NZ211994A - Wound healing preparation containing ca 2+ and k + ions - Google Patents

Description

2 1 1994 Priority Date(s): . .*7.. .<*.. $5^......... > 2^-85 Complete Spaciricatioq Filed: £ Class: J"'"'""''' 2 9 APR 1988 Puliation Date: P.O. Journal, Nc: ......

N.Z.No, NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION "TOPICAL PHARMACEUTICAL PREPARATIONS, PROCESS FOR THE .MANUFACTURE THEREOF, AND THEIR APPLICATION TO PROMOTE GRANULATION AND EPITHELIZATION OF WOUNDS." o We, GODECKE AKTIENGESELLSCHAFT, a company organised under the laws of the Federal Republic of Germany, of Salzufer 16, 1000 Berlin 10, Federal .Republic of Germany, do hereby declare the invention, for which we pray that a Patent may be granted to ug and the method by which it is to be performed, to be particularly described in and by the following statement : - (followed bv 1A) 2 1199 - ifl- Description It is known from in vitro experiments on cell cultures that Ca^+ions exert an influence on the division of cells. (Proc. Nat. Acad. Sci. 7£, p. 675-679). It is also known that the depolarization of the membrane potential may be realized by raising the extracellular K+-conzentration. So far, however, there have been no studies investigating the influences these effects exert on the granulation and epithelization of wounds. 2+ Extensive investigations have shown that neither the Ca concentrations established as being optimal for the growth of cell cultures, nor increased Ca2+ 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. 2+ + Surprisingly it has now first been found that Ca 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 wound healing.

These concentration ratios can be achieved if we ensure that the wound surface comes into immediate contact with an aqueous electrolyte which, in relation to the water contained in this electrolyte, contains 20 to 100 mM Ca^+ions and 25 to 60 mM K+ ions. 2 n 99 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 ionizable 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 exudation which show the above concentrations. However, the amount of wound exudation being variable and not exactly assessable, dry gels, powders, ion exchangers, fleeces, impregnated wound dressings, polysaccharides 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 marketed 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 dissociation of the Ca2+ and K+salts. Examples of this group of 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 preparations 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 v i 7 ■ V- ' ■•■■if - v - -v"-' •//•-.-■'••'v. ■ 2 119 A" 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 Ca^+ions and 35 to 45 mM K+ions. Particularly preferred is the ratio of 30 mM Ca2+ ions and 40 mM K+ions. r ^ As aqueous preparations may serve all pharmaceutical prepara tions having a water content which allows to adjust the molar ion concentration according to the invention. Besides simple aqueous solutions, lotions or oil-in-water emulsions these ^5 .could also be viscous solutions, dispersed systems or foams. i Above mentioned gel-like preparations, such as e.g. poly-acrylamide/agar gels are particularly well tolerated on i wounds and therefore preferred. i I Preferred is a process for the production of the above mentioned - ■ preparations to pranote the granulation and epithelization of wounds, which is J characterized by the fact that in a generally known manner "-i 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 anorganic 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. urn fi • ■ •;* & 2 1 1 9 9 / 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 electrolytes added in addition 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 ingredients will be desirable, such as e.g. antibiotic or fungistatic agents or surface anestetics.

By investigating the mechanism of the above mentioned preparations, it has been found that the positive effects for the promotion of wound granulation and epithelisation does not only occur *f+ + in the presence of Ca and K ions but in all cases, where the calcium permeability of the cell membrane is increaaed and thus Ca^-ions can flow into the cell.

This can not only be achieved as described above by uniformly distributing - possibly in addition to other active or auxiliary 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 •f K -ions for the opening of the so-called calcium channels in the plasma membrane) , but also by uniformlv distributina - instead 2+ of the mixture of active substances described - either a Ca _ 8 — g ionophore at a concentration of 3 x 10 to 3 x 10 M in combination with calcium salts at a concentration of 0.1 to 50 mM -5 -9 or a calcium-agonist at a concentration of 10 to 10 M in the ,SPV I 1 1994 + -3 -2 presence of K -ions at a concentration of 5 x 10 to 2 x 10 M 2+ -3 —2 and Ca at a concentration of 10 M to 3 x 10 Mina pharmacologically tolerated carrier.

Ca^+-ionophores are compounds which selectively render plasma membranes permeable to calcium.

Such an ionophore is, for example, the antibiotic 6S £6«C(2S*» 3S*) ,8j5(K*) ,9^,11^-5-methylamino-2-[3,9,ll-trimethyl-8- pL-methyl-2-oxo-3-(lH-pyrrol-2-yl)ethyl]]*l,7-dioxaspiro[5,5]undec-2-ylJmethy]1Jibenzoxazole-4-carboxylic acid, which has become known as A 23 187 (Hoechst Doc. no. 8154-1082).

In combination with calcium, A 23 187 according to the invention -8 -6 acts at a concentration of 3 x 10 to 3 x 10 M, the necessary concentration of calcium salts in the extracellular space being only 2+ 0.1 to 50 mK in order to ensure adequate inflow of Ca into the cell.

Since, of course, the principal object is calcium supply to the cell through the plasma membrane, the effect of more rapid wound healing can., according to the invention, be equally achieved with all other Ca-ionophores.

Ca-agonists act similarly, since they also increase the calcium permeability of the plasma membrane. The calcium channels are opened by them in the presence of potassium, i.'ith the use of Ca-agonists according to the invention, however, only very little potassium need be applied, so that, especially in the case of wounds of large area, the potassium balance is not disturbed.

This is an improvement compared with the above described use of j I pure Ca and K ions.

The most effective concentration range of calcium agonists is -5 -9 to 10 i-i. The required potassium concentration is in the -3 -2 range 5 x 10 to 2 x 10 ri. The calcium concentration should -3 -2 be between 10 H and 3 x 10 I..

Such a calcium agonist is, for example, the substance Bay K 8644, of which it is already known that it stimulates calcium inflow into the cell £cf. e.g. Arzneimittelforschung/Drug Res. 33 (II) No. 9 (1983) and Biochem. and Biophys. Research Communic. (1984) 118, No. 3, p. 842-47]]. 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 space has no effect whatsoever on wound healing without the agents for increasing the permeability according to the invention. Neither removal of calcium by the addition of a calcium chelator such as ethylene glycol bis(p-aminoethyl)-N,N,N',W'-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 pharmaceutical 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 substance which increases the calcium-permeability of the plasma membrane consists either of a) 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 1:3 to 4:1 or 2+ 8 b) a Ca -ionophore at a concentration of 3 x 10 to 3 x 10 ^ M in combination with calcium salts at a concentration of 0.1 to 50 mM or 2 1 199 c) a calcium-agonist at a concentration of 10 to — 9 + M in the'presence of K -ions at a concentration -3 -2 2+ of5x10to2x10 M and Ca at a concentration -3 -2 of 10 M to 3 x 10 M.

A further subject of the present invention is a process for the preparation of a topical pharmaceutical preparation for the promotion of wound granulation and epithelization, which is ^ characterized by the fact that within the pharmacologically ^ compatible carrier, optionally in addition to other active substances and excipients, there is uniformly distributed an active substance a), b) or c).

—. The following figures illustrate the invention: From figure 1 it can be seen that the formation of granulation tissue is essentially independent of the extracellular calcium concentration.

From figure 2 it is clear that the permeability increasing 2+ suostance 23 1S7 in the presence of Ca -ions in the extracellular space significantly increases the wound granulation compared with controls (test no. 5) at concentrations between 10 and 3 :: 10 ^ i.;, especially at a concentration of 3 x 10 ^ H. 2-~ If the Ca*"'-concentration in the cell is reduced by removal of 2+ Ca by means of Z3TA in the extracellular space and use of A 23 187 as permeability increasing agent, granulation is significantly reduced. This experiment shows that the formation of granulation tissue, a prerequisite for wound healing, is n > strongly dependent on the Ca content ox the cells and that - 2+ granulation can be significantly increased if the Ca content o of the cell is increased by the permeability increasing agents of the invention. 2119 The tests forming the basis of figures 1 and 2 were carried out as follows : 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, ^n 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 tissue at the time of the operation, for a period of 3 days, ^fter 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 for isotonic reasons, the volume of granulation tissue had significantly increased to up to about 180 %.

The histologic controls shewed a true proliferation of cells, and not only an increase in the cellular volume.

The following examples serve to further illustrate the invention. 2 1 199 Example 1 Isotonic Solution 80 ml of purified water are introduced into a 100-ml volumetric 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 20°C in a water bath, and the flask then filled to volume with purified water which also has a temperature of 20°C. Subsequently the solution is filtered sterile through a membrane filter of 0.2 (im pore size and bottled sterile.

[O.29822 g KC1 40 mMol K+ 0.44106 g CaCl2 30 mMol Ca2+J •Jv o • y-, ■_ •_ •; • - "€!i* j^st '■: - • 2 1199 Example 2 Oil-in-Water Emulsion In a first batch 7 g of a mixture consisting of saturated fatty acids, fatty 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 70°C in a water bath. j b r In a second batch 80 g purified water are mixed with 3 g f propylene glycol while stirring and heated to 70°C. The [ mixture so obtained is then mixed with 0.3 g potassium j chloride, 0.44 g calcium chloride and 0.2 g of a preser- j vative. The resultant clear solution is emulsified into the first batch while stirring at 70°C. The resultant emulsion is cooled to 40°C and the water loss from evaporation is made up. When cooled to 30°C the emulsion is bottled. 4 — : "" V . . ■ -j \ - r' •• •- "v' ' '-'I* -..j-.: ■; 2 119 z. g a ^ ^ / Example 3 Transparent Liquid Dressing Material (Gel Disk) Batch A: 3.5 g acrylamide and 0.091 g bisacrylamide are dissolved in ^ 100 ml purified water. The temperature of the solution is adjusted to 60°C.

Batch B: 0.3 g potassium chloride and 0.44 g calcium chloride are dissolved in 100 ml purified water and mixed with 0.2 g preservative. After adding 2 g agar-agar (OAB9) the solution is heated to boiling while stirring with a magnetic stirrer, and afterwards cooled to 60°C.

Subsequently batches A and B are mixed at 60°C while stirring. Then 0.045 g ammonium peroxydisulfite and 0.045 g (60 jil) tetramethylene diamine are added. After a brief period of vigorous stirring the mixture is poured into Petri dishes previously heated to 60°C in the heating cabinet. The filled Petri dishes are then placed into a heating cabinet set to 56°C 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 4°C for 24 hrs. for maturing. The disks so obtained may immediately be used to cover wounds. 2 119 Example 4 Ready-to-Use Gel 94 g purified water are heated to 70°C and mixed with 0.3 g potassium chloride and 0.44 g calcium chloride. After adding 0.2 g preservative, 5 g methylhydroxyethylcellulose are dispersed in the solution so obtained. Then the mixture is cooled while stirring. When cool the product is a highly :s viscous gel interspersed with air bubbles and of a viscosity ^ of 90 Pa.s, which is ready to use on wounds. j G ' 2 1 199-? - 13 -Example 5 ! Textile Wound Dressing I 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.

'X. } / jv -vo:i 2 119 Example 6 *"• 1 I & & Spreadable gel with ^ 23 187 1000 g of purified water are heated to 70°C and treated with 1.6 mg of A 23 1S7 and 560 mg of calcium chloride. After the addition of 2 g of preservative 50 g of methyl hydroxycellulose are dispersed into the solution obtained. The solution is then cooled with stirring, z^fter cooling a highly viscous gel with viscosity 90 Pa.s, interspersed with air bubbles, is obtained which can immediately be used for the treatment of wounds. k BBS "T ".' * - :**j v .! ^T^"- • *; "• ^y ,y..< ry -*-.. ••—•,- v~- ^ • ' 2 1J99 Example 7 Spreadable gel with 2>ay K 6644 1000 g of purified water are heated to 70°C and treated with 1.49 g of KC1, 3.3 g of CaC^ and 3.56 mg of Bay K 8644. After the addition of 2 g of preservative 50 g of methyl hydroxyethylcellulose are dispersed into the solution obtained and the mixture is worked up as described in example 1. v-> o

Claims (12)

MM;:? ■ '■ v- - 16- m — -i. V o' i'JHAT WE CLAIM IS:

1. A topical pharmaceutical preparation for the promotion of granulation and epithelization of wounds, containing a pharmacologically compatible carrier and an active substance, characterized by the fact that the active substance which increases the calcium-permeability of the plasma membrane consists of a) 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 1:3 to 4 :1, or 2 i M 0 b) a Ca -ionophore at a concentration of 3 x 10 .to 3 x 10 ' M in combination with calcium salts at a concentration of 0.1 to 50 mM, or c) a calcium-agonist at a concentration of 10 ^ to — 9 + 10 M in the presence of K -ions at a concentration of 5 x 10~3 to 2 x 10~2 M and Ca2+ at a concentration of 10~3 M to 3 x 10~2 M.

2. A pharmaceutical preparation according to Claim 1 characterized by the carrier consisting mainly of water.

3. A pharmaceutical preparation according to Claim 1 or 2 characterized by the carrier being a hydrated gel.

4. A pharmaceutical preparation according to Claim 3 wherein the hydrated gel contains methylhydroxyethyl cellulose.

5. A pharmaceutical preparation according to Claim 1 characterized by t the carrier being a dry gel. - 17 - ( > * f ■si. J 1

6. A pharmaceutical preparation according to Claim 1 characterized by the fact that in relation to the water content, the preparation contains 20 to 100 mM Ca2+ ions and 25 to 60 mM K+ ions.

7. A pharmaceutical ; preparation according to Claim 6 characterized by the fact that it contains about 30 mM Ca2+ ions and about 40 mM K+ ions.

8. A process, for the preparation of a topical pharmaceutical preparation for the promotion of the granulation and epithelization of wounds characterized by the fact that within the pharmacologically compatible carrier, option-ally in addition to other active substances and exci- ^ pients, there is uniformly distributed an active substance 'f " according to claim 1, 6 or 7.

9. A process according to Claim 8 characterized by the fact that the carrier mainly consists of water, and that, in addition to other active substances and excipients, and in relation to the water content, concentrations of between 20 and 100 mM Ca2+ ions and 25 to 60 mM K+ ions are added, and that these ions are uniformly distributed in the aqueous pharmaceutical carrier.

10. A process according to claim 8 or 9 wherein the carrier is a hydrated gel.

11. A process according to Claim 10, wherein methylhydr oxyethyl cellulose is a gel forming agent.

12. A preparation according to Claim 1 substantially as herein described or exemplified. GODECKE AKTIENGESELLSCHAFT 3y Their Attorneys HENRY HUGHES LIMITED ^