GB2208598A

GB2208598A - Antimicrobial preparations for external use

Info

Publication number: GB2208598A
Application number: GB8719375A
Authority: GB
Inventors: Alain Li Wan Po; Beat Willi Muller; Eileen Margaret Scott
Original assignee: Ciba Geigy AG
Current assignee: Novartis AG
Priority date: 1987-08-15
Filing date: 1987-08-15
Publication date: 1989-04-12
Also published as: JPS6466121A; FI883747A0; AU2065588A; IT1224701B; ZA885969B; LU87312A1; NL8802008A; IT8848276A0; BE1003229A3; NO883609D0; FI883747A; DE3827515A1; NO883609L; PT88253B; FR2619311A1; DK453688A; ES2020012A6; GR880100526A; DK453688D0; SE8802814D0

Description

Pharmaceutical Preparations For External Use 4-16635 This invention

relates to new antimicrobially active pharmaceutical preparations for external use, e.g. for topical application. More specifically, this invention relates to antimicrobially active pharmaceutical preparations for external use on the basis of forming a pharmaceutical preparation using as antimicrobial ingredient a compound selected from the group of compounds having in their molecule a lower alkyl substituted imidazole in combination with one or more optionally substituted benzoic acids, the combination resulting in synergistic antimicrobial activity.

n f) r)o 8 59 0 ú. le- The European Patent Application (EPA) No. 0007595 describes a pharmaceutical preparation for external use of the antifungal ingredient clotrimazole (1-[(2-chloropbenyl)diphenylmethyll-lH-imidazole), more particularly a novel gel, cream and liquid preparation using crotamiton (N-crotonyl-N-ethyl-o-toluidine) as solvent.

Moreover, the EPA No. 0070525 describes a pharmaceutical preparation for external use having as antifungal ingredient a compound having an imidazole ring in its molecule, such as miconazole, econazole or isoconazole, which is dissolved in crotamiton, peppermint oil or methylsalicylate, each of which by itself has pharmaceutical activities as skin preparations for external use, but none has antifungal activities.

As a result of further investigations detailed in the present invention it has been found surprisingly and unexpectedly that the combination of an antimicrobially active compound selected from the group of compounds having in their molecule a lower alkyl substituted imidazole ring, being a compound represented by the general formula I 11- 0 0 Y w H2-YH-O-CH?-/' ---R2 1;\ 11 /Cl R3 /=.

a a , \\ 0 / L R1 (I) Z1 wherein at least one of R1, R2 and R3 is halogen and the rest thereof are hydrogen atoms, as well as pharmaceutically acceptable acid addition salts thereof or a compound of the formula II /R4 (C6Hs) 2 (11) wherein R4 is halogen, with one or more optionally substituted benzoic acids in a pharmaceutical preparation for external use exhibits an extraordinary synergistic antimicrobial effect.

Optionally substituted benzoic acids are those containing one or more halogen atoms, hydroxyl and lower alkoxy groups as substituent in different positions of the benzene ring. Preferred are optionally monosubstituted benzoic acids with the substituent in ortho-position to the carboxy group, in the benzene ring.

Especially of interest in above combination is the o-hydroxy benzoic acid, the salicylic and/or the non-substituted benzoic acid.

Halogen is bromine, iodine or fluorine, especially chlorine. Lower alkoxy is ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy or tertbutoxy, especially methoxy.

The mixture results in unexpected synergistic antimicrobial activity of the constituents.

The invention relates especially to pharmaceutical preparations for external use, which contain the combination of a compound of formula I, wherein at least one of RI, RZ and R3 is chlorine and the rest are hydrogen atoms as well as pharmaceutically acceptable acid addition salts thereof, such as miconazole (R,=R2Cl, R3=H), econazol (R2=Cl, Rl=R3=H) and isoconazole (R,=R3Cl, R2=H) or a compound of the formula II wherein R4 is chlorine, such as clotrimazole with one or more optionally substituted benzoic acids.

The invention preferably relates to especially effective preparations for external use with regard to its synergistic antimicrobial activity which contain the combination of miconazole selected from the group of alkyl substituted imidazole of formula I, in which R, and R2 are chlorine and R3 is hydrogen as well as pharmaceutically acceptable acid addition salts thereof with salicylic acid and/or benzoic acid.

Most preferably the invention relates to very effective preparations for external use which contain the combination of miconazole nitrate with salicylic acid and/or benzoic acid.

Of particular use for producing acid addition salts of compounds of formula I are those acids which are suitable for the formation of pharmaceutically acceptable salts. The following may be mentioned as examples of suitable acids: hydrohalic acids, sulfuric acids, phosphoric acids, nitric acid, perchloric acid, aliphatic, alicyclic, aromatic or heterocyclic carboxylic or sulfonic acids, such as formic acid, acetic acid, propionic acid, succinic acid, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic. acid, or pyruvic acid; phenylacetic acid, benzoic acid, paminobenzoic acid, anthranilic acid, p-hydroxybenzoic acid, salicyclic acid or p-aminosalicYclic acid, embonic acid, methanesulfonic acid, ethanesulfonic acid, hydroxyethane- I sulfonic acid or ethylenesulfonic acid; halogenobenzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid or sulfanilic acid; and methionine, trypthophane, lysine or arginine.

Pharmaceutical preparations according to the invention exhibit antimicrobial activity, especially antifungal and antibacterial activity. Thus they are active against microorganisms causing or complicating dermal lesions, and are especially active against dermatophytes, such as Trichophyton, e.g. Trichaphyton mentagrophytes, Epidermaphyton, e.g. Epidermaphyton floccosum, Microsporon, e.g. Microsporon felineum, Aspergillus species, e.g. Aspergillus niger and Aspergillus funigatus, yeasts, such as Pytyrosporum species, Candida species, e.g. Candida albicans and grampositive bacteria, such as Staphylococcal species, e.g. Staphylococcus aureus, Pseudomonas aeruginosa, Propioni bacterium and Coryne bacterium acnes.

Pharmaceutical preparations according to the invention can be used as an antifungal agent for the treatment of fungal infections of the skin, e.g. dermatomycoses and systemmycoses and as an antibacterial agent, for the treatment of acne and dandruff and other related skin ailments. Moreover, the combinations mentioned above have also antipruritic activity and can be used for the treatment of prmritis.

The pharmaceutical preparations of this invention for topical application contain as active ingredient compounds of formulae I or II in combination with one or more optionally substituted benzoic acids together with a pharmaceutically acceptable carrier or excipient. The daily dosage of the active ingredients depends on the age and individual condition of the patient and also on the mode of application.

The preferred concentrations of the combined active ingredients in the formulated preparation are 0.01 - 10 7. by weight of the lower alkyl substituted imidazoles of formula I or II, preferably 0.1 - 2 % and 0.5 25 % of the one or more optionally substituted benzoic acids as sum, preferably 1 - 15 % by weight.

Suitable pharmaceutical preparations for topical applications are primarily creams, ointments and gels, as well as powders, pastes, solutions, tinctures, foams, sprays, aerosols, collodions, lotions and salves.

Creams or lotions are oil-in-water emulsions which contain more than 50 % of water. Fatty alcohols are chiefly used as oleaginous base, for example lauryl, cetyl or stearyl alchohol, fatty acids, for example palmitic or stearic acid, liquid to solid waxes, for example isopropyl myristate, wool wax or bees-wax, and/or hydrocarbons, for example petroleum jelly (petrolatum) or paraffin oil. Suitable emulsifiers are surface-active substances with primarily hydrophilic properties, such as corresponding non-ionic emulsifiers, for example fatty acid esters of polyalcohols or ethylene oxide adducts thereof, such as polyglycerol fatty acid esters or polyoxyethylene sorbitan fatty acid esters (Tweens); polyoxyethylene fatty alcohol ethers or ester; or corresponding ionic emulsifiers, such as alkali metal salts of fatty alcohol sulfates, for example sodium lauryl sulfate, sodium cetyl sulfate or sodium stearyl sulfate, which are customarily used in the presence of fatty alcohols, for example cetyl alcohol or stearyl alcohol. Additives to the aqueous phase include agents which reduce water loss through evaporation, for example polyalcohols, such as glycerol, sorbitol, propylene glycol and/or polyethylene glycols, as well as preservatives, perfumes etc.

Ointments or lotions are water-in-oil emulsions which contain up to 70 %, preferably however about 20 % to 50 %, of water or aqueous phase. The oleaginous phase comprises mainly hydrocarbons, for example petroleum jelly, paraffin oil and/or hard paraffins, which preferably contain hydroxy compounds suitable for improving the water-adsorption, such as fatty alcohols or esters thereof, for example cetyl alcohol or wool wax alcohols, or wool wax. Emulsifiers are corresponding lipophilic substances, such as sorbitan fatty acid esters (Spans), for example sorbitan oleate and/or sorbitan isostearate. Additives to the aqueous phase include humectants, such as polyalcohols, for example glycerol, propylene glycol, sorbitol and/or polyethylene glycol, and preservatives, perfumes etc.

Microemulsions are isotropic systems based on the following four components: water, an emulsifier such as a surfactant, e.g. Eumulginw, a lipid such as a non-polar oil, e.g. paraffin oil, and an alcohol containing a lipophilic group, e.g. 2-octyldodecanol. If desired, other ingredients can be added to the microemulsions.

Greasy ointments are anhydrous and contain as base in particular hydrocarbons, for example paraffin, petroleum jelly and/or liquid paraffins, and also natural or partially synthetic fat, for example coconut fatty acid triglycerides, or preferably hardened oils, for example hydrated ground nut or castor oil, and also fatty acid partial e9ters of glycerol, for example glycerol mono- and distearate, and, for example, the fatty alcohols, emulsifiers and/or additives for increasing the water-adsorption mentioned in connection with the ointments.

In the case of gels a distinction is made between aqueous gels, anhydrous gels, and gels having a low water content, and which consists of swellable gel-forming materials. Primarily transparent hydrogels based on inorganic or organic macromolecules are used. High molecular inorganic components with gel-forming properties are chiefly water-containing silicates such as aluminium silicates, e.g. bentonite, magnesium aluminium silicate, e.g. veegum, or colloidal silica, e.g. aerosil. High molecular organic substances are used e.g. natural, semi-synthetic or synthetic macromolecules. Natural and semi-synthetic polymers are derived e.g. from polysaccharides with carbohydrate components of the most widely different kind, such as celluloses, starches, tragacanth, gum arabic, agar-agar, gelatin, alginic acid and salts thereof, e.g. sodium alginate, and their derivatives such as lower alkylcelluloses, e.g. methyl or ethyl cellulose, carboxy- or hydroxy-lower alkyl cellulose, e.g. carboxymethyl cellulose or hydroxyethyl cellulose. The components of synthetic gel- formng macromolecules are e.g. correspondingly substituted unsaturated aliphatics such as vinyl alcohol, vinyl pyrrolidine, acrylic or methacrylic acid. Examples of such polymers are polyvinyl alcohol derivatives such as polyviol, polyvinyl pyrrolidines such as collidone, polyacrylates and polymethacrylates such as Rohagit SO or EudispertO. Conventional additives such as preservatives or perfumes can be added to the gels.

Pastes are creams and ointments containing powdered ingredients which absorb secretions, such as metal oxides, for example titanium oxide or zinc oxide, and talc and/or aluminium silicates whose purpose it is to bind moisture or secretion present.

Foams are administered from pressurised dispensers and are liquid oil-inwater emulsions in aerosol form, with halogenated hydrocarbons, such as chlorofluoro-lower alkanes, for example dichlorodifluoromethane and dichlorotetrafluoroetbane, being used as propellants. For the oleaginous phase there are used, inter alia, hydrocarbons, for example paraffin oil, fatty alcohols, for example cetyl alcohol, fatty acid esters, for example isopropyl myristate, and/or other waxes. As emulsifiers there are used, inter alia, mixtures of those emulsifiers with primarily hydrophilic properties, such as polyoxyethylene sorbitan fatty acid esters (Tweens), and those with primarily lipophilic properties, such as sorbitan fatty acid esters (Spans). In addition, the conventional additives are used, such as preservatives etc.

1 Tinctures and solutions generally have an aqueous ethanolic base to which are added, inter alia, polyalcohols, for example glycerol, I glycols, and/or polyethylene glycol, as humectants for reducing water loss, and fat-restorative substances, e.g. fatty acid esters with lower polyethylene glycols, i.e. lipophilic substances which are soluble in the aqueous mixture as substitute for fatty substances which are removed from the skin by the ethanol, and, if necessary, other assistants and additives.

The pharmaceutical preparations for topical application are obtained in known manner, for example by dissolving or suspending the active ingredient in the base or in a part thereof, if necessary. When processing the active ingredient in the form of a solution, it is usually dissolved in one of the two phases before the emulsification, and when processing the active ingredient in the form of a suspension, it is mixed with a part of the base before the emulsification and then added to the remainder of the formulation.

The superiority in performance of the formulated product consisting, e.g. of a combination of miconazole, salicylic acid and/or benzoic acid which is part of the subject of this invention is based on synergistic antimicrobial activity of the ingredients.

Claims

Evidence for this claim is provided below. Synergy When several active components are added to a formulation their effects can be inhibitory, additive or synergistic. A synergistic effect is clearly most desirable as it enables use of lower dosages and improvement of activity over and above the individual drug effects. With the antimicrobial, especially antifungal compounds or combinations thereof studied here, the ability of these compounds to inhibit fungal growth was studied by measuring minimum inhibitory concentrations and rate of kill by zone of inhibition measurements. If the term Fractional Inhibitory Concentration (FIC) is defined as Minimum inhibitory concentration (MIC) of the active components in the combination product (i.e. test is done with two or three active components in solution at the same time) FIC = Minimum inhibitory concentration of the active component on its own (i.e. test is done with only one active component in solution) then EFIC, the sum of FIC for each component equals unity when there is no interaction between the components. On the other hand, if there is synergy, the sum will be less than unity and the magnitude of EFIC will give an indication of the strength of interaction. Minimum Inhibitory Concentrations The minimum inhibitory concentration (MIC) in pg/ml for miconazole (miconazole nitrate used throughout), salicylic acid and benzoic acid alone and in combination are shown in Table 1. The detailed data gathered, results from methods described below. Table 1 Organism MIC (jig/ml) miconazole S. nitrate Fractional sum Inhibitory Conc FIC (FIC) B. 4 - s - - 1365 680 0.25 200 0.5 - 200 0.25 100 100 0.0625 + 0.146 0.2 0.125 + 0.294 0.389 0.063+0.07+0.15 0.28 Table 1 (continued) Organism T. mentagrophytes MIC (Vglml) miconazole S. nitrate 1 4 0.5 0.06 - 0.06 0.25 B. 670 310 Fractional sum Inhibitory Conc FIC (FIC) - 0.5 + 0.3 0.06 + 0.65 - 0.8 = 0. 71 0.06+0.04+0.65 = 0.75 0.25+0.3+0.16 - 0.71 S. = salicylic acid B. = benzoic acid The MIC values of individual compounds is as expected, with miconazole showing good activity, while much higher concentrations of benzoic acid and salicylic acid, approaching the limit of their aqueous solubility are required to produce a MIC. However, subcultures of wells showing no growth, indicated that concentrations of benzoic and salicylic acids at or near MIC values were also cidal, whereas the effects of miconazole were mainly static, with regrowth occurring on subculture. MICs of combinations of miconazole with salicylic acid or benzoic acid or both indicate more than just an additive effect of antimicrobial activity, and against C. albicans the combined action was much better than simply additive as indicated by the fractional inhibitory concentrations (FICs). It was also observed that concentrations well below MIC values were having a significant effect on growth. This observation that all components act together to contribute to an antimicrobial effect is particularly important in the formulated product, where diffusion of individual components is limited. The methodology of the assessment of in vitro activity of miconazole, salicylic acid and benzoic acid against Candida albicans, Trichophtyton mentagrophytes and Staphylococcus aureus is described below. Minimum Inhibitory.Concentration 1. Inoculum. C. albicans N.C.Y.C. 610, T. mentagrophytes N.C.P.F. 80 and S. aureus N.C.T.C. 8532 were used. C. albicans was grown overnight in SLM (Sabouraud Liquid Medium) at 32' and the inoculum diluted to give approx. 2 x 105, based on a total count. T. mentagrophytes microconidia were harvested from a 12 day culture grown on SDA at 32', a washed aqueous suspension being stored frozen until required, when an appropriate dilution was made in SLM based on a total count, to give 2 x 105 spores/ml. An overnight culture (37') of S. aureus in Isosensitest Broth (ISB) was diluted 1 in 500 with ISB. The viable count of all inocula was determined. 2. Solutions Doubling dilutions of miconazole, salicylic acid and/or benzoic acid were prepared in broth. At the higher concentrations of salicylic acid and benzoic acid, the concentration of drug in solution was checked by preparing a saturated solution, which was filtered through a 0.45 jim membrane filter. The filtrate, which was used to prepare doubling dilutions for MIC testing, was analyzed by HPLC (high pressure liquid chromatography). DMF (0.5 %) was used as solvent for miconazole and was present at this concentration in all media. - l') - 3. In-vitro testing procedure MICs of individual drugs were determined by inoculating 100 ul volumes into the wells of a microtitre plate, an equal volume of inoculum was added and the plates incubated at 32' for both fungi and 37' for S. aureus. For MICs the combinations of two compounds, compound solutions were prepared at 3 times the required concentrations, and 75 jil of each compound and inoculum were added to the wells of a microtitre plate and incubated as described. A three component system was also tested using 50 jil volumes to inoculate plates. Results Results were read after 24h incubation for C. albicans and S. aureus and after 6 - 7 days for T. mentagrophytes. End points were determined visually and by measuring "Optical Density" using a vertical scanning photometer at 450 nm. Optical density readings in particular made it possible to study the effects of sub MIC concentrations on growth. The antimicrobial activity of the cream formulations was also studied by the estimation of zones of inhibition by the following procedure: Measurement of Zones of Inhibition (spread plate) Approximately 1 x 104 c. f.u.lml of each microorganism was inoculated onto the surface of agar plates (S.D.A for the yeast and fungi and T.S.A. for bacteria) and left to dry for 30 min. Wells were cut into the agar with a number 3 cork bover and filled with approximately 100 mg of cream. Plates were incubated overnight (longer for fungi) at 32'C and the diameters of the zones of inhibition were measured (Table 2). Organisms employed: Candida albicans, Trichophyton mentagrophytes, Staphylococcus aureus, Pseudomonas aeruginosa. A comparison of all agar diffusion experiments (Table 2) indicates that MSB (Miconazole + salicylic acid + Benzoic acid) is significantly more active than the others tested, namely M (miconazole) or SB (Salicylic acid + Benzoic acid). Composite results of Cream Formulations XSB = cream containing miconazole salicylic and benzoic acids SB = cream containing salicylic and benzoic acids D = DaktarinO (2 % miconazole) (cream prepared by Janssen Pharmaceuticals, Belgium) X = miconazole 2 % Table 2 Organism cream formulation Zone of inhibition (Diameter, mm) MSB SB m D C.albicans (NCYC610) (1) 27 17.7 9.2 17.2 (2) 33.7 24.7 16.7 39.8 (3) 29.3 11.3 24.0 T. mentagrophytes 55 38 30.5 43 NCOF80, pregerminated T. mentagrophytes (1) 54.3 52.5 37.8 43 NCM0, microconidia (2) 49.0 55.3 45.3 40.8 (3) > 80 43.5 54 T. mentagrophytes ATCC 18748 52.3 50.3 41.8 37 (Spread Plate results) Table 2 (continued) Organism cream formulation Zone of inhibition (Diameter, mm) MSB SB m D T. mentagrophytes 56 54.6 28 C. albicans 24 20 15.6 T. mentagrophytes 44.6 34.9 30.9 MSB v D and M significant at 0.05 % level (abbreviations see above) MSB v SB significant at 2.5 % level = not taken Diffusion Studies Most pharmaceutical systems consist of the active ingredient plus the excipients and it is therefore important to validate the activity of the compound in the intended formulations. With topical applications diffusion of the compound out of the formulations are required and a plate diffusion method was therefore used to evaluate the performance of the products. The results are shown in Table 3. Formulations I to 7 are all of the same base composition but the ingredient compositions are different. The codes should be self explanatory with M2S3 standing for 2 % Miconazole and 3 7. Salicyclic acid. DJ stands for DaktarinO Janssen and AJ stands for AcnidazilO Janssen. Examination of the data demonstrates that on average for the range 1 of microorganisms studied the miconazole, benzoic acid and salicylic acid formulation performed better than the other formulations. Note 1 1 that in the comparisons all formulations were tested at the same time because tests repeated on different days yield different results but relative rankings remain essentially the same. Antimicrobial activity of cream formulations against a range of microorganisms Table 3 Organis Zones of Inhibition (Diameter mm= of cream formulation as given on pages 16 - 20 1. 2. 3. 4. 5. 6. 7. 8. 9. M2S3 M2 S3B6 S3 (C. albicans) 15.3 9.2 17.7 0 (N.C.Y.C.610) T. mentagrophytes (N.C.P.F.80) 34 Pregerminated T. menta- B6 M2B6 M2S3B6 W AJ 11.2 23 27 17.2 30.5 38 27.3 37.3 47.5 55 43 grophytes (N.C.P.F.80) 31.5 37.8 52.5 22.5 42 Microconidia 51.3 54.3 43 S. aureus 49.3 24.5 42.8 43.2 34.2 36 48.9 30.220.3 (N.C.T.C.6749 S.D.A.plates) Organisms 1. 2. 3. 4. 5. 6. 7. 8. 9. Ps.aerugi nosa 11.8 0 21.3 14.3 14.8 16.7 20.8 12.7 0 (N.C.T.C.10332 S.D.A. plates) C. albicans 33.3 16.7 24.7 11.2 21.5 33.3 33.7 39.8 20.3 (N.C.Y.C.610) T. mentagrophytes (A.T.C.C.18748) 50.5 41.8 50.3 28.3 47.8 54.2 52.3 37 32 T. menta grophytes 61.8 45.3 55.3 36.8 45.8 60.3 49.0 40.8 33 (N.C.P.F.80) = not taken M-= miconazole (%), B = benzoic acid (%) S = salicylic acid (%), DJ = DaktarinO (Jansen) AJ = AcnidazilO (Jansen) Cream Formulations tested (prepared as described in Examples 4 - 6) Cream 1 Composition White Soft Paraffin Propylene Glycol Cetostearyl Alcohol Cetrimide Methyl Paraben Propyl Paraben Water Miconazole Nitrate Salicylic Acid Benzoic Acid 23.75 11.4 23.75 0.95 0.025 0.015 35.15 2.0 3.0 0.0 Cream 2 Composition White Soft Paraffin Propylene Glycol Cetostearyl Alcohol Cetrimide Methyl Paraben Propyl Paraben Water Miconazole Nitrate Salicylic Acid Benzoic Acid 24.5 11.75 24.5 0.98 0.025 0.015 36.25 2.0 0 0 Cream 3 coPPSI White Soft Paraffin Propylene Glycol Cetostearyl Alcohol Cetrimide Methyl Paraben Propyl Paraben Water Miconazole Nitrate Salicylic Acid Benzoic Acid Composition White Soft Paraffin Propylene Glycol Cetostearyl Alcohol Cetrimide Methyl Paraben Propyl Paraben Water Miconazole Nitrate Salicylic Acid Benzoic Acid Cream 4 22.75 10.92 22.75 0.91 0.025 0.015 33.67 0.0 3.0 6.0 24.25 11.64 24.25 0.97 0.025 0.015 35.89 0.0 3.0 0.0 Cream 5 Composition White Soft Paraffin Propylene Glycol Cetostearyl Alcohol Cetrimide Methyl Paraben Propyl Paraben Water Miconazole Nitrate Salicylic Acid Benzoic Acid Composition White Soft Paraffin Propylene Glycol Cetostearyl Alcohol Cetrimide Methyl Paraben Propyl Paraben Water Miconazole Nitrate Salicylic Acid Benzoic Acid Cream 6 23.5 11.28 23.5 0.94 0.025 0.015 34.78 0.0 0.0 6.0 23.0 11.04 23.0 0.92 0.025 0.015 34.04 2.0 0.0 6.0 Cream 7 Composition White Soft Paraffin Propylene Glycol Getostearyl Alcohol Cetrimide Methyl Paraben Propyl Paraben Water Miconazole Nitrate Salicylic Acid Benzoic Acid Cream 8 Daktarins (Janssen) 2 % w/w Miconazole Nitrate 22.25 10.68 22.25 0.89 0.025 0.015 32.93 2.0 1 3.0 6.0 Cream 9 AcnidazilO (Janssen) 2 7. w/w Miconazole Nitrate and 5 % w/w Benzoyl Peroxide Fungicidal activity of miconazole with salicylic and/or benzoic acids Subcultures from MIC tests to determine the MBC (Minimum bactericidal concentration) suggested that benzoic acid and salicylic acid exhibited fungicidal activity at concentrations only slightly higher than MICs. The cidal action of active constituents was tested against microconidia using a rate of kill procedure. Concentrations of k 20 jiglml of miconazole were fungicidal within 24 h while benzoic and salicylic acids were fungicidal at 400 - 600 jiglml. 1 1 changed to 0.1 % of miconazole without loss of efficacy 1 1 Combinations of lower concentrations over a 6 hr period of study indicated that salicylic acid enhanced ftngicidal activity of both 20 and 10 ygIml miconazole. Combinations of benzoic and salicylic acids with miconazole gave improved fungicidal activity. Activity against spores may be sporostatic or sporicidal. The evidence for this can be gained by examining % germination in the presence of active ingredients compared with colony forming ability once the active agents are removed (Table 4). Miconazole alone (10 jiglml) and salicylic acid (200 1LgIml) or combinations of these were sporostatic, since germination was inhibited but spores were still viable. Effect of salicylic acid and miconazole on viability and % germination of T. mentagrophytes microconidia Table 4 Combination Viability (cfu/ml) % Germination at time (h) at time (h) 6 24 6 24 control 3.5 x 105 2.4 x 105 12 65 - M10 5.9 X 105 7.0 x 104 9 11 5.88 M101S 200 8.3 x 10' 2.3 x W1 6 16 5.41 M101S 400 0 0 4 13 5.06 M20 3.3 x 101 4.6 x 105 7 5 5.84 M201S 200 2.2 X 104 5.3 x 103 8 13 5.41 M201S 400 0 0 9 12 5.07 S 200 4.8 x 105 1.2 x 105 12 41 5.43 S 400 0 0 14 14 5.08 M = miconazole S = salicylic acid Initial count at T was 7.9 X 105CfUlMJ. Results read on day 0 1 Different concentrations of miconazole in the cream formulation were tested against T. mentagrophytes (Table 5). The formulations con taining MSB were all significantly better than SB. The 2 % MSB cream was significantly better than all the others tested, while concentrations of miconazole in the range 0.1 - 1 % were not significantly different from each other but were significantly better than 5B and proprietary products containing 2 % miconazole (DaktarinO). Effect of concentration of miconazole in cream formulation containing salicylic (3 %) and benzoic (6 %) acids against T. mentagrophytes Table 5 Cream Zones of Inhibition Diameter (mm) 1 2 3 4 5 MeanS.D. 2 1. Miconazole 2 % 46 44 45.5 45 41.5 44.6 1.39 2. Miconazole 1 % 40 40.5 43 44.5 43.44 42.2 1.89 3. Miconazole 0.5 % 40.5 40.5 40.5 44 42 41.8 1.44 4. Miconazole 0.25 42.5 40.5 40 42 39 40.8 1.44 5. Miconazole 0.11% 42.5 40.5 40.5 40 42.5 41.2 1.20 t 6. Miconazole 0 % 34 36 35.5 35 34 34.9 0.89 7. Miconazole 2 % 33.5 30 31 29 NT 30.91.93 (Daktarin) T. mentagrophytes challenge was 7.5 x W1 Significance (0.05%) Multiple Range Test (Neuman Keuls) for significance (0.05) 1 vs 2,3,4,5,6,7 2 vs 3,4,5 2 vs 6, 7 significant difference no significant difference significant difference 3 vs 4, 5 3 vs 6, 7 4 vs 5 4 vs 6,7 vs 6,7 6 vs 7 no significant difference significant difference no significant difference significant difference significant difference significant difference Table 5 shows that the synergistic effect is maintained over a very wide range of miconazole concentration. 2. Further evidence for the synergy is provided by the kinetic data shown in Table 6 Rate of kill of T. mentagrophyte microconidia by miconazole (M) alone and in combination with benzoic acid (B) and salicylic acid (S) Table 6 Time M20 M20+S 200 M20+B 200 M10 M10+S 200 M10+B 200 (hrs) 1 1X105 1.1x105 6.7x10I 2.0x105 1.3x105 1.2x105 2 3.3x104 1.2x104 2.3x104 9.2xl 04 5.8x104 4.2x104 4 2.2x104 1.8X103 1.9x10,1 1.6x105 1.5x104 2.5x1T 6 1.4x10I 5.0x102 1.2x104 7.5x10' 8.3xl 03 2.6x104 24 8.3x102 0 4.2xl 02 2.3x10I 3.3x102 1.7x1T pH before 5.98 5.57 5.50 after 5.95 5.54 5.50 5.94 5.54 5.49 5.93 5.53 5.48 S = salicylic acid B = benzoic acid Initial count at T 5.9 X 105 CfUlM1 All solutions made up in SUM (SLM + 0.5 % DMF) the previous evening to allow for complete dissolution. 0.1 ml Trichophyton mentagrophytes microconidia added to 9.9 ml of sample (assuming T. ment. at 1x108 efulml). Viable count performed on spore solution to check count. All 0.1 ml volumes measured with a 100 ul pipetman. Samples kept at 32C throughout the experiment. 1 ml removed at 1, 2, 4, 6, 24 hours, and viable count performed. Serial dilutions in 0.1 % peptone water and plated out using the method of Miles and Migra onto SDA. Plates read after 6 and 14 days. Sample 1 2 3 4 5 6 Concentration M20 jiglml M20 + S 200 iicrlml 0 M20 + B 200 jiglml M10 vg/M1 M10 + S 200 gglml M10 + B 200 jiglml S = salicylic acid B = benzoic acid. MIC also performed at the same time (in SLM) on miconazole. Miconazole concentrations: - 4, 2, 1, 0.5, 0.25, 0.125 gglml (duplicates) 1 h 0.1 ml T. ment. micronidia added to give a final concentration of approximately 1 x 105 cfulml (same spore batch as above). The invention will be illustrated by the following examples although it is not limited thereby. Example 1: A powder containing 2 % of miconazole nitrate, 2 % of salicylic acid and 3 % of benzoic acid is prepared as follows: Composition Miconazole nitrat Salicylic Acid Benzoic Acid Amorphous silica Talc purified and micronised 2 % 2 % 3 % 0.1 % % Miconazole nitrate, salicylic acid and benzoic acid are micronised in the presence of the amorphous silica. The microfined mixture is then added in small amounts to the sterilised micronised and purified talc. Mixing is carried out until a homogeneous product is formed. Example 2: An aerosol formulation containing 3 % of miconazole nitrate, 5 % of salicylic acid and 6 % of benzoic acid is prepared as follows: Composition Miconazole nitrate Salicylic Acid Benzoic Acid Tween 80 Amorphous Silica Deodorised hydrocarbon gs. 6 % 0.1 % 0.1 % - 26 A microfined mixture of miconazole nitrate, salicylic acid, benzoic acid and amorphous silica is prepared as for the powder formulation. Tween 80 is then sprayed on as a weak alcoholic solution at intervals to allow even deposition and evaporation of carrier. The tween 80 coated powder is then suspended in the deodorise d hydrocarbon mixture and packaged using standard techniques. The blend of hydrocarbon used is chosen as to be non flammable with suitable additives. 1 Example 3: A lotion formulation containing 1 % of miconazole nitrate, 3 % of salicylic acid and 6 % of benzoic acid is prepared as follows: Composition Miconazole nitrate Salicylic Acid Benzoic Acid Butylhydroxvtoluene Diethyltoluamide Polyethylene glycol 400 1 % 3 % 6 % 2 % 2 % to 100 % The miconazole nitrate, salicylic acid and benzoic acid are dissolved in half of the polyethylene glycol 400 and the butylhydroxytoluene and diethyltoluamide added until a clear solution is obtained. The remaining polyethylene glycol 400 is then added to the final weight. h Example 4: A cream formulation containing 2 % of miconazole nitrate, 3 % of salicylic acid and 6 % of benzoic acid is prepared as follows: Composition White Soft Paraffin Propylene Glycol Cetostearyl Alcohol Cetrimide Methyl Paraben Propyl Paraben Water Miconazole Nitrate Salicylic Acid Benzoic Acid 22.25 % 10.68 % 22.25 % 0.89 % 0.025 % 0.015 % 32.93 % 2.0 % 3.0 % 6.0 % A melt or a dispersion technique may be used. The white soft paraffin, propylene glycol and cetostearyl alcohol are mixed and heated until a uniform viscous fluid is obtained. Miconazole nitrate, salicyclid acid and benzoic acid are dissolved in the liquid together with the parabens. The cetrimide is dissolved in some of the water heated to 6CC and added with rapid stirring to the oil phase. A homogeneous cream is obtained. The remaining water is added and the mixture further homogenised. Example 5: A cream formulation containing 2 % of miconazole nitrate and 3 % of salicylic acid is prepared as described in Example 4 with following Composition White Soft Paraffin Propylene Glycol Cetostearyl Alcohol Cetrimide Methyl Paraben Propyl Paraben Water Miconazole Nitrate Salicylic Acid 23.75 11.4 23.75 0.95 0.025 0.015 35.15 2.0 3.0 Example 6: A cream formulation containing 2 % of miconazole nitrate and 6 % of benzoic acid is prepared as described in Example 4 with following Composition White Soft Paraffin Propylene Glycol Cetostearyl Alcohol Cetrimide Methyl Paraben Propyl Paraben Water Miconazole Nitrate Salicylic Acid Benzoic Acid 23.0 11.04 23.0 0.92 0.025 0.015 34.04 2.0 0.0 6.0 What is claimed is:

1. An antimicrobiallY active pharmaceutical preparation for external use comprising the combination of a compound selected from the group of compounds having in their molecule a lower alkyl substituted imidazole ring, being a compound of the general formula I 11- 0 0 Ri\ YH2-YH-O-CH2-.// -R2 cl 0=.

0 \ 0 / R 3 / 1 11 0 \\. / a L (I) wherein at least one of R,, R2 and R3 is halogen and the rest thereof are hydrogen atoms, as well as pharmaceutically acceptable acid addition salts thereof or a compound of the formula II /A4 (C6Hs)2 wherein R4 is halogen, with one or more optionally substituted benzoic acids together with conventional carriers and/or excipients for external use.

2. An antimicrobially active pharmaceutical preparation for external use according to claim 1, comprising the combination of a compound of formula I, wherein at least one of R1, R2 and R3 is chlorine and the rest are hydrogen atoms as well as pharmaceutically acceptable acid addition salts thereof, such as miconazole (Rj=R2=Cl, R3H), econazol (R2=Cl, RI=R3=H) and isoconazole (R,=R3=Cl, R2=H) or a compound of the formula II wherein R4 is chlorine, such as cl;trim- azole with one or more optionally substituted benzoic acids, together with conventional carriers and/or excipients for external use.

3. An antimicrobially acti-e pharmaceutical preparation for external use according to claim 1, comprising the combination of miconazole selected from the group of alkyl substituted imidazole of formula I, in which R, and R2 are chlorine and R3 is hydrogen as well as pharmaceutically acceptable acid addition salts thereof with salicylic acid and/or benzoic acid, together with conventional carriers and/or excipients for external use.

4. An antimicrobially active pharmaceutical preparation for external use according to claim 1, comprising the combination of miconazole nitrate with salicyclic acid and/or benzoic acid together with conventional carriers and/or excipients for external use.

5. An antimicrobially active pharmaceutical preparation for external use according to claims I to 4, comprising 0,01 - 10 % of the lower alkyl substituted imidazoles of formula I or II by weight and 0,5 - 25 % of the one or more optionally substituted benzoic acids as sum by weight, together with conventional carriers and/or excipients for external use.

6. An antimicrobially active pharmaceutical preparation for external use according to claims I to 4, comprising 0,1 - 2 % of the lower alkyl substituted imidazoles of formula I or II by weight and 1 - 15 % of the one or more optionally substituted benzoic acids as s um by weight, together with conventional carriers and/or excipients for external use.

7. An antimicrobially active pharmaceutical preparation for external use according to claims 1 to 6, comprising a synergistically effective antimicrobially active amount of the lower alkyl substituted 1 X t 1 imidazoles of formula I or II and one or more optionally substituted benzoic acids, together with conventional carriers and/or excipients for external use.

8. An antimicrobially active pharmaceutical preparation for external use according to any of claims 1 to 7, which is in a gel, cream, ointment, paste, powder, solution, tincture, foam, spray, aerosol. collodion or lotion form.

9. A process for the production of an antimicrobially active pharmaceutical preparation for external use, which process comprises mixing a compound selected from the group of compounds having in their molecule a lower alkyl substituted imidazole ring, being a compound of the formula I 11- 0 0 Y 2-YH-O-CH2-c/ -R2 /,\ / cl 0 R3 1 11 6 ,\, 0 / i R1 (I) wherein at least one of R1, R2 and R3 is halogen and the rest. thereof are hydrogen atoms, as well as pharmaceutically acceptable acid addition salts thereof or a compound of the formula II /R4 0-, \\1. - (C6H5)2 (II) 11 -A wherein R4 is halogen, with one or more optionally substituted benzoic acids, together with conventional carriers and/or excipients for external use.

10. Use of the antimicrobially active preparation according to any of claims 1 to 8 as antifungal agent for the treatment of fungal infections of the skin, as antibacterial agent for the treatment of acne, dandruff and other related skin ailments and as antipruritic agent for the treatment of pruritis.

FO 7.4 HFOINVIcs1w Published 1988 w. Tte Patent Office. Statte Hv.:sc. 667, 1 'H;Cborr-. London IXiR 4TF Purthcreopies inky be obminedfrcm The Patent Office.

sales Branch, St Mary Cray, Orpington, Kent BR5 3RD. Printed ky Multiplex techniques ltd, St Mary Cray. Kent. Con. 1/87