Note: Descriptions are shown in the official language in which they were submitted.
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BACKGRO~ND OF INVENTION
This inven-tion relates to novel hydrolytic enzyme
products, therapeutically useful compositions containing such
materials, and to methods of utilizing such products especially
in debridement of eschar tissue.
Considerable efforts have been made to discover
materials capable of distinguishing between viable and non-
viable tissue. The discovery of materials which would digest
devitalized tissue while not attacking viable tissue would make
it possible to remove the devitalized tissue without surgery.
It would be a beneficial therapeutic agent in virtually all
disease processes where topically devitalized tissue needs to
be removed from the viable organism such as decubitus ulcers,
pressure necroses, incisional, traumatic and pyogenic wounds,
and ulcers secondary to peripheral vascular disease.
One area that has attracted considerable attention is
the use of proteolytic enzymes and other chemicals to effect
the early debridement of eschar tissues resulting from burns.
Such devitalized tissue is an excellent culture mediurn and the
principal source of the speticemia which is the proximate cause
of death in the majority of severely burned patients.
Intensive investigations with such agents as tannic acid,
salicylic acid, and pyruvic acid as well as papain, pinguinain,
trypsin, streptokinase and other enzymes have not led to
satisfactory results. Chemical agents such as tannic acid were
found to cause further injury to already damaged tissue. The
proteolytic enzymes were found to be too slow, to have toxic
side effects or to attack viable as well a8 devitalized
tissue.
It is important that debridement of eschar tissue
take place early, i.e. in a period which is pre~erably no
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longer than four days, and by an agen-t which e~fects debride~
ment rapidly. If debridernent is postponed for too ex-tensive a
period, there results septicemia from invasion of the wound by
infectious microorganisms and toxernia from absorption by viable
tissue of toxic degradation products from the devitalized
tissue. Rapid debridement is essential since the environment
normally encountered is one which serves as an ideal culture
media for the growth of infectious colonies of microorganisms.
As a result suryical debridement with its attendant
pain and heavy bleeding continues to be the principal method
for the removal of eschar.
The enzyme bromelain which is, in fact, a complex
mixture containing materials including a number of hydroly-tic
and proteolytic enzymes has been used in the treatment of
burns. In fact, hydrated bromelain powder and some crude
extracts of bromelain have been employed previously for
debridement of eschar tissue; see Journal of the Maine Medical
Association, September 1964; Research in Burns, EIans Huber,
Publishers Bern Stuttgart Vienna 1971. These materials,
however, have not proved to be satisfactory, principally
because the results were not reproducible.
SUMMARY OF THE INVENTIO~
One aspect of the present invention is directed to a
process of preparing a novel hydrolytic enzyme product useful
for the dissection of devitalized tissue from a mammalian host.
The product comprises a water soluble, heat labile protein,
free of caseinolytic activity, having a peak isoelectric poink
at about 6, comprising at least 2 subunits, each subunit having
a molecular weight of from about 1~,300 to abollt 15,000 daltons
with a characteristic absorption peak in the ultraviolet region
of the spectrum at 280 nm.; the product being active in the
absence of sulfhydryl activation and in the presence of
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sulfhydryl deactivating quantities oE phenylmercuric ~cetate,
and the physiologically acceptable alkali metal and aci~
addition salts thereof. The proces~ comprises extracting
bromelain source material in acetate buffer 0.1 M, pH 5.5 made
up to l percent thioglycolic acid at a solution pH of about 4,
and expressing the bromelain source material through an
ultrafilter having a molecular weight cut off of about 50,000,
and when their physiologically acceptable alkali metal and acid
addition salts are required, treating the filtrate with a
suitable alkali metal reagent or acid.
In this process, the ultrafilter may be an ultra-
filter obtainable under the trade mark XM 50 Amicon Diaflo.
The hydrolytic enzyme may be purified by subjecting
the resultant mixture to molecular exclusion chromatography,
which may be performed on a column of polysaccharide gel
available under the trade mark Sephadex G75. The resultant
macro-molecular mixture may be further purified by fractiona-
tion by isoelectric focusing, and may be still further purified
by subjecting the mixture to polyacrylamide gel analytical
electrophoresis.
Another aspect of the present invention is directed
to a hydrolytic enzyme product useful for the dissection of
devitalized tissue from a mammalian host which comprises a
water soluble, heat labile protein, free of caseinolytic
activity, having a peak isoelectric point at about 6,
comprising at least 2 subunits, each subunit having a molecular
weight of from about 14,300 to about 15,000 daltons with a
characteristic absorption peak in the ultraviolet region of the
spectrum at 280 mm.; the said product being act:Lve in the
absence of sulfhydryl activation and in the presence of
sulfhydryl deactivating quantities of phenylmercuric acetate,
and the physiologically acceptable alkali metal and acid
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addition salts thereof, whenever prepared by the foregoiny
process.
DETAILED DESCRIPTION OF THE INVENTION
Novel hydrolytic enzyme materials useful therapeuti-
cally for dissection of devitalized tissues have now been
discovered. The novel products of this invention can be
employed for dissection and to facilitate removal of devital-
ized tissue from a mammalian host. The products comprise water
soluble, heat labile proteinaceous materials, free of
caseinolytic activity. The peak isoelectric point is about 6
and generally ranges from about 5.85 to 6.10. The protein is
comprised of at least two and most likely
three subunits, each of which has a molecular weight from about
14,300 to 15,000 daltons. There is a characteristic absorption
peak in the ultraviolet region of the spectrum at 280 nm. A
characteristic of the products of the invention is their
hydrolytic activity, even in the absence of sulfhydryl
activation or the presence of sulfhydryl deactivating
quantities of phenylmercuric acetate. The invention also
includes physiologically acceptable alkali metal and acid
addition salts of these products. The salts can be prepared by
reaction in an aqueous medium between the hydrolytic enzyme
substrate and preferably a slight molar excess of the selected
dilute alkaline metal base or acid, normally a mineral acid or
a low molecular weight aliphatic carboxylic acid. Typically
useful bases include sodium and potassium hydroxide. Acids
which can be employed include hydrochloric and acetic acids.
The products of this invention may be obtained by
appropriate treatment of commercially available brornelain
preparations whi.ch may be obtained**. In the presently
preferred procedure or ob-taining bromelain from the stem of
the pineapple plant, the juice ~rom -the stem is first adjusted
**from Castle and Cooke, Inc. of San Francisco, California
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to a pH of about 3 or 4 with phosphoric acid an~ ~odiwn
sulfhydride is added to protec-t against sulfhydryl oxidation.
A precipitate is formed by the addition of sufficient acetone
so that the solution is 30% in acetone and, after filtration,
the clarified fluid is again precipitated by the addition of
sufficient acetone so that the fluid is 70% in acetone. This
precipitate is collected by centrifugation and either
redissolved in water containing sodium sul~hydride which has
been acidified with phosphoric acid and reprecipltated, or
dried in a vacuum oven directly. If the material is
reprecipitated, 70 ~ acetone is utilized. The dried material
from either process is suitable as a starting material to
obtain the hydrolytic material of this invention.
Either of these source materials is extracted (10
grams per 200 ml.) in acetate buffer O.lM, pH 5.5 which has
been made up to 1~ in thioglycolic acid. The pH of this
solution is approximately 4. The solution is expressed through
~M 50 Amicon Diaflo*ultrafilter (Amicon Corp., Boston, Mass.).
This product is an anisotropic, ultrafiltration, polyacrylic
20 membrane with a molecular weight cut off of about 50,000. The
class is described in U.S. Patent No. 3,615,024.
The macromolecular mixture thus obtained is further
purified to obtain the hydrolytic enzyme products of this
invention. The mixture is first subjected to molecular
exclusion chromatography as a phenyl mercuric salt [prepared by
combining the mixture with an aqueous 0.2 M citrate buffer
saturated with the phenyl mercuric acetate salt in accordance
with the procedure of Ota et al in Biochern. 3:180 (1960)] on a
co].umn of Sephadex G 75*. The elution oE the de~ired enæyme
product from this column preceded the elution of pure stem
bromelain, and therefore must have a molecular weiyht in excess
of bromelain, which is known to be about 32,000.
*Trade Marks
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Sephadex G 75* is a polysaccharide gel available frorn
Pharmacia of Upsala, Sweden. It is employed for molecular
exclusion chromatography in accordance with procedures well
known in the art.
In fur~her separation and purification experiments
the macromolecular mixture was fractionated by isoelectric
focusing and subjected to polyacrylamide gel analytical
electrophoresis in 1% sodium diodecyl sulfate (SDS).
For isoelectric focusing -the mixture was mixed in a
sucrose gradient with LKB Ampholine* ampholytes initially from
pH 3 to 10, and subsequently at pH 5-8. The active material
was concentrated at a peak isoelectric point of pH 6.04, with a
range from 5.85 to 6.12. This isoelectric point is markedly
different from those described for the proteases called
bromelain (pH 4.7 and 9.9). See Vestberg Acta.Chem.Scand
20:820 ~1966).
LKB Ampholine* is available from the LKB Company of
Sweden for isoelectric focusing. It is believed to bè a
mixture of small ampholytes.
The products isolated by isoelectric focusing have an
extremely high order of useful hydrolytic activity.
The isoelectric focused active material is ln turn
subjected to polyacrylamide gel electrophoresis at pH 9 in 1%
SDS (Weber et al J. Biol. Chem. 244:4406 (1969). Only one
protein staining band can be visualized with a measured
electrophoretic mobility which, when compared with standard
proteins of known molecular weight, evidences a molecular
weight oE between 14,300 and 15,000 daltons. Since SDS is
known to dissociate proteins into their various suhunits, if
any, it is apparent that the enzyme products of this invention
comprise at least two, and most likely three, subunit~ of
substantially the same molecular weight.
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The material isola-ted Erom isoelectric focusiny was
subjected to ultraviolet spectropho-tometry in wa-ter and
exhibited a maximum absorption at 2~0 nm. This absorption is
characteristic oE aromatic amino acids.
It is concluded, as a result of the foregoing
studies, that the products of the invention are proteinaceous
in nature and contain aromatic amino acids. They were found to
be water soluble and hear labile. While the materials of the
invention appear to be substantially pure by the procedures
studied, there are most likely small quantities of other
materials still present. ~he activity does not require
absolute purity and molecular homogeneity.
The products of the nvention, as isolated by iso-
electric focusing, were demonstrated to contain no caseinolytic
activity when incubated under standard conditions with casein,
in accordance with the procedure of Ota et al referred to
hereinabove. Bromelain exhibits a high order of caseinolytic
activity under these conditions.
The void volume from the G-75 Sephadex* column
chromatography described above which contained the hydrolytic
enzyme product also contained phenylmercuric acetate which
inactivates sulfhydryl enzyme. The fact that this enzyme
activity occurred in the presence of the mercuric compound
indicates that, unlike bromelain, it does not require free
sulfhydryl groups for its biological effect. However, it was
found that when crude acetone precipitated bromelain was
subjected to molecular ultrafiltration in the absence of
thioglycolic acid (a sulfhydryl protector), the activity was
substantially reduced.
The molecular weight range of the active products in
the compositions of this invention is such -tha-t pathoyenic
organisms which are known to be of much higher molecular weight
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are excluded. The products of the invention therefore are
inherently ster.ile provi~ed, oE course, that they are prepared
under sterile conditions.
It is clear that the fraction is di~erent ~rom any
previously reported materials. Moreover, unlike any previously
reported materials it is safe, reliable and effective. The
therapeutic results arising from its proper utilization are
predictable and reproducible.
While the most active materials of this invention, on
a weight basis, are those which are obtained from isoelectric
focusing as described above, it is not necessary nor is it
practical to carry the purification procedure to this point.
The unexpected and most beneficial properties of the products
of this invention are obtained with products which are not
necessarily so completely purified.
For most purposes, useful products can be provided in
two ~orms. The lyophilized product is generally less dense
than the fraction obtained by acetone precipitation. Either
fraction can be used alone or in conjunction with the usual
pharmaceutically acceptable excipients such as petrolatum,
isotonic saline, polysaccharide gels or other stable, inert
hydrocarbon bases. Such compositions should be prepared
immediately prior to use since the products of the invention
are not stable in the presence of moisture.
In certain situations, it may be desirable to add
other active ingredients to the therapeutic compositions of the
invention. For example, antibiotics may be added to the
mixture. These agents or other antimicrobial agents, such as
bacitracin, may be useful to control bacteria and fungi and to
control possible in~ection. A keratolytic agent such a~ urea
may be added to aid in the breakup of the e~char tissue.
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To determine the bioloyical effects of the pro~ucts
of this invention, experimental full thickness hurns were
produced on anesthetized piglets by radiant heat. Six
different piglets were tested. Each experimental burn was
soaked with normal saline for periods of up to four hours after
the passage of time indicated in the table below:
Table 1
Post Burn Duration of
Period Saline Soak
1 hour 0 hour
24 " 1 "
36 " 1.5 "
48 " 2 "
72 " 4 "
Each burn was punctured with a series of small holes
to permit easy passage of the hydrolytic activity through the
eschar tissue to the underlying demarcation line between the
eschar -tissue and the viable tissue. The burns are then
separately coated with a powder of the invention and then with
agar. The agar is coated with a flexible, plastic sheet, and
the edges o~ the sheet are tightly adhered to the surrounding
flesh to protect against the oxygen in the air. A small amount
of normal saline is then injected through the plastic and into
the agar. This moisture has two effects. It swells the agar
to further protect the enzyme from oxygen and it activates the
enzyme.
The active powder was applied to the eschar tissue in
a quantity o~ 0.1 mg/mm2. The hydrolytic composition is
effective in quantities as low as 0.1 mg/mm2, but is
preferably used in quantities up to 10 mg/mm2, or even higher
to insure contact of the enzyme with the non viable tissue
substrate.
At the end of a one-hour period, the covering~ were
removed and it was ~ound that the non--viable tissue could be
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removed from the viable tissue with rninimal bleediny and wi-th
no apparent toxic efects. ~he bed of viable tissue remaining
after removal of the eschar is suitahle for acceptance of a
graft.
The graft was applied to the experimental burns from
which the eschar tissue was removed after first washing with
hydrogen peroxide to neutralize remaining enzymatic activity,
and then washing with normal saline. The grafts took success-
~ully.
In a subsequent test with a human subject, a full
thickness burn 5 cm2 in area was formed on the skin surface
of the anterior thigh under local anesthesia by exposure to
radiant heat for thirty seconds at 360 Celsius. At the end of
one hour, a paste formed from a 1:1 mixture of -the powdered
product of this invention in physiological saline solution was
applied to the burn. The paste was covered with a transparent
plastic sheet to seal out oxygen and to maintain humidity, and
then with a pressure bandage to secure good contact between the
burn substrate and the therapeutic material. The dressing was
removed after one hour and the eschar tissue was found to be
partially digested. There was no adherence of the wound bed,
and the remains of the eschar tissue were completely removed by
simple wiping, leaving a wound bed suitable for acceptance oE a
graft.
It was observed that even after the effects of the
local anesthetic had subsided, there was no pain associated
with the removal of the partially digested tissue, or any other
manipulation of the wound bed. The bleeding was minimal.
I'he wound bed was covered with a spl:it thickness
autograft. The autograft was inspected after four days. I-t
was found that the graEt bed was almost Eull~ covered. At the
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end of seven days, the wound be~ was 100~ covered with a viable
skin autograft.
It has been observed that the active rnaterial does
not digest the eschar -tissue appreciably when exposure is of
relatively short duration, for example, one hour. Rather, it
dissects the eschar tissue from the underlying tissue by
cleaving the connecting tissue. This reaction takes place
extremely rapidly.
The rapid dissolution of the connecting tissue was
illustrated by another experiment of the nature described above
with a piglet having an experimental burn. The entire eschar
and the surrounding tissue was covered with an adhesive plastic
spray and then a small vertical hole about 1 mm in diameter and
5 mm deep was drilled in the center of the eschar. The hole
was filled with a powdered product of the invention and then
moistened with a ~ew drops of saline colored with Evans slue
dye. Care was taken so that none of the enzymatic material
spilled over the edges of the hole, and it was sealed with
plastic. After one hour, bluish discoloration was apparent
around the periphery of the burned area (about 2 x 2 inches).
When the plastic seal was removed, it was found that the
diameter of the drilled hole had increased to about 10 mm.
Furthermore, the eschar tissue could easily be lifted away from
the viable tissue with little or no bleeding. This experiment
was repeated several times with both lyophilized and acetone
precipitated material in undiluted powdered form and in various
carriers such as physiological saline, vanishing cream and agar
and with substantially identical results.
What has been described are water soluble hydroly-tic
enzyme compositions containing proteins having a molecular
weight of from about 30,000 to 50,000 daltons. It is activated
by moisture. However, if exposed -to a mois-t atmosphere Eor an
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extended period of time, say for exampJe a day or rnore, it is
subject to autodiges-tion with loss o~ activity. The rnixture
also loses its useful activity when exposed to a temperature of
100 Celsius for about five minutes. It is therefore heat
labile.
In still further experiments, four to six weeks old
piglets anaesthesized with diethyl e-ther were shaved in small
areas taking care not to create superficial abrasions. Radiant
heat of 360 Celsius applied for twenty seconds with the heat
source being an electrically heated rod in an insulated chamber
with a variable opening~ There was no contact of the heating
rod with the skin. The variability of contact pressure was
therefore eliminated. rrhe standard assay burn size was two
centimeters by five centimeters, with the edges shielded
against excess heat by heavy asbestos plate which has to be
cooled to room temperature before each new application in order
to limit the burn injury to the actual opening in this asbestos
plate w~ich also can be varied in size but in the standard
assay was chosen to be two by five centimeters. This size was
chosen in order to channel the efficacy of the active material
along a narrow strip of eschar, facilitating at a distance of
separation from the injection edge, the actual efficacy of the
injected fraction.
One hour after the burn injury, the test fraction was
injected at the smaller side of the rectangle (2 cm.), 1 cm.
from each corner of the eschar with the needle inserted at the
edge o~ the burn injury and tangentially with the slight 10
angulation advanced parallel to the longer sides of the
rectangle about one centimeter into the sub-eschar space. The
instrument with which the fraction was injected hest. was a TB
syringe with a 25-gauge short needle. The bevel of the needle
was directed towards the viable base during the injection. rrhe
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amount of test fraction injected was dissolved in 0.1 cc.
volume of distilled wa-ter. This -technique places a pre-
measured amount of test fraction dissolved in 0.1 cc. of
- distilled water in the sub-eschar space one centimeter ~rom
each side of one end of the 2 x 5 cm. rectangle burn.
One hour after the fraction had been placed in the
sub-eschar space with the needle left in the injection canal in
order to avoid loss of the active material by reflux through
the injection canal, the needle was removed and a small rake, 2
centimeters in width with about five teeth sharpened at the
point, pushed in a 45 angle into the junction line hetween
eschar and adjacent non-burned tissue along the smaller side of
the rectangular burn where one hour before the test fraction
had been inserted into the sub-eschar space. Imitating a
mechanical separation in an actual burn debridement, the rake
was pulled in the direction of the longer axis of the burn
wound after the teeth of the rake were engaged in the edge of
the eschar. With active fractions it is possible to peel the
eschar out of the burn wound leaving a viable slightly bleeding
base. The amount of mechanical pull with which the eschar
strip can be peeled off this base depends on the loosening or
partial softening of the collagen strands with which the eschar
is anchored to the wound bed. The ease with which this peeling
process can be effective can be measured by a weight attached
to the handle of the rake with a fine string lead over a pulley
with the animal anchored against a board to which the pulleys
are attached. The second measure of the efficacy of the text
extract is the dis-tance a non-constant weight pull is able -to
peel the eschar strip measured from the edge of the inser-tion
of the rake towards the opposi-te small edge of the rectangular
eschar. Both the pulley weight recIuired -to peel off -the
eschar, and the length in which -the eschar strip of 2 x 5
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centimeters can be peeled o~f along the lonyer axis of this
rectangular burn starting ~rom the smaller border where the
test fraction had been injected, demonstrates the efficacy of
the particular test fraction in accelerating the demarcation of
the heat predetermined plane between irreversibly devitalized
eschar and viable wound bed.
Using this technique it has been found that aqueous
compositions, particularly physiological saline solutions
containing from about 0.1 to 2% by weight of active material
can be usefully employed.
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