WO2004022034A1

WO2004022034A1 - Dermatological preparation for the treatment of skin lesions

Info

Publication number: WO2004022034A1
Application number: PCT/PL2002/000105
Authority: WO
Inventors: Jerzy Maslanky
Original assignee: DERMAPHYT Ltd
Current assignee: DERMAPHYT Ltd
Priority date: 2002-08-01
Filing date: 2002-12-18
Publication date: 2004-03-18
Anticipated expiration: 2005-02-01
Also published as: PL355298A1; AU2002368219A1

Abstract

Dermatological preparation for treating skin lesions, especially burns, containing extract of propolis, extract of plant material, vegetable oil, antibiotics and vitamins, and a pharmaceutically admissible vehicle, said preparation having the following properties : 1) antiseptic, 2) anti-inflammatory, 3) analgesic, 4) fast wound healing, 5) preventing against formation of hypertrophied scars.

Description

Dermatological preparation for the treatment of skin lesions

The object of the present invention is a dermatological preparation for the treatment of skin lesions, particularly burns, thermal or chemical, and a method of preparing the preparation. Known dermatological preparations for treating skin lesions, particularly burns, usually have the form of creams or ointments, of solid or semisolid consistency. Known preparations are most often based on biologically active substances contained in the extract of one kind of natural raw material, mainly a plant extract, particularly from a medicinal plant. Biologically active substances contained in onion extracts form the basis of such preparations as (trade names) Contractubex Compositum, Cepan and Alcepal.

Polish patent application P.324236 describes an onion preparation for treating open wounds and burns and some forms of cutaneous carcinoma. The preparation consists of at least 22 parts by weight of onion extract and ointment base in an amount making up to 100 parts or the same amount of oil- water type emulsion.

Biologically active substances of standardized marigold (Calendula ofβcinalis) extract are part of the composition of known dermatological preparations such as

Calendula, Calendumed or Ekzevonen.

Standardized extract from Arnica montana or Arnica chamissonis, having anti- inflammatory and antioedematous activities associated with a group of flavonoids (also contained in marigold extract) and sesquiterpene lactones, is part of such preparations as

(trade names) Arniflor-N, Hyzum-N or Vasotonin.

The international patent application WO 91/15218 discloses a therapeutic composition for treatment of psoriasis, containing in its composition plant extracts of camomile, celandine, achillea, St. John's wort and marigold. The vehicle of the composition comprises a mixture of cholesterol, petrolatum, anhydrous lanolin, linseed oil and distilled water. There are also dermatological preparations containing the extracts of both marigold and arnica, such as (trade names) Ceffawell or Cesrasonal.

There are also preparations in the form of tincture or propolis ointment containing standardized propolis extract along with specific flavonoid and polyphenol compounds, having strong antimicrobial activity, accelerating tissue restoration, having analgesic activity and enhancing granulation and scar formation. External application of these preparations on wounds speeds up angiogenesis.

In some dermatological preparations, such as Aknin, Regepithel, Solan, the addition of a vitamin, most often vitamin A, is used to enliance the proper epidermis restoration, recommended to be applied to ageing or sun-injured skin as well as in excessive dryness, hyperkeratosis, psoriasis and allergic dermatitis.

The international patent application WO 99/55349 describes a preparation for treating sun burns, wounds, acne, haemorrhoids, varices, etc., which contains extract of propolis and aloe, tincture of camomile as well as sage, thyme, lavender.

In order to enhance the antibacterial and microbiological activities of dermatological preparations an addition of an antibiotic is used, most often bacitracin, e.g. in such drugs as (trade names) Batrax, Neobac, Nebacetin. Bacitracin is most often combined with neomycin, an aminoglycoside antibiotic, and with polymyxin B, a peptide antibiotic, e.g. in Polyspectran HC.

In the drugs mentioned above the vehicle for the biologically active substances is a neutral one, such as paraffin, soft white paraffin, cholesterol ointment or vegetable oil.

According to patent specification RU-2128500, an ointment for treatment of burns contains such active ingredients as aloe extract, vitamins A and E and levomicetine antibiotic.

A preparation protected by patent DE-19904801, designed for topical application in treatment of skin diseases, has a composition that varies depending on application and includes marigold extract, tincture of propolis/juniper, aloe, vitamins A and E and gentamicin anitbiotic. The method of introducing active ingredients into ointment vehicle obtained by melting lipophilic vehicle components on a water bath and then cooling it in order to avoid overheating is known. The mixture is cooled and stirred until a homogenous mass is obtained. Then the medicinal compound is introduced and the mixture is stirred until an ointment of homogenous consistency is obtained.

Treatment of skin lesions, particularly those associated with burns, still involves many problems. The methods of treating severe burns have not changed in principle over the past 20 years. Early debridement and grafting shorten the duration of hospital stay and improve the appearance of the wound after healing. However, such treatment is not always possible. It may be constrained, for instance, by patient's poor general condition or by the unavailability of skin for grafting. It may also be difficult, because it requires precise assessment of the depth and extent of the burn. Such an assessment is only possible several days after the injury, when severe haemodynamical disorders and sepsa might have already occurred. Another complication is local wound infection and general effects resulting from it. That is why it is very important to protect the wound against infection as soon as possible. Particularly in case of grade I and II burns finding an alternative, readily applicable treatment method free of the described shortcomings is desirable.

Dense consistency of the dermatological preparations mentioned above is one of the factors that considerably limit their penetration and efficacy. In such drugs their active part is most often the bottom layer that directly acts on the skin, whereas the other part acts like a barrier obturating skin pores and impairing free air circulation within the skin lesion. Moreover, the barrier inhibits transpiration through the skin, which may result in considerable inhibition of tissue restoration and cell breakdown within the lesion. Additionally, transpiration is reduced by the necessity of bandaging.

The application of such preparations to the wound, especially the burn, is not easy, apart from being painful for the patient.

Therefore the aim of the invention is to provide a dermatological preparation designed for application in the treatment of skin lesions, particularly burns, that preparation having the following properties: 1) antiseptic, 2) anti-inflammatory, 3) analgesic, 4) fast wound healing, 5) preventing against formation of hypertrophied scars. In particular, the preparation should display desired activity independent of the type of burn, acting effectively in the case of both thermal and chemical burns. The desirable preparation should be easily applicable and ready for immediate use after injury, particularly a burn.

The aim defined above has unexpectedly been achieved by the dermatological preparation of the present invention. Substance of the invention

The object of the present invention is a dermatological preparation for the treatment of skin lesions, particularly burns, the said preparation containing extract of propolis, a plant extract, a vegetable oil, antibiotics and vitamins forming a mixture with pharmaceutically admissible vehicle, characterized in that it contains 1 to 10 parts by weight of standardized extract of propolis with at least 4%^' of quercetin-equivalent flavonoids, 0.1 to 5 parts by weight of standardized plant extract, a small amount of 0 to 0.1 parts by weight of vitamin supplement, evenly dispersed in a neutral vehicle making up to 100 parts by weight, the said vehicle preferably comprising 0-30 parts by weight of liquid paraffin, 0-30 parts by weight of solid paraffin, 0-30 parts by weight of white petrolatum, 0-10 parts by weight of cholesterol, 0-70 parts by weight of untreated vegetable oil and 0-80 parts by weight of anhydrous lanolin.

The dermatological preparation according to the invention is advantageously characterized in that the untreated vegetable oil contained therein is linseed oil and/or sesame oil. The dermatological preparation according to the invention is advantageously characterized in that the vehicle comprises a mixture consisting of 30-80 parts by weight of anhydrous lanolin, 10-30 parts by weight of white petrolatum, 10-30 parts by weight of solid paraffin and 1-8 parts by weight of cholesterol.

The dermatological preparation according to the invention is advantageously characterized in that the vehicle comprises a mixture consisting of 64 parts by weight of anhydrous lanolin, 18 parts by weight of white petrolatum, 15 parts by weight of solid paraffin and 3 parts by weight of cholesterol. The dermatological preparation according to the invention is advantageously characterized in that the plant extract contained therein comprises a standardized extract of marigold, standardized extract of mountain and/or meadow arnica, standardized extract of St. John's wort, standardized extract of aloe or a combination thereof.

The dermatological preparation according to the invention is advantageously characterized in that the dermatological antibiotic contained therein comprises bacitracin, neomycin, polymyxin B, gentamycin, oxyteracin, erythromycin or staphylomycin or a combination thereof.

The dermatological preparation according to the invention is advantageously characterized in that the vitamin supplement contained therein comprises vitamin A, C or E or a combination thereof.

The dermatological preparation according to the invention is advantageously characterized in that it contains 50.06-54.28 parts by weight of untreated vegetable oil and anhydrous lanolin.

A beneficial variation of the dermatological preparation according to the invention is characterized in that 100 grams of it contain 5-10 grams of a standardized extract of marigold flowers, 1-5 grams of propolis extract, 0.1-2 grams of bacitracin, 0.01-0.1 gram of vitamin A made up to 100 grams with vehicle, whereas the vehicle comprises 20-25 grams of liquid paraffin, 2-8 grams of solid paraffin, 1-10 grams of white petrolatum, 0.2-2 grams of cholesterol, 30-70 grams of linseed oil. Preferably 100 grams of the preparation contain 9 grams of standardized extract of marigold flowers, 3 grams of propolis extract, 1 gram of bacitracin, 0.03 gram of vitamin A made up to 100 grams with vehicle, whereas the vehicle comprises 22 grams of liquid paraffin, 5 grams of solid paraffin, 6 grams of white petrolatum, 1 gram of cholesterol and 51 grams of linseed oil.

Another beneficial variation of the dermatological preparation according to the invention is characterized in that 100 grams of it contain 5-10 grams of a standardized extract of marigold flowers, 1-5 grams of propolis extract, 0.1-2 grams of bacitracin, 0.01- 0.1 gram of vitamin A made up to 100 grams with vehicle, whereas the vehicle comprises 10-40 parts by weight of solid paraffin, 5-30 parts by weight of white petrolatum, 0.2-5 parts by weight of cholesterol, 20-80 parts by weight of linseed oil, 30-80 parts by weight of anhydrous lanolin. Preferably 100 grams of the preparation contain 9 grams of standardized extract of marigold flowers, 3 grams of propolis extract, 1 gram of bacitracin, 0.03 gram of vitamin A made up to 100 grams with vehicle, whereas the vehicle comprises 10 parts by weight of solid paraffin, 12 parts by weight of white petrolatum, 2 parts by weight of cholesterol, 42 parts by weight of anhydrous lanolin and 43 parts by weight of linseed oil. The preparation preferably has the form of an ointment.

Another beneficial variation of the dermatological preparation according to the invention is characterized in that 100 grams of it contain 5-20 grams of a standardized extract of marigold flowers, 1-5 grams of propolis extract, 0.1-2 grams of bacitracin, 30-70 grams of linseed oil and 10-50 grams of anhydrous lanolin. Preferably 100 grams of the preparation contain 9 grams of standardized extract of marigold flowers, 3 grams of propolis extract, 1 gram of bacitracin, 0.03 gram of vitamin A, 52-53 grams of linseed oil and 33 grams of anl ydrous lanolin. The preparation preferably has the form of an aerosol. One of the described methods of preparing the dermatological preparation according to the invention consists in introducing into 53.50 - 56.50 parts by weight of neutral vehicle of 30.06 - 34.28 parts by weight of untreated vegetable oil at 40°C with preferably 0.02 - 0.04 parts by weight of vitamin dissolved in it beforehand and the entire mixture is stirred until homogenised. Then a solution is added obtained by dissolving 0.5 - 1.3 part by weight of antibiotic in 5.20 - 10.20 parts by weight of standardized extract of vegetable raw material containing not more than 1.5% quercetin-equivalent flavonoids, adding 3.5 - 4.9 parts by weight of propolis extract containing not more than 6% quercetin-equivalent flavonoids and heating to 40°C, stirring and homogenising, afterwards the whole preparation is stirred and homogenised under reduced pressure and cooled. One implementation of the described method of preparing the dermatological preparation of the invention consists in introducing into 33.50 - 36.50 parts by weight of neutral vehicle of 50.06 - 54.28 parts by weight of untreated vegetable oil at 40°C with preferably 0.02 - 0.04 parts by weight of vitamin dissolved in it beforehand and the entire mixture is stirred until homogenised. Then a solution is added obtained by dissolving 0.5 -

1.3 part by weight of antibiotic in 5.20 - 10.20 parts by weight of standardized extract of vegetable raw material containing not more than 1.5% quercetin-equivalent flavonoids, adding 3.5 - 4.9 parts by weight of propolis extract containing not more than 6% quercetin-equivalent flavonoids and heating to 40°C, stirring and homogenising, afterwards the whole preparation is stirred and homogenised under reduced pressure and cooled, after which a propellant gas, tetrafluoroethane for instance, is introduced under pressure in order to obtain a pressurized preparation that can be applied in the form of an aerosol.

In the described method the neutral vehicle is formed by a mixture comprising 64 parts by weight of anhydrous lanolin, 18 parts by weight of white petrolatum, 15 parts by weight of solid paraffin and 3 parts by weight of cholesterol.

In a variation of the described method of preparing the dermatological preparation of the invention the neutral vehicle preferably contains anhydrous lanolin.

In the numerous variations of the implementation of the described method of preparing the dermatological preparation linseed oil and/or sesame oil is used as the untreated vegetable oil, extract of marigold, extract of mountain and/or meadow arnica, aloe extract, extract of St. John's wort or any combination thereof is used as the standardized plant extract, bacitracin, neomycin, polymyxin B, gentamycin, oxyteracin, erythromycin, staphylomycin or any combination of two or three of these antibiotics is used as the antibiotic, and vitamin A, C or E or any combination thereof is used as the vitamin component.

The preparation obtained has the form of a soft ointment with a characteristic propolis scent and yellow to brown colour. Advantages

The dermatological preparation of the invention is an agent of very advantageous medicinal properties. Unexpectedly the present composition provided a preparation having the following properties: 1) antiseptic, 2) anti-inflammatory, 3) analgesic, 4) fast wound healing, 5) preventing against formation of hypertrophied scars.

The present preparation inhibits development and growth of bacteria and viruses giving rise to inflammatory condition. The rich set of flavonoids contained in it, in conjunction with other biologically active substances, provides it with very effective anti-inflammatory and antibacterial activity and speeds up the healing of burn wounds. An exceptional antiseptic effect has been produced by the incorporation of propolis, which has strong antibacterial properties, and an unexpected beneficial 2-3 -fold intensification of the antibacterial effect has been attained by adding bacitracin.

Beneficial physical properties have been unexpectedly attained by using experimentally selected vehicle. In the preferable variation of the invention, the neutral vehicle consists of a modified cholesterol ointment, where instead of liquid paraffin anhydrous lanolin has been used. The latter significantly strengthens the gel structure, thereby improving the cohesiveness of the ointment, which prevents the tendency of liquid ingredients to separate. High water number of anhydrous lanolin, ca. 200, makes the ointment capable of absorbing large amounts of water and aqueous solutions and, when applied to burned skin or a wound that heals with difficulty, it absorbs exudates forming a water/oil system. Moreover, lanolin enhances resorption of medicinal substances through skin, which is of great advantage in case of burns and wounds that heal with difficulty. Unexpectedly, changing linseed oil content has provided further improvement in the spreadability of the ointment.

Unexpectedly, it has occurred that replacing liquid paraffin with anhydrous lanolin led to improvement of consistency, optimum spreadability and skin adhesion, homogeneity, stability of internal structure, enhanced penetration of active ingredients into the skin. Further improvement of physical parameters has unexpectedly been attained by changing linseed oil content in the composition. Moreover, these alterations did not hinder the possibility of thermal sterilization and preparation of a sterile ointment.

Soft consistency of the preparation in the form of ointment and/or aerosol enables its fast and easy application directly onto the skin, particularly onto burns, which in the first place reduces the pain suffered by the patient, and at the same time enables immediate commencement of treatment, particularly in the case of grade I and II burns. Immediate application of the medication prevents the formation of blisters, which can reduce the healing time even by several days. In case of severe and extensive burns classified as grade II or III burns, when skin grafting is necessary, it has been unexpectedly observed that the preparation had a beneficial effect in postponed treatment of residual wounds and autogenic graft donor sites.

In the cases of less severe and less extensive burns, the preparation according to the invention makes it unnecessary to dress the wound and reduces or even eliminates the hospitalisation period, because the patient can apply the preparation on his own or with the help of persons attending. It has unexpectedly been observed that the preparation according to the invention reduces the risk of scar formation and speeds up regression of skin discolouration after burns. The preparation also protects against plasma loss, which makes it possible to avoid the necessity of administering infusions. Unexpectedly it has been found that the preparation of the invention speeds up and regulates healing processes (granulation tissue growth, angiogenesis and epithelialization) and epidermis and derma regeneration by intensifying the enzymatic activity of glucose-6- phosphate dehydrogenase (G6PD), c cytochrome oxidase (OCC) and electron transport enzyme dehydrogenase (NADHD), increasing RNA concentration in fibroblasts, intensifying collagen generation and accelerating transformation of fibroblasts into fobrocytes.

The figures shown here supplement this specification. Figure 1 depicts a sagittal section of a specimen of K group rat skin. 21 -days series. Response to RNA. Note the prevailing area of burn-affected skin showing generally poor development of granulation tissue (Z), lack of regenerative epidermis and presence of protective layer of exudate cells. On both sides of this area there are symptoms of regeneration in the form of regenerative epidermis (NR) growing from the epidermis (NO) of the area not affected by the burn. RNA reaction is strong in epidermis and hair follicles of unaffected skin. Within granulation tissue area (Z) sites of poor or moderate RNA reaction are present indicating poor or moderate growth of granulation tissue. Magnification measure = 100 micrometers. Figure 2 depicts a sagittal section of a specimen of Di group rat skin. 21 -days series. Response to RNA. In comparison to Fig. 1, dynamic progression of regeneration of burn- affected skin is conspicuous evidenced by growth of fresh regenerative epidermis (NR)- The area not covered by this fresh epidermis indicates the presence of protective leucocytal layer on skin surface. Strong response to RNA in epidermis (N) and hair follicles is evident in skin of proper structure on both sides of the area shown. Reaction in regenerative epidermis (NR) is evidently strong. Signs of poor, although distinct response to RNA occur in the granulation tissue (Z) under the NR epidermis. The arrow points to the fragment of the image shown in Figure 3.

Figure 3 depicts a fragment of the image shown in Figure 2 with spreading regenerative epidermis consisting of basic layer cells of high RNA content and other cells of the epidermis of much lower RNA content. In the lower part of the image there are granulation tissue cells of moderate response to RNA. Magnification measure = 50 micrometers. Figure 4 depicts a sagittal section of a specimen of D group rat skin. 21 -days series. Response to RNA. Skin of proper structure can be seen on both sides of the image with some mastocytes (m). The remaining part of the image presents skin in an advanced stage of regeneration. The arrow points to a fragment shown in Fig. 5.

Figure 5 depicts a fragment of the image shown in Figure 4. The regenerative epidermis which forms epidermal cones penetrating the granulation tissue and transforms into fresh connective tissue presents the final stages of mature epidermis regeneration. Magnification measure = 50 micrometers. Figure 6. Sagittal section of a specimen of K₂ group rat skin. 21 -days series. Evident strong

NADHD activity on both sides of the image in unaffected skin. The epidermis (N) of moderate NADH activity transforms into short segments of regenerative epidermis (NR) with low enzyme activity. Strong skin emaciation and necrosis can be seen within the area of the burn. Magnification measure = 100 micrometers.

Figure 7 depicts a sagittal section of a specimen of Di group rat skin. 21 -days series.

NADH dehydrogenase is evident over the entire area. Early healing stage showing lack of occlusion by regenerative epidermis (NR) of poor enzymatic activity. Skin of proper structure and enzymatic activity can be seen to the left. Epidermis with significant

NADHD activity is indicated. Magnification measure = 100 micrometers.

Figure 8 depicts a sagittal section of a specimen of D₂ group rat skin. 21 -days series.

NADH dehydrogenase is evident over the entire area. The central area of the image shows indications of the final stage of healing of the entire wound. Still some fresh epidermis (NR) is observed, however underneath it is a fresh loose connective tissue proper (TL) of low NADHD activity of fibroblasts transforming into fϊbrocytes. Magnification measure =

100 micrometers.

Figure 9 shows the average percentage of regenerative epidermis within the burn wound.

The object of the invention is described below with the use of examples in which the composition of the dermatological preparation is given in various possible and optimum proportions of its components as well as the methods of its preparation from those components.

Example 1. Composition of the preparation and list of auxiliary substances

100 parts by weight of the preparation contain, according to a general formula: - Standardized propolis extract: 1 to 10 parts by wt. (adjusted to 100% extract basis)

- Standardized plant extract: 1 to 20 parts by wt. (adjusted to 100% extract basis)

- Dermatological antibiotic: 0.1 to 5 parts by wt. (5,000 to 300,000 IU)

- Vitamin: 0 to 0.1 parts by wt. (< 500,000 IU)

- Vehicle: make up to 100 parts by wt. The composition of the vehicle is as follows (in parts by its weight):

Liquid paraffin 0-30 parts by wt.

Solid paraffin 0-20 parts by wt.

White petrolatum 0-30 parts by wt.

Cholesterol 0-10 parts by wt.

Untreated vegetable oil 0-70 parts by wt.

Anhydrous lanolin 0-80 parts by wt.

The "standardized plant extract" is an extract of a plant, such as marigold, mountain and/or meadow arnica, St. John's wort or aloe or a combination thereof, with the content of flavonoids (in terms of quercetin) not less than 0.4%.

The "standardized propolis extract" is an extract that contains not less than 4% flavonoids (in terms of quercetin) and meets the antibacterial activity standard (not less than 3,300 AU in 1 gram of extract, determined against standard strain, e.g. Staphylococcus aureus FDA 209 P). The "dermatological antibiotic" means bacitracin, neomycin, polymyxin B, gentamycin, oxyteracin, erythromycin or staphylomycin or a combination thereof. The "vitamin" is vitamin A, C or E or a combination thereof.

The "untreated vegetable oil" means linseed oil or sesame oil or a combination thereof. All ingredients should meet the respective pharmacopeal standards. The preparation formulated should have the form of an ointment or an aerosol. In the case of aerosol the composition may be supplemented with appropriate propellant gas. Variant 1 100 grams of preparation contain:

- standardized extract of marigold flowers (e.g. tincture of marigold flowers 1 :3): 9.00 g - standardized propolis extract (e.g. tincture of propolis 1:10): 3.00 g (adjusted to 100% extract)

- Bacitracin (USP XXIII): 1.00 g (50,000 IU)

- Vitamin A (FP N): 0.03 g (100,000 IU) -Vehicle, make up to 100 g:

Liquid paraffin (FP N) 22.080 g

Solid paraffin (FP N) 5.175 g

White petrolatum (FP N) 6.210 g

Cholesterol (FP N) 1.035 g

Linseed oil (FP N) 51.00 g where the listed vehicles are designated as:

FP N - meets standards specified in the Polish Pharmacopoeia N, USP XXIII - meets standards specified in US Pharmacopoeia XXIII.

The "standardized extract of marigold flowers" is an extract containing not less than 0.4% flavonoids (in terms of quercetin), which may be obtained by extracting herbal raw material with 70% (v/v) aqueous solution of ethanol at a ratio of 1:3 at 60°C, pressing the material and filtering the product (PΝ-84/R-87013). The "standardized propolis extract" with the characteristics described above may be obtained by extracting propolis with 70% (v/v) aqueous solution of ethanol at a ratio of 1:10 at room temperature, filtering and concentrating under reduced pressure until 67% dry matter content is attained (PN-A-77626). The preparation obtained is used in the form of an ointment. Variant 2

The preparation is obtained from ingredients that meet the standards specified above. The composition of 100.00 g of the preparation is as follows:

1. Propolis extract 3.0 g (adjusted to 100% propolis extract)

2. Marigold extract 9.0 g 3. Bacitracin 1.0 g

4. Vitamin A 0.03g (adjusted to 0.055 g retinol palmitate)

5. Vehicle, make up to lOOg

- Cholesterol 3 parts by wt.

- Solid paraffin 15 parts by wt. - White petrolatum 18 parts by wt.

- Anhydrous lanolin (FA N) 64 parts by wt. The preparation obtained is used in the form of an ointment. Variant 3

1. Propolis extract 3.0 g (adjusted to 100% propolis extract)

2. Marigold extract 9.0 g 3. Bacitracin 1.0 g

4. Vitamin A 0.03g (adjusted to 0.055 g retinol palmitate)

5. Vehicle, make up to lOOg

- Cholesterol ca. 2 parts by wt.

- Solid paraffin ca. 10 parts by wt. - White petrolatum iala ca. 12 parts by wt.

- Anhydrous lanolin (FA V) ca. 42 parts by wt.

- Linseed oil ca. 43 parts by wt. > The preparation obtained is used in the form of an ointment.

Variant 4 The preparation is obtained from ingredients that meet the standards specified above. The composition of 100.00 g of the preparation is as follows: Marigold extract 9.0 g

Propolis extract (100%) 3.0 g

Bacitracin 1.0 g Vitamin A 0.03 g

Linseed oil 52.5 g

Anhydrous lanolin 33.0 g

The preparation obtained is designed for use in the form of an aerosol. To this end a propellant gas, preferably tetrafluoroethane, is introduced under pressure into the emulsion. obtained (see also example 9).

Other advantageous variants of the preparation may be obtained as described in examples 2-9. Example 2. Method of preparing the preparation a. Preparation of vehicle

1. Melt (temp. 60-90°C) and combine the weighed ingredients, i.e. white petrolatum, anhydrous lanolin and solid paraffin.

2. Add the prescribed amount of cholesterol and stir until completely dissolved. 3. Heat the prepared vehicle to 90°C with exposition time of 1 hour and pressure reduced by 40%.

4. Cool the vehicle to 40°C while maintaining pressure reduced by 40%. b. Preparation of the mixture of active ingredients

5. Add the prescribed amount (adjusted to retinol palmitate quantity) of vitamin A to weighed portion of linseed oil, heat to 30°C and stir until completely dissolved (stirring time 10 min.). Heat to 40°C prior to introducing into vehicle.

6. Dissolve the prescribed amount of bacitracin in weighed portion of marigold extract until completely dissolved (stirring time ca. 15 min.). Then add the prescribed amount of propolis extract (watch the percentage content of propolis in the extract used) and stir at room temperature until dissolved and homogenised (stirring time ca. 15 min.). Heat to 40°C prior to introducing into vehicle. C. Introduction of active ingredients into vehicle

7. Introduce linseed oil with dissolved vitamin A (temp. 40°C) to vehicle cooled to 40°C. Stir to homogenise for 15 min. at pressure reduced by 40%. 8. Now add solution of bacitracine, marigold extract and propolis extract heated to

40°C. Stir to homogenise for 15 min. while continuously maintaining pressure reduced by 40%.

9. Homogenise the entire mixture at: - temperature 40°C - speed 1500 rp

- duration not less than 20 minutes

- pressure reduced by 40%. 10. After homogenising cool to 25°C while stirring (speed 300 rpm, time 15 min., pressure reduced by 40%). Transfer to sterile containers and store at room temperature (ca. 25 °C).

11. Before packing stir the preparation (300 rpm, time 15 min., pressure reduced by 40%o) and fill into metal or plastic tubes. Example 3.

Measure 18 parts by weight of white petrolatum, 64 parts by weight of anhydrous lanolin and 15 parts by weight of solid paraffin into a mixing vessel, heat to 60°C, then add 3 parts by weight of cholesterol and stir until dissolved. Heat the mixture to 90°C for 1 hour under reduced pressure, then cool to 40°C. Introduce into a vacuum homogenizer 56.50 parts by weight of neutral vehicle obtained above and add 34.28 parts by weight of linseed oil heated to 40°C with 0.02 part by weight of vitamin A dissolved in it beforehand, then add 5.20 parts by weight of standardized extract of marigold with 0.50 part by weight of bacitracin and 3.50 parts by weight of standardized propolis extract dissolved in it beforehand. The entire mixture is homogenised at 40°C under reduced pressure until homogenous emulsion is obtained, then cooled to room temperature. Example 4.

54.87 parts by weight of neutral vehicle obtained as described in example 3 are introduced into a mixing vessel. Then 32.00 parts by weight of linseed oil at 40°C with 0.03 part by weight of vitamin E dissolved in it beforehand are added followed by 8.10 parts by weight of standardized extract of marigold with 0.9 part by weight of neomycin dissolved in it beforehand and 4.10 parts by weight of standardized propolis extract. The entire mixture is homogenised at 40°C under reduced pressure until homogenous emulsion is obtained, then cooled to room temperature. Example 5.

53.50 parts by weight of neutral vehicle obtained as described in example 3 are introduced into a mixing vessel. Then 30.06 parts by weight of linseed oil at 40°C with 0.04 part by weight of vitamin A dissolved in it beforehand are added followed by 10.2 parts by weight of standardized extract of mountain arnica with 1.3 part by weight of polymyxin B dissolved in it beforehand and 4.90 parts by weight of standardized propolis extract. The entire mixture is homogenised at 40°C under reduced pressure until homogenous emulsion is obtained, then cooled to room temperature. Example 6.

55.20 parts by weight of neutral vehicle obtained as described in example 3 are introduced into a mixing vessel. Then 33.96 parts by weight of linseed oil at 40°C with 0.03 parts by weight of vitamin A and 0.01 part by weight of vitamin E dissolved in it beforehand are added followed by 6.00 parts by weight of standardized extract of mountain arnica with 1.00 part by weight of bacitricm dissolved in it beforehand and 3.80 parts by weight of standardized propolis extract. The entire mixture is homogenised at 40°C under reduced pressure until homogenous emulsion is obtained, then cooled to room temperature. Example 7.

56.00 parts by weight of neutral vehicle obtained as described in example 3 are introduced into a mixing vessel. Then 31.00 parts by weight of linseed oil at 40°C with 0.03 parts by weight of vitamin E dissolved in it beforehand are added followed by 5.50 parts by weight of standardized extract of marigold and 2.10 parts by weight of aloe extract with 1.17 part by weight of neomycin dissolved in it beforehand and 4.20 parts by weight of standardized propolis extract. The entire mixture is homogenised at 40°C under reduced pressure until homogenous emulsion is obtained, then cooled to room temperature. Example 8.

33.60 parts by weight of lanolin are introduced into a mixing vessel and heated at 90°C under reduced pressure for 1 hour, then cooled to 40°C. Afterwards 52.60 parts by weight of linseed oil at 40°C with 0.04 parts by weight of vitamin C dissolved in it beforehand are added followed by 5.10 parts by weight of standardized extract of mountain arnica and 2.20 parts by weight of aloe extract with 1.16 part by weight of polymyxin B dissolved in it beforehand and 4.50 parts by weight of standardized propolis extract. The entire mixture is homogenised at 40°C under reduced pressure until homogenous emulsion is obtained, then cooled to room temperature. A propellant gas, preferably tetrafluoroethane, is introduced under pressure into the emulsion obtained in order to formulate an aerosol preparation. Example 9. 33.60 parts by weight of lanolin are introduced into a mixing vessel and heated at 90°C under reduced pressure for 1 hour, then cooled to 40°C. Afterwards 50.80 parts by weight of sesame oil at 40°C with 0.02 parts by weight of vitamin A and 0.02 parts by weight of vitamin E dissolved in it beforehand are added followed by 6.20 parts by weight of standardized extract of marigold, 2.95 parts by weight of standardized extract of mountain arnica and 1.15 part by weight of aloe extract with 0.15 part by weight of neomycin dissolved in it beforehand and 4.80 parts by weight of standardized propolis extract. The entire mixture is homogenised at 40°C under reduced pressure until homogenous emulsion is obtained, then cooled to room temperature. A propellant gas, preferably tetrafluoroethane, is introduced under pressure into the emulsion obtained in order to formulate an aerosol preparation.

Example 10. Physicochemical properties of ointments obtained. Selection of vehicle. a. Testing of spreadability of ointment vehicle and preparations obtained with its use

(rheological tests).

Spreadability was determined by means of an extensometer at a temperature of 25°C. The measurement consists in the determination of the degree of area increase (in cm²) of vehicle placed between two glass plates subjected to increasing load (in grams).

The extensometer applied consisted of the following parts: base plate (glass plate 200mm x 200mm with millimetre ruler), cover plate (glass plate with centre point), dispenser, weights. In order to make the measurements a layer of vehicle (about 10 mm) was placed by means of the dispenser on the marked surface of base plate. Then the cover plate was placed over the vehicle so that the centre point was located in the centre of the vehicle layer. After 1 minute the effects of load in four directions were read, mean radius ( r ) and area obtained were calculated. The upper plate was further subjected to load increased in equal time intervals by placing subsequent weights and the surface area of the layer was calculated. Correlation equations listed in table 1 were used in the load range m =20-1000 g after integrating the relation J ( a + bx) dx = ax + ^l/₂ bx² to an applicable form, e.g.: ₂oJ¹⁰⁰⁰ (a + bx) dx = 1000a + ! b (1000)² - [20a + V₂ (20)²] to calculate surface area under spreadability curves in conventional units P_c.u.

The introduction of i(P) index, which is equal to the quotient of surface areas expressed in conventional units of the preparation and the vehicle, enables assessment of the effect of biologically active ingredients on the internal structure of the preparation. It was observed that the content of linseed oil, apart form the presence of lanolin, had the decisive effect on the physical properties of the ointment. The composition of the preparation used in the test, designated as sample "P", corresponded to that of variant II preparation in example 1. Linseed oil was added to that preparation in order to obtain a composition containing 20% (sample "P+20%"), 25% (sample "P+25%") or 30% (sample "P+30%") by weight of linseed oil, the mixture was homogenised and measurements were carried out.

Test results are presented in table 1.

Table 1. Correlation equations describing the spreadability of vehicle and ointments obtained with its use.

b. Determination of pharmaceutical availability of biologically active ingredients using the membrane method. 5 Calibration of measurement system.

2-propanol solutions of ethanol extract of propolis with marigold extract were prepared and spectrophotometric analysis was carried out. Ethanol extracts were used in the same quantitative ratios as in the preparations. The results obtained were used to plot standard curves. To this end 0.2463 g of ethanol extract of propolis and 0.5154 g of ethanol extract of 0 marigold were weighed, transferred quantitatively to a 50 cm³ measuring flask and made up to the mark with 2-propanol. Then 1 cm³ of the solution obtained was taken and diluted with solvent to 50 cm³. Then 1 cm³, 2 cm³, 3 cm³, 4 cm³ of the second solution was transferred to 50 cm³ measuring flasks, which were made up to mark with 2-propanol. Spectrophotometric analysis of these solutions was carried out. 5 The resultant values of absorbance (A) were used to formulate a correlation equation in the form y = a + bx and to compute the quantity of biologically substances - C_s. Assuming that in the y = a + bx equation, y = A and x = C_s, then the equation takes on the following form: ■ for ethanol extract of propolis ( λ=290.0nm )

A = 5.5437-10^"2 + 621.6588 C_s, at r = 0.9999 0 ■ for ethanol extract of marigold ( λ=290.0nm )

A = 5.5437-10^"2 + 297.0787 C_s, at r = 0.9999 Determination procedure:

Appropriate quantity of ointment obtained (~ 5 g) was introduced into a plastic container that performed the role of a therapeutic system. The composition of test preparations was identical with that of the previous experiment. The outer surface was covered with hydrated cellophane with pores of statistical size of 25 A. The container was placed in a beaker containing 0.5 dm³ solvent, the latter being water in the first series of tests and 2-propanol in the second series. At defined intervals of time (after 15, 30, 45, 60, 90, 120, 150, 180, 210, 240 minutes) 5 ml of the solution was sampled and its absorbance was measured directly. Results are presented in Table 2 (designations identical to those in table 1). After each sample was taken, solvent was added to make up to 0.5 dm³ so that diffusion proceeded in constant volume at any moment. Absorbance values (A) were used to compute concentrations of biologically active substances in the propolis extract and the marigold extract from correlation equations: ^■ for ethanol extract of propolis ( λ=290.0nm )

A = 5.5437-10^"2 + 621.6588 C_s, at r = 0.9999 ^■ for ethanol extract of marigold ( λ=290.0nm )

A = 5.5437-10^"2 + 297.0787 C_s, at r = 0.9999

Table 2. Concentration of substances diffusing from ointments to 2-propanol vs. container exposure time

Summary

Ointment with its composition defined as variant 1 (in example 1) showed insufficient physicochemical stability, high tendency to separate out liquid constituents, affecting thereby its homogeneity. After a certain period, inadequate consistency of the ointment made its application on skin difficult (when the tube was pressed, liquid ingredients of brown colour splashed in every direction) and its appearance unpleasant. The ointment described as valiant 3 (in example 1) was diametrically opposed to that of variant 1. The experiments carried out unexpectedly indicated that replacing liquid paraffin with anhydrous lanolin and experimental determination of proper amount of linseed oil are of key significance to the physical properties of the ointment obtained. Good spreadability of vehicle and high homogeneity and stability of the ointment were attained.

Tests of kinetics of the release of biologically active substances through cellophane to outer compartment (2-propanol) in vitro indicate that diffusion of lipophilic components from ointment of variant 1 is much slower. What more, it unexpectedly occurred that the modified vehicle not only improves physical properties of the preparation, but also promotes mass transfer on phase boundary. Unexpectedly the optimum values of these parameters were obtained for the ointment designated as variant 3 (cref. table 2, samples P+30%). Example 11. Microbiological tests of preparations of the invention

The following preparations (modifications of variant 1 preparation of example 1) were used in the tests: preparation A containing 1% ethanol extract of propolis, and preparation B containing 3% standardized ethanol extract of propolis, standardized extract of marigold and vehicle comprising cholesterol ointment and linseed oil. Bee venom at a concentration of 10 μg/ml was used as the irritant.

Additionally microbiological assessment of preparation B ointment containing no bacitracin was made.

Test results presented in table 3 indicate that preparation A showed low microbiological activity (40 AU/g), whereas microbiological activity of preparation B without bacitracin was 15 times stronger, and the same preparation containing bacitracin had 38 times stronger microbiological activity as compared to preparation A. Microbiological activity of preparation B containing bacitracin (1500 AU/g) was 2.5 times stronger than that of the same preparation containing no bacitracin (600 AU/g). Table 3. Microbiological activity of preparation A and B

* The preparation contained 1% ethanol extract of propolis ** The preparation contained 3% concentrated extract of propolis The effect of the preparations of the invention on micro-organisms that cause wound infections was also investigated. An extract of the tested preparation B containing bacitracin was made. The extract obtained was diluted in Penassay (Difco) liquid vehicle to concentrations ranging from 10 to 600 μg/ml.

The strains used in the test were of the type that cause wound infections, including burn infections. The tests included 5 Gram-positive coccus strains and 5 Gram-negative bacillus strains.

Liquid 18-hour cultures of the tested strains were diluted in Penassay vehicle to a concentration of 10 -10⁵ bacteria in 1 ml. Then 0.1 -ml samples of the diluted culture were introduced into each of the series of diluted preparations. The lowest concentrations of biologically active substances in the preparation B ointment that affected the growth of wound-infecting micro-organisms (MIC) were determined after 18 hours of sample incubation at 37°C.

The results of these tests presented in table 4 indicate strong effect of the tested preparation on wound-infecting micro-organisms. For Gram-positive coccus strains MIC was within 20 to 100 μg/ml, while for Gram-negative bacillus strains it was within 60 to 420 μg/ml. Table 4. Effect of preparation B on wound-infecting micro-organisms.

It may therefore be assumed that the concentration of biologically active substances contained in preparation B (30,000 μg/ml) is 70 to 1500 times higher in comparison with the lowest concentrations (20-420 μg/ml) of this preparation that affect the growth of the tested micro-organisms.

Conclusions

In order to determine the effect of bacitracin on microbiological activity of the preparation of the invention, 3 preparations were compared: - preparation A with 1% ethanol extract of propolis

- preparation B with 3% concentrated extract of propolis

- preparation B as above with bacitracin added.

Test results indicate that preparation A showed low microbiological activity (40 AU/g), whereas microbiological activity of preparation B without bacitracin was 15 times stronger, and the same preparation containing bacitracin had 38 times stronger microbiological activity as compared to preparation A. Microbiological activity of ointment B containing bacitracin was 2.5 times stronger than that of the same preparation containing no antibiotic. It must be stressed that the tests described proved that propolis shows strong antibacterial activity, and the addition of bacitracin enhances that activity 2-3 times, showing evidence of an unexpected advantageous strengthening of the antibacterial effect and justifying the proposed composition of the preparation. Further tests were carried out with preparation B containing bacitracin of the highest microbiological acivity. Test results have proven strong effect of the tested preparation on wound-infecting micro-organisms. For Gram-positive coccus strains MIC was within 20 to 100 μg/ml, while for Gram-negative bacillus strains it was within 60 to 420 μg/ml. It may therefore be acknowledged that the concentration of biologically active substances contained in preparation B (30,000 μg/ml) is 70 to 1,500 times higher in comparison with the lowest concentrations (20-420 μg/ml) of this preparation that affect the growth of the tested micro-organisms. It has also been confirmed that the addition of bacitracin to preparation B clearly enhances its microbiological activity and that the preparation of the invention effectively destroys wound-infecting bacteria. Example 12. Pharmacological, cytomorphological and histoenzymatic assessment of the preperation of the invention on a rat model of burn wound. MATERIALS and METHODS

Preparation as described in example 1 as variant 1 was subject to the assessment. Test were performed on white Wistar rats and white guinea pigs, to which the tested preparation was always administered in the form of skin application. 1. Assessment of the primary irritating action of the preparation.

Tests of irritating action of the preparation were carried out on white male guinea pigs weighing 250-300 g. The animals were given a standard diet and water without restrictions. Experiments were performed on two groups of animals: the studied group (15 animals) and the control group with vehicle (10 animals). Every day, for a period of 10 subsequent days, 0.5 g of the preparation or the vehicle was spread (not rubbed in) on the shaved and degreased with ethyl alcohol and ether skin of the right flank of each guinea pig. Reaction was evoked on a surface 5 x 5 cm under a loose dressing of sterile gauze and cellophane. Results were checked every day for a period of 10 days. An erythema reaction in 50%) of the tested animals or an increase in the thickness of the skin fold was determined as positive result. The thickness of the skin fold was measured using skin fold callipers (measuring accuracy 0.05 mm) before the experiment and on its last day [Farmakometria. Doswiadczalne metody badania lekόw (Pharmacometry. Experimental methods in drug testing). PZWL Warsaw, 1982].

2. Assessment of allergenic properties of the preparation - ability to generate delayed contact allergy.

Tests were carried out on white male guinea pigs weighing 250-300 g in two groups: control group (vehicle per se - 10 animals) and studied group (preparation - 15 animals). The animals were given a standard diet and free access to water. The tested compound preparation (as the potential contact allergen) and the vehicle were used in the skin (contact) tests. For 8 subsequent days, 0.1 g of the preparation (ointment) or the vehicle was spread on a surface of ca. 2 cm of the skin (5 x 5 cm) shaved and degreased with ethyl alcohol and ether on the right flank of each guinea pig (induction phase). When no reaction was seen in the observed area, then on the 15th day of the experiment, an elicitation dose of the tested preparation or the vehicle was exposed to the left flank of the guinea pig, the dose being diluted 10-fold in an amount of 0.01 g (expression phase). Erythema, skin lesion and increase in the thickness of the skin fold measured with skin fold callipers, occurring after 24, 48 and 72 hours after applying the elicitation dose was determined as positive result [Maibach H.J., Maguire H.C. Int. Arch. Allergy, 1969, 35, 535]. The aim of the test was to determine whether: a) the tested preparation may be an antigen that induces a delayed hypersensitivity reaction in healthy guinea pigs; b) the vehicle of the preparation produces a delayed skin reaction. 3. Cytomorphological and histoenzymatic assessment of burn wound healing in rats.

Tests were carried out on adult male white Wistar rats weighing 170-210 g. Experiments consisted in inducing a burn on shaved skin of the right flank on an area of 2 cm² by means of copper bar heated to 100°C and contacted with the skin for 12 seconds. Experiments were performed on four groups of animals.

Table 5. Outline of procedures and number of animals used in the experiment

The control group Kj consisted of rats not subjected to any procedures. A. Control group Ki - skin of healthy animals with no burns.

B. Control group K - skin of animals with burns, no treatment provided.

C. Studied group O_\ - skin of animals with burns, treated with vehicle.

D. Studied group D₂ - skin of animals with burns, treated with the preparation of the invention. The tested preparation or the vehicle was applied to the burn wound in an amount of 0.1 g, once a day for a period of 7 or 21 days from the day of inducing the burn. Skin specimens were taken from the burn area at the boundary of the zone around the wound and from unaffected zone after 7 and 21 days of the experiment.

Skin specimens were always taken with a wide margin of healthy skin (ca. 0.5 cm). Average dimensions of the specimens taken were 2 cm x 2 cm. Each specimen was cut into halves, set on cryostatic table, with the cross section facing upwards, and immediately frozen with dry ice. The specimens were cut on a cryostatic microtome into 6μm thick slices representing sagittal sections of the skin together with subcutaneous muscle layer.

Tissue sections were subjected to histochemical tests for assessing activity of selected oxidative enzymes:

- glucose-6-phosphate dehydrogenase (G6PD) as an indicator of the intensification of the pentose cycle participating in the synthesis of nucleotides [ Romeis B.R., Oldenburg Verlag, Munchen-Wien, 1968; Maibach H.J., Maguire H.C, Int. Arch. Allergy, 1969, 35, 535], - electron transport enzyme dehydrogenase (NADHD), which provides information on metabolism intensification and overall vitality of cells [ Romeis B.R. ibid., Maibach H.J. ibid.],

- c cytochrome oxidase (OCC) respiratory chain enzyme as an indicator of highly energetically efficient aerobic metabolism in cells [ Romeis B.R. ibid., Maibach H.J. ibid.]. Response to RNA was also tested in tissue sections to determine intensity of RNA biosynthesis and also, indirectly, of protein biosynthesis [Maibach H.J. , ibid.]. Pappenheim staining [Romeis B.R., ibid.] was applied to detect defensive cells (mastocytes in particular) and reconstructive cells (fibroblasts). Van Gieson staining was applied to observe the growth of fibres of the connective tissue [ Maibach H.J., ibid.]. Microscopic specimens containing tissue sections on which tests had been performed were subjected to microdensitometric analysis (in order to determine precisely the activities of the enzymes studied and RNA content) and to histomorphometric analysis (in order to calculate areas of tissue regeneration and the count of some defensive cells [Bagiήski S., Technika mikroskopowa (Microscopic technique), PWN, Warsaw, 1965]. The important results are supported by photographic and graphical documentation.

The ointment was administered every day in the form of skin application onto the burn wound, starting from the first day after inducing the burn, throughout the 21 -days period of the experiment. RESULTS 1. Assessment of the primary irritating action.

The tested preparation in the form of ointment, the composition of which and vehicle contained therein were presented in example 1 (variant 1), applied onto the skin in an amount of 0.5 g ( 5x5 mm ) every 24 hours for a period of 10 days did not produce inflammatory changes in any of the animals. Dermotoxic reaction (erythema, increase of the thickness of the skin fold) on the skin under contact with the preparation did not occur both after first contact with the skin as well as after 10 days of observation. The ointment vehicle applied in parallel for comparison did not produce primary irritating action as well. Appearance of skin treated with the preparation and untreated skin is illustrated in Figures 1-8.

In order to determine to what degree the preparation is able to induce symptoms of delayed immunological reaction, an assessment has been made of the effect of the preparation and of the ointment vehicle on the formation and expansion of erythema and on the generation of an exudate and formation of skin oedema, i.e. the principal symptoms of the delayed-type hypersensitivity.

Skin test performed with the preparation of the invention, as a potential contact allergen, has not produced positive reaction (erythema) in any of the animals of the studied group. The animals of the control group, to which the ointment vehicle was applied, have not reacted positively as well. In the control group (vehicle) and the studied group (preparation) skin oedema, another assessment criterion, has not occurred in any of the animals. Appearance of skin treated with the preparation and untreated skin is illustrated in

Figures 1-8.

3. Cytomorphological and histoenzymatic assessment of burn wound healing in rats.

Results of cytomorphological tests are presented in Fig. 1-8, and detailed results of histoenzymatic and histochemical tests are summarized in tables 6-8. The sagittal sections of normal skin specimens not subjected to burns present a typical morphological structure of integument hair-bearing skin consisting of thin epidermis and thick derma. Other integument components can also be seen in the specimens: subcutaneous tissue, muscle layer and fascia (loose connective tissue proper covered by peritoneal epithelium on the abdominal cavity side). The derma contains very dense entangled bundles of collagen fibres with hair between them, sebaceous glands, quite numerous connective tissue cells of various types (fibrocytes, mast cells, single histocytes and fibroblasts) and blood vessels, ranging from capillaries, located mainly near the epidermis, to arteries and veins located near the subcutaneous tissue. The subcutaneous tissue contains much less collagen fibres than the derma. The muscle layer consists of muscle fibres with a limited number of connective tissue collagen fibres entwining the muscular tissue. The epidermis-generating layer is characterised by distinctly strong activity of reduced NAD dehydrogenase (NADHD) and cytochrome oxidase (OCC) and moderate activity of glucose-6-phosphate dehydrogenase (G6PD). Results and interpretation of morphological changes in the burn wound zone subjected to treatment with the preparation and treated with vehicle only are illustrated on microphotographs (Figs. 1-8).

Table 6. Mean intensity of activity of oxidative enzymes and RNA response (in units of optical density) in selected components of skin specimens of group Ki rats

Table 7. Mean intensity of activity of oxidative enzymes and RNA response (in units of optical density) in selected components of skin specimens of group K₂ rats

Table 8. Mean intensity of activity of oxidative enzymes and RNA response (in units of optical density) in selected components of skin specimens of group D₂ rats

DISCUSSION OF RESULTS

The results of pharmacological, cytomorphological and histoenzymatic tests of the effects of the preparation of the invention on the process of wound healing have been presented. The assessment of the pharmacological properties of the preparation was designed to provide data for determining its pharmacological value in treatment and healing of a burn wound.

The preparation and its vehicle do not produce any primary irritating action and do not induce an inflammatory condition of skin. No toxico-allergic reactions of the skin have been observed in any of the tested animals, both after first contact with the skin as well as after repeated application of the preparation and the vehicle. The ointment and its vehicle do not cause irritating action and display no tendency to induce contact-type allergic reactions. The tests performed indicate that the vehicle and the preparation, as well as possible products of their catabolism, are not capable of inducing contact-type immunological reaction of the skin in guinea pigs.

When investigating the process of burn wound healing and new natural substances with potential therapeutic ability in relation to that process it is necessary to describe the changes occurring on the level of cells. The results obtained give a quite clear picture of the behaviour of the morphological structure of skin in the process of burn wound healing under the conditions of application of the preparation.

Morphological observation of skin specimens of the tested animals proves that the process of burn wound healing is very harmonious and much faster when the preparation is applied. This process was less advanced in the control group K₂ and group Di, i.e. the group treated with the vehicle only. In the latter group the abundance of regenerative epidermis in the burn wound area was much lower (Fig. 9).

The regenerative epidermis in the burn wound area was highly developed in the skin of animals of the group treated with the preparation (group D₂) and it displayed higher intensity of activity of the tested enzymes and higher content of RNA (Tables 4-7). The growth of the regenerative epidermis was enhanced by intensified cellular metabolism. This was manifested by intensified activity of OCC and NADHD in the regenerative epidermis and in hair follicles in the burn wound area after both 7 as well as 21 days after inducing the burns.

We must stress that most of the migrating epithelium after 21 days of the experiment, when the preparation was applied, transformed into a fresh epidermis. In the control group K and group Di (vehicle), such epidermis was rare. The causes of the effects described above are the more favourable conditions for the wound healing process provided by the action of the ingredients of the preparation.

Faster and more harmonious growth of the granulation tissue in animals of group D₂ (vehicle) was indicated by more intensive formation of collagen fibres in the area. These fibres are generated by the fibroblasts of the granulation tissue.

Characteristic intensification of OCC activity and increased RNA concentration in the cells of the regenerative epidermis and in hair follicles of the wound area were observed in skin specimens of animals treated with the preparation. These results indicate particularly prominent synthesis of nucleotides and proteins, associated with intensified cell division processes and with the differentiation of cells that reconstruct the area of skin and subcutaneous tissue destroyed by the burn. This conclusion is supported by the high OCC activity observed in this group of animals treated with the preparation, and also by high RNA activity. Intensified aerobic metabolism, indicated by the observed OCC activity, and intensified transport of electrons demonstrated by NADHD activity, provide evidence for the optimisation of conditions for proper healing of the wound treated with the preparation.

Comparison of the data collected indicates that 21 days after inducing of the burn only the animals treated with the preparation had their wounds completely or nearly completely occluded by fresh epidermis and the derma completely reconstructed in the burn zone. Example 13. Toxicological tests of the preparations of the invention

Tests on the irritating (dermatotoxic) effect of the preparations were based on the carrageenan method of assessing antiedematous effect of biologically active substances. The basis adopted for these tests was the effect of enhancing carrageenan-induced oedema in rats in the presence of irritating (toxic) substances.

The following preparations (modifications of variant 1 preparation of example 1) were used in the tests: preparation A containing 1% ethanol extract of propolis, and preparation B containing 3% standardized ethanol extract of propolis, standardized extract of marigold and vehicle comprising cholesterol ointment and linseed oil. Aqueous solution of bee venom at a concentration of 10 μg/ml was used as the irritant.

Wistar rats weighing 250-300 g were used in the tests, without restricting access to water and food. The number of animals in each studied and control group was 6. The tests followed the methodology described by Winter C.A., Risley E.A., Nuss G.W.: Carrageenan - induced oedema in hind paw of the rat as an assay for anti- inflammatory drugs . Proc. Soc. Exp. Biol. Med. 1962 ; 111 : 544 - 547.

Before carrageenan was administered, the volume of the rat's right hind paw was measured by means of a plethysmometer (Hugo Sachs Elektronik). Then 0.1 ml of a l-%> aqueous solution of carrageenan (Carrageenan Lambda, supplied by Sigma) was injected into that paw by means of a micro-syringe. The tested ointments and semi-products were applied onto sterile gauze in an amount of 100 g and placed on the rat's paw where carrageenan had been administered. Calculations for each of the group were based on the mean swelling of the rat's paw induced after 4 hours.

Measurement results presented in table 9 indicate that the tested ointments of preparation A and preparation B, as well as semi-products used for the formulation of the ointment of preparation B, reduced the swelling of the rat's paw induced by carrageenan. This means that the tested products have no irritating effect. Bee venom, used as a control substance for increasing the carrageenan-induced oedema of rat's paw, had an irritating effect. Therefore a conclusion may be inferred that the tested ointments and semi-products used for their formulation are non-toxic when applied topically.

Table 9. Effect of tested preparations and semi-products on the carrageenan-induced oedema of rat's paw

* LD₅₀ for both extracts upon oral administration to mice is over 15 g/kg body weight, which, according to Hodge and Sterner's classification, may be considered to be relatively harmless to humans. Conclusions

Toxicological tests, which included tests of the irritating effect of the preparation of the invention, were carried out on rats according to the carrageenan method, applied to assess the antiedematous effect of biologically active substances. Aqueous solution of bee venom, which at a concentration of 10 μg/ml is an irritating (toxic) substance, was used as positive control substance.

The tests performed indicate that the preparation is non-toxic and has no irritating effect when applied topically and demonstrates anti-inflammatory and antiedematous properties. CONCUSIONS FROM PRECLLNICAL ASSESSMENT

1. The preparation of the invention does not show any primary irritating, allergic or allergenic effects on skin.

2. It has an anti-inflammatory, antiedematous and antibacterial effect.

3. It speeds up the healing of burn wounds by sterilizing, intensifying regeneration of epidermis, accelerating the growth of granulation tissue, intensifying the formation of collagen fibres and formation of capillary vessels.

4. The conclusions above provided grounds for carrying out clinical tests (on humans).

Example 14. Assessment of allergenic action of the preparation.

Preparations and vehicles of the invention were assessed using patch tests on 30 patients suffering from chronic allergic illnesses, including contact eczema and atopic skin inflammation.

Samples were applied in the form of patch tests in 30 patients. Soaked paper discs were applied in occlusive dressing for 24 hours. Results were noted immediately after removing the occlusive dressing and after 48 and 96 hours.

Results

Positive results of patch tests 48 and 96 hours after applying the tested preparations have not been observed in any of the 30 patients. Conclusion

The tested preparations and vehicles of the invention do not display any allergenic effect. Example 15. Assessment of the primary irritating action of the preparation

The preparations and vehicles of the invention were applied to 30 healthy persons. Paper discs soaked with the tested preparations were applied in occlusive dressing for 48 hours. Results were noted immediately after removing the occlusive dressing and after 72 and 96 hours.

Results

Positive reaction (+++) to the tested preparation, in the form of a flare and oedema and blisters, was observed in patient P.K. no. 14 after 48 hours. That reaction was sustained after 72 hours. After 90 hours the effect was mainly erythema, which was still observed 6 days after application. No positive results have been observed in any of the remaining 29 volunteers.

Conclusion

Because of the occurrence of the primary toxic reaction, the preparations of the invention may have a primary irritating effect under occlusion conditions. Therefore we recommend including in the information materials a warning against complications that may arise when applying the preparation under an occlusive dressing. Example 16. Assessment of phototoxic action of the tested aerosol preparation and vehicle.

Tests were carried out on 30 healthy persons of both sexes aged 20 to 44.

The preparations of the invention and vehicles were transferred onto application spots and subsequently irradiated with ultraviolet light (UVB) - 0.25 minimum erythema dose (MED). Then the application spots were covered with paper discs in an occlusive dressing for 48 hours. MED dose was determined beforehand. Results were noted immediately after removing the occlusive dressing and after 72 and 96 hours.

Results : No positive results of skin tests have been observed in any of the examined volunteers after carrying out the phototoxic tests. Conclusion :

The tested preparations and vehicles of the invention do not display any phototoxic effect. Example 17. Use of the preparation for treating burns in children

Preparation of the invention (example 1, variant 3) was applied in the treatment of 45 children, aged 4 weeks to 15 years, suffering mainly grade Ila burns with an extent not exceeding 15% TBS A.

The medication was administered in various clinical situations, for instance: 1) first intention, onto minor burn wounds (Ila, up to 10% TBS A) after first change of occlusive dressing, on the second or third day after primary debridement of the burn wound on the admission day (Ila, up to 15% TBS A), or 2) on residual wounds after taking and implanting autogenic skin grafts on wounds of delayed healing.

The preparation was applied to wounds in the following manner: during the first 3 days the preparation was applied to the wounds every 2-3 hours, during the next 3-10 days the preparation was applied 3 times a day, afterwards the child had an overall bath made every 12 hours in lukewarm water with a small amount of Betadine added.

The principle of treating burn wounds using the preparation of the invention is that after initial debridement of the wound, further treatment is carried out in "open" conditions, without the use of occlusive dressings. Patients were placed without clothes, gauze or dressings under a scaffold covered with sterile bed sheets protecting them against heat loss and reduced skin evaporation. Sometimes, particularly in the case of children 2 years old or less, they were dressed in sterilized pyjamas to constrain their motility. Special care was taken to isolate eyes and mouth from contact with the medication. Temperature in rooms where the children were treated was maintained at the required level and the air was humidified.

Treatment results

The ease of medication application has been confirmed. The ointment was always spread over the surface of the burn wound exclusively by the doctor's hand, nurse's hand (initially under the doctor's supervision) or parent's hand (initially under the nurse's supervision) covered with a sterile surgical glove, without the use of any tools or other materials (gauze swabs, for instance). Because of the consistency of the ointment and its lack of tendency to flow down the wound, this was a simple and easy operation. Children usually showed anxiety only during the first application. This can also be contributed to the proven strong analgesic action of the preparation. The wound becomes painless ca. 12- 24 hours after the start of application. Until that time typical pharmacological pain-killing treatment is recommended, later its intensity is reduced and the doses of analgesic adjusted to individual needs of the patient.

A clear advantage of such treatment is the ease of continuous control of the depth and cleanliness of the burn wounds and ease of swabbing the wound in order to make bacteriological tests.

It has been observed that the preparation had a beneficial effect in postponed treatment of residual wounds in case of extensive and deep burns and autogenic skin graft donor sites.

However, the main positive surprise in the treatment performed using the preparation of the invention is the time after which the different wounds, caused by or associated with burns, heal. The time taken by grade Ila burn wounds to heal ranged from 4 days (minor wounds) to 12 days (large wounds). Complete healing of residual wounds described above took 3 to 6 days, whereas the healing of wounds that were difficult to heal before lasted 5 to 10 days. Change in the reaction (appearance of the surface) within the area of fresh burn wound was observed after just 24 hours. Within several successive days the appearance of the wound changes - within the first dozen to 24 hours it takes on a bright red colour, which is emphasized by the yellowish colour of the ointment, which in conjunction with the unavoidable covering of the surrounding healthy skin by the ointment may at first create a misguiding disconcerting impression. However, on the second or third day after application, the advance of the healing process of the wound can be easily seen. The inflammatory reaction and congestion, natural for this type of wounds, start to weaken. A thin, initially translucent pseudo-crust is formed, which at the beginning is firmly attached to the substrate. Its brownish colour may also be misleading to an inexperienced person. Forced detachment at this time should be avoided - after the next 2 or 3 days the crust can be separated easily, revealing a healed surface underneath, covered by fresh epidermis. No scar if formed on that surface. The area requires usual care and occasional application of grease. The process of healing residual wounds and wounds that heal with difficulty is somewhat different. After a short time on the surface of such wound a layer of epidermis cells appears. That layer, initially veiy thin, grows thicker, its colour changing from bright red to pale pink. In this case a thin pseudo-crust (on residual wounds) or a real crust (on some wounds that heal with difficulty) may also be formed. The procedure is similar to that described above, although if the crust is thick one may try to speed up its detachment, which may contribute to faster healing of the wound and minimisation of the scar forming area.

Summary and conclusions:

Only in the case of one child there was a suspicion of local allergic reaction. That reaction, however, subsided in a very short time without the need to administer any therapeutic agents. No tendency to form hypertrophied scars was observed in any of the children treated in the above manner, at least within the period of several months. Moreover, pharmacoeconomic analysis indicates that the use of the preparation of the invention is at least twice cheaper (grade I and Ila burns), and in cases of other burns (mosaic grade Ila and Ila/b) 2.5-3.5 times cheaper than treatment with the use of other preparations, which in addition usually involve occlusive treatment.

Claims

1. Dermatological preparation for treating skin lesions, especially burns, containing extract of propolis, extract of plant material, vegetable oil, antibiotics and vitamins, making up a mixture with a pharmaceutically admissible vehicle, characterized in that it contains 1 to 10 parts by weight of standardized propolis extract with not less than 4% of quercetin-equivalent flavonoids, 1 to 20 parts by weight of standardized plant extract with not less than 0.4% of quercetin-equivalent flavonoids and at least one dermatological antibiotic in the amount of 0.1 to 5 parts by weight and 0 to 0.1 part by weight of a vitamin supplement, evenly dispersed in the vehicle, the quantity of which makes up to 100 parts by weight, whereas the vehicle preferably consists of 0 to 30 parts by weight of liquid paraffin, 0 to 30 parts by weight of solid paraffin, 0 to 30 parts by weight of white petrolatum, 0 to 10 parts by weight of cholesterol, 0 to 70 parts by weight of untreated vegetable oil and 0 to 80 parts by weight of anhydrous lanolin.

2. Dermatological preparation according to claim 1, characterized in that the vegetable oil is preferably linseed oil and/or sesame oil.

3. Dermatological preparation according to claim 1, characterized in that the vehicle consists of a mixture comprising 30-80 parts by weight of anhydrous lanolin, 10-30 parts by weight of white petrolatum, 10-30 parts by weight of solid paraffin and 1-8 parts by weight of cholesterol.

4. Dermatological preparation according to claim 1 or 3, characterized in that the vehicle consists of a mixture comprising 64 parts by weight of anhydrous lanolin,

18 parts by weight of white petrolatum, 15 parts by weight of solid paraffin and 3 parts by weight of cholesterol.

5. Dermatological preparation according to claim 3 or 4, characterized in that the plant extract contains standardized extract of marigold, standardized extract of mountain and/or meadow arnica, standardized extract of St. John's wort, standardized extract of aloe or a combination thereof.

6. Dermatological preparation according to any of the claims above, characterized in that the dermatological antibiotic contained therein is bacitracin, neomycin, polymyxin B, gentamycin, oxyteracin, erythromycin or staphylomycin or a commination thereof.

7. Dermatological preparation according to claim 1, characterized in that the vitamin supplement contained therein is vitamin A, C or E or any combination thereof.

8. Dermatological preparation according to claim 1, characterized in that it contains 50.06-54.28 parts by weight of untreated vegetable oil and anhydrous lanolin.

9. Dermatological preparation according to claim 1, characterized in that 100 grams of it contain 5-10 grams of standardized extract of marigold flowers, 1-5 grams of propolis extract, 0.1-2 grams of bacitracin, 0.01-0.1 gram of vitamin A, made up with vehicle to 100 g, whereas the vehicle comprises 20-25 grams of liquid paraffin, 2-8 grams of solid paraffin, 2-10 grams of white petrolatum, 0.2-2 grams of cholesterol, 30- 70 grams of linseed oil.

10. Dermatological preparation according to claim 9, characterized in that 100 grams of it contain 9 grams of standardized extract of marigold flowers, 3 grams of propolis extract, 1 gram of bacitracin, 0.03 gram of vitamin A, made up with vehicle to 100 g, whereas the vehicle comprises 22 grams of liquid paraffin, 5 grams of solid paraffin, 6 grams of white petrolatum, 1 gram of cholesterol and 51 grams of linseed oil.

11. Dermatological preparation according to claim 1, characterized in that 100 grams of it contain 5-10 grams of extract of marigold flowers, 1-5 grams of propolis extract, 0.1-2 grams of bacitracin, 0.01-0.1 gram of vitamin A, made up with vehicle to 100 g, whereas the vehicle comprises 10-40 parts by weight of solid paraffin, 5-30 parts by weight of white petrolatum, 0.2-5 parts by weight of cholesterol, 20-80 parts by weight of linseed oil, 30-80 parts by weight of anhydrous lanolin.

12. Dermatological preparation according to claim 11, characterized in that

100 grams of it contain 9 grams of extract of marigold flowers, 3 grams of propolis extract, 1 gram of bacitracin, 0.03 gram of vitamin A, made up with vehicle to 100 g, whereas the vehicle comprises 10 parts by weight of solid paraffin, 12 parts by weight of white petrolatum, 2 parts by weight of cholesterol, 42 parts by weight of anhydrous lanolin and 43 parts by weight of. linseed oil.

13. Dermatological preparation according to claim 12, characterized in that it has the form of an ointment.

14. Dermatological preparation according to claim 1, characterized in that 100 grams of it contain 5-20 grams of standardized extract of marigold flowers, 1-5 grams of propolis extract, 0.1-2 grams of bacitracin, 30-70 grams of linseed oil and 10-50 grams of anhydrous lanolin.

15. Dermatological preparation according to claim 14, characterized in that

100 grams of it contain 9 grams of standardized extract of marigold flowers, 3 grams of propolis extract, 1 gram of bacitracin, 0.03 gram of vitamin A, 52-53 grams of linseed oil and 33 grams of anhydrous lanolin.

16. Dermatological preparation according to claim 15, characterized in that it has the form of an aerosol.