Note: Descriptions are shown in the official language in which they were submitted.
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Proteolytic cover of wounds
The invention pertains to a proteolytic cover of wounds
in the form of a powder which serves for coating and treat-
ment of ulcerous and necrotic wounds.
The powders consisting of various bases (starch, talc,
etc.) and bactericides or bacteriostatics, in particular
antibiotics, sulfonamides and antimycotics, are recommended
for this purpose in the common pharmaceutical practice.
Recently, absorbing covers in the form of powders, which
contain a hydrophilic polymer as a main component, proved
suitable in the treatment of ulcerous wounds. Their effect
consist in the removal of liquids from the surface of wound
by sorption in a porous structure of the effective polymeric
component and by suction of capillary forces into voids between
particles of the powder layer. The exudate together with
bacteria, substances causing inflammation, or also toxins are
removed from the wound in this way. In particular, prepara-
tions based on crosslinked dextrans (B.S. Jacobsson et al.,
Scand. J. Plast. Reconstr. Surg. 10, 65-72 (1976)) are used
in medical practice. Promising results were attained also with
polymer compositions based on cellulose, which contain a highly
hydrophilic polymeric component, for example carboxymethyl-
cellulose as an additive (H. Dautzenberg et al., Absorbing
cover of wounds; Czechoslovak certificate of authorship No. 237,604).
Further development of powders with cleaning effect in
3~
treatment of ulcerous wounds has been directed to cellulose
powders containing immobilized enzymes of protease type
(Collection of papers: Immobilized proteolytic ferments in
heating of ulcerous-necrotic processes (in Russian), AN SSSR,
Sibirskoe Otdel., Institut tsitol gii i Genetiki, Novosibirsk,
1981). In this case, the enzymatic action is used to attain
cleaning effects. The proteolytic covers of wounds cause
a hydrolytic decomposition and dissolution of necrotic tissues
and pus on the wound surface thus removing also the medium
for growth of bacteria and interrupting suction of toxic
products from the wound.
Practical utilization of the proteolytic principle requires
a solution of numerous problems in the preparation of the
efficient cover which would not develop harmful side effects.
This means, in human medicine, above all to prevent cellulose
or derivatized cellulose from entering into the blood circula-
tion, because the human organism does not have enzymatic systems
for the removal of such compounds at its disposal. However,
the preparations of proteolytic cover used so far employ only
conventional cellulose types, i.e. ground ion-exchanging dusts
with a fibrous structure containing fine fractions of dust
which may penetrate into blood.
To attain the maximum efficiency of covers, the proteolytic
principle should not be employed without using the previously
known absorption principle at the same time. Recent solutions
comprise, however, only the conventional type of cellulose
with a high crystallinity and low porosity as the starting
material, which provides covers with a relatively low absorp-
tion capacity.
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In addition, such ~rocedures for the immobiliza-
tion of proteases in cellulose have to be found for the
utilization of proteolytic principle, which would exclude
a release of the enzyme from matrix and penetration of its
soluble form into blood circulation, where it would develop
an alergic reaction as an antigen.
The above said shortcomings in the utilization
of proteolytic principle are met by a new cover for wounds
according to the invention.
The invention pertains to a proteolytic cover of
wounds in the form of powder, which serves for covering
and treatment of ulcerous and necrotic wounds. It is based
on the original combination of the proteolytic and absorp-
tion principles of cleaning of infected ulcerous wounds.
According to the invention, the cover consists of porous
spherical cellulose with particle size 0.05 to 0.5 mm, which
contains an immobilized protease. The method of production
warrants that the obtained products contain only the cova-
lently bonded protease and no undesirable toxic compounds
or soluble portions with the proteolytic activity, which
could penetrate into blood circulation and develop an alergic
reaction as antigens. The immobilized enzymes act in clean-
ing of the wound by dissolving undesirable proteins, in
particular fibrin, necrotic tissues, pus and others. The
porous structure of hydrophilic spherical particles of
cellulose warrants a high absorption capacity, which is
exhibited by sorption of an exudate into the material and
suction of pus infected with bacteria from the wound into
the interparticular space of powder layer. The combination
of proteolytic and absorption principles enables to achieve
the fast cleaning of infected necrotic defects, formation
of clean granulations, and fast healing of the wound.
In particular the present invention relates to
a composition of matter which can be used as a proteolytic
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cover for wounds, which consists of spherical particles of
diameter 0.05 to 0.5 mm, preferably o 1 to 0.3 mm, based
on (a derivatized) cellulose provided with one or more
immobilized enzymes of protease type e.g. selected from
the group which comprises chymotrypsine, trypsine, and
subtilisine. In another aspect the present invention pro-
vides a method for producing a composition o~ matter,
useful as a proteolytic cover for wounds, from bead cellulo-
se, prepared by the procedure according to Czechoslovak
Patent no. 172,640, where the bead cellulose swollen in
water, which never has been dried, of particle size 0.07
to 0.7 mm, prefera~ly o.14 to 0.4 mm, is perfectly freed
of toxic contaminants by washing, distillation with steam
or both activated for bonding of an en~.yme, modified by
immobilization of one or more proteases selected from the
group compri.sing chymotrypsine, trypsine, and subtilisine,
alternately washed with a buffer of pH 8.5 to 9.5 and a
buffer of pH 4 to 5 until the washings have proteolytic
activity zero, then washed with a buffer of pH 7.5 to 8.5,
and dried in this medium to 0.1 to 15~ of the residual
water content in th~ dry substance, preferably by lyophili-
sation,a composition of matter of the above defined particle
size thereafter being recovered by any suitable means.
The regular spherical shape of individual particles,
which form the new cover for wounds, and the chosen particle
size and the distribution of particle size (0.05-G.5 mm,
preerably 0,1-0.3 mm), warrant an easy handling both in
production and in application, a smooth flow of powder
during its spreading on wounds, and, in particular, meet
the requirement to prevent penetration of cellulose into
blood circulation.
The production of new cover advantageously employs
the regenerated spherical cellulose prepared according to
Czechoslovak Patent no. 172,640. Its advantage is a high
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porosity which facilitates the activation for enzyme bind-
ing and the immobiliæation of proteases. The porous hydro-
philic character of the bead cellulose carrier is retained
even after activation, immobilization and drying. Lyophili-
sation proved a suitable drying method which removes waterin a considerate way with respect to the bonded enzyme and
provides the dry derivatized cellulose with a sufficient
absorption power.
The bead cellulose prepared by the procedure
described in Czechoslovak Patent no. 172,640 is thoroughly
freed of soluble portions, in particular of all impurities
with toxic effects which cause its cohtamination during
preparation (decomposition products of xanthogenate groups,
residues of a disperse medium, e.g. of chlorobenzene). For
this purpose, it is washed with water at 50-90C and/or
with ethanol. An effective removal of chlorobenzene occurs
by distillation with steam.
Several methods known from literature can be
used to activate the cellulose for protease binding
(Handb ok of
, / _ . _ . . . .
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Enzyme Biotechnology, Ed. ~. Y~iseman; E. ~orwood Ltd~ Chich~-
ster 1975), but such methods should be chosen which form
a sufficiently stable bond between enzyme and cellulose and
do not contaminate the product with toxic co~pounds becoming
effective during application of the coYer, for example; by
releasing or action in the direct contact. A suitable method
is the periodate oxidation~ which is relatively simple and
easy to carry out and provides products of a suffi~ient
porosity which exhibit a high absorption effectO
Yarious proteases are suitable for immobilization, parti-
cularly trypsine, chymotrypsine and subtilisine, but also
thermolysine5 papaine, and others. Binding of the enzyme to
cellulo~e activated by periodate oxidation, î.eO ~he cellulose
containing reactiYe dialdehyde groups, is carried out in two
steps: the compounds of cellulose with the enzyme of a Schiff-
-base type are formed first~ which are then stabilized by
reduction of the ~nreacted aldehyde groups with sodium boro-
hydride.
An important stage in the preparation of the new type o~
coYer is the perfect removal of solub~e portions containing
proteases from the modified cellulo~e beads. I~ is attained
by the repeated alter~ation o~ a wea~ly alkaline (pH 8~5-9.5)
~nd a weakly acidic buffer (pH 4-5) in a static or column
arrangement. ~or this purpose7 thsy are suitable~ e.g~ the
0.1 M borate buffer ~ontaining 1 M NaCl tp~ 9~ and the 0.1
acetate buf~er containing 1 M NaCl 5pH 4.53~ or both these
buffer~ with~ut NaCl, which are applied on the cellulose
prsduct after immobilization until the lîquid phase ha~
.
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a proteolytic activity ~ero. ~he alternation of buf'~er~ of
lower and higher pH and variable ionic strength ~acilitates
the elution of ccvalently non~onded protein by the alternating
suppression of electrostatic and hydrophobic interactions.
To obtain a perfectly stable preparation with the immobilized
enzyme, the beads are even-tually washed with a borate buf~er,
e.gO, of pH 8, and lyophilized in a suspDnsion with this
bu~fer~ Under ~uch conditions~ the resulting product contains
only the covalently bonded enzyme and no ~oluble portion ~f
it, which could penetrat~ i~to blood during treatment and
could become an antigen.
The proteolytic co~er with immobilized chymotrypsine was
tested in clinical practiceO It exhibited the proteolytic
acti~ity to necrotic tissue, pus and fibrin and, at the s~me
time, did not harm a healthy tissue at all~ The cover ~YaS
noted for the hi~h absorption capacity in agreement with the
hydrophilicity and porous structure of cellulo~e matrixO It
sucked up the exudate ~rom a wound, which contained clea~ed
tissue necroses and bacteria. Binding o~ the enzyme t~ carrier
pre~ent~ its autolysiæ and los~ of its acti~ity during appli-
caticn~ The extended proteolytic acti~ity of chymotryp~ine
led t~ the release of necroses from wound in a large extent
and to the stepwise cleani.ng of wo~nd~ The early in~ected
secretion wa~ actively absorbed between the particles of powder
thu~ preventing the circumference of wound from maceration
and reducing the nutrient ~ur~ace ~or the ~rowth of bacterial
in~ection. The original combination of the proteolytic and
abssrption principle~ enabled to achieYe the ~a~t cleaning o~
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infected necrotic defects, formation of clean granulations
and fast healing of the wound.
It has been proved that the new cover is suitable for
all kinds of ulcerous and necrotic wounds, including the surgi-
cal wounds healing per secundam, for abcesses, prolonged andnonhealing ulcers of various origin (varicose, X-ray and trophic
ulcers~, defects after decubital necroses, infected and necro-
tic defects after acral amputations, incisions and necrecto-
mies at diabetic gangrenes and gangrenes of arteriosclerotic
origin, carbuncles, ulcerous burns of the 2nd and 3rd degree,
infected open fractures, treatment of amputation stumps, decom-
posed and ulcerous tumours. No harmful side effects were
observed in the application of the new cover.
The following examples of performance illustrate the method
for production of covers according to the invention and their
applications without limiting the scope of invention.
Example 1
Preparation of the cover with bonded chymotrypsine
a) Introduction of aldehyde groups by oxidation with sodium
periodate
The bead cellulose swollen in water and never dried before
with porosity P = 90~ (i.e. the overall content of pores in
volume ~), which was prepared according to Czechoslovak Patent
no. 172,640, was used as the starting material. Cellulose
was perfectly freed of impurities by washing with hot water
(90, 5h, twentyfold excess) and by distillation with steam
(4h). The filtered cellulose (1000 g) was dispersed in 5
1 0.1 M sodium periodate and stirred at laboratory temperature
for 45 min. After completion of the oxidation, the oxidized
cellulose was immediately washed with about 20 1 distilled
water (on Buechner funnel), transferred into a column and
washed with distilled water until the electric conductivity
of effluentbecame equal to the conductivity of distilled water
(overnight).
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b) Binding of chymotrypsine to oxydized cellulose
The washed oxidized cellulose (1000 g) was dispersed in
1 litre 0.1 M borate buffer (pH 9) containing 5 g chymotrypsine
(the proteolytic activity of the solution was 5.35 JA280/min-ml
as determined by means of a solution of denaturated haemo-
globin, pH 8). The suspension was stirred at laboratory tempe-
rature. The progress of binding was followed as the decreasing
proteolytic activity of the binding solution. After an hour,
the proteolytic activity decreased to o.l JA2~0/min.ml and
bonding was stopped by suction off the binding solutionO
The rate of chymotrypsine binding increases with increasing
pH, however, the solubilization of cellulose increases at
the same time depending on time of oxidation~ The chosen
procedure was worked out on the base oE numerous comparative
experiments and compromises between the amount of bonded enzyme
and a loss of cellulose caused by solubilization.
c) Reduction of cellulose with bonded chymotrypsine to stabilize
Schiff bond between the carrier and enzyme and remove
unreacted aldehyde ~roups
The cellulose with bonded chymotrypsine was suspended
in 1 litre 0.1 M borate bufEer (pH 9) with dissolved 500 mg
NaBH4. The reduction was stopped after 20 min of stirring
at laboratory temperature by suction oEf the reducing solution
.
~: `
lQ
and once more rep~ated in the sa~e way with the new ~olution
of sodium borvhydride. The reduction vras carried out twice
by addition of the l~aBH4 solution of lower concentration to
prevent the loss of proteolytic activity of chymotrypsine by
the contingent reduction o~ disulfide bridges.
d ) Washing and lyophilisation o~ cellulose with bonded
chymotrypsine
The cellulose with bonded chymotrypsine was washed, a~ter
completion o~ reduction, alternately with 2 1 Ool M borate
buffer containing 1 ~ NaCl (pH 9~, 2 1 0~1 ~ acetate buffer
containing 1 ~ NaCl (pX 4.5~, and further with the same volumes
of both buffers without NaC10 The cellulose ~Jith bonded
chymotrypsine ~as then trans~erred into a column~ where it was
washed with all above mentioned buffers repeatedly alY~ays
until the proteolytic activity of e~luent was zero. The cellu-
lose with bonded chymotrypsine was eventually washed with
0.25 M borate buffer o~ pH 8 and lyophilised also in the
su~pension with this buffer. The obtained preparation contained
5 mg active chymotrypsine on 1 g dry cellulose~ The comparative
amino acid analysis o~ the prepared sample and a ~ample addi-
tionally washed with 6 M guanidine hydrochloride and distilled
water proved that all chymotrypsine is covalently bonded to
cellulose and therefore cannot penetrate into blood during
application and become an antigen.
Example 2
Preparation of the cover with covalently bonded trypsine
I~obilization of trypsine ~ras carried out in the same way
~2~3~
as in example 1. The obtained preparation contaIned 8.2 mg
actiYe trypsine in 1 g lyophilised cellulose. Because of it~
narrower specificity, trypsine clea~es proteins at lower
number of sites (it cleaves proteins only behind basic amino
acids lysine and arginine).
Example 3
Preparation o~ the coYer with covalently bonded subtilisine
The immobilization of bacterial p~oteinase was carried out
in the same way as in example lo The obtained preparation
contained 11~3 mg active enzyme in 1 g lyophilised preparation.
~xample 4
The proteolytic cover with chymotrypsine bonded according
~o example 1 was ap?lied to 7 patients ~or the period ~f 1 to
weeks with a very good eflec~ at 6 patients. The effect is
ch~racterized by retreat of purulent secre~ion, cleaning o~
the de~ect base and release of adhering necroses of which those
of larger extent were surgically removed by necrectomy. The
defect was rinsed with hydrogen peroxide solution and 2 per~
mille solution of Chloramine before further application of the
preparationO Vi~id red granulation~ and epithelisation of the
defects from circumference,:vllowed by their reduction in size,
were achieved by this procedure. ~ local application o~
Panthenol spray int~ epithelising defect was mostly used
after the treatment ~va3 ~inished~,
One case showed ~ decrease of purulent secretion, o~ fifth
of the def~ct was cleaned to vivid granulations ? but deeply
reacting adhering necroses occurred in the r~maining parts of
de~ect in the terrain of chronic ischaemia caused by the
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combination of diabetic microangiopathy and oblitering arte-
riosclerosis of peripheral arteries, so that an insufficient
blood supply of neighbouring tissues wa~ the limiting factor
of healing o~ the defectO ~our weeks after the ~pplication of
~he preparation was stopped, the defect made progress in the
sense of abcessing diabetic phlegmon~
Example~ of application of the preparation:
1) J~., man, age 72~ report no. 8357/83; a defect of diameter
3 cm after amputation of the 4th toe of right leg for the
diabetic gangrene with wet necroses on the bottom and a puru
lent secretion. After 2-week application of the preparation~
predominan~ly clean granulations occur with isolated adhering
necrose~ in the medial edge of wound ~ without purulent secretion.
2) V.S~, man, age 60, report no. 6417/83; the state after
a~putation of the right shin ~or diabetic gangrene, where
the defect of diameter 5 cm and depth 20 5 cm arose af~er
abce~sing phlegmon, which had necroses on the lower lateral
part of amputation stump-and al30 medially scabby granulat;ons
in ~he whole range of wound 10 cm long and 1 cm wide with
purulent secretion. After 4-week application of the prepara-
tion, almost complete healing was achie~ed, except a de~ect
of diameter 2 cm and depth 1 cm in the lateral part of stump
with clean granulations and progressi~e epithelisation of the
G ircumference~
3) F.N~, man, age ~6, report no. 3376/83; a defect after ampu-
tation of 1st to 3rd toes including the heads of corresponding
metatarses for diabetic gangrene, ~ize 5-4 cm~ partly with
scabby granulations and partly with adhering necroses;
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the depth of defect was in it~ lateral part up to 1 cm. After
4-week application o~ the preparation, clean granulations
were obtained in the whole region of defect with the progressive
epithelisation of circu~ference and reduction to diameter of
3 cm. The same patient, ~ N., man, age 66, after amputation
of the distal half of 4th toe including the head of 4~h meta-
tarse; a defect aro~e of diameter 3 cm with a scabby lower
part; after a week application of the preparation, the defect
cleanly granulates without secretion and with sound surroundings.
4) F.Yi~ man7 ag~ 77, report ~o. 2~4/84, with the defect after
amputation of the 2nd, 3rd and 4th toe for diabetic gangrene
of size 5x4 cm and depth up to 3 cm with an abundant purulent
secretion and extensive adhering necroses in the whole ran~e
o~ wound; after 4 week application of the preparation, clean
granulations were formed in one ~ifth of the defect; the necro-
tic process remained Ylithout limits in other parts due to poor
blood supply in leg periphery at the diabetic microangiopathy
and oblitering arteriosclerosis, but the purulent secretion
in the defect ceased; the progress in defect occurred first
after the application of preparation was stopped.
5) P~. M.K3~ man, age 56, report noO 9568/83, with the defect
after incision of an abc~ssing phlegmon of the interphalangal
joint of the 1st to~ 7 ~hich re~ched intraarticularly, of
diameter 205 cm, with necroses and purulent secretion; after
a week application of ~he preparation, the secretion receded~
the defect was cleaned to clean granulations and the surrounding
of defect get calm~ ~
6) ~,.P~ 3 woman, age ~8~ report no. 1994~83, with a defect aftar
amputation of the 1st and 2nd toes including the heads o~
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corresponding metatarses for diabetic gangrene of size
Sx3 cm, with a scabby lower part, adhering necroses and
purulent secretion in the distal field of wound. After
4-week application of the preparation, clean granulations
were obtained in the whole range of wound, the purulent
secretion was suppressed and the defect was reduced to 3x2
cm by the advanced epithelisation.
7) M.T., woman, age 77, report no. 17741/83, with two defects
in an amputation stump of shin after amputation for a diabetic
gangrene followed by the abcessing phlegmon of stump, which
caused liqhefaction of the wound in whole region and defects
of the size lOx2-4 cm with a purulent secretion, scabby
granulations and extensive necroses. After 4-week application
of the preparation, the granulations became clean, necroses
were released, the secretion suppressed and epithelisation
advanced from the circumference of the defects so that these
; were medially reduced to diameter of 3 cm and laterally to
diameter of 1.5 cm.
