such as sirolimus and tacrolimus, and other immunosuppressant
agents. Dexamethasone is a preferred auxiliary therapeutic agent
for inclusion in the compositions described herein. The amount of
the auxiliary agent included in the composition will be understood
to be an effective amount and will depend upon the agent selected.
For purposes of the present invention, an effective amount shall be
understood to be an amount which is sufficient to enhance the
therapeutic effect of the protease(s). It is contemplated that the
auxiliary agents will be present in amounts of from 0.001 to about
10% by weight of compositions.
[0041] In those embodiments where the composition of the invention
is combined with additional therapeutic entities such as
anti-inflammatory agents to further increase its efficacy, a
purpose for doing so is to minimize the inflammation at the site of
hypertrophic scarring. Inflammation is believed to be a
contributing factor in overproduction of collagen in these
conditions. As mentioned above, the anti-inflammatory agent can be
incorporated in the microparticles containing one or more protease
enzymes, or it can be included as a separated microparticle system
mixed with the microparticles of the protease enzymes.
Compositions/Formulations
[0042] Pharmaceutical compositions containing the protease
containing microparticles described herein may be manufactured by
processes well known in the art. The compositions containing the
microparticles may be formulated in conjunction with one or more
physiologically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the microparticles into
final formulations or preparations which can be used
pharmaceutically. It is contemplated that the compositions will in
some embodiments be provided in pre-filled syringes containing an
amount of the inventive compositions for a single or unit dose.
Proper formulation is dependent upon the route of administration
chosen, i.e. injection or topical administration.
[0043] For injection, including, subcutaneous injection, the
microparticles of the invention may be formulated as part of an
aqueous suspension, and may preferably include physiologically
compatible preservatives, buffers, etc. Aqueous injection
suspensions may also contain substances that increase the viscosity
of the suspension, such as sodium carboxymethyl cellulose,
sorbitol, or dextran. Optionally, the suspension may also contain
suitable stabilizers and/or agents that increase the solubility of
the compounds to allow for the preparation of highly concentrated
solutions. Alternatively, the microparticles may be lyophilized in
powder form for constitution with a suitable vehicle, e.g.,
sterile, pyrogen-free water, before use.
[0044] The microparticles of the invention may also be formulated
in topical compositions such as creams or ointments, using, e.g.,
conventional excipients and bases such as petrolatum, etc.
Methods of Treatment
[0045] In yet another aspect, the present invention provides
methods of treating hypertrophic scars (or tissue) in a mammal. The
methods include administering an effective amount of a composition
containing the protease microparticles described herein to a
patient, i.e. mammal or human in need thereof. In alternative
aspects, the methods of treatment using the compositions described
herein generally include any condition calling for administration
of a protease. In many aspects of the invention, the hypertrophic
scar is a keloid and the treatment is carried out by injecting the
composition to an area requiring the treatment. Alternatively, the
administering of the compositions described herein is carried out
by topically applying the composition to an affected area.
Determination of a therapeutically effective amount is well within
the capability of those skilled in the art, especially in light of
the disclosure herein. Moreover, the amount required for each
administration will vary somewhat depending upon the protease or
proteases selected and the type of polymeric matrix used. For
example, in some aspects of the invention the compositions are
administered by injection at the site of need. i.e.
intra-lesionally, once a week. In other aspects, the composition is
administered once a month or every two months.
[0046] Generally speaking, it is contemplated that administration
of compositions capable of delivering from about 25 U to about 200
U of collagenase, about 0.05 U to about 2.0 U of elastase and from
about 0.2 U to about 5.0 U of papain per administration as needed
or until the desired improvement in the condition being treated is
observed. Based on the units/mg activity values provided by the
supplier, these concentrations correspond to about 200
micrograms/ml to about 1600 micrograms/ml of collagenase, about
12.5 micrograms/ml to about 500 micrograms/ml of elastase and from
about 10 micrograms/ml to about 250 micrograms/ml of papain per
administration as needed or until the desired improvement in the
condition being treated is observed. These concentrations will vary
somewhat according to the specific condition and size of the
treatment area and such variations will be apparent to those of
ordinary skill without undue experimentation.
[0047] One use of this invention is in the treatment of
hypertrophic scars resulting from a variety of reasons. These
reasons include post-surgical scars, wounds from injury or burns,
formation of keloids, scarring due to acne and related conditions
and tissue adhesion in the organs. Further, it has been discovered
that protease enzymes when injected or applied in a slow release
form in combination with each other work in a synergistic fashion,
thereby exerting a higher degree of protease activity than is
expected from application of a single protease enzyme. This
invention allows one to produce a customized combination of the
slow release microparticle form of protease by mixing individual
enzyme microparticles in various proportions. This is a distinct
advantage over the existing systems because it allows customization
of the composition to overcome the need for individual disease
condition.
EXAMPLES
[0048] The following examples serve to provide further appreciation
of the invention but are not meant in any way to restrict the
effective scope of the invention.
Methods of Preparing Compositions
Example 1 Preparation of Collagenase Microparticles
[0049] 0.5 gram of polylactic co-glycolic acid polymer (called 1A)
with an average molecular weight of about 10 kilo Dalton and which
consisted of a 50:50 mixture of lactic acid and glycolic acid
monomers is dissolved in 10 milliliters of dichloromethane. In a
separate vessel, 20 milligrams of collagenase powder is added to
0.5 milliliter of purified water to obtain a clear solution. This
solution is filtered through a 0.2 micron filter to remove any
insoluble material. 0.4 milliliters of the filtered protease
solution is added to the polymer solution. This mixture is vortexed
at a high rate for 60 seconds to disperse the protease solution in
the polymer solution. The dispersion is sonicated using a 3 mm
sonication tip at 40% amplitude for 60 seconds to prepare a fine
emulsion containing the protease solution emulsified in the polymer
solution. This first emulsion is added to 40 milliliters of a 2%
solution of polyvinyl alcohol (PVA) in water. A homogenizer with 7
mm tip is dipped in the above mixture and the mixture is
homogenized at a setting of 3 for 90 seconds to obtain a second
emulsion. This second emulsion is poured in a beaker containing 200
milliliters of purified water stirred by a magnetic stirrer. The
mixture is stirred at room temperature for 4 hours to allow
evaporation of dichloromethane.
[0050] The above mixture is transferred to large centrifuge tubes
and subjected to centrifugation for 5 minutes to concentrate the
particles as a pellet at the bottom of the tubes. The concentrated
particles are pooled in a single tube and washed 3 times with
purified water to remove excess PVA solution. Washings are carried
out by adding 20 milliliters of purified water, vortexing for 10
seconds, followed by centrifugation. After the final washing, the
particle pellet is washed out in a glass vial using about 2
milliliters of purified water and frozen at -40.degree. C. See FIG.
1. The process of this example was repeated two further times to
provide Compositions 3 and 4 mentioned in Table 1 below.
Example 2 Preparation of Papain and Elastase Microparticles
[0051] Papain and elastase microparticles were made following the
process of example 1 except that 10 mg each of these enzymes was
added to purified water followed by stirring at 37.degree. C. for
one hour. This mixture was filtered through a 0.2 micron syringe
filter to remove undissolved enzyme. The filtered solution was used
to prepare the microparticles as described in example 1 starting
with the vortexing step.
[0052] The processes of this example were repeated two further
times to provide Compositions 6 and 8 for papain and Compositions
10 and 12 for elastase mentioned in Table 1 below.
Example 3
[0053] Collagenase, papain and elastase microparticles were made
following the processes of Examples 1 and 2, except that the
polylactic co-glycolic acid polymer is listed as grade 3A and has
an average molecular weight of about 25 kilo Dalton. The
compositions are identified below in Table 1 as Composition Numbers
2, 5 and 9 respectively. See FIGS. 2 and 3
Example 4 Preparation of Dexamethasone Particles
[0054] 0.5 grams of the polylactic co-glycolic acid polymer used in
Example 1 is dissolved in a mixture of 14 milliliters of
dichloromethane and 7 milliliters of ethyl alcohol. 100 mg of
dexamethasone are added to this mixture in a 50 ml tube. 30
milliliters of 4% PVA solution are added to the tube, followed by
vortexing for 30 seconds. The mixture is then homogenized using a 7
mm tip at setting of 2 for 30 seconds. The mixture is immediately
poured in 60 milliliters of purified water in a beaker and stirred
with a magnetic stirrer for 4 hours.
[0055] The above mixture is transferred to large centrifuge tubes
and subjected to centrifugation for 5 minutes to concentrate the
particles as a pellet at the bottom of the tubes. The concentrated
particles are pooled in a single tube and washed 3 times with
purified water to remove excess PVA solution. Washings are carried
out by adding 20 milliliters of purified water, vortexing for 10
seconds, followed by centrifugation. After the final washing, the
particle pellet is washed out in a glass vial using about 2
milliliters of purified water and frozen at -40.degree. C. See FIG.
4.
Example 5 Freeze Drying of Particles
[0056] The material of Examples 1-4 was subject to freeze drying by
applying a vacuum of less than 200 millitorrs for 24 hours while
the temperature of the vial was maintained at 0.degree. C.
Following this initial phase of drying, the temperature of the vial
was increased to 25.degree. C. and vacuum continued for another 2
hours. The vial was then removed from the freeze dryer and the
material was removed with a spatula and mixed to obtain a uniform
solid material. The material was transferred to a weighing paper
and weighed to calculate the yield.
Determination of Drug Loading in the Microparticles
Example 6 Drug Loading of Protease Enzymes
[0057] 5 mg of accurately weighed microparticles were added to 5
milliliters of dichloromethane in a 50 ml tube. As a reference, 5
mg of polymer and about 1 mg of each enzyme was added to another
tube in 5 milliliters of dichloromethane. A blank was prepared by
adding 5 mg of polymer to a tube in 5 milliliters of
dichloromethane. The mixtures were stirred until the microparticles
and the polymer dissolved. To each tube, 10 milliliters of purified
water was added followed by vortexing at high speed for 60 seconds.
The mixtures were allowed to stand at room temperature for 15
minutes followed by centrifugation for 2 minutes. 100 microliters
of the aqueous layer was transferred from each tube to a labeled
vial and diluted with 900 ul of purified water. These solutions
were subjected to a protein assay using the BCA assay protocol
using bovine serum albumin as protein standards. Protein content in
the microparticles was determined from this procedure and the value
was used to calculate the loading efficiency (percent of total
added enzyme incorporated in the polymer) and percent loading
(protein content as percent of total weight of microparticles).
Example 7 Drug Loading of Dexamethasone
[0058] 15 milligrams of accurately weighed dexamethasone
microparticles were added to 5 milliliters of 0.1 molar sodium
hydroxide solution in a labeled vial. This vial was subjected to
gentle shaking at 37.degree. C. for 24 hours. 5 milligrams of
dexamethasone powder was similarly processed as a standard sample.
After shaking, the samples were cooled to room temperature and 10
milliliters of methyl alcohol was added to each vial. This solution
was centrifuged at 2000 g for 3 minutes. 100 microliters of the
supernatant from the vials was diluted with 900 microliters of
methyl alcohol. Absorbance of these solutions was read at 240 nm in
a UV spectrophotometer. The amount of dexamethasone in
microparticles was calculated by comparing the absorbance with the
standard containing 5 milligrams of dexamethasone. Percent loading
of dexamethasone was calculated based on the amount of
dexamethasone in the microparticles.
Example 8 Microscopic Examination and Size Measurement
[0059] About 5 milligram of microparticles was suspended in 100
microliters of purified water. A drop of this suspension was placed
on a clean glass slide and spread as a thin layer. The layer was
observed under the microscope. Digital pictures were taken for the
microparticles and the diameter of 20 microparticles in a set field
was measured to calculate the average diameter of the
particles.
TABLE-US-00001 TABLE 1 composition, percent yield and drug loading
of microparticles Composition Polymer Average diameter % % drug #
Material grade Micrometers Yield loading 1 Collagenase 1A 11 59 3.6
2 Collagenase 3A 10 50 4.91 3 Collagenase 1A 12 68 4.06 4
Collagenase 1A 11 81 3.85 5 Papain 3A 11 69 0.82 6 Papain 1A 9 65
0.68 7 Papain 1A 9 58 0.59 8 Papain 1A 5 72 0.66 9 Elastase 3A 6 36
0.49 10 Elastase 1A 6 59 0.21 11 Elastase 1A 6 52 0.16 12 Elastase
1A 9 50 0.17
[0060] As seen in Table 1, the amount of protease enzymes in the
microspheres calculated as percent on w/w basis ranges from 0.16 to
4.91. This amount varies for each protease enzyme, ranging from
0.16-0.49 for elastase, 0.59-0.82 for papain, and 3.60-4.91 for
collagenase. This difference in percent content of protease enzymes
is due to different solubility of these enzymes in water or buffer
solution. Collagenase being highly soluble can be loaded in higher
concentration.
Example 9 Tissue Preparation
[0061] A. Pig Tendon
[0062] Fresh pig feet were washed with phosphate buffer saline
solution containing 0.2% sodium azide as preservative. Tendons in
the feet were removed with scissors and were scrapped with a sharp
razor blade to remove any remnants of muscle and fat. The cleaned
tendons were cut into pieces weighing about 20 mg each. These
pieces were added to enough volume of phosphate buffer saline
solution containing 0.2% sodium azide in a beaker to cover the
tissue and the mixture was stirred for 12 hours to remove any
soluble protein. The buffer was decanted from the beaker, replaced
with fresh buffer, stirred for 5 minutes and decanted again. This
process was repeated 2 more times to thoroughly wash the tissue.
The washed tissue was transferred to a stainless steel tray and
blotted with a clean lint free tissue paper to remove excess
liquid. The tissue was then weighed in individual labeled tubes for
protease activity studies.
[0063] B. Dermal Collagen
[0064] Fresh pig skin was excised from the pig feet in about 2
square inch sections. The skin was washed with phosphate buffer
saline solution containing 0.2% sodium azide as preservative and
kept soaked in the buffer solution when not being processed. The
dermal collagen was removed by carefully scrapping about 2 mm thick
layer of the skin from the dermal tissue. This tissue was pooled in
a beaker containing enough volume of phosphate buffer saline
solution containing 0.2% sodium azide and the mixture was stirred
for 12 hours to remove any soluble protein. The buffer was decanted
from the beaker, replaced with fresh buffer, stirred for 5 minutes
and decanted again. This process was repeated 2 more times to
thoroughly wash the tissue. The washed tissue was transferred to a
stainless steel tray and blotted with a clean lint free tissue
paper to remove excess liquid. The tissue was then weighed in
individual labeled tubes for protease activity studies.
[0065] C. Pig Skin
[0066] Fresh pig skin was excised from the pig feet in about 2
square inch sections. The skin was washed with phosphate buffer
saline solution containing 0.2% sodium azide as preservative and
kept soaked in the buffer solution when not being processed. For
protease studies, the skin was cut into square pieces measuring
about 4 square millimeters. This tissue was pooled in a beaker
containing enough volume of phosphate buffer saline solution
containing 0.2% sodium azide and the mixture was stirred for 12
hours to remove any soluble protein. The buffer was decanted from
the beaker, replaced with fresh buffer, stirred for 5 minutes and
decanted again. This process was repeated 2 more times to
thoroughly wash the tissue. The washed tissue was transferred to a
stainless steel tray and blotted with a clean lint free tissue
paper to remove excess liquid. The tissue was then weighed in
individual labeled tubes for protease activity studies.
[0067] D. Lyophilized Collagen
[0068] Lyophilized bovine collagen was obtained from commercial
sources. This collagen is a pure form of collagen and was used as
received. For protease activity studies, the collagen fibers were
weighed as dry material into individual tubes followed by the
addition of protease solutions or microparticles.
Example 10 Protease Activity Protocol
[0069] All tissues were prepared fresh for each study as described
above. Stock solutions of each protease enzyme were prepared in the
buffer at a concentration of 100-500 ug/ml. Appropriate volume of
each solution was added to a 2 milliliter tube to allow the final
concentration to be at the desired level as mentioned in Table 2
(below in Example 13) after the volume in the tube was made up to 1
milliliter using the buffer. The buffer contained a 0.2%
concentration of sodium azide to protect from microbial growth.
Microparticles were suspended in the buffer solution at a
concentration of 150 milligrams in 4.5 milliliters. 0.5 milliliters
of this suspension was added to each tube to obtain the final
concentration of 15.5 mg/ml of each microparticle. Table 2 contains
the details of each protease and its microparticle concentration in
the study. All studies were set up by mixing appropriate volumes of
the stock solutions of protease in solution and microparticle form
with enough volume of buffer to have a final volume of 1
milliliter.
[0070] Once all samples were prepared, the tubes were loaded on the
racks and placed in an incubator containing a rotary shaker. The
temperature in the incubator was maintained at 37.degree. C. and
the racks were stirred at 100 rpm in a rotary shaker. The samples
were continuously stirred during the entire duration of study
except when they were removed for sampling. All studies were
carried out in duplicate and the values reported are an average of
the two values.
[0071] At the designated time points, the tubes were removed from
the rack and were subjected to centrifugation at 2000 g for 3
minutes. The tubes were opened and a 10 microliter sample of clear
supernatant solution was transferred to a labeled 96-well plate.
The tubes were then sealed and replaced in the incubator.
[0072] The content of free (soluble) protein in samples removed
from the tubes was measured using a commercially available BCA
protein assay kit. Bovine serum albumin (BSA) was used as a
standard protein. Several concentrations of BSA were prepared for
this purpose in order to obtain a regression line for the standard
curve. The regression equation was used to calculate the
concentration of soluble protein in each sample. The protein
content in each tube was determined based on the volume of the
solution in the tube and the protein concentration. Since
successive sampling reduces the volume in each tube by 10
microliters, all protein content values were corrected for this
volume change. Cumulative amount of soluble protein in each sample
was obtained and was normalized for the weight of the tissue. The
final protein content was calculated as milligrams of soluble
protein for one gram of the tissue in the sample.
Example 11
[0073] In order to study the efficacy of the compositions of the
invention relative to the injection or application of a solution of
the same protease enzymes, the protease enzymatic activity of the
prepared microparticles was studies in a variety of tissues and
materials. The materials used in this study include a pure form of
collagen obtained from bovine sources. The tissues included in this
study include the whole skin of pig, tendon from pig and the dermal
collagen from pig. Pig skin has been extensively used as a model
for screening of therapeutic agents for use in humans. The
selection of these tissues was based on their collagen content. Pig
tendon consists almost entirely of type 1 collagen with small
amounts of type 3 collagen. Skin dermal collagen is a good model
for studying the activity of protease enzymes because it mimics the
type of collagen matrix found in hypertrophic scars and keloids. In
addition, whole skin of pig was included to study the effect of the
composition on the entire organ.
[0074] Preliminary studies were carried out using the pure bovine
collagen to select appropriate concentration of each protease
enzyme. The enzyme activity was determined by a method developed in
our laboratory to study the conversion of native collagen into a
soluble form of protein that can be absorbed by the body. Native
collagen is practically insoluble in body fluids and other aqueous
solutions similar to body fluids. When collagen in any form is
suspended in an aqueous solution of pH (pH value of 7.4) close to
the body fluids, it is insoluble and stays insoluble over a period
of several weeks. When a clear sample of this suspension is
analyzed for soluble protein content, the amount of soluble protein
content does not change over a period of several weeks as shown in
FIG. 5. The amount of soluble protein in buffer without any
protease is within the experimental variation of the soluble
protein content over a period of 27 days. However, when a protease
is added to the suspension, the protease activity begins to degrade
the insoluble collagen into soluble protein, thereby increasing the
amount of soluble protein in the solution phase of the
suspension.
[0075] As seen in FIG. 5, in the presence of papain solution, the
amount of soluble protein increases steadily over a period of about
4 weeks. The soluble (also called as free protein) can be measured
by a variety of protein assay methods. The method used in this
study is called as the BCA protein assay kit that is commercially
available from several sources. This assay can reliably measure the
soluble protein amount in an aqueous system at concentrations
ranging from a few micrograms to several hundred micrograms per
milliliter.
[0076] FIG. 5 also shows that the activity of papain in
microparticle form has much higher activity against collagen in the
tendon tissue. As a comparison, the amount of soluble protein
present in the sample increases by 300% with papain microparticles
when compared with the papain solution.
[0077] As seen in FIG. 5 when pig tendon was exposed to buffer,
there was no significant increase in the amount of soluble protein
the solution phase of the tendon suspension over a period of about
4 weeks. This amount stays in range of 6-10 milligrams of protein
per gram of tendon tissue. When papain enzyme at a concentration of
100 micrograms per milliliter is added to the suspension, the
amount of soluble protein increases steadily over a period of about
4 weeks. After 27 days, the total amount of soluble protein in the
solution phase is about 14 milligrams per gram of tendon tissue.
However when papain microparticles are added to the suspension
without the papain solution, the amount of soluble protein
increases at a much faster rate. At 27 days, the amount of soluble
protein is about 65 milligrams per gram of tendon tissue. Since the
only source of soluble protein the solution is from degradation of
collagen in the tendon, the soluble protein amount correlates with
degradation of the collagen in tendon tissue, and hence with
protease activity of the enzyme. The microparticles show
approximately 450% increase in the extent of collagen degradation
when papain is added as microparticle composition described in this
invention.
Example 12
[0078] In order to confirm that these results are due to protease
activity on collagen tissue, the study was repeated on collagen
tissue harvested from dermal layer of the pig skin as described in
Example 9B. Similar results can be seen with the collagen from
dermal layer as shown in FIG. 6. The amount of soluble protein
produced in this study after about 4 weeks is about 700% higher
from the microparticle form of papain compared with the solution
form of papain. The results of this example also confirm that the
composition described in this invention is effective on collagen
from more than one source or organ.
Example 13
[0079] Similar results can be seen in FIG. 7 where addition of
collagenase enzyme as microparticles results in 880% and 178%
increase in the extent of collagen degradation compared with buffer
and collagen solution, respectively. Similarly, as shown in FIG. 8,
elastase enzyme results in 1200% and 584% increase in the extent of
collagen degradation compared with buffer and collagen solution,
respectively. These results clearly show that application of
protease enzymes as composition of this invention offers
significant advantage in degrading collagen.
[0080] In the above studies, the elastase and collagenase enzymes
were added as solutions in the concentration of 100 micrograms per
milliliter. 15 milligrams of microparticles of each enzyme were
added in the microparticle study. Based on average percent enzyme
loading in microparticles (calculated as percent of protease enzyme
in total amount of microparticles on w/w basis) (0.52% and 0.70% of
enzyme in the microparticles for elastase and papain, respectively)
the total amount of elastase and papain added as microparticles
were 61 and 105 micrograms, respectively. Since the release of
these enzymes is typically linear with time from the
microparticles, their average concentration in the solution can be
approximated by half of the total amount added, assuming that all
the enzyme is released in 27 days. When the data is normalized to
the average amount of protease in solution from microparticles
(expressed as .mu.g soluble protein/g tissue/.mu.g enzyme),
elastase and papain microparticles increase the amount of collagen
degradation by 1016% and 484%, respectively. For collagenase, there
is a decrease of 86% in collagen degradation when the data is
normalized to total amount of protease in the solution. Although
this decrease is observed, the overall advantage of collagenase on
collagen degradation can be obtained by adding proportionately
larger amount of collagen in microparticle form. Further, it can be
noted that the extent of collagen degradation in collagen solution
treated studies from day 14 to day 27 increases by 1.89 fold, while
the corresponding value for collagen in microparticle form when
normalized for protease amount is 4 fold. Therefore, it is clear
that over a period of about 4 weeks, all protease enzymes perform
significantly better when used as composition of this invention
compared with the solutions of the protease.
[0081] It must be noted that in these studies, none of the protease
solutions are removed from contact with the tissue because there is
no clearance of protease from the suspensions. In a biological
system, the soluble part of the protease will be removed by
absorption in the circulatory system whereas the rate of removal
from microparticle form will be smaller due to the fact that most
of the protease is protected by the polymer matrix. This will
result in a much higher efficacy of protease enzymes in an in-vivo
situation.
[0082] The above studies were carried out for all protease enzymes
for 3 different sources of collagen. These sources included pig
tendon, whole pig skin and the dermal layer of pig skin. The
results of these studies are summarized in Table 2.
TABLE-US-00002 TABLE 2 Comparison of protease activity in
microparticle and solution forms in different collagen tissues
Protease Protease Percent Ratio Protease conc. conc. increase
Microspheres/ Tissue Compositions Enzymes ug/ml* units/ml** Days
1-27 Solution Tendon Solutions Collagenase 10 1.25 222.6 Elastase
50 0.2 22.8 Papain 50 1.05 44.2 Microparticles Collagenase 600 75
365.7 1.6 Elastase 45 0.18 599.4 26.3 Papain 90 1.87 352.4 8.0 Skin
Solutions Collagenase 10 1.25 154.9 Elastase 50 0.2 47.3 Papain 50
1.05 40.8 Microparticles Collagenase 600 75 465.6 3.0 Elastase 45
0.18 600.3 12.7 Papain 90 1.87 253.9 6.2 Dermal Solutions
Collagenase 10 1.25 46.3 Layer Elastase 50 0.2 -18.1 Papain 50 1.05
-22.6 Microparticles Collagenase 600 75 50.8 1.1 Elastase 45 0.18
596.6 high Papain 90 1.87 287.8 high *protease amount in
microparticles calculated from percent loading of enzymes
**units/ml calculations based on vendor supplied enzymes activity
(units/mg)
[0083] This table shows that there is an increase ranging from
about 50% to about 600% for all protease enzymes in tendon and skin
tissues when compared with exposure of the tissues to buffer alone.
In the dermal layer, elastase and papain enzymes show no
significant increase in protease activity when added to the tissue
as solutions. The microparticle containing proteases on the other
hand did. In order to assess the relative activity of enzymes in
microparticle and solution forms, a ratio of the protease activity
is calculated by dividing the activity from microparticles with
activity from solution. As seen in the Table, this ratio ranges
from 1.6 to 26.3 for collagen tissue, similar results are observed
for the other tissues as well. Therefore it can be concluded that
microparticle form of enzymes performed significantly better in all
tissues.
Example 14
[0084] In order to study the possible synergistic action of
protease enzymes from microparticle compositions, various
combinations of enzyme containing microparticles were studies in a
similar way as the above studies in pig tendon and bovine collagen.
As seen in FIG. 9, when collagenase and papain microparticles are
added together to the pig tendon tissue, the resulting protease
enzymatic activity is higher than the activity of individual
enzymes. This shows that there is a synergistic action of the
enzymes in microparticle form. In order to confirm that this
phenomenon occurs in collagen from other sources as well, this
study was also carried out on lyophilized form of bovine collagen.
As shown in FIG. 10, the results are very similar to the ones in
FIG. 9, confirming a synergistic action of the two enzymes in
microparticle form.
Example 15
[0085] The unexpected synergistic activity of protease enzymes in
microparticle form was further studied in a more comprehensive
study where a larger number of combinations were studied over a
period of 96 hours. The results of this study confirm that the
synergistic action of protease enzymes is observed. As can be seen
in FIG. 11, the combination of elastase protease enzyme
microparticles show a much higher activity than the individual
protease enzymes as microparticles. FIG. 12 shows the same study
carried out in the pig dermal collagen, once again showing that the
combination enzyme microparticles work better than individual
microparticles. FIG. 13 shows a study of the comparison of papain
microparticles alone and in combination of microparticles of the
other enzymes. Once again, the combination microparticles show up
to 208% increase in enzyme activity when all 3 enzyme
microparticles are present in the system as compared to the papain
microparticles alone. Together, these studies show that in a
variety of collagen sources, protease enzymes work better when
added in combination as slow release microparticles than when added
alone as slow release microparticles.
Example 16
[0086] In this Example, Pap 1A of Example 2 which was prepared with
polymer with an average molecular weight of about 10 kilo Dalton
was compared to Pap 3A of Example 3 which was prepared with polymer
with an average molecular weight of about 25 kilo Dalton. The
activity is expressed as the amount of soluble protein per gram of
collagen tissue as a function of time. Similarly, Elastase 1A of
example 2 which was prepared with polymer with an average molecular
weight of about 10 kilo Dalton was compared to Elastase 3A of
Example 3 which was prepared with polymer with an average molecular
weight of about 25 kilo Dalton. The activity is expressed as the
amount of soluble protein per gram of collagen tissue as a function
of time.
[0087] FIG. 14 shows the protease activity of papain from 1A and 3A
polymers. The activity from 1A polymer (low molecular weight grade)
is higher and starts faster than the activity from higher molecular
weight polymer. The release of the papain from the higher molecular
weight polymer is lower but is released over a longer period of
time. This is an example of one way in which the protease activity
profile can be varied using differing molecular weight polymers.
FIG. 15 shows the protease activity of elastase from 1A and 3A
polymers. As was the case with the papain, the elastase activity
from 1A polymer (low molecular weight grade) is higher and starts
faster than the activity from higher molecular weight polymer which
has a prolonged delivery profile.
Example 17
[0088] A ready to use final composition for treatment is prepared
by mixing appropriate amounts of each microparticle alone or in
combination with each other, along with one or more inactive
ingredients. Examples of inactive ingredients include suspending
agents such as carboxymethyl cellulose and other such water soluble
polymers, polyhydric sugars such as mannitol, and wetting agent
such as a non-ionic surfactant, as needed. A suitable method of
sterilization is used to obtain a sterile dry material that is
reconstituted with a suitable aqueous solvent prior to injection.
Examples of such compositions comprise of the following:
[0089] A vial containing lyophilized collagenase microparticles is
made containing from about 50 to about 150 units of collagenase
activity along with 5-20 mg of carboxymethyl cellulose, 50-200 mg
of mannitol and 5-20 mg of polyvinyl pyrrolidone. This composition
may also contain microparticles containing dexamethasone or another
similar anti-inflammatory agent containing 2-10 mg of the active
medicament.
[0090] A vial containing lyophilized elastase microparticles
containing from about 0.1 to about 0.5 units of elastase activity
along with 5-20 mg of carboxymethyl cellulose, 50-200 mg of
mannitol and 5-20 mg of polyvinyl pyrrolidone. This composition may
also contain microparticles containing dexamethasone or another
similar anti-inflammatory agent containing 2-10 mg of the active
medicament.
[0091] A vial containing lyophilized papain microparticles
containing from about 1 to about 5 units of papain activity along
with 5-20 mg of carboxymethyl cellulose, 50-200 mg of mannitol and
5-20 mg of polyvinyl pyrrolidone. This composition may also contain
microparticles containing dexamethasone or another similar
anti-inflammatory agent containing 2-10 mg of the active
medicament.
[0092] A vial containing lyophilized collagenase microparticles
containing from about 50 to about 150 units of collagenase
activity, from about 0.1 to about 0.5 units of elastase activity,
from about 1 to about 5 units of papain activity, along with 5-20
mg of carboxymethyl cellulose, 50-200 mg of mannitol and 5-20 mg of
polyvinyl pyrrolidone. This composition may also contain
microparticles containing dexamethasone or another similar
anti-inflammatory agent containing 2-10 mg of the active
medicament.
* * * * *