ESCRIPTION
ENZYMATIC WOUND DEB DING COMPOSITIONS WITH ENHANCED
ENZYMATIC ACTIVITY
5
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No.
61/267,730, filed 8 December 2009. The contents of the referenced application are
incorporated by reference.
10 BACKGROUND OF THE INVENTION
A. Field of the Invention
[0002] The present invention relates generally to topical enzymatic wound debriding
compositions and methods of treating wounds in need of debridement.
B. Background
15 [0003] The healing of wounds is a complex process which is often further complicated
by the presence of non-viable, necrotic tissue in the wound area. Debridement is the process
of removing the non-viable tissue from a wound to prevent or diminish infection and facilitate
healing. Topical compositions containing proteolytic enzymes such as trypsin, papain,
bromelain, subtilisin, sutilains, and collagenase have been used for enzymatic wound
20 debridement. Generally, the standard of care is to apply the composition to the wound in need
of debridement once daily (once every 24 hours) or more often if the composition becomes
soiled. Because many proteolytic enzymes are susceptible to degradation in water-based,
compositions, many wound debriding compositions are made with anhydrous, hydrophobic
bases such as petrolatum, mineral oil and/or vegetable oil as disclosed in US 3,821,364 and
25 US 6,479,060, both of which are herein incorporated by reference. However, enzymatic
wound debriding compositions based on hydrophobic bases are generally not miscible in the
aqueous environment of a wound bed, and thus contact of the proteolytic enzyme with the
wound bed is generally hindered. Some other compositions are made with anhydrous,
hydrophilic bases such as propylene glycol or poloxamers as disclosed in US 6,548,556, US
30 2003/0198631 and US 2003/0198632, all of which are herein incorporated by reference.
90753719.1 1
SUMMARY OF THE INVENTION
[0004] The present invention is directed to topical enzymatic wound debriding
compositions with enhanced enzymatic activity. These compositions comprise a dispersed
phase comprising at least one proteolytic enzyme and at least one hydrophilic polyol; and a
5 continuous phase comprising a hydrophobic base. The wound debriding compositions of the
present invention possess enhanced enzymatic activity over wound debriding compositions of
the prior art.
[0005] In one aspect of the present invention, there is disclosed a wound debriding
composition comprising a dispersed phase comprising a liquid hydrophilic polyol and at least
10 one proteolytic enzyme; and a continuous phase comprising a hydrophobic base; wherein the
amount of liquid hydrophilic polyol is within l 10% w/w of the optimum amount of the liquid
hydrophilic polyol. For example, if the optimum amount was about 30% w/w, the amount of
liquid hydrophilic polyol that could be used would be between about 20% w/w and about 40%
w/w of the total formulation to achieve enhanced enzymatic activity of the formulation. In
15 another aspect, the amount of liquid hydrophilic polyol is within ± 9%, 8%, 7%, or 6% w/w of
the optimum amount of the liquid hydrophilic polyol. In still another aspect, the amount of
liquid hydrophilic polyol is within ± 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% w/w of the
optimum amount of the liquid hydrophilic polyol.
[0006] The "optimum amount of liquid hydrophilic polyol" in a composition
20 comprising (a) a dispersed phase including a liquid hydrophilic polyol and at least one
proteolytic enzyme; and (b) a continuous phase comprising a hydrophobic base can be
determined by the method described in Section A of the Detailed Description section of this
specification, which is incorporated into this section by reference.
[0007] A method for determining whether a composition is within + 10% w/w of the
25 optimum amount of a liquid hydrophilic polyol is described in Section B of the Detailed
Description section of this specification, which is incorporated by reference.
[0008] The optimum amount of liquid hydrophilic polyol for compositions with
different proteolytic enzymes can differ. Additionally, the optimum amount of liquid
hydrophilic polyol for compositions with a specific proteolytic enzyme can differ depending
30 on the ingredients of the composition. For example, the optimum amount of liquid
hydrophilic polyol in a collagenase composition containing PEG-400 and petrolatum can be
90753719.1 _ 2
different from the optimum amount of liquid hydrophilic polyol in a collagenase composition
containing PEG-600 and petrolatum, or different from a collagenase composition containing
poloxamer®124 and petrolatum.
[0009] The term "hydrophilic polyol" means water-soluble, polar aliphatic alcohols
5 with at least two hydroxyl groups and includes, but is not limited to, polymeric polyols (e.g.,
polyethylene glycols and poloxamers).
[0010] The term "liquid" in the context of describing "hydrophilic polyol",
"polyethylene glycol", or "poloxamer" means that the material is in the liquid state at 25 °C.
[00111 The term "solid" in the context of describing "hydrophilic polyol",
10 "polyethylene glycol", or "poloxamer" means that the material is in the solid state at 25 °C.
[0012] In another aspect of the present invention, there is disclosed a method of
treating a wound in need of debridement comprising : applying to the wound a composition
comprising a dispersed phase comprising a liquid hydrophilic polyol, and an effective
debriding concentration of at least one proteolytic enzyme; and a continuous phase
15 comprising a hydrophobic base ; wherein the amount of liquid hydrophilic polyol is within ±
10% w/w of the optimum amount. In another aspect, the amount of liquid hydrophilic polyol
is within 1 9%, 8%, 7%, or 6% w/w of the optimum amount. In still another aspect, the
amount of liquid hydrophilic polyol is within ± 5%, 4%, 3%, 2%, 1%, 0.5%, or U 'Xn w/w of
the optimum amount.
20 [0013] In some embodiments, the proteolytic enzyme is a metalloprotease, a cysteine
protease, a serine protease, or an aspartic peptidase. Generally, the optimum amount of
hydrophilic polyol for compositions comprising a metalloprotease, a cysteine protease or a
serine protease is from about 10%, 11%, 12%, 13%, 14%, 15%, 16% 17%, 18%, 19%, 20%,
21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,
25 37%, 38%, 39% w/w to about 40% w/w, or any range or numerical amount derivable therein.
The optimum amount of hydrophilic polyol for compositions comprising an aspartic peptidase
is from about 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,
61%, 62%, 63%, 64%, 65%, 66%, 67% w/w to about 68% w/w or any range or numerical
amount derivable therein. In one embodiment the metalloprotease is collagenase. In another
30 embodiment the metalloprotease is collagenase and the optimum amount of the hydrophilic
polyol is from about 10%, 11%, 12%, 13%, 14%, 15%, 16% 17%, 18%, 19%, 20%, 21%,
90753719.1 3
22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,
38%, 39% w/w to about 40% w/w or any range or numerical amount derivable therein. In one
embodiment, the metalloprotease is thelmolysin. In another embodiment, the metalloprotease
is thermolysin and the optimum amount hydrophilic polyol is from about 19%, 20%, 21 %,
5 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,
38% w/w to about 39% w/w or any range or numerical amount derivable therein. In one
embodiment, the cysteine protease is papain. In another embodiment the cysteine protease is
papain and the optimum amount of the hydrophilic polyol is from about 19%, 20%, 21%,
22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%,
10 38% w/w to about 39% w/w or any range or numerical amount derivable therein. In one
embodiment, the serine protease is trypsin. In another embodiment the serine protease is
trypsin and the optimum amount of hydrophilic polyol is from about 4%, 5%, 6%, 7%, 8%,
9%, 10%, 11%, 12%, 13%, 14%, 15%, 16% 17%, 18%, 19%, 20%, 21%, 22%, 23% w/w to
about 24% w/w or any range or numerical derivable therein. In one embodiment, the aspartic
15 peptidase is pepsin. In another embodiment the aspartic peptidase is pepsin and the optimum
amount of hydrophilic polyol is from about 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,
56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67% w/w to about 68% w/w
or any range or numerical amount derivable therein. In some embodiments, the proteolytic
enzyme is suspended in the dispersed phase. In other embodiments the proteolytic enzyme is
20 dissolved in the dispersed phase.
[0014] In some embodiments, the liquid hydrophilic polyol is a liquid polyethylene
glycol or a liquid poloxamer, or mixtures thereof.
[0015] In some embodiments of the present invention, the dispersed phase may further
comprise a -solid hydrophilic polyol in order to help physically stabilize the composition or
25 reduce or prevent phase separation. In some embodiments, the solid hydrophilic polyol is a
solid poloxamer, or a solid polyethylene glycol, or mixtures thereof.
[0016] In various embodiments of the present invention, the hydrophobic base
comprises petrolatum, mineral oil, or vegetable oil, or mixtures thereof. In one embodiment,
the base comprises petrolatum. In another embodiment, the hydrophobic base comprises a
30 vegetable oil. In still another embodiment, the hydrophobic base comprises mineral oil. In a
further embodiment, the hydrophobic base comprises petrolatum and mineral oil, petrolatum
and vegetable oil, mineral oil and vegetable oil, or petrolatum, mineral oil, and vegetable oil.
90753719.1 4
In still another embodiment, the hydrophobic base comprises a vegetable oil, wherein the
vegetable oil is castor oil.
[0017] In one embodiment, the composition is a semisolid. In another embodiment,
the composition is a liquid. In other embodiments, the composition is impregnated on a pad,
5 gauze, or sponge. In one embodiment, the composition is sterile or anhydrous or both.
[0018] The composition can be packaged in any package appropriate for dispensing a
wound debrider. The compositions can be packaged in multi-use, single-dose, or metered
dose packages. Non-limiting examples include a tube, bottle, jar, pump container, pressurized
container, bladder container, aerosol container, aerosol spray container, non-aerosol spray
10 container, syringe, pouch, or sachet.
[0019] In another embodiment of the present invention there is disclosed a method of
determining the optimum amount of liquid hydrophilic polyol to add to a target composition
comprising a dispersed phase including a proteolytic enzyme and a continuous phase
including a hydrophobic base, the method comprising: (1) obtaining a series of compositions
15 comprising the dispersed phase and the continuous phase, wherein the dispersed phase further
includes a liquid hydrophilic polyol, and wherein each composition in the series of
compositions include an identical amount of proteolytic enzyme and a different amount of the
liquid hydrophilic polyol; (2) determining the enzymatic activity of each composition in the
series of compositions; (3) determining the highest point on a graph that plots the enzymatic
20 activity versus the amount of liquid hydrophilic polyol(s) included in each composition of the
series of compositions, wherein the highest point on the graph correlates to the optimum
amount of liquid hydrophilic polyol to add to the target composition. In one aspect, the
enzymatic activity of the series of compositions can be determined by using the in-vitro
artificial eschnr testing model as described in this specification.
25 [0020] In a further aspect of the present invention there is disclosed a method of
increasing enzymatic activity in a target composition comprising a dispersed phase including
a proteolytic enzyme and a continuous phase including a hydrophobic base, the method
comprising: (1) obtaining a series of compositions comprising the dispersed phase and the
continuous phase, wherein the dispersed phase further includes a liquid hydrophilic polyol,
30 and wherein each composition in the series of compositions includes an identical amount of
proteolytic enzyme and a different amount of the liquid hydrophilic polyol; (2) determining
90753719.1 - 5
the enzymatic activity of each composition in the series of compositions; (3) determining the
highest point on a graph that plots the enzymatic activity versus the amount of liquid
hydrophilic polyol(s) included in each composition of the series of compositions, wherein the
highest point on the graph correlates to an optimum amount of liquid hydrophilic polyol to
5 add to the target composition, and (4) adding ± 10% w/w of the optimum amount of liquid
hydrophilic polyol to the target composition, thereby increasing the enzymatic activity in the
target composition. In one aspect, the enzymatic activity of the series of compositions can be
determined by using the in-vitro artificial eschar testing model as described in this
specification.
10 [0021] The amount of polyol in the series of compositions can vary from each
composition randomly or by a selected amount. In one embodiment, the amount of polyol in
each composition of the series of compositions can be 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,
0.6%,0.7%,0.8%,0.9%,1%,2%,3%,4%,5%,6%,7%, 8%, 9%, 10%, 11 %, 12%, l 3 %o,
14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,
15 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,
46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%,
20 99.8%, 99.9%, or 100% by weight or volume of the composition.
[0022] The term "anhydrous" means that the compositions contain less than about 5%
w/w, or less than about 3% w/w, or less than about 1% w/w, or less than about 0.5% w/w, or
less than about 0.1 % w/w in relation to the total composition, or 0%, of free or added water,
not counting the water of hydration, bound water, or typical moisture levels present in any of
25 the raw ingredients of the compositions.
[0023] Unless otherwise specified, the percent values expressed herein are weight by
weight and are in relation to the total composition.
[0024] The use of the word "a" or "an" when used in conjunction with the term
"comprising" or "containing" in the claims and/or the specification may mean "one," but it is
30 also consistent with the meaning of "one or more," "at least one," and "one or more than one."
90753719.1 0 6
[0025] Throughout this application, the term "about" is used to indicate that a value
includes the inherent variation of error for the device obtaining the value, the method being
employed to determine the value, or the variation that exists among the objects being
evaluated.
5 [0026] As used in this specification and claim(s), the words "comprising" (and any
form of comprising, such as "comprise" and "comprises"), "having" (and any form of having,
such as "have" and "has"), "including" (and any form of including, such as "includes" and
"include") or "containing" (and any form of containing, such as "contains" and "contain") are
inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
10 [0027] The terms "treating," "inhibiting," "preventing, or "reducing" or any variation
of these terms, when used in the claims and/or the specification includes any measurable
decrease or complete inhibition to achieve a desired result.
[0028] The term "effective," as that term is used in the specification and/or claims,
means adequate to accomplish a desired, expected, or intended result.
15 [0029] The compositions and methods for their use can "comprise," "consist
essentially of," or "consist of' any of the ingredients or steps disclosed throughout the
specification. With respect to the transitional phase "consisting essentially of," and in one
non-limiting aspect, a basic and novel characteristic of the compositions and methods
disclosed in this specification includes the composition's enhanced enzymatic activity.
20. [0030] Other objects, features and advantages of the present invention will become
apparent from the following detailed description. It should be understood, however, that the
detailed description and the specific examples, while indicating specific embodiments of the
invention, are given by way of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent to those skilled in the art
25 from this detailed description.
90753719.1 - 7
BRIEF DESCRIPTION OF THE RAWINGS
[0031] FIG. 1. Plot of the in-vitro collagenolysis activity (mg/ml) of a series of
compositions comprising a dispersed phase comprising collagenase and PEG-400, dispersed
in a hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the
5 PEG-400 comprised in the series of compositions (x-axis).
[0032] FIG. 2. Plot of the in-vitro collagenolysis activity (mg/ml) of a series of
compositions comprising a dispersed phase comprising collagenase and PEG-600, dispersed
in a hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the
PEG-600 comprised in the series of compositions (x-axis).
10 [0033] FIG. 3. Plot of the in-vitro collagenolysis activity (mg/ml) of a series of
compositions comprising a dispersed phase comprising collagenase and poloxamer-124,
dispersed in a hydrophobic phase comprising white petrolatum (y-axis) versus the percentage
of the poloxamer-124 comprised in the series of compositions (x-axis).
[0034] FIG. 4. Plot of the in-vitro collagenolysis activity (mg/ml) of a series of
15 compositions comprising a dispersed phase comprising trypsin and PEG-400, dispersed in a
hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the PEG-
400 comprised in the series of compositions (x-axis).
[0035] FIG. 5. Plot of the in-vitro collagenolysis activity (mg/ml) of a series of
compositions comprising a dispersed phase comprising papain and PEG-400, dispersed in a
20 hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the PEG-
400 comprised in the series of compositions (x-axis).
[0036] FIG. 6. Plot of the in-vitro collagenolysis activity (mg/ml) of a series of
compositions comprising a dispersed phase comprising thermolysin and PEG-400, dispersed
in a hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the
25 PEG-400 comprised in the series of compositions (x-axis).
[0037] FIG. 7. Plot of the in-vitro collagenolysis activity (mg/ml) of a series of
compositions comprising a dispersed phase comprising pepsin and PEG-400, dispersed in a
hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the PEG-
400 comprised in the series of compositions (x-axis).
90753719.1 - 8
[0038] FIG. S. Plot of the physical release of collagenase (mg) from of a series of
compositions comprising a dispersed phase comprising collagenase and PEG-400, dispersed
in a hydrophobic phase comprising white petrolatum (y-axis) versus the percentage of the
PEG-400 comprised in the series of compositions (x-axis).
5 [0039] FIG. 9. Enzyme stability in PEG-in-white petrolatum dispersion compared
with oil-in-water emulsion cream.
[0040] FIG. 10. Debridement efficacy in Eschar removal in pig burn wound.
DETAILED DESCRIPTION
[0041] One aspect of the present invention provides for topical enzymatic wound
10 debriding compositions with enhanced enzymatic activity. These compositions comprise a
dispersed phase comprising at least one proteolytic enzyme and a hydrophilic polyol; and a
continuous phase comprising a hydrophobic base. In one aspect of the invention, the
hydrophilic polyol is a liquid hydrophilic polyol.
[0042] It was found that the enzymatic activity (e.g., in vitro collagenolysis) of the
15 compositions of the present invention, which are dispersions of a hydrophilic polyol and a
proteolytic enzyme in a hydrophobic base, not only was higher than the enzymatic activity of
enzyme compositions based solely on a proteolytic enzyme and hydrophob , base
combination (i.e., no hydrophilic phase such as a hydrophilic polyol), but also surprisingly
higher than those enzyme compositions based solely on a proteolytic enzyme and hydrophilic
20 base combination (i, e., no hydrophobic phase such as petrolatum). Since enzymes are
activated in the presence of moisture, it would have been expected to see the highest
enzymatic activity in compositions based solely on a proteolytic enzyme and hydrophilic base
combination, where the base would be completely miscible in moisture and conditions would
be the most favorable for release and activation of the enzyme. However, the dispersion
25 composition of hydrophilic and hydrophobic phases of the present invention had the highest
enzymatic activity correlating to an optimum amount of the hydrophilic polyol which was
more than 0% and less than 100% of the hydrophilic polyol in the composition.
[0043] It was found, expectedly, that the physical enzyme release in compositions
based solely on a hydrophilic vehicle was greater than the release of the enzyme in
30 compositions based solely on a hydrophobic vehicle, and also more than compositions of the
90753719.1 -9
present invention. As seen in FIG. 8, the enzyme release profile generally increased with the
increasing percentage of hydrophilic polyol (PEG-400), with the highest release at 100% and
the lowest release at 0%. However, surprisingly, the enzymatic activity was greater with the
dispersion compositions of the present invention (see FIGS. 1 - 7). Thus the enzymatic
5 activity profile of these dispersion compositions does not correlate with the physical enzyme
release profile as would be expected.
[0044] The compositions of the present invention are suitable for treatment of a
wound in need of debridement by applying to the wound a composition comprising a
dispersed phase comprising a hydrophilic polyol, and an effective debriding concentration of
10 at least one proteolytic enzyme; and a continuous phase comprising a hydrophobic base;
wherein the amount of hydrophilic polyol is within 1 10% w/w of the optimum amount, or 1
9%, 8%, 7%, or 6% w/w of the optimum amount, or ± 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%
w/w of the optimum amount of hydrophilic polyol.
[0045] These and other non-limiting aspects of the present invention are discussed in
15 further detail in the following sections.
A. Method for Determining the Optimum Amount of Liquid Hydrophilic Polyol
[0046] The following protocol can be used to prepare a series of compositions
(referred to as "Series of Compositions") and to subsequently determine the optimum amount
of liquid hydrophilic polyol that can be used in a dispersion of the present invention:
20 [0047] Eleven (11) compositions can be used to create the Series of Compositions.
Note that the amount (% w/w) of proteolytic enzyme in the series of compositions is held
constant. ThQfollowing steps can be used to prepare the eleven (11) compositions:
[0048] (i) Determine the ingredients (i.e., liquid hydrophilic polyol, proteolytic
enzyme, and hydrophobic base) to be used in the Series of Compositions and select the
25 amount of proteolytic enzyme to be used. By way of example, liquid hydrophilic polyol (e.g.,
PEG 400), proteolytic enzyme (e.g., collagenase at 1% w/w), and hydrophobic base (e.g.,
white petrolatum).
[0049] (ii) For composition one in the Series of Compositions, use 0% of the
liquid hydrophilic polyol, use the selected amount of proteolytic enzyme, and q.s the batch
90753719.1 m 10 -
with the hydrophobic base to 100%. For example, and referring to step (i) above, composition
one of the Series of Compositions would have: 0 % w/w PEG 400, 99% w/w of white
petrolatum, and 1% w/w of collagenase.
[0050] (iii) For composition two in the Series of Compositions, use 10% w/w of the
5 liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch with
the hydrophobic base to 100%. (Note that it is permissible to use some solid hydrophilic
polyol in the makeup of the liquid hydrophilic polyol as necessary to produce a physically
stable dispersion for compositions in the Series of Compositions).
[0051] (iv) For composition three in the Series of Compositions, use 20% w/w of
10 the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch
with the hydrophobic base to 100%.
[0052] (v) For composition four in the Series of Compositions, use 30% w/w of
the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch
with the hydrophobic base to 100%.
15 [0053] (vi) For composition five in the Series of Compositions, use 40% w/w of
the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch
with the hydrophobic base to 100%.
[0054] (vii) For composition six in the Series of Compositions, use 50% w/w of the
liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch with
20 the hydrophobic base to 100%.
[0055] (viii) For composition seven in the Series of Compositions, use 60% w/w of
the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch
with the hydrophobic base to 100%.
[0056] (ix) For composition eight in the Series of Compositions, use 70% w/w of
25 the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch
with the hydrophobic base to 100%.
[0057] (x) For composition nine in the Series of Compositions, use 80% w/w of
the liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch
with the hydrophobic base to 100%.
90753719.1 11
[0058] (xi) For composition ten in the Series of Compositions, use 90% w/w of the
liquid hydrophilic polyol, the same amount of the proteolytic enzyme, and q.s. the batch with
the hydrophobic base to 100%.
[0059] (xii) For composition eleven in the Series of Compositions, use 0% of the
5 hydrophobic base, the same amount of the proteolytic enzyme, and q.s. the batch with the
hydrophilic polyol.
[0060] (xiii) determine the enzymatic activity of each of the eleven compositions in
the Series of Compositions by using the in vitro artificial eschar testing model for the
following sample collection times: 6, 12, 18 and 24 hours, as described in Section H of the
10 Detailed Description section of this specification.
[0061] (ivx) plot a curve of the enzymatic activity of each composition versus the
correlating amount of liquid hydrophilic polyol(s) present in each composition of the Series of
Compositions cumulatively for each data collection time. The highest point on the curve for
the cumulative 24-hour data collection time correlates to the optimum amount of liquid
15 hydrophilic polyol that can be used in a dispersion.
[0062] Further, given that multiple ingredients can be included in the Series of
Compositions (e.g., polyol(s) proteolytic enzyme(s), hydrophobic base, and additional
ingredients within the dispersed phase, and/or additional ingredients within the con sinuous
hydrophobic phase), the Series of Compositions can be created by (1) varying the amount of
20 hydrophilic polyol as discussed above for each composition in the series, (2) using the
determined amount of proteolytic enzyme, and (3) q.s.-ing the batch to 100% with the amount
of the additional ingredients including the hydrophobic base; except for composition eleven,
where the batch would be q.s.-ed to 100% with the amount of the additional ingredients
including the hydrophilic polyol.
90753719.1 - 12-
B. Method for Determining Whether A Composition Has f/- 10% w/w of the
Optimum Amount of Liquid Hydrophilic Polyol
[0063] It can be determined if a composition comprising (a) a dispersed phase
including a liquid hydrophilic polyol and at least one proteolytic enzyme; and (b) a continuous
5 phase comprising a hydrophobic base (referred to as "Composition of Interest") is within ±
10% of the Optimum Amount of liquid hydrophilic polyol by using the following protocol:
[0064] Step One: Obtain a Composition of Interest that includes: (i) a dispersed phase
including a liquid hydrophilic polyol(s) and a proteolytic enzyme and (ii) a continuous phase
including a hydrophobic base.
10 [0065] Step Two: Prepare a series of compositions (referred to as "Series of
Compositions") based on the Composition of Interest. Note that the amount (% w/w) of
proteolytic enzyme in the Series of Compositions is held constant and is the same as the
amount (% w/w) present in the Composition of Interest. The following steps can be used to
prepare the Series of Compositions:
15 [0066]
(% w/w).
(i) Determine the amount of all ingredients in the Composition of Interest
[0067] (ii) Determine the total amount of the continuous phase in the Composition
of Interest (% w/w). By way of example, if the Composition of Interest includes I YA w/w
liquid hydrophilic polyol (e.g., PEG 400), 1% w/w proteolytic enzyme (e.g., collagenase), and
20 84% w/w hydrophobic base (e.g., white petrolatum), then the Composition of Interest would
be 84% w/w continuous phase and 16 %w/w dispersed phase.
[0068] Step Three: Prepare the Series of Compositions in a manner described above
in Section A-of this specification (e.g., this would include preparing 11 compositions in a
manner described in Section A of this specification).
25 [0069] Step Four: Determine the enzymatic activity of each of the eleven
compositions in the Series of Compositions by using the in vitro artificial eschar testing model
for each of the following sample collection times: 6, 12, 18 and 24 hours as described in
Section H of the Detailed Description section of this specification.
[0070] Step Five: Plot a curve of the enzymatic activity of each composition
30 versus the correlating amount of liquid hydrophilic polyol(s) present in each composition of
90753719.1 - 13 -
the Series of Compositions cumulatively for each data collection time. The highest point on
the curve for the cumulative 24-hour data collection time correlates to the optimum amount of
liquid hydrophilic polyol for the Composition of Interest.
[0071] Step Six: Compare the amount of liquid hydrophilic polyol present within
5 the Composition of Interest to determine whether it is within 1 10% w/w of the optimum
amount of liquid hydrophilic polyol for the Composition of Interest.
C. Proteolytic Enzymes
[0072] Any proteolytic enzyme useful for wound debridement is suitable for the
present invention. Proteolytic enzymes (proteases) break down protein by hydrolysis of the
10 peptide bonds that link amino acids together in the polypeptide chain of a protein. They are
divided into four major groups on the basis of catalytic mechanism: serine proteases, cysteine
proteases. metalloproteases, and aspartic proteases. Some proteases have been identified with
other catalytic amino acids in the active site, such as threonine and glutamic acid; however,
they do not form major groups.
15 1. Serine proteases
[0073] Serine proteases depend upon the hydroxyl group of a serine residue acting as
the nucleophile that attacks the peptide bond. The major clans found in humans iiicAude the
chymotrypsin-like, the subtilisin-like, the alpha/beta hydrolase, and signal peptidase clans. In
evolutionary history, serine proteases were originally digestive enzymes. In mammals, they
20 evolved by gene duplication to serve functions in blood clotting, the immune system, and
inflammation. These proteases have a broad substrate specificity and work in a wide pH
range. Non4imiting examples of serine proteases include trypsin, chymotrypsin, subtilisin,
sutilains, plasmin, and elastases.
2. Cysteine Proteases
25 [0074] Peptidases in which the nucleophile that attach the scissile peptide bond in the
sulfhydryl group of a cysteine residue are known as cysteine proteases. Cysteine proteases are
commonly encountered in fruits including papaya, pineapple, and kiwifruit. Cysteine
proteases have a broad specificity and are widely used under physiological conditions. In this
family, papain has been used extensively for wound debridement for a long time. Other
90753719.1 - 14 -
cysteine proteases, such as bromelain and analain, have also been investigated for the
applications in wound debridement. Other non-limiting examples of cysteine proteases
include calpain, caspases, chymopapain, and clostripain.
3. Metalloproteases
5 [0075] Metalloproteases are among the proteases in which the nucleophilic attach on a
peptide bond is mediated by a water molecule, while a divalent metal cation, usually zinc but
sometimes cobalt, manganese, nickel or copper, activates the water molecule. The metal ions
are extremely important for the activity. Any compounds that have potential to interact with
the metal ion, chelating or oxidation, will affect the enzymatic activity. Non-limiting
10 examples of metalloproteases in this family include thermolysin, collagenases, matrix metallo
proteinases (MMPs), bacillolysin, dispase, vibriolysin, pseudolysin, stromelysin, and various
bacterial derived neutral metalloproteases.
4. Aspartic Peptidases
[0076] Aspartic peptidases are so named because aspartic acid residues are the ligands
15 of the activated water molecule. In most enzymes in this family, a pair of aspartic residues act
together to bind and activate the catalytic water molecule. All or most aspartic peptidases are
endopeptidases. Most aspartic peptidases have a broad specificity. However, the optimum pH
of most aspartic peptidases is in the acidic range. Non-limiting examples of aspartic
peptidases are pepsin, chymosin, beta-secretase, plasmepsin, plant acid proteases and
20 retroviral proteases.
5. Collagenase
[0077] A suitable proteolytic enzyme for wound debridement is the metalloprotease
collagenase. The collagenase can be substantially pure or it may contain detectable levels of
other proteases.
25 [0078] The potency assay of collagenase, and meaning of "collagenase unit:" as used
herein, is based on the digestion of undenatured collagen from (bovine Achilles tendon) at pH
7.2 and 37°C for 24. hours. The number of peptide bonds cleaved is measured by reaction
with ninhydrin. Amino groups released by a trypsin digestion control are subtracted. One net
90753719.1 - 15 -
collagenase unit will solubilize ninhydrin reactive material equivalent to 1 nanomole of
leucine equivalents per minute.
[0079] The amount (potency or concentration) of collagenase in the compositions of
the present invention is at an effective level to debride the wound. Generally, the potency of
5 collagenase in the compositions can vary from about 1 to about 10,000 collagenase units per
gram of product, based on the activity of the collagenase used in the product. In various
embodiments, the potency, expressed as collagenase units per gram of product, is from about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,
100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230,
10 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,
950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500,
8000, 8500, 9000, 9500 to about 10000, or any range or numerical amount derivable therein.
[0080] The concentration of collagenase in the compositions generally can vary from
about 0.001% w/w to about 8% w/w. In various embodiments, the concentration, expressed
15 as percentage weight by weight, is from about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007,
0.008, 0.009, 0.010 0.015, 0.020, 0.025, 0.030, 0.035, 0.040, 0.050, 0.055, 0.060, 0.065,
0.070, 0.075, 0.080, 0.085, 0.090, 0.095, 0.100, 0.125, 0.150, 0.175, 0.20, 0.25, 0.30 ,0.35,
0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90, 0.95, 1, 2, 3, 4, 5, 6, 7 to about 8
or any range or numerical amount derivable therein.
20 [0081] In one embodiment, the collagenase is derived from Clostridium histolyticum;
however, in other embodiments the collagenase can be derived from other sources. Methods
for producing a suitable collagenase are disclosed in US patents 3,705,083; 3,821,364;
5,422,261; 5,332,503; 5,422,103; 5,514,370; 5,851,522; 5,718,897; and 6,146,626 all of
which are herein incorporated by reference.
25 6. Trypsin
[0082] Another suitable proteolytic enzyme for wound debridement is the serine
protease trypsin. Typically, trypsin is derived from the pancreas of healthy bovine or porcine
animals, or both. Trypsin can also be derived from recombinant sources. The pharmaceutical
grade (USP/NF) of trypsin is known as Crystallized Trypsin. It contains not less than 2500
30 USP Trypsin Units per mg, calculated on the dried basis, and not less than 90.0% and not
more than 110.0% of the labeled potency. The potency assay of trypsin as well as the
90753719.1 -16-
definition of a USP Trypsin Unit are found in the Crystallized Trypsin monograph of the USP
31 (Official August 1, 2008) herein incorporated by reference.
[0083] The amount (potency or concentration) of trypsin in the compositions of the
present invention is at an effective level to debride the wound. Generally, the potency of
5 trypsin in the compositions can vary from about 90 to about 60,000 USP Trypsin Units per
gram of product. In various embodiments the potency of trypsin, expressed as USP Trypsin
Units per gram of product, is from about 90, 100, 150, 200, 250, 300, 320, 350, 375, 400, 500,
600, 675, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 10000, 20000, 30000, 40000, 50000
to about 60000, or any range or numerical amount derivable therein.
10 [0084] The concentration of trypsin in the compositions generally can vary from about
0.0025% w/w to about 1% w/w. In various embodiments, the concentration of trypsin,
expressed as percent weight by weight, is from about 0.0025, 0.0050, 0.010, 0.015, 0.020,
0.025, 0.030, 0.035, 0.040, 0.045, 0.050, 0.055, 0.060, 0.065, 0.070, 0.075, 0.080, 0.085,
0.090, 0.095, 0.10, 0.15, 0.20 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75,
15 0.80, 0.85, 0.90, 0.95 to about 1, or any range or numerical amount derivable therein.
D. Hydrophilic Polyols
[0085] Hydrophilic polyols of the present invention are water-soluble, polar aliphatic
alcohols with at least two hydroxyl groups, and include polymeric polyols, e.g., pol ycihylene
glycols and poloxamers. In one aspect of the invention, the hydrophilic polyol in the
20 dispersed phase is a liquid hydrophilic polyol. In some embodiments, the liquid hydrophilic
polyol is a liquid polyethylene glycol or a liquid poloxamer, or mixtures thereof. Solid
hydrophilic polyols such as solid polyethylene glycols or solid poloxamers can also be added
to the dispersed phase of the invention to help physically stabilize the dispersion. Other
examples of liquid hydrophilic polyols include but are not limited to propylene glycol,
25 butylene glycol, pentylene glycol, hexylene glycol, glycerin, hexylene glycol, methoxy
polyethylene glycol, propylene carbonate, and ethoxydiglycol, and these may also be added to
the dispersed phase.
90753719.1 - 17-
1. Polyethylene Glycols
[0086] Polyethylene glycols are homo-polymers of ethylene glycol and water
represented by the formula:
H(OCH2CH2)„ OH
5 in which n represents the average number of oxyethylene groups. Polyethylene glycols can be
either liquid or solid at 25 °C depending on their molecular weights.
[0087] The following suitable non-limiting examples of liquid polyethylene glycols
are described using USP nomenclature: polyethylene glycol 200, polyethylene glycol 300,
polyethylene glycol 400, polyethylene glycol 500, and polyethylene glycol 600.
10 [0088] The following suitable non-limiting examples of solid polyethylene glycols are
described using USP nomenclature: polyethylene glycol 700, polyethylene glycol 800,
polyethylene glycol 900, polyethylene glycol 1000, polyethylene glycol 1100, polyethylene
glycol 1200, polyethylene glycol 1300, polyethylene glycol 1400, polyethylene glycol 1450,
polyethylene glycol 1500, polyethylene glycol 1600, polyethylene glycol 1700, polyethylene
15 glycol 1800, polyethylene glycol 1900, polyethylene glycol 2000, polyethylene glycol 2100,
polyethylene glycol 2200, polyethylene glycol 2300, polyethylene glycol 2400, polyethylene
glycol 2500, polyethylene glycol 2600, polyethylene glycol 2700, polyethylene glycol 2800,
polyethylene glycol 2900, polyethylene glycol 3000, polyethylene glycol 3250, polyethylene
glycol 3350, polyethylene glycol 3750, polyethylene glycol 4000, polyethylene glycol 4250,
20 polyethylene glycol 4500, polyethylene glycol 4750, polyethylene glycol 5000, polyethylene
glycol 5500, polyethylene glycol 6000, polyethylene glycol 6500, polyethylene glycol 7000,
polyethylene glycol 7500, and polyethylene glycol 8000.
[0089] The liquid and solid polyethylene glycols are available commercially from the
DOW Chemical Company under the CARBOWAXTM tradename and from the BASF
25 Corporation under LUTROL® E and PLURACARE® E tradenames. Both pharmaceutical
grade (USP/NF) and cosmetic grade polyethylene glycols are suitable for the present
invention.
90753719.1 - 18 -
2. Poloxaners
[0090] Poloxamers are synthetic block copolymers of ethylene oxide and propylene
oxide represented by the formula:
HO(C2H40)a(C3H60) b(C2H40)aH
5 in which formula a and b represent the number of repeat units. Generally a is from 2 to 150
and b is from 15 to 70 depending on the particular poloxamer. Poloxamers can be either
liquid or solid at 25 °C depending on their molecular weights.
[0091] The following suitable non-limiting examples of liquid poloxamers are
described using CTFA/INCI nomenclature: poloxamer 101, poloxamer 105, poloxamer 122,
10 poloxamer 123, poloxamer 124, poloxamer 181, poloxamer 182, poloxamer 183, poloxamer
184, poloxamer 212, poloxamer 231, poloxamer 282, poloxamer 331, poloxamer 401, and
poloxamer 402.
[0092] The following suitable non-limiting examples of solid poloxamers are
described using CTFA/INCI nomenclature: poloxamer 108, poloxamer 188, poloxamer 217,
15 poloxamer 237, poloxamer 238, poloxamer 288, poloxamer° 338, poloxamer 407, poloxamer
185, poloxamer 215, poloxamer 234, poloxamer 235, poloxamer 284, poloxamer 333,
poloxamer 334, poloxamer 335, and poloxamer 403.
[0093] The liquid and solid poloxamers are available commercially from the BASF
Corporation under the PLURONIC® and LUTROL® tradenames and from the UNIQEMA
20 Corporation under the SYNPERONIC® trademark. Pharmaceutical grade (USP/NF)
poloxamers are poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and
poloxamer 407. Both pharmaceutical grade and cosmetic grade poloxamers are suitable for
the present invention.
90753719.1 _19-
E. Hydrophobic Bases
[0094] The hydrophobic bases of the present invention can comprise, but are not
limited to, plant, animal, paraffinic, and synthetic derived fats, butters, greases, waxes,
solvents, and oils; mineral oils, vegetable oils, petrolatum, water insoluble organic esters and
5 triglycerides, silicones, or fluorinated compounds; or mixtures thereof. In one embodiment of
the present invention the hydrophobic phase comprises petrolatum.
[0095] Plant derived materials include, but are not limited to, arachis (peanut) oil,
balsam Peru oil, carnauba wax, candellila wax, castor oil, hydrogenated castor oil, cocoa
butter, coconut oil, corn oil, cotton seed oil, jojoba oil, macadamia seed oil, olive oil, orange
10 oil, orange wax, palm kernel oil, rapeseed oil, safflower oil, sesame seed oil, shea butter,
soybean oil, sunflower seed oil, tea tree oil, vegetable oil, and hydrogenated vegetable oil.
[0096] Non-limiting examples of animal derived materials include beeswax, cod liver
oil, emu oil, lard, mink oil, shark liver oil, squalane, squalene, and tallow.
[0097] Non-limiting examples of paraffinic materials include isoparaffin,
15 microcrystalline wax, heavy mineral oil, light mineral oil, ozokerite, petrolatum, and paraffin.
[0098] Suitable non-limiting examples of organic esters and triglycerides include C12-
15 alkyl benzoate, isopropyl myristate, isopropyl palmitate, medium chain triglycerides,
trilaurin, and trihydroxystearin.
[0099] Non-limiting examples of silicones are dimethicone and cycloinethicone. A
20 non-limiting example of a fluorinated compound is polytetrafluoroethylene (PTFE).
1. Petrolatum
[00100] Petrolatum is a purified mixture of semisolid hydrocarbons obtained from
petroleum and varies from dark amber to light yellow in color. White petrolatum is wholly or
nearly decolorized petrolatum and varies from cream to snow white in color. Petrolatum and
25 White Petrolatum can also vary in melting point, viscosity, and consistency.
[0100] Various grades are available commercially from the PENRECO Corporation
under the tradenames : PENRECO°ULTIMA, PENRECO°SUPER, PENRECO®SNOW,
PENRECO°REGENT, PENRECO°LILY, PENRECO't CREAM, PENRECO°ROYAL,
90753719.1 -20-
PENRECO "BLOND, and PENRECO®AMBER. Various grades are also available
commercially from the SONNEBORN Corporation under the tradenames : ALBA®, SUPER
WHITE PROTOPET®, SUPER WHITE FONOLINE®, WHITE PROTOPET 1 S®, WHITE
PROTOPET 20, WHITE PROTOPET 3C®, WHITE FONOLINE®, PERFECTA®,
5 YELLOW PROTOPET 2A®, YELLOW FONOLINE®, PROTOLINE", SONOJELL #40 ,
SONOJELL #9®, MINERAL JELLY # 10®, MINERAL JELLY # 14®, MINERAL JELLY
#17®, AND CARNATION TROUGH GREASE®.
[0101] Petrolatum and White Petrolatum are available in cosmetic grade and
pharmaceutical (USP/NF) grade and both are suitable for the present invention.
10 F. Topical Compositions
[0102] The topical compositions of the present invention are dispersions comprising a
hydrophilic' dispersed phase in a hydrophobic continuous phase. The dispersed phase
comprises a proteolytic enzyme and a hydrophilic polyol. In an aspect of the invention, the
hydrophilic polyol is a liquid hydrophilic polyol. In some embodiments, the liquid
15 hydrophilic polyol is a liquid polyethylene glycol or a liquid poloxamer, or mixtures thereof.
The continuous phase comprises a hydrophobic base. The hydrophobic base can be
petrolatum. The compositions are useful for treatment of wounds for wound debridement.
[0103] The compositions can be anhydrous as defined herein. The compose ! ions can
be semisolid or liquid. The composition can be impregnated on a pad, gauze, or sponge. The
20 compositions can also be, sterile.
[0104] The compositions can include additional materials known in the art that are
suitable for topical compositions of this nature, e.g., absorbents, deodorizers, surfactants,
solvents, rheology modifiers, film formers, stabilizers, emollients, moisturizers, preservatives,
antimicrobials, antioxidants, chelating agents, fragrances, and colorants.
25 [0105] The compositions can also include additional pharmaceutical active ingredients
known in the art that are suitable for topical compositions of this nature, e.g., antimicrobial
agents, wound healing agents, anesthetic agents, vulnerary agents, and haemostatic agents. A
non-limiting example of a vulnerary agent is balsam Peru.
[0106] The compositions can be packaged in any package suitable for dispensing a
30 wound debrider. The compositions can be packaged in multi-use, single-dose, or metered
90753719.1 - 21 -
dose packages. Non-limiting examples include a tube, bottle, jar, pump container, pressurized
container, bladder container, aerosol container, aerosol spray container, non-aerosol spray
container, syringe, pouch, or sachet.
G. Manufacturing Process
5 [0107] The compositions of the present invention can be prepared by techniques and
methods known by one of ordinary skill in the art by dissolving or suspending the proteolytic
enzyme in part or all of the available hydrophilic polyol. The resulting solution or suspension
can be mixed with a hydrophobic base to form a dispersion, wherein the hydrophobic base
becomes the continuous phase and the hydrophilic polyol/enzyme phase becomes the
10 dispersed phase. These compositions can be prepared using processing equipment known by
one of ordinary skill in the art, e.g., blenders, mixers, mills, homogenizers, dispersers,
dissolvers, etc.
H. In vitro Artificial Eschar Testing Model
[0108] Enhancement of the enzymatic activity of the compositions was established by
15 testing the compositions using an in vitro artificial eschar model as described below and in the
publication "Study on the debridement efficacy of formulated enzymatic wound debriding
agents by in vitro assessment using artificial wound eschar and by an in vivo pig model", Shi
et. al., Wound Repair Regen, 2009, 17(6):853, herein incorporated by reference . Bovine
collagen (Type I), bovine fibrinogen, and elastin were used to make an Artificial Wound
20 Eschar (AWE) substrate . Collagen-FITC labeled, elastin-rhodamine, and fi brin-coumarin
were the raw materials used for producing the AWE substrate . To prepare 1 gram of AWE
substrate , 650 mg Collagen-FITC and 100 mg each of elastin-rhodamine and fibrin-coumarin
were weighed into a 50 mL tube and homogenized in 10 mL of Tris buffer saline. In a
separate tube, 10 mL of fibrinogen solution was prepared at 15 mg /mL with Tris buffer saline.
25 The two solutions were combined and thoroughly mixed. A thrombin solution (0.25 mL at 50
U/mL) was added, quickly mixed, and the solution was poured into a Petri dish containing a
90 mm nonreactive membrane filter. As a result of the thrombin-induced fibrinogen
polymerization, the material began to form a soft sheet on top of the membrane filter by
clotting the dyed proteins into a solid matrix. The clotted AWE substrate was allowed to
30 solidify for 30 minutes and then rinsed with water for 15 minutes to remove the thrombin.
The AWE substrate was further dehydrated to 75% moisture content in preparation for use.
90753719.1 -22-
[0109] With the AWE substrate still attached to the membrane, a 35 mm diameter
piece was punched out using a hole punch. The AWE substrate punch was placed on the top
flat face of a Franz Diffusion Cell System (Hanson Research, Chatsworth, CA), and a
TEFLONOO sample holder placed on top. The debriding ointment samples were loaded in the
5 center of the sample holder, and any excess sample was removed by scraping. The solution in
the receptor cells was Tris buffer at a pH of 7.4 for samples containing collagenase, papain,
thermolysin, or trypsin; and was sodium acetate buffer at a pH of 2 for samples containing
pepsin. The solution in receptor cells was sampled in 1 mL increments at the following
sample collection times: 0, 1, 2, 3, 6, 12, 18 and 24 hours. Once finished, the samples were
10 analyzed by fluorescence measurement of FITC dye at 485nm (excitation wavelength) and
520nm (emission wavelength) to determine the digestion of collagen (collagenolysis) reported
in mg/ml.
1. In-vitro Physical Enzyme Release Test
[0110] The release of enzyme from the compositions was determined by a Franz cell
15 diffusion study using PVDF (0.45 micron) filters. This study was performed at 35°C and
lasted for 6 hours. The solution samples in the receptor cells were subjected to a total protein
analysis.
[0111] The protein concentration was determined by a BCA assay (Peirce) while the
same collagenase was used as the reference standard. The details are described as follows.
20 [0112] The BCA Protein Assay combines the well-known reduction of Cu2+ to Cul+
by protein in an alkaline medium with the highly sensitive and selective colorimetric detection
of the cuprous cation (Cu1+) by bicinchoninic acid. The first step is the chelation of copper
with protein in an alkaline environment to form a blue-colored complex. In this reaction,
known as the biuret reaction, peptides containing three or more amino acid residues form a
25 colored chelate complex with cupric ions in an alkaline environment containing sodium
potassium tartrate. This became known as the biuret reaction because a similar complex forms
with the organic compound biuret (NH2-CO-NH-CO-NH2) and the cupric ion. Biuret, a
product of excess urea and heat, reacts with copper to form a light blue tetradentate complex.
In the second step of the color development reaction, BCA, a highly sensitive and selective
30 colorimetric detection reagent reacts with the cuprous cation (Cu') +that was formed in step 1.
The purple-colored reaction product is formed by the chelation of two molecules of BCA with
90753719.1 - 23 -
one cuprous ion. The BCA/copper complex is water-soluble and exhibits a strong linear
absorbance at 562 nm with increasing protein concentrations. The purple color may be
measured at any wavelength between 550 nm and 570 nm with minimal (less than 10%) loss
of signal. See the following reference herein incorporated by reference: Smith, P.K., Krohn,
5 R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K.,
Goeke, N.M., Olson, B.J. and Klenk, D.C. (1985). Measurement of protein using
bicinchoninic acid. Anal. Biochem. 150, 76-85.
EXAMPLES
[0113] The following examples are included to demonstrate certain non-limiting
10 aspects of the invention. It should be appreciated by those of skill in the art that the
techniques disclosed in the examples which follow represent techniques discovered by the
applicants to function well in the practice of the invention. However, those of skill in the art
should, in light of the present disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or similar result without
15 departing from the spirit and scope of the invention.
Example 1: Dispersions of Collagenase/PEG 400 in Petrolatum
[0114] The dispersions in TABLE 1 were prepared with varying concentrations of
Polyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.
TABLE 1
20
Dispersion PEG 400
%w/w
PEG-1450
%w/w
Wht Petrolatum
%w/w
Poloxamer 407
%w/w
Collagenase
%w/w
A 0 0 99.8 0 0.2
B 10 0 89.8 0 0.2
C 15 0 84.8 0 0.2
D 20 0 79.9 0.4 0.2
E 30 0 69 0.9 0.2
F 50 0 49.3 1.2 0.2
G 68 0 29.8 2.0 0.2
H* 83 12.5 4.5 0 0.2
I* 70 29.8 0 0 0.2
and 100% respectively.
[0115] The enzymatic debridement activity of each dispersion was determined by the
in-vitro artificial eschar model described above and the results plotted in FIG. 1. As can be
90753719.1 -24-
seen by the results in FIG. 1, the optimum amount of PEG-400 based on the 24 hour curve is
about 20% w/w PEG-400.
Example 2: Dispersions of Collagenase/PEG 600 in Petrolatum
[0116] The dispersions in TABLE 2 were prepared with varying concentrations of
5 Polyethylene Glycol 600 (PEG-600) dispersed in Petrolatum.
TABLE 2
Dispersion PEG 600
%w/w
Wht Petrolatum
%w/w
'Poloxamer 407
%w/w
Collagenase
%w/w
J 0 99.8 0 0.2
K 10 89.525 0.275 0.2
L 20 79.248 0.552 0.2
M 30 68.973 0.827 0.2
N 50 48.42 1.38 0.2
0 80 17.59 2.21 0.2
P 97 0 2.8 0.2
[0117] The enzymatic debridement activity of each dispersion was determined by the
10 in-vitro artificial eschar model described above. The results are plotted in FIG. 2. As can be
seen by the results in FIG. 2, the optimum amount of PEG-600 based on the 24 hour curve is
about 30% w/w PEG-600.
Example 3 Dispersions of Collagenase/Poloxamer 124 in Petrolatum
[0118] The dispersions in TABLE 3 were prepared with varying concentrations of
15 Poloxamer 124 dispersed in Petrolatum.
TABLE 3
Dispersion Poloxamer 124
%w/w
Wht Petrolatum
%w/w
Poloxamer 407
%w/w
Collagenase
%w/w
Q 0 99.8 0 0.2
R 10 89.8 0 0.2
S 20 79.8 0 0.2
T 30 69.8 0 0.2
U 50 48.14 1.66 0..2
V 80 17.14 2.66 0.2
W 85* 0 15 0.2
* Poloxamer 407 was added to Poloxamer 124 to form a semi-solid resulting in approximate
total of Poloxamer of 100%
20
90753719.1
-25-
[0119] The enzymatic debridement activity of each dispersion was determined by the
in-vitro artificial eschar model described above. The results are plotted in FIG. 3. As can be
seen by the results in FIG. 3, the optimum amount of Poloxamer 124 based on the 24 hour
curve is about 30% w/w Poloxamer 124.
5 Example 4: Dispersions of Trypsin/PEG 400 in Petrolatum
[0120] The dispersions in TABLE 4 were prepared with varying concentrations of
Polyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.
TABLE 4
Dispersion PEG 400
%w/w
PEG 1450
%w/w
Wht Petrolatum
%w/w
Poloxamer 407
%w/w
Trypsin
%w/w
X 0 0 99.8 0 0.2
Y 14 0 84.9 0.4 0.2
Z 29 0 69.8 0.9 0.2
AA 59 0. 39.16 1.64 0.2
BB 80 0 17.06 2.74 0.2
CC 82* 15.2 0 2.6 0.2
10 *PEG- 1450 was added to PEG-400 to forma semi-solid resulting in approximate total PEG of
97%
[01211 The enzymatic debridement activity of each dispersion was determined by the
in-vitro artificial eschar model described above. The results are plotted in FIG. 4. As can be
seen by the results in FIG. 4, the optimum amount of PEG-400 based on the 24 hour curve is
15 about 14% w/w PEG-400.
Example 5: Dispersions of Papain/PEG 400 in Petrolatum
[0122] The dispersions in TABLE 5 were prepared with varying concentrations of
Polyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.
TABLES
20
Dispersion PEG 400
%w/w
PEG 1450
% w/w
Wht Petrolatum
%w/w
Poloxamer 407
%w/w
Papain
%w/w
DD 0 0 99.85 0 0.15
EE 15 0 85.05 0.4 0.15
FF 29 0 69.82 0.83 0.15
GG 43 0 54.85 1.24 0.15
HH 59 0 39.694 1.636 0.15
II 82* 15.01 0 2.67 0.15
*PEG-1450 was added to PEG-400 to form a semi-solid resulting in approximate total PEG of 97%
90753719.1 -26-
[0123] The enzymatic debridement activity of each dispersion was determined by the
in-vitro artificial eschar model described above. The results are plotted in FIG. S. As can be
seen by the results in FIG. 5, the optimum amount of PEG-400 based on the 24 hour curve is
about 29% w/w PEG-400.
5 Example 6: Dispersions of Thermolysin/PEG 400 in Petrolatum
[0124] The dispersions in TABLE 6 were prepared with varying concentrations of
Polyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.
TABLE 6
Dispersion PEG 400
%w/w
PEG 1450
% w/w
Wht Petrolatum
%w/w
Poloxamer 407
%w/w
Thermolysin
%w/w
if 0 0 99.85 0 0.15
KK 14 0 85.05 0.4 0.15
LL 29 0 69.82 0.83 0.15
MM 59 0 39.694 1.636 0.15
NN 82* 15.01 0 2.67 0.15
10 *PEG-1450 was added to PEG-400 to form a semi-solid resulting in approximate total PEG of 97%
[0125] The enzymatic debridement activity of each dispersion was determined by the
in-vitro artificial eschar model described above. The results are plotted in FIG. 6. As can be
seen by the results in FIG. 6, the optimum amount of PEG-400 based on the 24 hour curve is
15 about 29% w/w PEG-400.
Example 7: Dispersions of Pepsin/PEG 400 in Petrolatum
[0126] The dispersions in TABLE 7 were prepared with varying concentrations of
Polyethylene. Glycol 400 (PEG-400) dispersed in Petrolatum.
TABLE 7
20
Dispersion PEG 400
%w/w
PEG 1450
% w/w
Wht Petrolatum
%w/w
Poloxamer 407
%w/w
Pepsin
%w/w
00 0 0 99 0
PP 15 0 84.2 0.4 1
QQ 29 0 68.97 0.83 1
RR 44 0 54.005 1.24 1
SS 58 0 38.844 1.636 1
TT 81* 15.01 0 2.67 1
*PEG-1450 was added to PEG-400 to form a semi-solid resulting in approximate total PEG of 96%
90753719.1 -27-
[0127] The enzymatic debridement activity of each dispersion was determined by the
in-vitro artificial eschar model described above. The results are plotted in FIG. 7. As can be
seen by the results in FIG. 7, the optimum amount of PEG-400 based on the 24 hour curve is
about 58% w/w PEG-400.
5 Example 8: Dispersions of Collagenase/PEG 400 in Petrolatum for Physical Release of
Enzyme
10
[0128] The dispersions in TABLE 8 were prepared with varying concentrations of
Polyethylene Glycol 400 (PEG-400) dispersed in Petrolatum.
TABLE 8
Dispersion PEG 400
%w/w
PEG 1450
% w/w
Wht Petrolatum
%w/w
Collagenase
%w/w
UU 0 0 99.8 0.2
W 5 0 94.8 0.2
WW 10 0 89.8 0.2
XX 15 0 84.8 0.2
YY 83* 12.5 4.5 0.2
ZZ 70* 29.8 0 0.2
*PEG-1450 was added to PEG-400 to form a semi-solid resulting in approximate total PEG of 83%
and 100% respectively.
[0129] The physical release of enzyme was determined by the In-vitro Physical
Enzyme Release Test as described above. The results are plotted in FIG. 8. As can he seen
15 by the results in FIG. 8, the physical release of collagenase generally increased as the
concentration of PEG-400 in the dispersion increased with the highest release at 100% and the
lowest release at 0% PEG-400.
[0130] As can be seen by the results shown herein, the physical enzyme release profile
of the dispersions as a function of increased concentration of hydrophilic polyol does not
20 correlate to the enzymatic activity profile of the enzyme as a function of increased
concentration of hydrophilic polyol.
90753719.1 - 28 -
Example 9: Stability and Efficacy Data
[0131] FIG. 9 provides data comparing the stability of collagenase in a dispersion of
the present invention ("30% PEG in WP dispersion") and an oil-in-water emulsion ("Aqueous
cream"). These data suggest that collagenase was more stable in the 30% PEG in WP
5 dispersion when compared to the Aqueous cream. Tables 9-10 provide descriptions of the
30% PEG in WP dispersion and Aqueous cream formulations.
TABLE 9 (30% PEG in WP dispersion)*
Ingredients wt%
PEG-600 30.059774
Poloxamer-407 1.5078044
White Petrolatum 68.309516
Collagenase 0.1228163
TOTAL 100
10
15
*PEG in WP dispersion was prepared as follows: (A) Active Phase: (1) 9.71 grams of PEG-
600 and 0.2361 grams of collagenase were mixed for 20 minutes at room temperature (20-
25°C) for 45 min. (B) Main Phase: (1) 102.784 grams of white petrolatum, 37.65 grams of
PEG-600, and 2.27 grams of poloxamer-407 were mixed at 70°C until uniform; (2) the
mixture was cooled to 40-45°C. Added 7.79 grams of the Active Phase was added to the
Main Phase followed by stirring for 30 minutes or until homogenous mixture obtained.
TABLE 10 (Aqueous cream)*
Ingredients wt%
Isopropyl Myristate 30.57437
Emulsifying Wax 4.502116
White Petrolatum 20.369574
Incro uat TMS 4.502116
Water 20.009404
Glycerin (96%) 19.839324
Collagenase 0.2030955
TOTAL 100
20
*Aqueous cream was prepared as follows: (A) Active Phase: (1) 0.2 grams of collagenase was
mixed with 20 grams of deionized water. (B) Main Phase: (1) 20.36 grams of white
petrolatum was mixed with 4.5 grams of emulsifying wax, 4.5 grams of Incroquate TMS, and
19.83 grams of glycerin (96%) at 70°C until uniform; (2) the mixture was cooled to 35-40°C.
Added Active Phase to Main Phase followed by stirring for 30 minutes or until homogenous
mixture obtained.
90753719.1 -29-
[0132] FIG. 10 provides data comparing enzyme debridement efficacy in eschar
removal in pig bum wounds of a dispersion of the present invention ("PEG-in-White
Petrolatum"-Table 11) to the following three formulations: (1) an Aqueous cream-Table
12; (2) SANTYLOO ("Commercial product", which is a mixture of collagenase and white
5 petrolatum); and a hydrogel formulation-Table 13. The burn wounds were created on pigs
and hard eschars formed after several days. Formulation was applied to the hard eschars one a
day for two weeks. Only fully debrided wounds were counted as "complete debridement."
There were a total of 20 wounds per treatment.
10
TABLE 11 (PEG-in-White Petr° olatum)*
ingredients wt%
Poloxamer-407 0.99891551
White Petrolatum 78.7544989
Thermolysin 0.20168104
PEG-600 20.0449046
TOTAL 100
15
*PEG-in-White Petrolatum was prepared as follows: (A) Active Phase: (1) 32.67 grams of
PEG-600 and 1,63 grams of Poloxamer-407 were homogenized at 70°C until mixture was
clear; (2) mixture was cooled to about 35°C.; and (3) thermolysin was and mixed for at least
30 min.. (B) Main Phase: (1) 236.52 grams of white petrolatum, 30.05grams of PEG-600, and
1.5 grams of poloxamer-407 were homogenized at 70°C; and (2) mixture was cooled to about
35°C. The Active Phase (B) was added to the Main Phase (B) and mixed at room temperature
(20-25°C) for 45 min.
TABLE 12 (Aqueous Cream)*
Ingredients wt%
Emulsifying Wax 14.993927
1% KH2PO4 in water (pH=7.5) 74.057507
Isopropyl Palmitate, NF 5.4571649
Glycerin 5 .0104708
Thermolysin 0.2001065
Methyl paraben 0 .2007937
Propyl paraben 0.0800301
TOTAL 100
20 *Aqueous cream was prepared as follows: (1) parabens were melted in buffer at high temperature
(>70°C) along with glycerin; (2) emulsifying wax and isopropyl palmitate were added; (3) the mixture
was mixed at high temperature for 45 min and then cooled to about 35°C; (4) thermolysin was added as a
slurry in the buffer; (5) the mixture was cooled to room temperature (20-25°C).
90753719.1 -30-
TABLE 13 (Hyrlrogel)*
Ingredients wt%
Hydroxy ro ylmethylcellulose 2.250621745
1% KH2PO4 in water (pH=7.5) 77.96851753
Thermolysin 0.202530294
Methyl paraben 0.244719829
Propyl paraben 0.0480663
Propylene glycol 19.28554438
TOTAL, 100
5
*Hydrogel was prepared as follows: (1) parabens and propylene glycol were solubilized in water at 70°C;
(2) HPMC was added at room temperature (20-25°C); (3) Thermolysin was added and a millcy viscous
solution formed.
90753719.1 -31

CLAIMS
1. A wound debriding composition comprising:
5
(a)
(b)
a dispersed phase comprising a liquid hydrophilic polyol and at least one
proteolytic enzyme; and
a continuous phase comprising a hydrophobic base;
wherein the amount of the liquid hydrophilic polyol is within 1 10% w/w of the
optimum amount of the liquid hydrophilic polyol.
2. The composition of claim 1, wherein the amount of liquid hydrophilic polyol is within
± 7% w/w of the optimum amount of the liquid hydrophilic polyol.
10 3. The composition of claim 1, wherein the amount of liquid hydrophilic polyol is within
± 5% w/w of the optimum amount of the liquid hydrophilic polyol.
4. The composition of any one of claims 1-3, wherein the proteolytic enzyme is a
metalloprotease.
5. The composition of claim 4, wherein the metalloprotease is collagenase.
15 6. The composition of claim 4, wherein the metalloprotease is thermolysin.
7. The composition of any one of claims 1-3, wherein the proteolytic en'yiue is a
cysteine protease.
8. The composition of claim 7, wherein the cysteine protease is papain.
9. The composition of any one of claims 1-3, wherein the proteolytic enzyme is a serine
20 - protease.
10. The composition of claim 9, wherein the serine protease is trypsin.
11. The composition of any one of claims 1-3, wherein the proteolytic enzyme is an
aspartic peptidase.
12. The composition of claim 11, wherein the aspartic peptidase is pepsin.
25 13. The composition of any one of claims 1-12, wherein the liquid hydrophilic polyol is a
liquid polyethylene glycol, or a liquid poloxamer, or mixtures thereof.
90753719.1 -32-
14. The composition of any one of claims 1-13, wherein the proteolytic enzyme is
suspended in the dispersed phase.
5 16. The composition of any one of claims 1-15, wherein the hydrophobic base comprises
petrolatum.
18. The composition of claim 17, wherein the solid hydrophilic polyol is a solid
10 polyethylene glycol, or a solid poloxamer, or mixtures thereof.
19. The composition of any one of claims 1-18, wherein the composition is a semisolid.
20. The composition of any one of claims 1-19, wherein the composition is anhydrous.
21. The composition of any one of claims 1-20, wherein the composition is sterile.
22. A method of treating a wound in need of debridement comprising: applying to the
15 wound a composition comprising a dispersed phase comprising a liquid hydrophilic
polyol, and an effective debriding concentration of at least one proteolytic enzyme;
and a continuous phase comprising a hydrophobic base; wherein the amount of liquid
hydrophilic polyol is within + 10% w/w of the optimum amount.
15. The composition of any one of claims 1-13, wherein the proteolytic enzyme is
dissolved in the dispersed phase.
17. The composition of any one of claims 1-16, wherein the dispersed phase further
comprises a solid hydrophilic polyol.
23. The method of claim 22, wherein the composition is any one of the compositions
20 described in claims 1-21.
25
24. A method of determining an optimum amount of liquid hydrophilic polyol to add to a
target composition comprising a dispersed phase including a proteolytic enzyme and a
continuous phase including a hydrophobic base, the method comprising:
(1) obtaining a series of compositions comprising the dispersed phase and the
continuous phase, wherein the dispersed phase further includes a liquid
hydrophilic polyol, and wherein each composition in the series of compositions
90753719.1 - 33 -
include an identical amount of proteolytic enzyme and a different amount of
the liquid hydrophilic polyol;
(2) determining the enzymatic activity of each composition in the series of
compositions;
5 (3) determining the highest point on a graph that plots the enzymatic activity
versus the amount of liquid hydrophilic polyol(s) included in each composition
of the series of compositions,
wherein the highest point on the graph correlates to the optimum amount of liquid
hydrophilic polyol to add to the target composition.
10 25. The method of claim 24, further comprising adding ± 10% of the optimum amount of
liquid hydrophilic polyol to the target composition.
26. The method of claim 24, wherein the series of compositions includes at least five
compositions.
27. The method of claim 26, wherein the amount of polyol in the series of compositions
15 varies in increments of at least 10% w/w.
28. The method of claim 27, wherein the first composition includes 10% w/w of liquid
hydrophilic polyol, the second composition includes 30% w/w of liquid hydrophilic
polyol, the third composition includes 50% w/w of liquid hydrophilic polyol, the
fourth composition includes 70% w/w of liquid hydrophilic polyol, and the fifth
20 composition includes 90% w/w of liquid hydrophilic polyol.
29. The method of claim 26, wherein the first composition includes 0 to 30% w/w of
liquid hydrophilic polyol, the second composition includes 10 to 50% w/w of liquid
hydrophilic polyol, the third composition includes 20 to 60% w/w of liquid
hydrophilic polyol, the fourth composition includes 30 to 80% w/w of liquid
25 hydrophilic polyol, and the fifth composition includes 40 to 100% w/w of liquid
hydrophilic polyol.
90753719.1 -34-
30. The method of any one of claims 24®29, wherein the target composition is any one of
the compositions described in claims 1-2 1.
31. A method of increasing enzymatic activity in a target composition comprising a
dispersed phase including a proteolytic enzyme and a continuous phase including a
5 hydrophobic base, the method comprising:
10
(1) obtaining a series of compositions comprising the dispersed phase and the
continuous phase, wherein the dispersed phase further includes a liquid
hydrophilic polyol, and wherein each composition in the series of compositions
includes an identical amount of proteolytic enzyme and a different amount of
the liquid hydrophilic polyol;
(2) determining the enzymatic activity of each composition in the series of
compositions;
(3) determining the highest point on a graph that plots the enzymatic activity
versus the amount of liquid hydrophilic polyol(s) included in each composition
15 of the series of compositions, wherein the highest point on the graph correlates
to an optimum amount of liquid hydrophilic polyol to add to the target
composition, and
20
(4) adding ± 10% w/w of the optimum amount of liquid hydrophilic polyol to the
target composition, thereby increasing the enzymatic, activity in the target
composition.
32. The method of claim 31, wherein the series of compositions includes at least five
compositions.
33. The method of claim 32, wherein the amount of polyol in the series of compositions
varies in increments of at least 10% w/w.
25 34. The method of claim 33, wherein the first composition includes 10% w/w of liquid
hydrophilic polyol, the second composition includes 30% w/w of liquid hydrophilic
polyol, the third composition includes 50% w/w of liquid hydrophilic polyol, the
90753719.1
-35 -
fourth composition includes 70% w/w of liquid hydrophilic polyol, and the fifth
composition includes 90% w/w of liquid hydrophilic polyol.
35. The method of claim 32, wherein the first composition includes 0 to 30% w/w of
liquid hydrophilic polyol, the second composition includes 10 to 50% w/w of liquid
5 hydrophilic polyol, the third composition includes 20 to 60% w/w of liquid
hydrophilic polyol, the fourth composition includes 30 to 80% w/w of liquid
hydrophilic polyol, and the fifth composition includes 40 to 100% w/w of liquid
hydrophilic polyol.
36. The method of any one of claims 31-35, wherein the target composition is any one of
10 the compositions described in claims 1-21.