NO851793L - PROCEDURE FOR PREPARING PHARMACEUTICAL PREPARATIONS. - Google Patents

Description

It is known from in vitro experiments on cell cultures that

2+

Ca ions exert an influence on cell division. (Proe. Nat. Acad. Sei. 70, pp. 675-679). It is also known that depolarization of the membrane potential can be achieved by raising the extracellular K<+> concentration. So far, however, no studies have been carried out regarding the influence these effects exert on the granulation and epithelialisation of wounds. Thorough investigations have shown that neither the Ca<2+->concentration established as optimal for the growth of cell cultures nor increased Ca 2+ concentrations exhibit an activating or inhibiting influence on wound healing.

The addition of physiological amounts of potassium to the extracellular space also does not activate wound healing, even if at the same time an optimal extra-2+ is provided

cellular Ca concentration.

Surprisingly, it has only now been found that Ca 2+ - and K<+-> ions together, in combination with very specific concentration conditions, exhibit a surprisingly good, and in ideal molar conditions even a very strong influence on granulation and epithelization of wounds and therefore represents a valuable means of activating wound healing.

These concentration ratios can be achieved if one ensures that the wound surface comes into immediate contact with an aqueous electrolyte which, in relation to the water contained in this

2+

electrolyte, contains 20 to 100 mM Ca ions and 25 to 60 mM K+ ions.

The electrolyte itself can consist of either wound fluid and/or an aqueous preparation that is additionally applied to the wound.

In the former case, physiologically compatible ionizable Ca and K salts are applied in a molar ratio of 1:3

to 4:1 and with a solid carrier, to the wound in such an amount that the Ca and K salts will form solutions in the wound fluid that exhibit the concentrations indicated above. However, the amount of wound fluid is variable and cannot be precisely determined/and dry gels, powders, ion exchangers, compresses, impregnated wound compresses, polysaccharides or similar dry preparations will not always ensure that the concentrations according to the invention are achieved and can be maintained for a longer time.

More applicable are such preparations which, even if they are marketed in dry form, are required to be mixed with a relatively precisely determined amount of water before they are used, and which are not applied to the wound until the required amount of water has been incorporated, and subsequent dissociation of Ca - and K - salts. Examples of this group of preparations are dry gels according to DE-OS 28 49 570.

It would also be possible to use mixed forms in which trout carriers are already moistened from the start with water containing Ca and K ions in the desired ratio. Examples of this group of forms are e.g. ion exchangers, gels suitable for gel chromatography (molecular sieves) or simple, solid materials, e.g. textile wound preparations that are first moistened and then sealed sterile.

The latter preparations are already typical aqueous preparations per se. These will be preparations which mainly have water as a carrier and contain Ca" ions and K ions, if desired in addition to other excipients and/or active ingredients, in a concentration of 20 to 100 mM and 25 to 60 mM. In this in this case the preferred range will be between 25 and 35 mM Ca<2+-> ions and 35 to 45 mM K<+-> ions.

2+

Particularly preferred is the ratio of 30 mM Ca ions and

40 mM K<+->ions.

All pharmaceutical preparations which have a water content which makes it possible to adjust the molar ion concentration according to the invention can be used as aqueous preparations. Besides simple, aqueous solutions, lotions or oil-in-water emulsions, these can also be viscous solutions, dispersed systems or foams.

The gel-like preparations mentioned above, such as, for example, polyacrylamide/agar gels, are particularly well tolerated on wounds and are therefore preferred.

Preferred is a method for producing the above-mentioned preparations to activate the granulation and epithelisation of wounds, which is characterized by the fact that Ca - and K - ions, which form pharmacologically compatible salts in a generally known manner, are added to an aqueous pharmaceutical carrier, possibly in addition to other excipients and/or active ingredients, and in relation to the water content to a concentration of 20 to 100 (mM) Ca" - ions and 25 to 60 (mM) K<+-> ions with an even distribution of 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 can also be used if they are tolerated by the tissue and are dissociable.

In the case of aqueous preparations, it is useful to make the aqueous phase isotonic. In order to avoid using foreign ions, the isotonization can preferably be carried out using glucose. The possible effects of cationic electrolytes added in addition to Ca<2+> and K<+> have not been investigated so far. In many cases, it will also be useful to add surfactants to the aqueous phase, which will increase the permeability of the skin to the electrolytes.

Since the effectiveness of the above preparations according to the invention depends exclusively on the required ratio between Ca and K ions in the extracellular space, such preparations will of course also be fully effective which contain the indicated ions as the only active component, In many cases, the addition of further active ingredients will be desirable, such as e.g. antibiotics or fungistatic agents or surface anesthetics.

When investigating the mechanism of the above-mentioned preparations, it has been found that the positive effects for activation and epithelization do not only take place in the presence of Ca++ and K<+-> ions, but in all cases where the calcium permeability of the cell membrane increases so that Ca - ions can flow into

the cell.

This can only be achieved as described above by uniform distribution - possibly in addition to other active or auxiliary substances - 0.2 to 10% by weight of a mixture of active substances consisting of pharmacologically acceptable ionizable Ca<2+-> and K<+ ->salts in the molar ratio Ca2+:K+ of 1:3 to 4:1, in a pharmacologically acceptable carrier (so that K ions are used to open the so-called calcium channels in the plasma membrane), but also with uniform distribution - instead of mixed-2 + none of the described active substances - of either a Ca

— R —fi

ionophore in a concentration of 3 x 10 to 3 x 10 Mi combination with calcium salts in a concentration of 0.1 to 50 mmol or a calcium agonist in a concentration of 10— 5 to 10 Mi presence of K ions in a concentration of 5 x 10- -3 - to 2 x 10 -2 M and Ca 2+ in a concentration of 10 -3 M

-2

to 3 x 10 M in a pharmacologically acceptable carrier.

2+

Ca ionophores are compounds that selectively make the plasma membranes permeable to calcium.

Such an ionophore is e.g. the antibiotic 6S-6S-[6a-(2S*,3Stf),83(R&),9 0,11a]-5-methylamino-2-[3,9,11-trimethyl-8-[l-methyl-2- oxo-3-(1H-pyrrol-2-yl)-ethyl]-1,7-dioxaspiro-[5,5]-undec-2-yl]-methyl]-benzoxazol-4-carboxylic acid which has been known as A 23 187 (Hoechst doc. no. 8154-1082).

In combination with calcium, A 23 187 works according to

— 8 —6 the invention at a concentration of 3 x 10 to 3 x 10 M, the necessary concentration of calcium salts in the extracellular space being only 0.1 to 50 mmol to ensure to-2+

extensive influx of Ca" into the cell.

As the principal goal is calcium supply to the cells through the plasma membrane, the effect of more rapid wound healing according to the invention can of course also be achieved with all other Ca-ionophores.

Ca agonists do not work in any way as these also increase the calcium permeability of the plasma membrane. The calcium channels are opened by these in the presence of potassium. With the use of Ca agonists according to the invention, however, very little calcium needs to be added so that - especially in the case of wounds with large surfaces - the calcium balance is not disturbed. This is an improvement compared to the above-described use of pure Ca and K ions.

The most effective concentration range for calcium-

-5 -9 agonists is 10 to 10 M. The required potassium concen--3 -2 tration is in the range 5 x 10 to 2 x 10 M. Calcium concentration -3 -9

tration should be between 10 M and 3 x 10 ~ M.

Such a calcium agonist is e.g. the substance Bay K 8644 which is already known to stimulate calcium influx into the cell [see e.g. Arzneimittelforschung/Drug Res. 33 (II), No. 9 (1983) and Biochem. and Biophys. Research Communic.

(1984) 118, No. 3, pp. 842-47]. It must be considered very surprising that calcium within the cell is an important factor for wound granulation. The calcium concentration in the extracellular space has another effect when it comes to wound healing without agents to increase permeability according to the invention. Neither removal of calcium by addition of a calcium chelator such as ethylene glycol-bis-(3-aminoethyl)-N,N,N<1>,N<1->tetraacetic acid (EDTA) nor an increase of the calcium concentration to 20 times the physiological concentration in the extracellular space leads to some change in wound granulation. From these data, the person skilled in the art would only be able to conclude that calcium ions do not participate significantly in wound granulation.

The aim of the present invention is therefore a topical pharmaceutical preparation for the activation of granulation and epithelialization of wounds containing a pharmacologically compatible carrier and an active substance which is characterized in that the active substances which increase the calcium permeability of the plasma membrane consist either of

a) a combination containing 0.2 to 10% by weight of an ionizable mixture of pharmacologically compatible

Ca - and K -salts in Ca:K molar ratios of 1:3 to 4:1, or

2+ -8

b) a Ca ionophore in a concentration of 3 x 10 to

—6

3 x 10 Mi combination with calcium salts in a concentration of 0.1 to 50 mmol, or

c) a calcium agonist in a concentration of 10 µl

10 — 9 Mi presence of K<+->ions in a concentration of

5 x 10 -3 to 2 x 10 -2 M and Ca 2+ in a concentration of. 10~<3>M to 3 x 10~<2>M.

The invention further relates to a method for producing a topical pharmaceutical preparation for activating wound granulation and epithelization, which is characterized by the fact that an active substance a) is evenly distributed within a pharmacologically compatible carrier, possibly in addition to other active substances and excipients. b) or c).

The following figures illustrate the invention:

From fig. 1, it appears that the formation of granulation tissue is mainly independent of the extracellular calcium concentration.

From fig. 2 it is clear that the permeability increaser

2+

substance-A 23 187 in the presence of Ca' ions in the extracellular space significantly increases wound granulation compared to

_7 controls (test no. 5) in concentrations between 10 and 3 x 10 ^ M, especially at a concentration of 3 x 10 ^ M.

2+

If the Ca concentration in the cell is reduced by removing

2+

Approximately with the help of EDTA in the extracellular space and the use of A 23 187 as a permeability-increasing agent, the granulation is significantly reduced. This experiment shows that the formation of granulation tissue, a prerequisite for wound

2+

ning, is strongly dependent on the Ca content in the cells, and that

2+

granulation can be increased significantly if the Ca content in the cell is increased with permeability-increasing agents according to the invention.

The tests that form the basis for fig. 1 and 2, were carried out as follows: The dorsal skin of the guinea pig was injured as far as the muscularis. A Teflon ring with a diameter of 21 mm was sewn into the wound. This was intended to prevent epithelial closure of the wound. An aqueous polyacrylamide agar gel containing the active substances in the indicated concentrations was applied to the muscle membrane of the back muscles which were free of granulation tissue at the time of surgery, for a period of 3 days. After 3 days when the optimal granulation stage had been reached, the entire granulation tissue was removed with a sharp spoon, weighed and examined histologically.

When carrying out method a) compared to the controls for which the specified part was mixed with 0.9% calcium chloride for isotonic reasons, the volume of granulation tissue had been significantly increased up to 180%.

The histological controls showed a real growth of the cells and not just an increase in the cellular volume.

The following examples illustrate the invention.

Example 1

Isotonic solution

80 ml of purified water was introduced into a 100 ml volumetric flask. 20 mg of benzalkonium chloride was added and dissolved while stirring with a magnetic stirrer. Then 0.3 g of potassium chloride, 0.44 g of calcium chloride (both salts in accordance with Ph. Eur. I.) and 2.62 g of glucose monohydrate (Ph. Eur. II.) were added successively. The temperature of the flask was adjusted to 20°C in a water bath, and the flask was then filled to volume with purified water which also had a temperature of 20°C. The solution was then sterile filtered through a membrane filter with a pore size of 0.2 µm and sterile corked.

[0.29822 g KCl 40 mmol K<+>

0.44106 g CaCl230 mmol Ca<2+>]

Example 2

01je- in- water emulsion

In a first batch, 7 g of a mixture consisting of saturated fatty acids, fatty alcohols, lanolin, mineral oils and non-ionic emulsifiers were homogeneously melted together with 2.5 g of polyethylene glycol-glycerol fatty acid esters, 3 g of mono-glycerides of stearic acid and palmitic acid, 0 .3 g of cetyl alcohol and 3.0 g of isopropyl palmitate by heating to 70°C in a water bath.

In a second batch, 80 g of purified water were mixed with 3 g of propylene glycol with stirring and heated to 70°C.

The mixture thus obtained was then mixed with 0.3 g of potassium chloride, 0.44 g of calcium chloride and 0.2 g of a preservative. The resulting clear solution was emulsified into the first batch with stirring at 70°C. The resulting emulsion was cooled to 40°C, and the water loss due to evaporation was compensated. When the emulsion had cooled to 30°C, it was filled into bottles.

Example 3

Transparent liquid dressing material (gel disk)

Batch A:

3.5 g of acrylamide and 0.091 g of bisacrylamide were dissolved in 100 ml of purified water. The temperature of the solution was adjusted to 60°C.

Batch B:

0.3 g of potassium chloride and 0.44 g of calcium chloride were dissolved

in 100 ml of purified water and mixed with 0.2 g of preservative. After adding 2 g of agar-agar (0AB9), the solution was heated to boiling while stirring with a magnetic stirrer and was then cooled to 60°C.

Batches A and B were mixed at 60°C with stirring. Then 0.045 g of ammonium peroxydisulfite and 0.045 g (60µl) of tetramethylenediamine were added. After a short period of vigorous stirring, the mixture was poured into Petri dishes that had previously been heated to 60°C in a heating cabinet. The filled Petri dishes were then placed in the heating cabinet set at 56°C for 30 minutes. The temperature was then reduced to room temperature, and the dishes with the transparent, solidified disks were placed in a cabinet set at a temperature of

4°C for 24 hours for ripening. The discs thus obtained could be used immediately to cover wounds.

Example 4

Gel ready for use

94 g of purified water was heated to 70°C and mixed with 0.3 g of potassium chloride and 0.44 g of calcium chloride. After adding 0.2 g of preservative, 5 g of methylhydroxyethyl cellulose was dispersed in the solution thus obtained. The mixture was cooled with stirring. When the product had cooled, it was a very viscous gel containing air bubbles and with a viscosity of 90 Pa.s, which is ready for use on wounds.

Example 5

Textile wound dressing material

Sterile gauze pads, 4x4 cm in size and 5 mm thick, were dipped in the sterile isotonic solution obtained according to Example 1 and then compressed just enough to no longer drip from the pads. The pads thus obtained were then sealed in polyethylene foils under sterile conditions.

Example 6

Spreadable gel with A 2 3 18 7

1000 g of purified water was heated to 70°C and treated with 1.6 g of A 23 187 and 560 mg of calcium chloride. After adding 2 g of preservative, 50 g of methyl-hydroxycellulose was dispersed in the resulting solution. The solution was then cooled with stirring. After cooling, a highly viscous gel with a viscosity of 90 Pa.s, containing air bubbles, was obtained, which can be immediately used for the treatment of wounds.

Example 7

Spreadable gel with Bay K 8644

1000 g of purified water was heated to 70°C and treated with 1.49 g of KCl, 3.3 g of CaCl 2 and 3.56 mg of Bay K 8644. After adding 2 g of preservative, 50 g of methyl-hydroxyethyl cellulose was dispersed in the solution was obtained, and the mixture was worked up as described in example 1.

Claims (6)

1. Process for the production of topical pharmaceutical preparations for activating granulation and epithelialization of wounds, containing a pharmacologically compatible carrier and an active substance, characterized in that an active substance that increases the calcium permeability of the plasma membrane consisting of a| a combination containing 0.2 to 10% by weight of an ionizable mixture of pharmacologically compatible Ca<2+->and K<+->salts in a Ca2+:K+ molar ratio of 1:3 to 4:1, or 2+ —8 b) a Ca ionophore in a concentration of 3 x 10 to — 6 3 x 10 Mi in combination with calcium salts in a concentration of 0.1 to 50 mfl, or c) a calcium agonist in a concentration of 10 ^ to 10 i presence of K ions in a concentration of 5 x 10 to 2 x 10 M and Ca in a concentration of 10~<3>M to 3 x 10~2 M distributed evenly within a pharmacologically compatible carrier, possibly with the addition of other active substances and excipients. 2. Method according to claim 1, characterized in that water is mainly used as the carrier. 3. Method according to claims 1-2, characterized in that the carrier is a hydrated gel. 4. Method according to claim 1, characterized in that the carrier is a dry gel. 5. Method according to claim la), characterized in that in relation to 2+ the water content uses 20 to 100 mM Ca ions and 25 to 60 mM K ions. 6. Method according to claim 5, characterized in that 30 mM Ca ions and 40 mM K ions are used.