Note: Descriptions are shown in the official language in which they were submitted.
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9785-15
D e s c r i p t i o n
It is known from in vitro experiments on cell cultures
that Ca2+ions exert an influence on the division of cells. (Proc.
Nat. Acad. Sci. 70, p. 675-679). It is also known that the depol-
arization of the membrane potential may be realized by raising the
extracellular K+-concentration. So far, however, there' have been
no studies investigating the influences these effects exert on the
granulation and epithelization of wounds. Extensive investiga-
tions have shown that neither the Ca2+ concentrations established
as being optimal for the growth of cell cultures, nor increased
Ca~+ concentrations exert a promoting or inhibiting influence on
the healing of wounds.
The addition of physiological amounts of potassium in
the extracellular space also does not promote the healing of
wounds, not even if at the same time there is provided an optimal
extracellular Ca2+ concentration.
Surprisingly it has now first been found that Ca2~ and
K+ ions together, in combination with quite specific concentration
ratios, exert a surprisingly good, and in ideal molar ratios even
highly potent, influence on the granulation and epithelization of
wounds, and therefore represent a valuable agent for the promotion
of wqund healing.
Accordingly, the present invention provides a topical
pharmaceutical preparation for the promotion of granulation and
epithelization of wounds, which preparation comprises a pharmaco-
logically compatible carrier and
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a mixture of ioni~able pharmacoloyically compatible Ca2 and K
salts in the Ca2 :K molar ratio of about 1:3 to 4:1.
The specific concentration ratios can be achieved i~ we
ensure that ~he wound surface comes into immedia~e contact wi~h an
aqueous electrolyte which, in relation to ~he water contained in
this electrolyte, contalns about 20 to 100 mM Ca2 ions and about
25 to 60 mM K ion~.
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The electrolyte itself may consist either of wound exudation
and/or an aqueous preparation additionally applied to the
wound.
In the former case physiologically compatible ioni~able Ca-
and K-salts in a molar ratio of 1:3 to 4:1 and with a solid
carrier are applied to the wound in a quantity that the Ca-
and K-salts will form solutions in the wound e~udation which
show the above concentrations. However, the amount o~ wound e~uda
tion being variable and not exactly assessable, dry gels,
powders, ion exchangers, fleeces, impregnated wound dressings, poly-
saccharides or similar dry preparations will not always ensure
that the concentrations according to the invention are
achieved and can be maintained for a prolonged period of
time.
More useful are such preparations which, though being mar-
keted in dry form, require to be mixed with a relatively
precisely determined amount of water before being used, and
which will not be applied to the wound before the required
amount of water has been incorporated, and following disso-
ciation of the Ca2+ and K+salts. Examples of this group oE
preparations are dry gels according to DE-OS 28 49 570.
Possible would also be mixed forms in which dry carriers
right from the start are wetted with water containing Ca2+
and K+ions in the desired ratio. Representatives of this
group of forms are, e.g., ion exchangers, gels suitable for
gel chromatography (molecular sieves), or simple solid, e.g.,
textile wound dressings which first are wetted and then are
sealed in a sterile way.
The latter preparations are already typical aqueous prepara-
tions per se. This would be preparations having mainly water
as the carrier and containing Ca 2+ions and K+ions, if
desired in addition to other excipients and/or active
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ingredients, in a concentration of 20 to 100 mM, and 25 to
60 mM, respectively. In this case the range of preference
would be between 25 and 35 mM Ca2+ions and 35 to 45 mM
K+ions. Particularly preferred is the ratio of 30 mM
Ca ions and 40 mM K ions.
As aqueous preparations may serve all pharmaceutical prepara-
tions having a~atercontent which allows to adjust the molar
ion concentration according to the invention. Besides simple
aqueous solutions, lotions or oil-in-water emulsions these
could also be viscous solutions, dispersed systems or foams.
Above mentioned gel-like preparations, such as e.g. poiy-
acrylamide/agar gels are particularly well tolerated on
wounds and therefore preferred.
Preferred is a process for the production of the above mentioned
preparations to prc~Lote the oranulation and epithelization of w~unds, which is
characterized by the fact that in a generally known manner
Ca2+ and K+ ions forming pharmacologically compatible salts
are added to an aqueous pharmaceutical carrier, optionally
in addition to other excipients and/or active ingredients,
and in relation to the water content up to a concentration
of 20 to 100 (mMol) Ca2+ ions and 25 to 60 (mMol) K+ ions by
uniformly distributing these ions in the aqueous phase.
Possible pharmacologically compatible salts are mainly
chlorides and phosphates, but other inorganic or organic
salts, such as e.g. citrates, maleates, succinates or
similar salts may also be used if they are tolerated by the
tissue and dissociable.
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~9785-15
In the case of aqueous preparations it is useful to
isotonize the aqueous phase. In order to avoid having to use
alien ions, isotonization may be brought about preferably by means
of glucose. So far unresearched are the possible effects of
cationic elec~rolytes added in additisn to Ca2 and K~. In many
cases it would also be useful to add tensides to the aqueous phase
which increase the permeability of the skin to electrolytes.
Since the effectiveness of the above preparations
according to the invention exclusively depends on the claimed
ratio of Ca2 to K ions in the extracellular space, of course
also such preparations would be fully effective which contain said
ions as the exclusive active ingredient. In some cases, however,
the addition of further active lngredients will be desirable, such
as e.g. antibiotic or fungistatic agents or surface anaesthetics.
By investigating the mechanism of the above mentioned
preparations, it has been found that the positive effects for the
promotion of wound granulation and epithelization does not only
occur in the presence of Ca~+ and K ions but in all cases, where
the calcium Permeabillt~ of the cell membrane is increased and
thus Ca2 ~ions can flow into the cell.
This can not only be achieved as de~cribed above by
uniformly distributing, possibly in addition to other active or
auxlliary substances, 0.2 to 10% by weight of a mixture of active
substances, consisting of pharmacologically tolerated ionizable
Ca2 - and K -salts in the molar Ca2 :K ratio of 1:3 to 4:1, in a
pharmacologically tolerated carrier (thus using K -ions for the
opening of the so-called calcium channels in the plasma n.embrane),
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69785-15
but also by uniformly distributing a calcium-agonist at a
concentra~ion of 10 5 ~o 10 9 M in the presence of K -ions at a
concentration of 5 x 10 3 to 2 x 10 2 M and Ca at a
concentration of 10 3 M to 3 x 10 2 M in a pharmacologically
tolerated carrier.
Ca-a~onists act to increase the calcium permeability of
the plasma membrane. The calcium channels are opened by them in
the presence of potassium. With the use of Ca-agonists according
to the lnvention, however, only very little potassium need be
applied, so khat, especially in the case o~ wounds o~ large area,
the potassium balance is not disturbed. This is an lmprovement
compared with the above clescribed use of pure Ca and K ions.
The most effective concentration range of calclum
agonists is 10 to 10 M. The required potassium concentration
is in the ranye 5 x 10 3 to 2 x 10 2 M. The calcium concentration
should be between 10 3 M and 3 x 10 2 M.
Such a calcium agonist is, for example, the suhstance
Bay K 8644, of which it is alraady known that it stimulates
calcium inflow into the cell [cf. e.g. Arzne:Lmittel~orschung/Drug
Res. 33 (II) No. 9 (1983) and Biochem. and Biophys. Research
Communic. l1984) 118, No. 3, p. 8~2-47]. It must be regarded as
extremely surprising that within the cell calcium is an important
factor for wound granulation. The calcium concentration in the
extracellular spaGe has no effect whatsoever on wound healing
without the agents for i.ncreasing the permeability according to
the invention. Neither removal of calclum by the addition of a
calcium chelator such as ethylene glycol bis-(~-aminoe~hyl)-
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N~N,N',N'-tetraacetic acid (EGTA) nor increase of the calcium
concentration to 20 times the physiological concentration in the
extracellular space leads to any change in wound granulation.
From these data the experts could only conclude that calcium ions
do not significantly participate in wound granulation.
The subject of the present invention is therefore a
topical pharmaceutlcal preparation for the promotion of
granulation and epithelization of wounds, containing a
pharmacologically compatible carrier and an active substance which
is characterized by the fact that the active suhstance which
increases the calcium-permeahillty of the plasma membrane consists
of a combination containing 0.2 to 10 percent in weight of an
ionizable mixture of pharmacologically compatible Ca2 and K
salts in the Ca2 :K molar ratio of about 1:3 to 4:1.
A further subject of ~he present invention is a pxocess
for the preparation of a topical pharmaceutical preparation for
the promotion of wvund granulation and epithelization, which is
characterized by the fact that within the pharmacologically
compatible carrier, op~ionally in addition to other active
substances and excipients, there is uniformly distributed the
active substances.
The following ~igures illustrate the invention:
From Figure 1 it can be seen that the ~ormation of
granulation tissue is essentially independent of the extracellular
calcium concentration.
The tests ~orming the basis of Figure 1 were carried out
as follows:
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The skin of the backs of guinea-pigs was severed as far
as the fascia~ A teflon ring of diameter 21 mm was sown into the
wound. This was intended to prevent epithelial closure of the
wound. An aqueous polyacrylamide agar gel containing the active
substances in the stated concentrations was applied to the fascia
of the back musculature, which was free from granulation tlssue at
the time of the operation, for a period of 3 days. After 3 days,
when the optimum stage of granulation had been attained, the
entire granulation tissue was removed with a sharp spoon, weighed
and examined histologically.
By carrying out process a~ compared with the controls,
for which said gel was mixed with 0.9 percent in weight sodium
chloride ~or isotonlc reasons, the volume of granulation tissue
had significantly increased to up to about 180%.
The hlstologic controls showed a true proli~eration of
cells, and not only an increase in the cellular volume.
The following examples serve to further lllustrate the
invention.
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Example 1
Isotonic Solution
80 ml of purified water are introduced into a 100-ml vol-
umetric flask. 20 mg benzalkonium chloride are added and
dissolved while stirring with a magnetic stirrer. Then are
added successively 0.3 g potassium chloride, 0.44 g calcium
chloride ~both salts in conformity with Ph.Eur.I.) and
2.62 g glucose monohydrate (Ph.Eur.II.). The temperature of
the flask is adjusted to 20C in a water bath, and the flask
then filled to volume with purified water which also has a
temperature of 20C. Subsequently the solution is filtered
sterile through a membrane filter of 0.2 ~m pore size and
bottled sterileO
rO.29822 g KCl 40 mMol K+
0.44106 g CaC12 30 mMol Ca2+~
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Example 2
Oil-in-Water Emulsion
In a first batch 7 g of a mixture consisting of saturated
fatty acids, ~a~ty alcohols, lanolin, mineral oils and
nonionogenic emulsifying agents are melted homogeneously
together with 2.5 g polyethylene glycol glycerol fatty acid
ester, 3 g monoglycerides of stearinic and palmitic acid,
0.3 g cetyl alcohol and 3.0 g isopropyl palmitate by
heating to 70C in a water bath.
In a second batch 80 g purified water are mixed with 3 g
propylene glycol while stirring and heated to 70C. The
mixture so obtained is then mixed with 0.3 g potassium
chloride, 0.44 g calcium chloride and 0.2 g of a preser-
vative. The resultant clear solution is emulsified into the
first batch while stirring at 70C. The resultant emulsion
is cooled to 40C and the water loss from evaporation is
made up. When cooled to 30C the emulsion is bottled.
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Example 3
Transparent Liquid Dressinq Material (Gel Disk)
Batch A:
3.5 9 acrylamide and 0.091 9 bisacrylamide are dissolved in
100 ml purified water. The temperature of the solution is
adjusted to 60C.
Batch B:
0.3 g potassium chloride and 0.44 9 calcium chloride are
dissolved in 100 ml purified water and mixed with 0.2 g
preservative. After adding 2 9 agar-agar (OAB9) the solution
is heated to boiling while stirring with a magnetic stirrer,
and afterwards cooled to 60C.
Subsequently batches A and B are mixed at 60C while stirring.
Then 0.045 9 ammonium peroxydisulfite and 0.0~5 9 (60 ~1)
tetramethylene diamine are added. After a brief period of
vigorous stirring the mixture is poured in~o Petri dishes
previously heated to 60C in the heating cabinet. The filled
Petri dishes are then placed into a heating cabinet set to
56C for 30 min. The temperature is then lowered to room
temperature and the dishes with the transparent solidified
disks placed into a cabinet set to a temperature of 4C for
24 hrs. for maturing. The disks so obtained may immediately
be used to cover wounds.
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Example 4
Ready-to-Use Gel
94 g purified water are heated to 70C and-mixed with 0.3 g
potassium chloride and 0O44 g calcium chloride. After adding
0.2 g preservative, 5 g methylhydroxyethylcellulose are dis-
persed in the solution 50 obtained. Then the mixture is
cooled while stirring. When cool the product is a highly
viscous gel interspersed with air bubbles and of a viscosity
of 90 Pa.s, which is ready to use on wounds.
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Example_5
Textile Wound Dressin~
Sterile gauze pads, 4 x 4 cm large and 5 mm thick, are dipped
into the sterile isotonic solution obtained according to
Example 1 and then squeezed out only so much that the pads
do no longer drip. The pads thus obtained are then sealed
into polyethylene foils under sterile conditions.
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69785-15
Example 6
Spreadable ~el_with_~c K 8644
1000 g of purified water are heated to 70C and treated
with 1.49 g of KCl, 3.3 g of CaC12 and 3.56 mg of Bay K 8644.
After the addition of 2 g of preservative 50 g of methyl hydroxy-
ethylcellulose are dispersed into the solution obtalned and the
mixture is worked up as descrlbed in Example 1.
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