U.S. patent application number 09/351985 was filed with the patent office on 2002-01-03 for medicinal product for the promotion of wound healing.
Invention is credited to BRAUN, FRIEDRICH, EIBL, JOHANN, SPANGLER, HANS-PETER.
Application Number | 20020001624 09/351985 |
Document ID | / |
Family ID | 3523799 |
Filed Date | 2002-01-03 |
United States Patent
Application |
20020001624 |
Kind Code |
A1 |
BRAUN, FRIEDRICH ; et
al. |
January 3, 2002 |
MEDICINAL PRODUCT FOR THE PROMOTION OF WOUND HEALING
Abstract
The invention relates to a medicinal product for topical use for
the promotion of wound healing, which comprises thrombocytes or
thrombocyte fragments, wherein the thrombocytes or thrombocyte
fragments contain growth factors and are capable of releasing the
same and are present in the lyophilized or deep-frozen state and
have been subjected to a process for virus partitioning and/or
virus inactivation.
Inventors: |
BRAUN, FRIEDRICH; (VIENNA,
AT) ; SPANGLER, HANS-PETER; (VIENNA, AT) ;
EIBL, JOHANN; (VIENNA, AT) |
Correspondence
Address: |
BAKER & BOTTS
30 ROCKEFELLER PLAZA
NEW YORK
NY
10112
|
Family ID: |
3523799 |
Appl. No.: |
09/351985 |
Filed: |
July 12, 1999 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09351985 |
Jul 12, 1999 |
|
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PCT/AT98/00278 |
Nov 12, 1998 |
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Current U.S.
Class: |
424/532 ;
424/533; 424/534; 435/374; 514/13.6; 514/14.7; 514/3.7; 514/9.4;
530/382 |
Current CPC
Class: |
A61K 38/39 20130101;
A61L 26/0066 20130101; A61K 2300/00 20130101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K 35/19
20130101; A61K 38/363 20130101; A61L 2300/252 20130101; A61K
2300/00 20130101; A61K 2300/00 20130101; A61L 2300/414 20130101;
A61K 35/54 20130101; A61K 35/19 20130101; A61K 35/36 20130101; A61L
2300/626 20130101; A61P 17/02 20180101; A61P 43/00 20180101; A61K
35/36 20130101; A61K 38/363 20130101; A61K 35/54 20130101; A61L
26/0057 20130101; A61L 24/0005 20130101; A61L 24/0015 20130101;
A61K 38/4833 20130101; A61L 2300/64 20130101; A61K 38/4833
20130101; A61K 38/39 20130101 |
Class at
Publication: |
424/532 ;
424/533; 424/534; 435/374; 514/2; 514/21; 530/382 |
International
Class: |
A61K 038/00; A61K
035/14; A61K 009/00; A61K 035/18 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 12, 1997 |
AT |
A 1916/97 |
Claims
1. A medicinal product for topical use for the promotion of wound
healing, which comprises thrombocytes or thrombocyte fragments,
wherein said thrombocytes or thrombocyte fragments contain growth
factors and are capable of releasing the same, are present in the
lyophilized or deep-frozen state, and have been subjected to a
process for virus partitioning and/or virus inactivation.
2. A medicinal product according to claim 1, characterized in that
the content of thrombocytes or thrombocyte fragments is such that
it corresponds to at least 10.sup.4, preferably at least 10.sup.5,
thrombocytes per ml after reconstitution of the lyophilisate or
thawing.
3. A medicinal product according to claim 1 or 2, characterized in
that the medicinal product comprises additional growth factors.
4. A medicinal product according to any one of claims 1 to 3,
characterized in that the medicinal product comprises
biomaterials.
5. A medicinal product according to claim 4, characterized in that
the biomaterials have been subjected to a process for virus
partitioning and/or virus inactivation.
6. A medicinal product according to claim 4 or 5, characterized in
that the biomaterials are present in the lyophilized or deep-frozen
state.
7. A medicinal product according to any one of claims 4 to 6,
characterized in that tissue adhesive and/or collagen are provided
as biomaterials.
8. A medicinal product according to claim 7, characterized in that
the tissue adhesive is composed of fibrinogen-containing proteins
and thrombin.
9. A medicinal product according to any one of claims 4 to 8,
characterized in that the medicinal product additionally comprises
epithelial cells and/or keratinocytes and/or embryonic and/or fetal
cells and/or liposomes.
10. The use of thrombocytes or thrombocyte fragments containing
growth factors and capable of releasing the same, for the
production of a medicinal product for topical use for the promotion
of wound healing.
Description
[0001] This invention relates to a medicinal product for topical
use for the promotion of wound healing.
[0002] It is known that the healing of a wound progresses in
several successive stages.
[0003] In stage I, the blood plasma protein fibrinogen is
precipitated by thrombin so as to induce the formation of a fibrin
clot, which solidifies in the presence of blood coagulation factor
XIII. In the first stage which takes only minutes bleeding is
controlled and the wound area is sealed.
[0004] In stage II, cells from the wound area migrate into the
fibrin clot, i.e., inflammatory cells, connective tissue cells and
endothelial cells. They form vessels and, as an extracellular
matrix, connective tissue primarily comprised of collagen. This
connective tissue, which is referred to as granulation tissue,
serves as the substratum for the formation of epithelial tissue and
is the substratum for the epidermis on the body surface. Stage II
lasts for days to weeks and is complete as soon as the wound area
has been closed by epithelium, and by the epidermis on the
skin.
[0005] Wound healing is complete by stage II, which lasts for weeks
to months. During that phase, the cellular elements are reduced and
the connective tissue is growing so as to form a firm and permanent
scar tissue. (Bennett N. T., Schultz G. S., Am. J. Surg. 1993,
165:728-737; Bennett N. T., Schultz G. S., Am. J. Surg. 1993, 166:
74-81).
[0006] The formation of granulation tissue in stage II of the wound
healing process is effected by growth factors promoting the
migration and the division of connective tissue cells as well as
the regeneration of vessels and, thereby, accelerating wound
healing. Of the known growth factors, platelet derived growth
factor (PDGF), transforming growth factor .beta. (TGF-.beta.),
epidermal growth factor (EGF) and insulin-like growth factor I
(IGF-I) are particularly involved in those processes. (Bennett N.
T., Schultz G. S., Am. J. Surg. 1993, 165: 728-737; Bennett N. T.,
Schultz G. S., Am. J. Surg. 1993, 166: 74-81; Bhora F. Y. et al.,
J. Surg. Res. 1995, 59: 236-244; Lynch S. E. et al., Proc. Natl.
Acad. Sci. USA 1987, 84: 640-646; Lynch S. E. et al., J. Clin.
Invest. 1989, 84: 7696-7700).
[0007] Also the regeneration of the epidermis is induced by growth
factors. They activate the epidermal cells (keratinocytes) that
have been detached from the cell association of the intact basal
cell layer due to the lesion, so as to form specific membrane
receptors enabling the adherence to the granulation tissue
substratum, in particular to fibrin-fibronectin, which constitutes
a provisional scaffold for keratinocyte migration (Brown G. L. et
al., J. Exp. Med. 1986, 163: 1319-1324; Brown G. L. et al., N.
Engl. J. Med. 1989, 321: 76-79).
[0008] Growth factors are synthesized in the human body by various
tissues and cell types and secreted into the surrounding body
liquid. In the context of wound healing, an important regulatory
role is attributed to thrombocytes, which are able to synthesize in
significant amounts and store growth factors PDGF, TGF-.beta., EGF
and IGF-I, which are essential to wound healing in cytoplasmic
granula. (Lynch S. E. et al., Proc. Natl. Acad. Sci. USA 1987; 84:
640-646; Ginsberg M. H. et al., Thromb. Haemostas. 1988, 59: 1-6;
Hyner O. R., Thromb. Haemostas. 1991, 66: 40-43).
[0009] In order to release or deliver the stored growth factors
from the thrombocytes, the latter must be activated by
physiological stimuli such as, e.g., collagen, thrombin, trypsin,
ADP, serotonin or adrenalin, which bind to specific receptors on
the external surface of the thrombocyte plasma membrane. Activation
results in a change of shape followed by the aggregation of
thrombocytes, whereupon the latter secrete the stored growth
factors into the surrounding body liquid. With most of these
physiological stimuli, the aggregation of thrombocytes following
activation is a prerequisite for the release of growth factors. By
stimulation with thrombin, growth factors may be released also
without thrombocyte aggregation. (Kaplan K. L. et al., Blood 1979,
53: 604-618; Holmsen H. et al., J. Biol. Chem. 1981, 256:
9393-9396; Philipps D. R., Baughan A. K., J. Biol. Chem. 1983, 258:
10240-10245).
[0010] The interactions between activated thrombocytes, which lead
to aggregation, and their adherence to surfaces are mediated by
extracellular adhesive matrix proteins such as, e.g., fibrinogen,
fibronectin and von Willebrand factor, which bind to a glycoprotein
receptor on the external side of the plasma membrane of the
activated thrombocytes. Strong binding of these matrix proteins to
the receptor is effected only where thrombocytes have been
activated by an appropriate stimulus as described above. These
complex procedures of thrombocyte activation and aggregation
followed by the release of growth factors constitute one of the
essential control elements in the wound healing process. (Ginsberg
M. H. et al., Thromb. Haemostas. 1988, 59: 1-6; Hyner O. R.,
Thromb. Haemostas. 1991, 66: 40-43; Landolfi R. et al., Blood 1991,
78: 377-381; Perschke E. I. et al., Blood 1980, 55: 841-847; Hynes
O. R., Cell 1992, 69: 11-25; Perschke E. I., J. Lab. Clin. Med.
1994, 124: 439-446; Savage B.,Ruggeri Z. M., J. Biol. Chem. 1991,
266: 11227-11233; Bennett J. S. et al., J. Biol. Chem. 1982, 257:
8049-8054; Cierniewski C. S. et al., Biochim. Biophys. Acta 1982,
714: 543-548; Philipps D. R., Baughan A. K., J. Biol. Chem. 1983,
258: 10240-10245).
[0011] Disturbances in wound healing as these occur, for instance,
in patients with diabetes, venous or arterial occlusions, but also
wound healing disturbances of other geneses such as, for instance,
irradiation with radioactive substances or after bums particularly
affect stage II of the wound healing process. It has been found
that in such cases growth factors are present to a reduced extent
so that no or only a low quality granulation tissue is formed.
(Dvonch V. M. et al., Surgery 1992, 112: 18-23; Matsuoka J.,
Grotendorst G. R., Proc. Natl. Acad. Sci. USA 1989, 86:
4416-4420).
[0012] In order to enhance wound healing in the case of wound
healing disturbances, growth factors are known to be applied to the
wound area, either individually or in combination, as a pure
substance or mixed in ointment bases (Knighton D. R. et al., Surg.
Gynecol. Obstet. 1990, 170: 56-60; Brown G. L. et al., J. Exp. Med.
1986,163: 1319-1324; Holmsen H. et al., J. Biol. Chem. 1981, 256:
9393-9396). The growth factors provided in this manner are,
however, rapidly inactivated or degraded and develop their
activities only over short periods of time (minutes) after
application. Thus, these preparations offer no satisfactory
enhancement of wound healing.
[0013] Other known therapeutic approaches consist in covering the
wound area with collagen sponges or other preparations aimed to
ensure permanent humidity of the wound area or in using
preparations degrading the superficial connective tissue layer of
the wound area by fermentation so as to enable new connective
tissue to re-grow from the wound bed (Nielsen P. G. et al., Acta
Dermato-Venerologica 1990, Suppl. 152: 1-12; Lippert P., Wolff H.,
Zent.bl. Chir. 1990, 115: 1175-1180). Yet, none of those hitherto
applied wound dressings and preparations or medicinal products have
brought satisfactory results in improving wound healing.
[0014] The object of the present invention is to provide a
medicinal product which efficaciously accelerates natural wound
healing processes and is capable of substantially improving wound
healing where wound healing is disturbed, in particular in severe
forms of wound healing disturbances, as compared to conventional
therapies.
[0015] In accordance with the invention, this object is achieved in
that a medicinal product for topical use for the promotion of wound
healing is provided, which comprises thrombocytes or thrombocyte
fragments, wherein said thrombocytes or thrombocyte fragments
contain growth factors and are capable of releasing the same, are
present in the lyophilized or deep-frozen state and have been
subjected to a process for virus partitioning and/or virus
inactivation.
[0016] "Thrombocyte fragments" is intended to denote any insoluble
thrombocyte constituents that are separable from the soluble
thrombocyte constituents either by filtration including
nano-filtration or by centrifugation including
ultracentrifugation.
[0017] Unless indicated otherwise, the term "thrombocytes" in the
following also encompasses "thrombocyte fragments".
[0018] The invention is based on the finding that the topical use
of thrombocytes containing growth factors and capable of releasing
the same can efficaciously accelerate wound healing processes. The
thrombocytes applied on the wound area constitute a natural
reservoir for the growth factors required for the promotion of the
wound healing processes. It has been found that the activation of
locally applied thrombocytes by physiological stimuli present in
the wound area and the subsequent aggregation and binding of the
matrix proteins present in the wound area lead the growth factors
stored in the thrombocytes to be released into the wound area
continuously over an extended period of time (several days). Due to
this fact, higher concentrations of growth factors are apparently
availabe in the wound area over a substantially longer period of
time than with the direct administration of growth factors, thereby
promoting the immigration of inflammatory cells, connective tissue
cells and endothelial cells and enhancing the propagation of said
cells in stage II of the wound healing process. In that manner, the
rapid and sufficient formation of granulation tissue is ensured,
which, in turn, renders possible the formation of epithelial tissue
and the final wound closure. The epithelization process, moreover,
is additionally accelerated by the released growth factors
promoting the immigration and proliferation of epithelial
cells.
[0019] To ensure that the medicinal product can be stored over an
extended period of time, the thrombocytes in the medicinal product
according to the invention preferably are present in the
lyophilized or deep-frozen state. In order to minimize the risk of
virus infections, the thrombocytes advantageously are subjected to
a process for virus partitioning and/or virus inactivation, whereby
a physical or a chemical or a combined process may be used.
[0020] In order to provide for a higher concentration of growth
factors, in particular in the treatment of wound healing
disturbances, it is preferred that the content of thrombocytes or
thrombocyte fragments of the medicinal product according to the
invention is such that it corresponds to at least 10.sup.4,
preferably at least 10.sup.5, thrombocytes per .mu.l after
reconstitution of the lyophilisate or thawing.
[0021] In order to obtain a particularly pronounced initial effect
of the medicinal product according to the invention immediately
upon application, it may be appropriate, in particular in the case
of severe disturbances of wound healing, that the medicinal product
comprises additional growth factors that are not derived from the
thrombocytes contained in the medicinal product. The additional
growth factors may be of the same type as those stored and released
by the thrombocytes of the medicinal product according to the
invention or belong to a different type. The growth factors may be
present in the same container with the thrombocytes or contained in
a separate container in the form of a solution or lyophilisate.
[0022] It has been found that it is advantageous, in particular in
severe cases of disturbed wound healing, that the medicinal product
comprises biomaterials. "Biomaterials" in the sense of the
invention is intended to comprise any materials which are
tissue-compatible and absorbable and assist in the promotion of
wound healing either in combination with the thrombocytes or growth
factors contained in the medicinal product or independently
thereof. Thus, substances activating thrombocytes as stimuli and/or
materials mediating thrombocyte aggregation may be contained as
biomaterials in the medicinal product according to the invention.
In that manner, the activity of natural substances present in the
wound area which activate thrombocytes and mediate their
aggregation is enhanced, which increases the release of growth
factors and promotes wound healing even further.
[0023] In order to minimize the risk of virus infections, the
biomaterials preferably are subjected to a process for virus
partitioning and/or virus inactivation, wherein a physical or
chemical process or a combined process may be applied. The
biomaterials may be subjected to such a process either individually
or mixed with other components of the medicinal product (e.g.,
thrombocytes).
[0024] To ensure that the medicinal product can be stored over an
extended period of time, the biomaterials in the medicinal product
according to the invention advantageously are present in the
lyophilized or deep-frozen state. In that case, the biomaterials
may be present in the same containers with the thrombocytes and/or
growth factors or contained in separate containers and
deep-freezing or lyophilization of the biomaterials may be effected
individually or in mixture with other components of the medicinal
product.
[0025] It is known that the activation and aggregation of
thrombocytes and hence the release of growth factors stored in the
thrombocytes is enabled by the attachment of matrix proteins.
Moreover, such proteins may form cross-linked structures to which
the thrombocytes adhere and firmly bind to the wound area, such
structures promoting the diffusion of growth factors to the wound
area and the immigration of cells from the wound area. Accordingly,
a preferred embodiment of the medicinal product according to the
invention is characterized in that tissue adhesive and/or collagen
are provided as biomaterials. Tissue adhesive in the sense of the
invention is intended to encompass biomaterials totally or
partially consisting of cross-linkable proteins suitable for tissue
adhesion.
[0026] Fibrinogen is a particularly active substance for triggering
the aggregation of activated thrombocytes, while thrombin
represents one of the most active substances for the activation of
thrombocytes. It is, therefore, advantageous for an increase in the
relase of growth factors and an enhancement of wound healing that
the tissue adhesive is composed of fibrinogen-containing proteins
and thrombin.
[0027] It has been shown that human cells such as keratinocytes,
epithelial cells, embryonic and fetal cells as well as cell
constituents such as liposomes are able to additionally accelerate
thrombocyte-promoted wound healing and cell propagation. It is,
therefore, preferred that the medicinal product additionally
comprises epithelial cells and/or keratinocytes and/or embryonic
and/or fetal cells and/or liposomes. The cells or the liposomes may
be present as a liquid or deep-frozen suspension or as a
lyophilisate in separate containers, or one or several of the
mentioned cell types or liposomes either without or with any of the
other components of the medicinal product in common containers.
[0028] In order to minimize the risk of virus infections, the cells
or the liposomes may have been subjected to a process for virus
partitioning and/or virus inactivation, whereby a physical or a
chemical process or a combined process may be used. The cells or
the liposomes may be subjected to such a process either
individually or mixed with other components of the medicinal
product.
[0029] The invention also relates to the use of thrombocytes or
thrombocyte fragments containing growth factors for the production
of a medicinal product for topical use for the promotion of wound
healing.
[0030] Preferred embodiments of the invention will now be explained
in more detail by way of examples.
EXAMPLE 1
[0031] Preparation of a Medicinal Product According to the
Invention
[0032] A human thrombocyte concentrate or concentrate of
thrombocyte constituents is anticoagulated by 3% sodium citrate and
centrifuged (1000 g/20 min) in order to eliminate plasma and other
cell constituents. The thrombocyte-rich supernatant, or supernatant
of thrombocyte constituents, is suspended in RPMI medium and washed
three times in RPMI medium (1000 g/20 min). The washed
thrombocytes, or the washed thrombocyte constituents, are suspended
in RPMI medium and adjusted to a concentration of at least
6.times.10.sup.5 thrombocytes or thrombocyte constituents per
.mu.l. After this, the thrombocyte suspension is subjected to a
virus inactivation process according to Example 3 and subsequently
deep-frozen or lyophilized in accordance with the methods described
below, thereby obtaining a medicinal product according to the
invention.
[0033] Deep-freezing:
[0034] 1 ml of the thrombocyte suspension is each shock deep-frozen
at -80.degree. C. within 30-40 minutes and stored in a deep-frozen
state. Before use, the thrombocyte concentrate is thawed at room
temperature.
[0035] Lyophilization:
[0036] 1 ml of the thrombocyte suspension is each shock deep-frozen
at -80.degree. C. for at least 24 hours and subsequently
lyophilized at -20.degree. C. to -40.degree. C. in vacuo for 20 to
24 hours. The lyophilized thrombocytes are stored at between
-20.degree. C. and -80.degree. C. and rehydrated with 1 ml RPMI
medium before use.
EXAMPLE 2
[0037] Preparation of a Medicinal Product According to the
Invention Comprising Biomaterials
[0038] The virus-inactivated thrombocyte suspension prepared
according to Example 1 is supplemented with a solution of
cross-linkable human protein (either fibrinogen, fibronectin, blood
coagulation factor XIII or collagen) which may have been subjected
to one or several processes for virus inactivation according to
Example 4, each protein type separately or together in combination,
wherein the concentration of the cross-linkable protein types in
the supplemented solution preferably is to amount to 70-90 mg/ml.
The mixing ratio of the thrombocyte suspension to the solution of
cross-linkable human protein preferably is to be 1:3. The thus
obtained mixture is deep-frozen or lyophilized in accordance with
the processes described in Example 1 in order to obtain suitable
storability.
[0039] Instead of carrying out virus inactivation on individual
components (thrombocytes or biomaterials), it is also possible to
effect virus inactivation on a mixture of thrombocyte suspension
and protein solution according to the process of Example 3.
EXAMPLE 3
[0040] Virus Inactivation of Thrombocyte Suspension (Photodynamic
Virus Inactivation)
[0041] To 50 ml of the thrombocyte suspension prepared according to
Example 1 is added 8-methoxypsoralen (dissolved in
dimethylsulfoxide [DMSO]) until a final concentration of 300
.mu.l/ml (final concentration of DMSO 0.3%) and irradiated with
ultraviolet light from below and above for 6 hours at 22-27.degree.
C. under an atmosphere of 5% CO.sub.2 and 95% N.sub.2 and at a
pressure of 2 psi such that the overall light intensity is 3.5 to
4.8 mW/cm.sup.2 (Lin L. et al., Blood 1989, 74: 517-525).
[0042] After photoinactivation has been completed, the thrombocyte
suspensions obtained in that manner are examined for their
finctional capacities. The functional capacity is determined by
measuring [.sup.3H]-thymidine incorporation in a fibroblast cell
culture.
EXAMPLE 4
[0043] Virus Inactivation of Biomaterials (Chemical Virus
Inactivation)
[0044] Biomaterials which are admixed to the thrombocyte suspension
prepared according to Example 1 are virus inactivated by a solvent
detergent method. To this end, a biomaterial suspension is
supplemented with 1% (w/w) tri(n-butyl) phosphate and 1% (w/w)
Triton X-100 at 30.degree. C. and the mixture is kept for 4 hours
under shaking. After this, the solvent detergent mixture under the
addition of 5% (v/v) soybean oil is removed from the biomaterial
suspension by chromatography on a C18-column (Waters Millipore)
(Horowitz B. et al., Blood 1992, 79: 826-831; Piet M. P. J. et al.,
Transfusion 1990, 30:591-598; Piquet Y. et al., Vox sang. 1992, 63:
251-256).
[0045] The biomaterials treated by the above-described chemical
virus inactivation method may subsequently be subjected to
photodynamic virus inactivation in addition.
EXAMPLE 5
[0046] Evaluation of the Promotion of Connective Tissue
Proliferation by the Medicinal Product According to the
Invention
[0047] The test was carried out on a fibroblast cell culture. The
medicinal product prepared according to Example 2 was applied on a
cell culture plate in an amount of 200 .mu.l per cm.sup.2 and
activated by 50 .mu.l of a thrombin solution (3.2 IU thrombin per
ml physiological saline). Human fibroblasts derived from the
4.sup.th to 10.sup.th passages of a primary culture were placed on
the applied suspension at a density of 4.times.10.sup.4 cells per
cm.sup.2 and cultivated in cell culture medium (RPMI) (culture 1).
On the third, fifth and seventh days of cultivation, the cell
mitotic rate was measured by measuring DNA synthesis via
[.sup.3H]-thymidine incorporation. The cell mitotic rate of culture
1 was compared to the cell mitotic rate of another fibroblast
culture (culture 2) realized in RPMI nutrient supplemented with 10%
by vol. of calf serum without addition of the medicinal product
according to the invention.
[0048] Results:
[0049] On day 3 of cultivation, culture 1 exhibited a
[.sup.3H]-thymidine incorporation (196645.+-.56864 cpm/ml) that was
seven times higher than that of culture 2. On days 5
(152749.+-.93951 cpm/ml) and 7 (77045.+-.27974 cpm/ml)
[.sup.3H]-thymidine incorporation in culture 1 still was 5 to 10
times higher than that of culture 2. These differences between
culture 1 and culture 2 statistically are highly significant
(p<0.01), demonstrating the ability of the medicinal product
according to the invention to promote connective tissue
proliferation and maintain that activity over an extended period of
time (at least 7 days).
EXAMPLE 6
[0050] Evaluation of the Binding of Matrix Proteins to Thrombocyte
Surfaces Resulting in the Thrombocyte Stored Growth Factors to be
Continuously Released
[0051] The test was carried out on a fibroblast culture (according
to Example 5). Culture 1--as in Example 5--was supplemented with
the medicinal product according to the invention. In culture 2, the
thrombocytes were treated with specific antibodies, against the
superficial binding sites for matrix proteins so as to prevent the
matrix proteins from binding to thrombocyte surfaces. On the third
day of cultivation, the cell mitotic rate was measured by measuring
DNA synthesis via [.sup.3H]-thymidine incorporation.
[0052] Results:
[0053] While culture 1 exhibited a thymidine incorporation rate
similar to that of Example 5, no thymidine incorporation could be
measured in culture 2. That difference proves that the binding of
matrix proteins to the thrombocyte surfaces is necessary for the
thrombocyte stored growth factors to be released.
EXAMPLE 7
[0054] Evaluation of the Promotion of Wound Healing by the
Medicinal Product According to the Invention
[0055] The clinical efficacy of the medicinal product according to
the invention was studied in six patients suffering from chronic,
non-healing cutaneous ulcera of the lower extremities and already
treated by surgical or conservative topical therapies for more than
six months without success. The ulcera were classified using a
wound score according to Knighton D. R. et al., Ann. Surg. 1986,
204:322-330. The wound score includes general parameters,
anatomical conditions and measurable variables of the ulcus. The
higher the scores, the poorer the preconditions for healing; the
highest score to be reached is 97 (=worst starting situation).
[0056] Treatment Plan:
[0057] The ulcera were cleaned, necrotic tissue was removed and
wetted with a thrombin solution (3.2 IU bovine thrombin/ml RPMI
medium). After this, the defect was filled up with the thawed
medicinal product according to the invention prepared according to
Example 2, and the above-mentioned thrombin solution was then
applied at a volume ratio of medicinal product suspension to
thrombin solution of 3:1 in order to activate the thrombocytes. The
ulcera eated in that manner were covered by a non-adhering wound
dressing (metal foil). Until healing, the ulcera were treated twice
a week in the above-identified manner. The healing progress was
documented photographically and histologically (fine needle
biopsies in the 2.sup.nd and 5.sup.th weeks of treatment).
[0058] Relults:
[0059] The demographics, causative vascular and metabolic diseases
of the patients and the evaluation of the wound scores at the
beginning of treatment are summarized in Table 1.
1 TABLE 1 Vascular Disease Metabolic Wound Patient Sex Age arterial
venous disease Score 1 male 67 + + diabetes 51 2 male 72 + - -- 65
3 male 69 + - diabetes 33 4 male 63 + - diabetes 49 5 male 78 + +
diabetes 63 6 female 74 - + -- 65.sup.a/63.sup.b .sup.a,btwo ulcera
on one leg: .sup.aproximal, .sup.bdistal ulcus
[0060] The time course of wound healing (indicated in weeks as of
the beginning of treatment) is illustrated in Table 2.
2TABLE 2 Beginning of Granulation Tissue Beginning of Completion of
Patient Formation Epithelization Epithelization 1 1st week 3rd week
8th week 2 1st week 3rd week 9th week 3 3rd week 8th week 12th week
4 1st week 4th week 10th week 5 1st week none none 6 .sup.a,b1st
week .sup.a6th/.sup.b3rd week .sup.a12th/.sup.b9th week .sup.a,btwo
ulcera on one leg: .sup.aproximal, .sup.bdistal ulcus
[0061] With the exception of patient 3, a granulation tissue well
supplied with blood formed starting from the bottom of the ulcus in
all of the patients already within the first week of treatment,
which granulation tissue increased upon further treatment with the
medicinal product according to the invention until approximately
two weeks after the beginning of the therapy and filled up the
ulcus. It was striking that already after the first days of
treatment the surrounding of the ulcus calmed down, the erythema
and the edema of the surrounding skin disappeared and also the edge
of the ulcus was no longer edematous and miscolored in all of the
patients. Histologically, cell-rich granulation tissue primarily
consisting of fibroblasts and fibrocytes and exhibiting intensive
new vascular formation and collagenous fiber formation and only a
slight infiltration of inflammatory cells and tissue necroses on
the surface was to be seen in all biopsies in the second week of
treatment. Epithelization of the skin defects after the third week
of treatment started from the edges of the wound and could then
also be detected histologically by the second biopsies in the fifth
week of treatment. In the further course of treatment, the size of
the ulcera declined due to epithelization, but also to cicatricial
shrinkage. With the exception of patient 5, they were scarred over
in the 12.sup.th week of treatment the latest.
[0062] The results indicated above demonstrate that the topical use
of the medicinal product according to the invention promotes wound
healing and, thus, is able to completely cure chemically
non-healing cutaneous ulcera in patients treated by conservative
therapies for at least six months without success and, thus,
offering extremely poor prognoses for wound healing.
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