Note: Descriptions are shown in the official language in which they were submitted.
oO 94/22470 PCT/IB94/00040
2159603
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FACTOR XIII FOR TREATMENT OF SKIN WOUNDS.
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The present invention relates to an external preparation for treatment of skin
wounds containing as an active ingredient human blood coagulation factor XIII.
Human coagulation factor XIII has been known in the past as a therapeutic
agent
for skin wounds, and has been administered intravenously. However, there are
problems with this administration route regarding delivery of the drug to the
wound
site. In addition, this administration requires the attendance of a physician.
In EP A-0 358 995 it is disclosed that factor XIII can be used in a process
for
preparing a topical medicament for immunsuppression.
It is an object of the present invention to provide a human coagulation factor
XIII
preparation for skin treatment that is free of the problems of the prior art
mentioned above.
The present invention provides a dermal preparation for treatment of skin
wounds
containing as an active ingredient human blood coagulation factor XIII. It is
preferable that the dermal preparation of the present invention contain
aprotinin
as well. The dennal preparation of the present invention is particularly
suitable for
local treatment of bums. Human blood coagulation factor XIII of human plasma
or
placenta origin is used preferably, and that produced by recombinant gene
technology can also be used.
Human blood coagulation factor XIII (hereinafter referred to as factor XIII)
is
widely found in plasma, placenta and the like and activated with thrombin and
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WO 94/22470 PCT/IB94/00040
Ca2+. The activated factor XIII catalyzes i;-(y-glutamyl)Iysil cross-links
between
specific protein pairs. The cross-linking between fibrin monomers to form y-y
dimers and a-chains to form a-polymers by activated factor XIII could provide
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fibrin with strength and elasticity and act on hemostatic mechanism.
Furthermore,
factor XIII catalyzes crosslinking of fibronectin to an a-chain of fibrin or
collagen,
and thus plays an important role in the process of wound healing [Matsuda,
Acta
Haematologica Japonica (in Japanese)(1977), Vol. 40, No. 6, pp. 995-1002].
Factor XIII preparation has been intravenously administered mainly for the
treatment of wound healing disturbances; now, the present inventors have made
various studies and, as a result, found that factor XIII could percutaneously
pene-
trate wounded skin and permeate into the affected skin tissue in a sufficient
amount to be required for the treatment. The present invention has been
completed to prove that factor XIII is effective as a dermal agent as well.
Factor XIII preparation does not cause any troubles in side-effects, toxicity
and
othe'rs usually at a dose of 20-50 units/kg/body/day, in view of the results
of acute
toxicity test as described below and intravenous applications of over 10,000
cases
at home and abroad.
As a dermal agent, one may administer the agent at 1-1000 units/site to be
given/body/day, depending upon the severity of disturbance at the damaged
portion and the damaged stage. One unit of factor XIII as used herein is
defined
as an amount of factor XIII contained in 1 ml of plasma of healthy person,
which is
usually about 15 g/mI.
The results of acute toxicity test of factor XIII are shown in Table 1.
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00 94/22470 PCT/IB94/00040
2159609
Table 1
Animal species Administration Sex LD50
route units/k
Mice (NMRI strain) i.v. Male >3,125
Male and female, Female
each 10
animals/group
Rats (Wistar i.v. Male >625
strain) Female
Male and female,
each 10
animals/ rou
The dermal agent of the present invention is applicable to treatment of a
variety of
skin wounds such as cut wound, stabbed wound, cracked wound, contused
wound, lacerated wound, decupitus, burn and the like and is particularly
effective
for local treatment of bum.
Factor XI11 preparation may be prepared by fractional purification of plasma
starting from human placenta or plasma according to a well-known method in the
art. The preparation as prepared by fractional purification of plasma might
contain
hepatitis virus, AIDS virus and others and such viruses should be inactivated
by
heat treatment and other means. Heat treatment is performed by dissolving
factor
Xill in an aqueous solution of sodium chloride containing EDTA and heating the
solution at about 60 C for approximately 10 hours.
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WO 94/22470 PCT/IB94/00040
Factor XIII may be also prepared according to genetic engineering techniques.
Factor XIII which is employed in the present invention may include those
factors
prepared by all available methods including not only fractional purification
but also
genetic engineering techniques and others.
As the dosage forms of the present dermal agent, there may be mentioned water-
soluble ointments, oleaginous ointments, lotions, sprays, oils, gels and the
like.
As representative bases, there may be mentioned macrogol bases for water-
soluble ointments, petrolatum bases for oleaginous ointments, olive oil,
sesame
oil, camellia oil and the like for oils, carboxyvinyl polymers, sodium
polyacrylate
and the like for gels.
Factor XIII is, for instance, dissolved in an aqueous solution of sodium
chloride
containing glucose, human serum albumin and the like, aseptically filtered
after its
titer is adjusted, poured into vials and then freeze-dried. At the time of
use, it is
reconstituted in distilled water for injection and the resultant solution as
such is
used as solutions or filled into a vessel equipped with a spray device and
then
used as sprays.
As assays for factor XIII activity, there are employed a fibrin-formation
method, a
transglutaminase activity assay, an immunological assay for antigen level and
others. There was employed in Examples of the present invention the dansyl-
cadaverine method by Y. Nishida et al., which can determine an amount of
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*0 94/22470 PCT/IB94/00040
ay f9
activated factor XIII with a high sensitivity [Y. Nishida et al., (1984),
Thromb. Res.,
Vol. 36, pp. 123-131]. Dansylcadaverine (available from IATRON Laboratories,
Inc.) is a fluorescent amine which may be a substrate for factor XIII, it may
form a
dansylcadaverine-casein complex with casein by the action of factor XIII and,
after
reaction, fluorescence intensity of the said complex recovered by gel
filtration may
be measured to determine the factor XIII activity.
This invention will be more illustratively explained by way of the following
Production Example, Preparation Examples and Test Examples.
Examples:
Production Example 1- Production of factor XIII from human placenta
Placenta was freezed, finely divided, stirred with an aqueous solution of
sodium chloride and then centrifuged to collect supernatant I. The supernatant
I
was confirmed to be negative for HBs antigen by enzyme immunoassay and an
acrinol (Rivanol) solution was added to form precipitate II, which was then
washed
and stirred with an aqueous solution of sodium chloride containing EDTA. After
removing undissolved substance (precipitate III), to the resultant supernatant
III
was added an N-cetyl-pyridinium chloride solution to precipitate concomitant
proteins and mucopolysaccharides and then an acrinol solution was added to
supernatant IV to form a precipitate V containing factor XIII. To the
precipitate V
was added an aqueous solution of sodium chloride containing EDTA. After
stirring, to supernatant VI, from which undissolved substance (precipitate VI)
was
removed, was added ammonium sulfate to form a precipitate VII containing
factor
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WO 94/22470 PCT/1B94/00040 ~
XIII. To the precipitate VII was added an EDTA solution and the resulting
mixture
was dialyzed against a Tris-hydrochioride buffer containing EDTA and sodium
azide. After pH was adjusted, the precipitate VIII thus formed was removed,
supernatant VIII was gel-filtrated to collect active fractions. To the
fractions was
added ammonium sulfate to form a precipitate IX containing factor XIII. The
precipitate IX was dissolved in a Tris-hydrochloride buffer containing EDTA,
dialyzed against the same buffer and pH was adjusted to precipitate factor
XIII as
an euglobulin. This euglobulin precipitate was dissolved in an aqueous
solution
of sodium chloride and then aminoacetic acid and sucrose were added. Then,
ammonium sulfate was added to form a precipitate X containing factor XIII,
which
was dissolved in an aqueous solution of sodium chloride containing EDTA and
dialyzed against the same solution. Thereafter, the titer of factor XIII was
adjusted
using an aqueous solution of sodium chloride containing glucose and human
serum albumin.
Preparation Example 1 - Preparation 1 of a therapeutic agent for dermal use
containing factor XIII
An dermal preparation was prepared using the factor XIII solution
prepared as described in the above Production Example 1. More specifically,
after factor XIII was adjusted to a titer of 240 units/mI and filtered
aseptically, the
so adjusted factor XIII was transferred to a glass vial together with 32 mg of
human serum albumin, 20 mg of glucose and 34 mg of sodium chloride, followed
by freeze drying. At the time of use, this freeze-dried factor XIII was
dissolved
with distilled water for injection so that the concentration of factor XIII at
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OD 94/22470 PCT/IB94/00040
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administration was 120 units/mi. This solution was used as a spray solution.
Preparation Example 2 - Preparation 2 of a therapeutic agent for dermal use
containing factor XIII
The freeze-dried factor XIII identical to that prepared in Preparation
Example 1 was dissolved in an aprotinin solution having a concentration of
1000
units/ml or more at the time of use so that the concentration of factor XIII
was 120
units/mi. This solution was used as a spray solution.
Test Example 1- In Vitro Diffusion Cell Study
The abdominal skin was excised from Sprague-Dawley rats (males, 9-10
weeks old, body weight: 300 g) after shaving, or heating for 45 seconds in a
60 C
water bath after shaving, and sandwiched between a lid and a receptor chamber
for measurement of percutaneous absorption having a surface area of approxi-
mately 8 cm2 (John J. Windheuser, et al. (1982), J. of Pharmaceutical
Sciences,
Vol. 71, No. 11, pp. 1211-1213). The preparation of Preparation Example 1 or 2
(concentration: 120 units/mI) was applied in an amount of 1 ml to the upper
surface of the skin specimens. The lower surface of each skin specimen was
brought into.contact in advance with 45 ml of 0.05 M Tris-HCI=0.15 M NaCi
solution (pH 7.5). The cells were maintained at 37 C to observe percutaneous
absorption for 8 hours. During the period, 20 l samples were withdrawn from
the
receptor solution through the side arm every 2 hours to determine the activity
of
factor XIII in those samples. The results for the skin excised after shaving
are
shown in Table 2, while the results for the skin heat treated after shaving
are
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WO 94/22470 PCT/IB94/00040
shown in Table 3, provided that the total amount of the preparation applied to
the
skin upper surface is 100%.
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0694/22470 PCT/IB94/00040
Table 2
Cumulative Vaiue of Activity of
Preparation Permeated Skin
Pre aration After 2 hrs. After,4 hrs. After 6 hrs. After 8 hrs.
Preparation
Example 1 0.2 0.3 0.2 0.3
Preparation
Example 2 0.4 0.5 0.5 0.5
(n=3)
Table 3
Cumulative Value of Activity of
Preparation Permeated Skin %
Preparation After 2 hrs. After 4 hrs. After 6 hrs. After 8 hrs.
Preparation
Example 1 0.7 5.9 9.5 12.5
Preparation
Example 2 3.2 9.8 16.6 23.0
(n=3)
When the skin excised after shaving was used, factor XIII had hardly
permeated the skin in the case of both Preparation Example 1 and Preparation
Example 2. However, when the skin that had been bumed by heat treatment was
used, factor Xill permeated well the skinboth in the case of Preparation
Example
1 and Preparation Example 2. Preparation Example 2 containing aprotinin
showed particularly excellent permeation.
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WO 94/22470 PCT/IB94/00040
Test Example 2 - Rat Burn Model Administration Test
The dorsal skin of Sprague-Dawley rats (males, 9-10 weeks old, body
weight: 300 g) was shaved, and a metal rod heated to 85 C (70 g, diameter: 18
mm) was pressed to the skin for 20 seconds under nembutal anesthesia to
provide burn models. Two bum models were formed on both sides of the
backbone of each animal. The preparation of Preparation Example 2 was sprayed
in an amount of 30 units/0.2 ml onto the surface of the burned skin each day.
Specimens of burned skin having a surface area of approximately 2 cm2 were
then excised after 2 days, washed with physiological saline solution and
homoge-
nized after addition of 0.05 M Tris-HCIØ15 M NaCi solution (pH 7.5). After
centrifugation of the homogenate, the activity of factor XIII in the
supernatant was
determined. As a control, 120 units of the preparation were administered
intravenously, and its dorsal skin having the same area as that of the burned
skin
was excised after 2 days. The specimen was washed with physiological saline
solution and homogenized after addition of 0.05 M Tris-HCI. 0.15 M NaCI
solution
(pH 7.5). After centrifugation of the homogenate, the activity of factor XIII
in the
supernatant was determined. As a result, the activity of factor XIII per 1 cm2
of
skin is shown in Table 4 in terms of the unit of factor XIII defined above.
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94/22470 PCT/1B94/00040
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4949
Table 4
Administration Factor XIII Activity in Skil~i
Method (units/cm)
Spray Adminis- 0.050
tration
Intravenous 0.023
Administration
(n=4)
According to the results shown in Table 4, although the total dose in the
case of spray administration is 1/2 that of intravenous administration, the
factor
XIII activity in the skin was roughly 2 times greater. This result suggests
that it is
more effective to administer the factor XIII preparation in the form of an
dermal
preparation rather than intravenous administration in terms of treatment of
wounds.
Production Example 2 - Expression of recombinant factor XIII
The amino acid sequence of the recombinant factor XIII used in the
present invention is that of human factor XIII from the first position, Ser,
to the
731 st position, Met,
and Phe is substituted for Leu in the 88th position in Figure 3 of Grundmann
(U.
Grundmann, et al. (1986), Proc. Natl. Acad. Sci. USA, Vol. 83, pp. 8024-8028.
The DNA sequence encoding the amino acid sequence described above was
inserted into the region from Sstl to HindIIl of the polylinker site of
pEMBLyex4 (J.
K. Selton, Genetic Engineering (1987), Plenum Publishing Co., Vol. 9, pp.
135-154) that is an expression vector for yeast, which was used as a plasmid
for
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WO 94/22470 PCT/IB94/00040
production of factor XIII. The yeast host cell, CL3ABYS86 (Offenlegungsshrift
DE
3804890 Al), was transfected by the above plasmid to produce recombinant
factor XIII in yeast cells. Then the recombinant factor XIII was purified from
the
supernatant of yeast cell destruction solution.
Preparation Example 3- Preparation 3 of a therapeutic agent for dermal use
containing factor XIII
The recombinant factor XIII solution produced in the above Production
Example 2 was diluted with a solution containing 1.6 % of human serum albumin,
1% of glucose and 1.7% of sodium chloridis so that the concentration of factor
XIII
at administration is 120 units/mi. This solution was used as a spray solution.
Test Example 3- In Vitro Diffusion Cell Study
The abdominal skin was excised from Sprague-Dawley rats (males, 9-10
weeks old, body weight: 300 g) after shaving, or heating for 45 seconds in a
60 C
water bath after shaving, and sandwiched between a lid and a receptor chamber
for measurement of percutaneous absorption having a diameter of 7 mm (above
described in Test Example 1). The preparation of Preparation Example I or 3
concentration: 120 units/mi) was applied in an amount of 0.1 ml to the upper
surface of the skin specimens. The lower surface of each skin specimen was
brought into contact in advance with 4.7 ml of 0.05 M Tris-
HCI=0.15M NaCi solution (pH 7.5). The cells were maintained at 37 C to observe
percutaneous absorption for 8 hours. During the period, 20 l samples were
withdrawn from the receptor solution through the side arm every 2 hours to
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determine the activity of factor XIII in those samples. The results for the
skin
excised after shaving are shown in Table 5, while the results for the skin
heat
treated after shaving are shown in Table 6, provided that the total amount of
the
preparation applied to the skin upper surface is 100%.
Table 5
Cumulative Value of Activity of
Preparation Permeated Skin !o
Preparation After 2 hrs. After 4 hrs. After 6 hrs. After 8 hrs.
Preparation
Exam le 1 0.1 0.2 0 0
Preparation
Example 3 0.3 0.3 0.3 0.5
(n=3)
Table 6
Cumulative Value of Activity of
Preparation Permeated Skin (%)
Preparation After 2 hrs. After 4 hrs. After 6 hrs. After 8 hrs.
Preparation
Exam le 1 0.1 1.2 4.3 7.9
Preparation
Example 3 0.3 3.2 6.5 12.7
(n=3)
The results above show that the dermal preparation containing the
recombinant factor XIII is as effective as that containing factor XIII of
human
placenta origin.
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WO 94/22470 PCT/IB94/00040
When the dermal preparation of the present invention is applied to the
wounded skin, particularly to burns or scalds, the active ingredient human
blood E
coagulation factor XIII shows excellent drug delivery to the wound compared
with
the case of intravenous administration. In addition, the preparation of the
present
invention can be administered without assistance of a physician.
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