AU2062495A - Topical preparation - Google Patents

Description

TOPICAL PREPARATION Technical Field

This invention relates to a topical formulation which comprises Bacitracin and Polymyxin in combination with an antiseptic. The combination of the three actives is suitable for the treatment or prophylaxis of infection in minor cuts, wounds and scrapes. The topical application of the combination also facilities healing of minor cuts, wounds and scrapes. Background Art

Bacitracin is a commercially available antibiotic and is a polypeptide complex produced by certain strains of Bacillus licheniformis and by B. subtilis var. Tracy. Commercial Bacitracin is a mixture of at least nine Bacitracins. On hydrolysis it yields the a ino acids L- cysteine, D-glutamic acid, L-histidine, L-isoleucine, L- leucine, L-lysine, D-ornithine, D-phenylalanine and DL- aspartic acid. Other reported forms of Bacitracin include Bacitracin methylenedisalicylic acid and its sodium salt and zinc Bacitracin.

Polymyxin is a commercially available antibiotic complex produced by the growth of Bacillus polymyxa (Prazmowski) igula, or a mixture of two or more such salts. To date, Polymyxins A, B, C, D, E, F, K, M, P, S and T have been reported. Polymyxin B is a mixture of Polymyxins B.^ and B₂. Available derivatives of Polymyxin B include the sulphate salt, ie Polymyxin B sulphate, Polymyxin B-methanesulfonic acid and its sodium salt.

The antiseptic suitable for use in the present combination include Cetrimide and Chlorhexidine both of which are commercially available. Cetrimide consists of tri ethyltetradecyl ammonium bromide and may contain smaller amounts of dodecyl- and hexadecyltrimethyl ammonium bromides. It contains not less than 96.0% and not more than 101.0% of alkyltrimethylammonium bromides, calculated as C₁₇H₃₈BrN (336.4) with reference to the dried substance. Chlorhexidine is N,N' -bis (4- chlorophenyl) -3, 12-diimino-2,4,11,13- etraazatetradecane- diimidamide. Available derivatives include the dihydrochloride salt and the diacetate and the digluconate.

The combination of Polymyxin and Bacitracin has previously been reported but no synergistic effect has previously been reported. The present inventors have found that the combination of Bacitracin, Polymyxin with an antiseptic has a synergistic effect on the treatment of wound infections. Disclosure of Invention

In one aspect, the present invention provides a pharmaceutical formulation comprising Bacitracin or a pharmaceutically acceptable derivative thereof, Polymyxin or a pharmaceutically acceptable derivative thereof and an antiseptic.

Preferably, the combination of the three actives is present in a pharmaceutically acceptable carrier.

The antiseptic is selected from Cetrimide and Chlorhexidine. Preferably, the Polymyxin is Polymyxin B sulphate.

Preferably, the combination of the three actives is presented is a gel, typically in a hydrogel for topical application. Other suitable forms of the formulation include ointment, cream, liquid, impregnated dressings or the like.

More preferably, the carrier is hydroxypropyl methylcellulose and water.

In another aspect, the present invention provides a method of treatment or prophylaxis in a mammal of wound infection which comprises topical application to the wound a pharmaceutical formulation comprising Bacitracin, Polymyxin and an antiseptic.

In a further aspect, the present invention provides the use of Bacitracin and Polymyxin in combination with an antiseptic in the manufacture of a medicament for the treatment or prophylaxis of wound infection.

The expression "wound" includes minor cuts and scrapes. Modes for Carrying Out the Invention

All of the constituents of the combination according to the present invention are commercially available or can be readily prepared according to literature procedure.

The combination of the active compounds of the present invention are applied in the form of an appropriate composition, in particular, compositions usually employed for topically administering drugs. These compositions take a wide variety of forms such as for example, solid forms such as powders, liquid forms such as solutions or suspensions in aqueous or oily mediums; semi-liquid formulations, such as creams, gels, pastes, ointments, salves. The compositions ideally contain the active compounds in a wound-acceptable carrier. If desired, further ingredients may be incorporated into the composition. For example, anti- inflammatory agents, disinfectants, antibiotics, etc. Furthermore, wound covers such as plasters, bandages, dressings, gauze pads, and the like containing the combination according to the present invention may also be used. In particular, plasters, bandages, dressings, gauze pads and the like which have been impregnated or sprinkled with the liquid formulation containing the combination of the present invention can be used. Preferred composition of the present invention is a gel.

The combination of the present invention is applicable to either humans or animals.

An advantage of the present invention resides in the fact that beside having a beneficial effect on the prevention or reduction of infection, there is also a beneficial effect on the healing process. Wounds treated using the combination of the present invention will be less subject to or show an increased resistance to infections and the wound will heal more rapidly.

The compositions of the present invention can be prepared following methods generally employed in the art of pharmaceutical formulation. A typical formulation according to the present invention comprises Cetrimide (1,024 μg/ml) , Bacitracin

(16 units/ml) and Polymyxin B sulphate (512 units/ml) .

In a preliminary clinical study such a combination showed significant antibacterial activity when topically delivered in a hydroxypropyl methylcellulose gel (35 mg/ml) .

The above drug ratios can be maintained regardless of the delivery form even though actual concentrations may vary.

Specific embodiments of the present invention are illustrated by the following examples. It will be understood, however, that the invention is not confined to the specific limitations set forth in the individual examples.

EXAMPLE 1.

Unit Dose Formula - Gel Formulation

Ingredient Per 1L

Cetrimide B.P. 1024 mcg/ml 1024.0 mg Bacitracin USP 16 μ/ml 250.4 mg

Polymyxin B Sulphate USP 512 μ/ml 58.4 mg

Hydroxypropyl Methylcellulose USP 35 mg/ml 35. .0 g

Purified Water B.P. qs qs 1L

Method of Preparation:

Purified water (800 ml) is heated to 80°C and hydroxypropyl methylcellulose (HPMC) USP (35g) is added slowly to the hot water with stirring until the dissolution of the powder is complete. The mixture is then allowed to cool to below 30°C. Bacitracin USP

(250.4 mg) is dissolved in purified water BP (30 ml) and is then slowly added to the solution of HPMC and water.

Polymyxin B sulphate USP (58.4 mg) is dissolved in purified water BP (30 ml) and to this solution is added, the solution comprising , HPMC and water. Cetrimide BP (1024 mg) is dissolved in purified water BP (50 ml) and to this is slowly added the solution of , HPMC and Polymyxin B sulphate. Finally, purified water BP is added to make the solution up to 1 litre. EXAMPLE 2.

Ointment Paraffin soft, white 736 mg/g Microcrystalline wax 40 mg/g

Paraffin liquid 200 mg/g

Cholesterol 5 mg/g

Methylhydroxybenzoate 0.2 mg/g

Butylhydroxybenzoate 1.8 mg/g Cetrimide 1.024 μg/ml

Bacitracin 16 units/ml

Polymyxin B sulphate 512 units/ml

EXAMPLE 3.

Pharmacological Example Table 1 shows the sensitivity of bacteria isolated from wound infections to Cetrimide, Bacitracin and Polymyxin B sulphate when used either individually or in combination. The results in the table clearly indicate that the combination of the three actives produces a synergistic effect.

Methodology Clinical Isolates

Epidemiological data from Royal Prince Alfred Hospital was used to identify the organisms most commonly associated with wound infections. All organisms isolated from 2% or more of wound infections during 1992 were examined for potential inclusion in bacteriological evaluations. Eight bacterial species - Ent . cloacae, E. coli , Pr. mirabilis, Ps . aeruginosa, Staph . aureus, Staph . epidermidis, group A Streptococcus, and group G Streptococcus, were considered to be potential causes of wound infections in both the hospital and community environs. These were included in all further in vi tro evaluations. Geigy binomial confidence limits tables were used to determine the sample size of bacteria necessary to confirm the validity of an MIC value (Geigy Scientific Tables, 1982) . To have 95% confidence that an antimicrobial concentration would inhibit 97.5±2.5% of the bacteria of a given species, it was calculated that a sample of 72 organisms were required. Therefore it was aimed to collect 72 isolates of each of the eight targeted bacterial species for inclusion in MIC determinations for the four antimicrobials.

Bacteria isolated from wound infections were collected from five hospitals - Royal Prince Alfred, Westmead, Concord Repatriation, Royal North Shore, and Royal Alexandria for Children, and two private pathology laboratories - Sydney Diagnostic Services, and Douglas Laboratories. These were conveniently selected. The bacterial collection period was from October 1992 to May 1993. Minimum Inhibitory Concentrations Growth Conditions

Mueller-Hinton (M-H) broth (Oxoid CM337) was prepared in deionised water, dispensed in 100ml volumes and sterilised at 121°C for 15 minutes. Each species of bacteria was subcultured into cooled broth and incubated at 35°C for 18 hours. Standardisation of Bacterial Inoculum

The bacteria were adjusted to a known concentration of colony forming units/ml using spectrophotometric methods at 540nm. A 0.5 MacFarland standard is known to have an optical density which corresponds to 10⁸ CFU/ml, and was prepared by adding 0.5ml of 0.048M BaCl₂ to 99.5ml of 0.18M H₂S0₄ (NCCLS, 1985) . The overnight bacterial cultures were diluted with M-H broth until absorbance readings were equivalent to this and then further diluted 1/50 to standardize to 2xl0⁶ CFU/ml. Antimicrobials

Concentration ranges of each of Cetrimide,

Bacitracin, and Polymyxin B sulphate were prepared for each organism. Starting points were identified from MIC values previously determined using reference strains and from the literature.

Each drug was prepared at the maximum required concentration in 100ml volumes of M-H broth, taking into account the standard units of activity associated with antibiotics. As assay units may differ widely from actual drug weight, the following formula was used to standardize solutions.

Weight (mg) = Volume (ml) x Concentration (μg/ml)

Assay potency (μg/mg)

MIC Evaluation

MIC evaluations were carried out using standard microdilution methods for bacteria that grow aerobically (NCCLS, 1985) . The microtitre trays were Cel-Cult, 96 well, plastic, individually packaged, and gamma sterilised (Sterilin, code no. 239275) with lids. The growth medium used throughout was Mueller-Hinton broth. An electronic multichannel pipette with a range of 25- 250μl, was used to dispense antimicrobials and bacteria.

150μl aliquots of each antibiotic dilution were dispensed in duplicate into wells in the microtitre trays. 50μl of bacterial suspension was added, giving a final bacterial concentration of 5xl0⁵ CFU/ml. The trays were incubated for 18-24 hours at 35°C and the MIC was recorded as the lowest antibiotic concentration which inhibited bacterial growth. This was obvious to the eye by the presence/absence of turbidity in the well. Results

Results were pooled and the concentration which would have inhibited all isolates of a bacterial species was selected as the overall minimum inhibitory concentration (Table I) . Three Drug Combinations

Antimicrobial drugs were evaluated in three drug combination with aims of

(i) gaining an understanding of how the drugs were affected by each other's presence, and (ii) selecting a combination of concentrations capable of inhibiting all eight target bacterial species.

The combination of Cetrimide-Bacitracin-Polymyxin B - 8 - sulphate was evaluated using clinical isolates. Selection of Bacterial Sample

The Geigy binomial confidence limits tables were used to determine the sample size of bacteria necessary to confirm the interactions of the three antimicrobial agents (Geigy Scientific Tables, 1982) . To have 95% confidence that a particular combination of drug concentrations would inhibit 85.5±14.5% of the bacteria of a given species, it was calculated that a sample of 11 organisms were required. These were collected from the hospitals and private pathology laboratories within the Sydney metropolitan area which were used for the MIC determinations. Growth Conditions Overnight cultures of bacteria were prepared as for the MIC evaluations. Antimicrobials

The antimicrobials were the same as for the MIC determinations of the clinical isolates. Drugs were weighed and doubling dilutions prepared as for the MIC work. Concentrations ranged from the MIC to four dilutions below the MIC (MIC x 1/16) . Solutions were four times the required concentrations to account for the dilution occurring in the wells. The maximum recorded MICs were used to initiate concentration ranges for those antibacterial agents that individual organisms were not sensitive to. Method for Microdilution Checkerboard Titrations

Microtitre trays were used as for the MIC determinations. An 8x8 matrix was established in the microtitre plates, with one antimicrobial increasing in concentration along the x-axis, and the other increasing along the y-axis. Each triple checkerboard was performed in duplicate. 40μl of Bacitracin was dispensed into the wells, with a new concentration in each column (i.e. increasing along the x-axis) , commencing at column 2. 40μl of Polymyxin B sulphate was dispensed into the wells, with a new concentration in each row (i.e. increasing along the y- axis) , commencing at row 2. Eight trays were prepared with the same two drug combination. One tray was left containing only this double combination and an additional 40μl of M-H broth was added to each well to make up the volume and to account for the antibiotic concentrations. Each of seven trays had a different concentration of Cetrimide added to them (40μl/well) , with one concentration held constant throughout one tray. 40μl of Mueller-Hinton broth was added to the wells of the first row and first column to make up the final volumes. 40μl bacterial suspension, standardized to 10⁶ CFU/ml, was added to all wells of the 8x8 checkerboard, diluting the cultures to 2.5xl0⁵ CFU/ml. The microtitre trays were incubated at 35°C for 18 hours, and results were read as presence or absence of bacterial growth. Results

Each plate allowed the confirmation of the MIC for Bacitracin (x axis) and Polymyxin B sulphate (y axis) . The Cetrimide MIC was evident since trays containing this concentration or greater had total bacterial inhibition, with no growth evident throughout the plate. A control plate allowed observation of the Bacitracin/Polymyxin B sulphate combination. From each microtitre tray a well was selected which showed bacterial inhibition with the combination of the lowest drug concentrations. Eleven isolates of each bacterial species were evaluated in this way. These figures were compiled to give a combination of concentrations which was capable of inhibiting the growth of each species (Table I) . The combinations observed for the eight bacterial species were compared and an overall combination was selected which appeared capable of inhibiting all target organisms. This combination was used for further in vitro evaluations which confirmed its appropriateness and in vivo evaluations which examined its topical delivery and clinical efficacy. Table 1 Sensitivity of bacteria isolated from wound infections to Cetrimide, Bacitracin and Polymyxin B sulphate when used either individually or in combination

BACTEa IA CETRIMIDE Polymyxin B

(μg/ml) (units/ml) SULPHATE (units/ml)

ALONE COMBINED ALONE COMBINED ALONE COMBINED

Enterobacter cloacae 128 128 n.s. 4 >512 32

Escherichia coli 128 16 n.s. 4 >256 16

Proteus mirabilis 256 128 >512 4 > 1 ,024 32

Pseudomonas aeruginosa n.s. 16 n.s. 4 >256 128

Staphylococcus aureus 16 1 32 2 n.s. 32 o

I

Staphylococcus 16 1 64 8 > 1 ,024 64 epidennidis

Group A streptococci 4 0.25 0.125 0.0078 n.s. 32

Group G streptococci >4 0.5 >2 0.25 n.s. 64 n.s. this organism is not sensitive to this antibacterial drug

Comparative Clinical Trial of Topical Antimicrobial Preparations

Clinical Trial Protocol

A double-blind comparative clinical trial evaluated a topical preparation containing 1,024 μg/ml cetrimide, 16 units/ml bacitracin, and 512 units/ml polymyxin B sulphate in a 3.5% hydroxypropyl methylcellulose gel. Positive and negative controls were Betadine^® antiseptic cream and placebo gel, respectively. The multi-centre study was conducted in 5 primary schools using children aged 5-12 years with parental consent. As accidental minor injuries occurred, the participating children were treated with a randomly assigned topical preparation. Injuries were examined by a physician on the third day of treatment and a clinical outcome of 'no infection' or 'suspected' infection was established. If an infection was suspected, the injury was swabbed for microbiological evaluation.

All those which were considered to be 'suspected infections' were recorded as clinical infections. Those clinical infections from which microbiological pathogens were isolated were classified as microbiological infections.

A total of 177 injuries were treated and outcomes are summarized in Table 2.

Table 2. Treatment groups and infection outcomes

Placebo Betadine^® Test Preparation

School Total Clinical Micro. Total Clinical Micro, Total Clinical Micro no. infect. Infect. no. infec . infect. no. infect. infect. treated treated treated

1 4 0 0 8 1 1 6 0 0

2 13 2 2 20 0 0 15 0 0

3 6 0 0 8 0 0 9 0 0

4 4 1 1 8 0 0 6 0 0

5 21 3 1* 23 1 1 26 1 1

TOTAL 48 6/48 4/47 67 2/67 2/67 62 1/62 1/62 (12.5%) (8.5%) (3.0%) (3.0%) (1.6%) (1/6%)

Clinical infect. clinical infection

Micro, infect. = microbiological infection * _ one suspected infection was not swabbed

Comparison of Outcomes 1. Clinical

Chi square analysis of proportions of clinical infections for the three treatment groups showed a significant difference (p<0.05). This difference was further analysed:

* A comparison of placebo to Betadine^® using a one-tail test (since a priori knowledge suggested that Betadine^® would have a definite antibacterial effect and placebo would not) showed no statistically significant difference in the incidence of clinical infections (p>0.05) .

* A comparison of placebo to the test preparation using a two-tail test showed a significant difference in the incidence of clinical infections (p<0.05).

* A comparison of test preparation to Betadine^® using a two-tail test showed no significant difference in incidence of clinical infections

(p>0.05) .

Therefore, it was concluded that the test preparation containing cetrimide, bacitracin and polymyxin B sulphate prevented significantly more clinical infections of wounds than placebo.

2. Microbiological

Chi square analysis of proportions of microbiological infections for the three treatment groups showed no significant difference (p>0.05). One of the injuries which had been clinically categorised as 'infected' showed no microbial pathogen on culture, and one of the clinical infections was not swabbed and was therefore classed as a missing variable. These two cases had both been treated with placebo and this small alteration in the small sample sizes was sufficient to alter outcomes of significance testing.

Claims (12)

1. A pharmaceutical formulation comprising Bacitracin or a pharmaceutically acceptable derivative thereof, Polymyxin or a pharmaceutically acceptable derivative thereof and an antiseptic.

2. A formulation according to claim 1 wherein the antiseptic is selected from Cetrimide, Chlorhexidine and pharmaceutically acceptable derivatives thereof.

3. A pharmaceutical formulation according to claim 1 or 2 wherein the Polymyxin is Polymyxin B sulphate.

4. A pharmaceutical formulation according to claim 4 additionally comprising a pharmaceutically acceptable carrier.

5. A pharmaceutical formulation according to claim 4 in the form of a gel, ointment, cream, liquid or impregnated dressing.

6. A pharmaceutical formulation according to claim 4 wherein the pharmaceutically acceptable carrier is a hydrogel.

7. A pharmaceutical formulation according to claim 6 wherein the hydrogel is hydroxypropyl methylcellulose in water.

8. A method of treatment or prophylaxis in a mammal of wound infection which comprises topical application to the wound a pharmaceutical formulation comprising Bacitracin, Polymyxin and an antiseptic.

9. A method according to claim 8 wherein the antiseptic is selected from Cetrimide, Chlorhexidine and pharmaceutically acceptable derivatives thereof.

10. A method according to claim -9 wherein the Polymyxin is Polymyxin B sulphate.

11. Use of Bacitracin and Polymyxin in combination with an antiseptic in the manufacture of a medicament for the treatment or prophylaxis of wound infection.

12. A method of preparation of a pharmaceutical formulation as defined in any one of claims 1 to 4, 6 or

7 which comprises dissolving each of the actives and the carrier in water and then combining them. INTERNATIONAL SEARCH REPORT International application No. PCT/AU 95/00159

A. CLASSIFICATION OF SUBJECT MATTER

Int. Cl.⁶ A61K 38/12, 31/165

According to International Patent Classification (IPC) or to both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols) IPC A61K 38/12

Documentation searched other than minimum documentation to the extent that such documents are mcluded in the fields searched AU: IPC as above

Electronic data base consulted during the international search (name of data base, and where practicable, search terms used) WPAT: (A61K/IC and bacitracm) and (Polymyxin) and (Antisep: or cetrimide or chlorhexidine) CASM: as above

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category Citation of document, with indication, where appropriate, of the relevant passages Relevant to Claim No.

X Hendley, J O et al, (1991) Effect of topical Antimicrobial treatment on aerobic 1 ,8, 11

A bacteria in the stratum corneum of human skin, Antimicrobial Agents and 2-7,9-10, 12 Chemotherapy, Vol. 35, No. 4, pages 627-631. Whole document.

X New animal drugs for ophthalmic and topical use. Zinc bacitracin, Polymixin B 1 ,8, 11 A Sulfate, neomycin sulfate ophthalmic ointment, vetennary, Federal Register, Vol. 2-7,9-10, 12 38, No. 191 , page 27353.

D Further documents are listed See patent family annex. in the continuation of Box C.

Special categories of cited documents : later document published after the international filing date or priority dale and not in conflict

"A" document defining the general state of the art which is with the application ^'but cited to understand the not considered to oe or particular relevance principle or theory underlying the invention "E" earlier document but published on or after the "X" document of particular relevance: the claimed international filing date invention cannot be considered novel or cannot be "L" document which may throw doubts on priority claιm(s) considered to involve an inventive step when die or which is cited to establish the publication date of document is taken alone another citation or other special reason (as specified) document of particular relevance: the claimed

"O" document referring to an oral disclosure, use, mvention cannot be considered to involve an itpn exhibition or other means mventive step when the document is combined document published prior to the international filing date with one or more other such documents, such but later than the priority date claimed combination bemg obvious to a person skilled in the art

"&'^• document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report 23 June 1995

Name and mailing address of me ISA/AU Authorized officer

AUSTRALIAN INDUSTRIAL PROPERTY ORGANISATION PO BOX 200 WODEN ACT 2606 AUSTRALIA S CHANDRA

Facsimile No. 06 2853929 Telephone No. (06) 2832264

Fo P / i f 2 Jul 19 2 co dia