WO1997000076A1

WO1997000076A1 - Compositions containing poly(hexamethylene biguanide) salts and uses thereof

Info

Publication number: WO1997000076A1
Application number: PCT/GB1996/001457
Authority: WO
Inventors: Nigel Morlet; Frank Larkin; John Dart; Victor Andrews
Original assignee: Moorfields Eye Hospital NHS Trust
Current assignee: Moorfields Eye Hospital NHS Trust
Priority date: 1995-06-16
Filing date: 1996-06-17
Publication date: 1997-01-03
Anticipated expiration: 1997-12-16
Also published as: AU6130896A; GB9512290D0

Abstract

Uses of poly(hexamethylene biguanide) salts, especially the hydrochloride salt. Especially the use of the salts in the manufacture of medicaments for, and methods of topical treatment of microbial infections. Also pharmaceutical preparations, antiseptics, disinfectants and liquid formulations comprising a poly(hexamethylene biguanide) salt.

Description

COMPOSITIONS CONTAINING P0LY(HEXAMETHYLENE BIGUANIDE) SALTS AND USES THEREOF

The present invention relates to poly(hexamethylene biguanide) salts, especially the hydrochloride salt (known as polyhexanide or PHMB) , especially to the use of the salts in the manufacture of medicaments for, and methods of, topical treatment of microbial infections and to their use as antiseptics.

Microbial infections, such as bacterial, amoebal and fungal infections, are common and may be generalised, i.e. throughout the body, or may be localised, i.e. restricted to one area, for example, a wound site or an organ. (The term "microbes" is taken herein to include bacteria, amoebae, fungi and obligate intracellular organisms.)

Infections of the eye are relatively common and may be very serious, even sight threatening. At the present time there is a very limited number of agents that have been developed for use in the treatment of infections in the eye.

Currently the main antibiotics or antimicrobial agents used in the treatment of the eye are tetracycline, erythromicin, poly ixin, trimethoprim, chloramphenicol, gentamicin, neosporin (a mixture of neomycin and bacitrasin) , fusidic acid, quinolones and anti-fungal agents such as the azole group of agents. Propamidine, an antimicrobial, is often used as an over-the-counter (OTC) medication. Less commonly used are the sulphonamide group and beta-lactam antibiotics (e.g. penicillin) . Although those agents are useful and safe, there is a continuing need for further antimicrobial agents which are safe for use in the eye. Alternative agents for treatment are required which are less likely to produce microbial resistance and that are more effective, that is, have a broader spectrum and activity without problems of toxicity. These treatment compounds and compositions should be stable and easy to administer. Inexpensive treatments which fulfil these requirements are especially desired.

Polyhexanide is known to be a broad spectrum antibacterial agent. At relatively low concentrations the antibacterial action is biostatic and at higher levels, dependent on particular species of bacteria, it is bactericidal. It is a "membrane-active" antibacterial. Polyhexanide has been found to be active against both Gram- negative and Gram-positive bacteria.

Polyhexanide is currently sold as a swimming pool disinfectant, a water system disinfectant and at a concentration of approximately 0.00005%, as a preservative in contact lens solutions. Polyhexanide has been used successfully in the treatment of Acanthamoeba keratitis. an uncommon but blinding amoebal infection of the cornea (the clear front surface of the eye), see, for example, Larkin et al., 1992, Hay et al., 1994, Seal 1994, Elder et al., 1994, and Bacon et al., 1993. The disease has a range of symptoms and resulting conditions including severe pain, ring abscesses, perforated corneas, corneal scarring and intumescent lens requiring keratoplasty, extracapsular lens extraction and posterior chamber lens implant. The infection can be difficult to treat and may be resistant to commonly used agents. Hence, polyhexanide was used in its treatment even though very little was known about the penetration of polyhexanide into the eye and what toxicity problems it might cause. In the treatment of Acanthamoeba keratitis polyhexanide has been recommended for use in very low concentrations: up to 0.02 % (w/v). In the treatment of an infection such as Acanthamoeba keratitis some relatively serious side effects of treatment may be acceptable when they would not usually be in the treatment of other, less serious, infections.

Various investigators have discussed and expressed concern about the toxicity of polyhexanide. For example, D. V. Seal in his paper in 1994 states that although polyhexanide has been applied topically in the treatment of Acanthamoeba keratitis it is not ideal for topical ocular treatment. Some toxic effects are discussed by Masaki Imayasu et al. 1992. Rabbit eyes were treated with sterilised phosphate buffered solutions of 0.01, 0.02 and 0.03 % polyhexanide and it was found, after fluorescein instillation, that there was superficial punctate staining on the cornea (showing lesions) and conjunctival chemosis. It has, however, been found by the present inventors that polyhexanide can be used safely and without toxicity problems on the eye and in other locations.

The present invention is concerned with the use of any physiologically acceptable salt of poly(hexamethylene biguanide) , for example, the hydrochloride, acetate or gluconate salt. Currently the only salt commercially available is the hydrochloride salt, which is known as "polyhexanide" or "PHMB". However, the invention is not limited to the use of the hydrochloride salt and, unless stated otherwise, references to polyhexanide or PHMB include all other physiologically acceptable salts.

The present invention provides the use of a poly (hexamethylene biguanide) salt, especially of poly (hexamethylene biguanide) hydrochloride, for the manufacture of a medicament for the topical treatment of microbial infection of the human or animal body, excluding amoebal infection of the eye.

The present invention especially provides the use of a poly(hexamethylene biguanide) salt, especially of poly (hexamethylene biguanide) hydrochloride, for the manufacture of a medicament for the topical treatment of microbial infection of the eye, excluding amoebal infection of the eye. The present invention further provides use of a poly (hexamethylene biguanide) salt for the manufacture of a medicament for the topical treatment of microbial infection of the human or animal body, including infection of the eye, the salt being present in a concentration of at least 0.1 % w/v or w/w (w/v for solutions, w/w for other formulations) .

It will be understood that the term "infection" does not imply the presence of a particular number of microbes; infection may be said to be present even if a relatively small number of microbes is present. The virulence of the organism, the host response and many intrinsic and environmental factors are all important factors.

The present invention also provides the use of a poly(hexamethylene biguanide) salt, especially poly (hexamethylene biguanide) hydrochloride, for the manufacture of an antiseptic for topical use on the human or animal body, including use on the eye.

The term "antiseptic", generally accepted, is used herein to have its generally accepted meaning of a preparation which is used for disinfecting, i.e. cleaning or sterilising, rather than for the treatment of infection. For example, an antiseptic may be used to clean the skin or the surface of the eye before surgery or may be used to clean a wound, made in surgery or in an accident, to prevent infection. Antiseptic preparations may be used in order to eradicate or prevent the spread of bacteria, amoeba or fungi or other microbes such as free obligate intracellular organisms. Polyhexanide when used as an antiseptic may also be able to kill certain free viruses (i.e. those coated in a cell membrane - enveloped viruses) even though it may not be a therapeutic antiviral agent.

Antiseptic preparations in accordance with the invention may also comprise further compounds having antimicrobial properties. The present invention further provides a pharmaceutical preparation comprising a physiologically acceptable amount of a poly(hexamethylene biguanide) salt, especially poly(hexamethylene biguanide) hydrochloride, in a form suitable for topical administration in or on the human or animal body, excluding administration on the eye. That is to say, the invention provides preparations of a type suitable for use in or on the body which are not suitable for application on the eye. Such preparation may, for example, comprise carriers or diluents which may not be used on the eye. The present invention also provides a pharmaceutical preparation comprising a physiologically acceptable amount of a poly(hexamethylene biguanide) salt in a form suitable for topical administration in or on the human or animal body, including on the eye, the salt being present in a concentration of at least 0.1 % w/v or w/w (w/v for solutions, w/w for other formulations) .

The present invention further provides an antiseptic preparation, comprising a physiologically acceptable amount of a poly(hexamethylene biguanide) salt, especially poly(hexamethylene biguanide) hydrochloride, in a form suitable for topical administration in or on the human or animal body, including use on the eye.

Preferably an antiseptic preparation comprises the poly(hexamethylene biguanide) salt in a concentration of at least 0.1 % w/v or w/w (w/v for solutions, w/w for other formulations) .

The invention further provides a method of treating microbial infection, other than amoebal infection on the eye, comprising administering a therapeutically effective amount of a poly(hexamethylene biguanide) salt, especially poly(hexamethylene biguanide) hydrochloride, topically to the human or animal body.

The invention especially provides a method of treating microbial infection of the eye, other then an amoebal infection on the eye, comprising administering a therapeutically effective amount of a poly(hexamethylene biguanide) salt, especially poly(hexamethylene biguanide) hydrochloride, topically to the eye.

The invention also provides a method of treating a microbial infection comprising administering a therapeutically effective amount of a poly(hexamethylene biguanide) salt, especially poly(hexamethylene biguanide) hydrochloride, topically to the human or animal body, including to the eye, the salt being present in a concentration of at least 0.1 % w/v or w/w (w/v for solutions, w/w for other formulations) .

- The invention further provides a method of disinfecting (asepticizing) a part of the human or animal body comprising topical administration of an effective amount of a poly(hexamethylene biguanide) salt, especially poly(hexamethylene biguanide) hydrochloride, to the human or animal body, including to the eye. Preferably the salt is present in a concentration of at least 0.1 % w/v or w/w (w/v for solutions, w/w for other formulations)

The invention further provides for the use of a poly (hexamethylene biguanide) salt, especially poly

(hexamethylene biguanide) hydrochloride, as a disinfectant for surfaces and for surface disinfectants which comprise a poly(hexamethylene biguanide) salt. Such a disinfectant may be used to disinfect medical equipment and medical and household surfaces. Disinfectants will usually comprise the salt in the form of an aqueous solution. The concentration of polyhexanide in a disinfectant will usually be relatively high and may be as high as 20 %. Preferably the concentration will be greater than 0.1 % w/v or w/w (w/v for solutions, w/w for other formulations) . Advantageously the antiseptic preparation, pharmaceutical preparation or method of treatment is in a form suitable for use on the skin; on or in a wound; on a mucosal surface, for example, in the nasal passages, the throat, in the bladder or in the vagina; as a colonic enema or lavage prior to or during surgery; in an ear cavity; in the mouth, for example, on the gums or on the teeth; on the scalp or on the hair.

Where the preparation is to be used to kill microbes on the skin surface or in a wound the preparation may take the form of an aqueous formulation, an oily formulation, an oil-in-water emulsion or a water-in-oil emulsion, a liposomal formulation or a gel formulation.

Where the preparation is to be used to kill microbes on mucosal surfaces the preparation may take the form of a mouth wash, a gargle, a slowly dissolving pastille or lozenge or a mouth, throat or nasal spray. A gel formulation may be, for example, suitable for application to the gums and both gel and paste formulations are suitable for use as toothpastes. In a liquid formulation such a preparation may be used in bladder irrigation to eradicate or prevent the spread of infection.

For treatment of the hair, scalp or skin, shampoo or soap formulations of polyhexanide may be especially useful.

The treatment of microbial infections may be carried out in order to eradicate the bacteria, amoeba, fungi or other microbe completely or may be used to restrict or prevent the growth or spread of the bacteria, amoeba, fungi or other microbe.

Microbial infections which may be treated with polyhexanide include the common organisms which cause infection of the' external eye and other parts of the body, such as, Gram positive cocci and rods, for example, Staphylococci species, Corvnebacteria species and

Steptococci species, Gram negative cocci and rods, for example, Haemophilus species, Pseudomonas species, Enterobacter species and Bacillus species, yeast forming fungi, for example, Candida, filamentary fungi, for example, Aspergillus and Fusarium. amoebae, for example, Acanthamoeba species, and obligate intracellular organisms, for example, Chlamydia species.

Common external conditions of the eye which may be treated are blepharitis, which is an infection of the eyelids and eyelid margins, conj nctivitis, which is an infection of the conjunctiva and the conjunctival sac, and keratitis, which is an infection of the clear cornea. In some cases a patient may suffer from all three infections at the same time.

The term "eye" is used herein to include not only the eyeball itself but also associated structures such as the eyelids, conjunctiva and conjunctival sac.

Administration of polyhexanide to the eye is to be topical administration. Use of the terms administration "to the eye" and "on the eye" are used to mean administration to the external surfaces of the eye. It is not intended that the polyhexanide be administered to internal tissues of the eye by means such as injection into the eye.

The invention also provides the use of a poly (hexamethylene biguanide) salt, especially poly(hexamethylene biguanide) hydrochloride, as a preservative in liquid formulations, for example, eye drops and contact lens solutions, for use on the eye, the polyhexanide being present in a concentration of greater than 0.01 %, preferably in the range of greater than 0.01 % to 2.0 % and more preferably in the range of greater than 0.01 % to 0.02 % w/v or w/w (w/v for solutions, w/w for other formulations; lg per 100 ml of solution iε 1% w/v) . When used therapeutically as an antimicrobial, polyhexanide will preferably be used in a concentration of at least 0.02 % and more preferably at least 0.1 %. Preferably the concentration will be in the range 0.02 % to 2.0 % and still more preferably in the range 0.1 % to 1.0 %. (Concentrations are calculated as w/v or w/w, w/v for solutions, w/w for other formulations.) When used as an antiseptic polyhexanide may be used in similar concentrations but will generally be used in higher concentration formulations.

Medicaments according to the present invention are provided in a pharmaceutical preparation form suitable for topical administration, for example, an emulsion, suspension, solution, cream, lotion, ointment, drops, foam, gel, a liposomal preparation, an aerosol or spray. Such preparations are generally conventional formulations, for example, as described in standard text books of pharmaceutics such as the British Pharmacopoeia or Martindale The Extra Pharmacopoeia.

Preparations for the treatment of eye infections may be in the form of aqueous formulations, oily formulations, liposomal formulations, gels or ointments. The polyhexanide may be mixed with other polymeric compounds such as polyacrylic acid. The pH of liposomal, aqueous and gel formulations may be adjusted to a pH in the range 6 to 8.5. Acid or alkali compounds may be used to adjust the pH, preferably with a buffer, especially a boric acid/borate buffer, a phosphate buffer or an acetic acid/acetate buffer.

Preparations for treatment of the eye may also comprise sodium chloride or another excipient to adjust the tonicity of the medicament.

Preparations, especially for treatment of the eye, may also comprise polyvinyl alcohol or another excipient, for example, hypromellose, to adjust the viscosity of the medicament. Ointments, especially for treatment of the eye, may also comprise polyethylglycol, paraffin oils or other excipients to adjust the volume and viscosity of the medicament.

Liposomal preparationε, especially for treatment of the eye, may comprise phosphatidylglycerol, phosphatidylcholine and/or chloresterol dissolved in chloroform.

Gel formulations may compriεe carbomer (polyacrylic acid) 940.

Antiseptic preparations according to the invention may take any of the forms discussed above. In addition antiseptic preparations may take the form of wipes, medicated plasters and other such forms which are commonly used for antiseptics.

Polyhexanide may be administered, for example, as a solution in physiological saline or in an artificial tear complex. An example of a εolution which iε εuitable for use in the eye is one comprising polyhexanide and 0.3% hypromellose, 0.45% NaCl, 0.37% KCI, 0.19% borax (sodium tetraborate decahydrate) and 0.19% boric acid (% are w/v). Further examples of formulations which may be used in accordance with the present invention are given in Examples 7 to 14.

The medicaments, and methods described herein may also compriεe further pharmaceutically active εubstances, for example, steroids, such as Dexamethasone, antivirals, such as aciclovir and other compounds having antimicrobial activity, such as bacitacin and trimethoprin (currently used in Polytrim™ drops) . The polyhexanide may be uεed in combination with other pharmaceutically active agentε either in the same formulation or they may be applied in separate formulations.

The appropriate dose and frequency of treatment will be dependent on the symptoms and phyεical condition of the individual patient. In order to assist in determining the suitability of treatment with polyhexanide and also the optimal concentration and frequency of treatment samples of the infecting agent may be taken and grown in culture and those cultures subjected to sensitivity testing. Examples of such tests are described in Elder et al. 1994, Larkin et al. 1992 and Hay et al. 1994.

Medicaments administered to the eye are usually diluted by tears. It may therefore be advantageous to use relatively high concentrations of polyhexanide when treating the eye. In that way a therapeutically effective concentration of polyhexanide may remain in the eye for longer.

Polyhexanide, especially in the form of drops, is suitable for general use as a first line therapy for external ocular infections such as blepharitis, conjunctivitis and keratitis. It may be used to replace chloramphenicol, for which there are few alternatives, and may be especially useful in the U.S.A. where chloramphenicol is not used because of the rare, but fatal, complication of aplastic anaemia with which its use has been linked. It is cheap to manufacture and may therefore be extremely valuable in treatment of patients in the Third World where corneal blindness from infection is prevalent. Apart from 5% Betadine™ (providone iodine) there are no ocular antiseptics currently available commercially.

Polyhexanide is composed of a mixture of polymeric compounds with the structure:

H H j 1 H

1

(CH₂) - N - - C - N - - C - N - (CH₂)- •HC1 II II NH NH n

where n is generally in the range 2 to 30 inclusive with a mean value of 5.5. If desired fractionation may be used to produce polyhexanide with a specific composition or mean value of n. The biguanide groups exist in the form of their εalts so polyhexanide is esεentially a polyelectrolyte. The hydrochloride εalt iε highly water εoluble.

The antibacterial activity of polyhexanide has been found to increase with chain length. This haε been rationaliεed through the mechaniεm of action, εee below; the longer chain being more effective in combining with acidic phosphoiipids such as phosphatidyl glycerol in the cell membrane. Advantageously, polyhexanide comprising a mixture of short and long polymer chains is used (see Gilbert et al.) .

Use of polyhexanide may overcome the drug resiεtance in bacterial infectionε εince it seems unlikely that resiεtance to polyhexanide will develop, it will broaden the limited spectrum of currently used antibiotics and cause fewer side effects. (Resiεtance is unlikely to develop to polyhexanide because of its mechanism of action: the polyhexanide attaches to the acidic phoεpholipidε in the cell wall and diεturbε the function of the membrane leading to leakage of intracellular contentε.) Although the primary uεe of the preεent invention is in the field of human medicine, it may also be desirable to treat other animals, especially mammals, for microbial infections, and the present invention therefore also includes uεeε of the deεcribed medicamentε and preparations for the treatment of animals other than humans and further includes preparations and medicamentε deεcribed above that are suitable for the treatment of non-human animals.

The contentε of the literature referenceε mentioned herein are hereby incorporated by reference. The following non-limiting Examples illustrate the invention. Examples 1 and 2 illustrate the efficacy of polyhexanide (PHMB) aε an anti-microbial. Examples 3 to 6 illustrate the effect of polyhexanide (PHMB) on the ocular surface of the eye and confirm that it is safe for use at the proposed concentrations. Examples 7 to 14 show a number of different formulations which are suitable forms in which to administer polyhexanide.

In Examples 1 to 6 the polyhexanide used waε the hydrochloride salt. For Examples 7 to 14, the formulationε exemplified may comprise any physiologically acceptable salt of poly(hexamethylene biguanide) but preferably comprise the hydrochloride εalt.

The Examples refer to the following Figures: Figure 1 is a diagrammatic representation of the results of experiments described in Example 2. It is a graph showing the colony forming units/mL of a Pseudomonaε aeruginosa clinical isolate in a test sample over time for a number of different treatment regimes with polyhexanide.

Figure 2 is a diagrammatic representation of the results of experiments described in Example 2. It is a graph showing the colony forming units/mL of NCTC 10662, a laboratory εtrain of Pεeudomonaε aeruginosa. over time for a number of different treatment regimes with polyhexanide.

Figure 3 is a diagrammatic representation of the results of experiments described in Example 4. It is a graph showing corneal thicknesε over time for a number of different experimental regimes. Example 1 - Efficacy

The efficacy of polyhexanide as a bactericide has been tested and the results are set out below. The European Pharmacopoeia criteria for evaluation of antimicrobial activity for Ophthalmic Preparations are given in terms of the log reduction in the number of viable micro-organisms against the value obtained for the inoculum. The "A criteria" that are usually required to be pasεed are:

Log Reduction

Organism Criteria 6h 24h 7d 14d 28d

C.albicans and A 2 NI

A.niger B 1 NI

P.aeruginosa and A 2 3 NR

S.aureus B 1 3 NI

NI:No Increase NR:No Recovery The "A criteria" express the recommended efficacy to be achieved. In juεtified caεeε where the "A criteria" cannot be attained, for example, for reasons of an increased risk of adverse reactionε, the "B criteria" (also set out above) must be satisfied. Polyhexanide eye drops of 0.01 and 0.02% concentrations, without preservatives, having the following compositions: polyhexanide 0.01 % or 0.02% hypromellose 0.3 % sodium chloride 0.45 % potasεium chloride 0.37 % borax 0.19 % boric acid 0.19 % purified water to 100 %

were tested and the resultε were aε followε,

Log Reduction

Product Storage Organiεm 6h 24h 7d 14d 28d

PHMB C.albicans NR NR NR NR NR 0.01% <25"C A.niger 0.60 1.30 2.22 2.85 3.02

P.aeruginosa NR NR NR NR NR

S.aureus NR NR NR NR NR

Log Reduction

Product Storage Organism 6h 24h 7d 14d 28d

PHMB C.albicans NR NR NR NR NR 0.02% <25°C A. iger 0.45 1.23 2.25 3.31 NR

P.aeruginosa NR NR NR NR NR

S.aureus NR NR NR NR NR As may be seen both of the solutionε teεted satisfied the A criteria.

Example 2 - Efficacy against a wide range of microorganisms The minimum inhibitory concentration (MIC) of polyhexanide was found for a broad range of microbes. Those microbes, listed below with the MIC values, are all known ocular pathogens and may also be found on or in other parts of the body. The experimental procedure used was the standard one used for calculating MIC values. The procedure iε εet out in the Clinical Microbiology Procedureε Handbook (Iεenberg 1992a) .

The microbes used in the tests were all stored clinical isolates. All MIC values are concentrations given in parts per million.

Gram positive organisms

(a) Staphylococcus aureus

14 isolateε tested - mean MIC 3.5 ppm range 1 ppm to 20 ppm

(b) Coagulase negative Staphylocci

8 isolates tested - mean MIC 2.5 ppm range 1 ppm to 5 ppm

(c) Streptococci species

14 isolates tested - mean MIC 2 ppm range 1 ppm to 5 ppm

Gram negative organisms

(d) Pseudomonas aeruginoεa

17 isolateε tested - mean MIC 15 ppm range 5 ppm to 35 ppm

(e) Enterobacter specieε

6 iεolates tested - mean MIC 16.5 ppm range 5 ppm to 30 ppm

(f) Bacillus species

2 isolates - MICs 10 ppm & 30 ppm Yeasts (g) Candida specieε 8 iεolateε teεted - mean MIC 2 ppm range 1 ppm to 5 ppm

Fillamentouε fungi (h) Aspergilluε εpecieε

4 iεolates tested - mean MIC 37.5 range 35 ppm to 40 ppm

(i) Fusarium

1 isolate tested - MIC 20 ppm

^■ Figures 1 and 2 show the kill time for a range of concentrations of polyhexanide against two isolates of

Pseudomonas aeruginosa. One isolate is a clinical isolate taken from a patient (Figure 1) and the other, NCTC 10662, is a standard laboratory strain. The figures show plots of colony forming units/mL (cfu/mL) against time for microbe populations treated with polyhexanide at the concentrations shown. The experimental procedure followed was the standard one used for kill time experiments, see the Clinical Microbiology Procedureε Handbook (Isenberg, 1992b). (The controlε uεed culture broth alone.) MIC for the pseudomonas aeruginosa strain in Figure 1 is 30 ppm and for NCTC 10662 in Figure 2 is 7 ppm of PHMB.

Figures 1 and 2 show that PHMB has a very rapid antimicrobial action.

Example 3 - Toxicity

In theεe εetε of experiments corneal hydration following direct application of a test solution to a whole eye was studied. The experiments were carried out in two sets: (A) Whole eyes were used with the epithelium intact. (B) Whole eyes were used but with the epithelium completely removed prior to exposure to the test solutions. Freεh unscalded pig eyes were kept on ice until dissected, usually within 4 hours of slaughter. After measuring the corneal thickneεs using an ultrasonic pachymeter (Pachpen, Oculab, Mentor) , the whole eye was placed face down in a deep diεh and the corneal epithelial surface was covered with the test εolution for one hour. After drug application (teεt εolution) the corneal thickness was remeasured then the corneal scleral buttons were dissected from the eye. The buttons were then weighed (weight 1) and placed epithelial surface down, covered with medium 199 (Sigma) and incubated at 37°C for four to six hourε. After incubation, the corneaε were re-weighed (weight 2) , air dried at room temperature for 24 to 48 hourε and re-weighed dry (weight 3) . That was Experiment Set A. The experiments were also performed using eyes that had the epithelium completely removed prior to exposure to the teεt εolution, Experiment Set B.

Controlε were expoεed to balanced salt solution (BSS (Alcon Laboratories)) only (BSS control). Test εolutions were made up from 20% stock solution (aqueouε) of PHMB diluted with BSS. The percentage change in weight was calculated by εubtracting the dry weight (weight 3) from each of the wet weights (weights 1 and 2) to give a pre¬ incubation net weight (weight 1 - weight 3) and a post- incubation net weight (weight 2 - weight 3) and dividing the post-incubation net weight by the pre-incubation net weight then multiplying by 100.

The percentage change represents the change in hydration of the eye tiεεues and gives an indication of changes in endothelial function, as does the change in corneal thicknesε. The greater the damage to endothelial function the greater the percentage change in weight. Poεitive controls were performed in which the endothelial cells of the eye were destroyed after exposure to the test solutions thereby causing complete dysfunction of the endothelium. These are shown below as " + control". For Experiment Set A only one positive control was carried out and that eye was treated with the BSS control solution. For Experiment Set B a positive control was performed with each of the four teεt εolutionε and with BSS control solution. The positive controls show the maximum change which would be measured if treatment with test εolution had caused total dysfunction of the endothelium. The reεults were as follows: Test solution change in thicknesε % change in weight mm (±SD) (±SD)

Set*A - epithelium intact

BSS control 0.026 mm (±0.028) 16 . 5% ( ±7 . 8 )

BSS + control 31. 3% (±10. 8 )

2% PHMB -0.011 mm (±0.01) 10. 1% (±4 . 3 )

0.2% PHMB -0.02 mm (±0.016) 2.7% (±5.6)

0.1% PHMB 0.006 mm (±0.025) 0.2% (±5.0)

0.02% PHMB -0.039 mm (±0.036) 11.8% (10.0)

Set B - epithelium removed BSS control 0.289 mm (±0.033) 6.9% (±5.0) BSS + control 29.0% (±1.3)

2.0% PHMB 0.057 mm (±0.058) 10.1% (±4.3)

2.0% PHMB + control 12.8% (±9.3)

0.2% PHMB 0.072 mm (±0.024) 22.8% (±4.3)

0.2% PHMB + control 45.0% (±13.8)

0.1% PHMB 0.033 mm (±0.05) 13.5% (±7.6)

0.1% PHMB + control 31.9% (±14.9)

0.02% PHMB 0.099 mm (±0.077) 21.6% (±29.3)

0.02% PHMB + control 43.1% (±7.7) Summary -

Set A - intact epithelium

The corneal thicknesε diminishes slightly after treatment with PHMB rather than thickening slightly as with the control. There was no evident endothelial damage, the percentage change in weight waε not significantly different from that observed for the control test solution, and no significant dose effect was discernible. Set B - epithelium removed The corneal εwelling obεerved with the control test solution was lesε than that obεerved with the polyhexanide (PHMB) test εolution, but no εignificant doεe effect was discernible. There was no evidence of endothelial damage.

The difference between the results of Set A and Set B show that there is little penetration of the PHMB through the epithelium.

Example 4 - Toxicity

In this example experiments were carried out to study the effect of polyhexanide on corneal thicknesε (a measure of endothelial cell function) . Theεe experimentε were carried out on isolated mounted corneaε.

Freεh unscalded pig eyes were disεected within 4 hours of slaughter. After removing the epithelium, the corneal scleral button waε mounted on an artificial anterior chamber and the endothelial εurface waε perfuεed with BSS Plus (Alcon Laboratories) at 23 cm H₂0, 37°C, with the anterior chamber volume replaced every 1/2 hour. Serial meaεurements of corneal thickness were made with an ultrasonic pachymeter. The epithelial surface was kept moistened with BSS and was not allowed to dehydrate. Where appropriate, the test εolution was used to bathe the epithelial surface continuously for 1 hour. (Perfusion of the endothelium with BSS Plus was continued throughout) . The test solution waε made up from 20% εtock εolution (aqueouε) of PHMB diluted with BSS .

Positive controls were produced by exposing the endothelium to 70% alcohol for 1 to 2 minutes and then continuing perfusion with BSS Plus. The poεitive controls show the reεults which would be obtained if complete dysfunction of the endothelium were to have been caused by the test solutions.

The results of these experiments are shown in Figure 3. Measurementε of the corneal thickness were taken over a period of εix hourε. Figure 3 shows corneal thickness against time for that period.

Measurements for three control cornea are shown (control) ; these were cornea which were continuously perfused with BSS Plus for the full six hours and had their epithelial εurfaces kept moistened with BSS. They received no other treatment. Measurements for a positive control are shown ("+" control) . That cornea was treated as a normal control but the endothelial surface was expoεed to 70% alcohol for 1 to 2 minuteε after 120 minutes of the test period had pasεed. Meaεurementε for a cornea which had the epithelial surface bathed in 0.2% PHMB solution are shown (0.2% PHMB). Exposure to PHMB took place between minutes 120 and 180 of the experiment. The endothelium was perfused with BSS Plus for the entire six hour period. Measurements were also taken for a cornea receiving exposure of the epithelial surface to 0.2% PHMB εolution for 1 hour followed by expoεure of the endothelium to 70% alcohol for 1 to 2 minuteε. This is shown as "0.2%' PHMB "+" control" on Figure 3.

The results show that the 0.2% PHMB solution reduced corneal thicknesε, but there was no evidence of endothelial damage.

Example 5 - Toxicity

A series of experiments was carried out in which the effect of polyhexanide on cornea was εtudied by electron microεcopy. Fresh unscalded pig eyeε were placed in deep disheε and the epithelial surfaces were exposed to test solutions for 5 minutes. The test solutions were 2.0%, 0.2% and 0.02% PHMB εolutionε. Test solutionε were made up from 20% PHMB stock solution (aqueouε) diluted with BSS. BSS waε uεed as the control solution. The corneal scleral buttonε were then dissected and fixed in gluteraldehyde and processed for scanning electron microscopy.

Dose dependent changeε in the morphology of the epithelium were seen but no discernible changes to the endothelium were observed (either when the epithelium was intact or removed). The 2.0% polyhexanide solution produced obvious dehydration of the epithelial cells with a large loss of cell volume and flattening of the cells, but no disruption of the cell membranes. The 0.2% polyhexanide solution produced an irregular but less complete losε of cell volume, producing an irregular appearance of the epithelial surface but not disruption of the cell membranes. The 0.02% polyhexanide solution produced changes that were similar but less pronounced than those produced by the 0.2% solution.

Conclusionε from Exampleε 3. 4 and 5

Polyhexanide (PHMB) had an oεmotic dehydrating effect on the cornea and limited penetration through the intact epithelium. There was some penetration of the PHMB into those cornea without epithelium but no endothelial damage was observed.

These resultε illuεtrate that polyhexanide may be εafely uεed on the external eye and other epithelial surfaces of the body such as the skin and mucous membranes, at concentrations εhowing antimicrobial activity.

Example 6 - Toxicity

150 subjects with keratitis were treated with polyhexanide 0.02 % drops (composition as in Example 1) for an average of six months (range 1 to 18 months) . Initial treatment of one drop per hour was given for 48 hours followed by 16 drops per day for one week and 4 to 6 drops a day thereafter. Although the subjects had large epithelial defectε, no delay in corneal epithelial healing due to the drops was observed.

Two subjects developed punctate corneal epithelial erosions requiring therapy to be stopped after one month and another had punctate corneal epithelial erosions caused by the drops but was able to continue with the treatment. No other side effects of therapy were recognised and the drops were well tolerated by all other subjectε. Thiε rate of epithelial toxicity iε mild compared with conventional treatment with propamidine and neomycin, which frequently cause punctate erosionε of the cornea or inflammation of the conjunctiva (10 times more frequent than the PHMB treatment) .

Examples 7 , 8 and 9 - Aqueous Solutions

Compositionε are given aε % w/v. (7) Phosphate buffered: polyhexanide 0.1 to 2.0 % hypromellose 0.3 % sodium chloride 0.42 % sodium acid 0.075 % phosphate (2H₂0) magnesium chloride (6H₂0) 0.03 % sodium acetate (3H₂0) 0.39 % acetic acid 0.001 % citric acid 0.00001465 % purified water to 100 %

(8) Acetate/citrate buffered: polyhexanide 0.1 to 2.0 % hypromellose 0.3 % sodium chloride 0.49 % potasεium chloride 0.075 % calcium chloride (2H₂0) 0.048 % magnesium chloride (6H₂0) 0.03 % sodium acetate (3H₂0) 0.39 % acetic acid 0.001 % citric acid 0.00001465 % purified water to 100 %

(9) Bicarbonate buffered: polyhexanide 0.1 to 2.0 % hypromellose 0.25 % sodium chloride 0.6 % - sodium bicarbonate 0.45 % purified water to 100 %

Examples 10. 11 and 12 - Ointment formulations

Compositionε are given aε % w/w.

(10) polyhexanide 0.1 to 2.0 % yellow εoft paraffin to 100 %

(11) polyhexanide 0.1 to 2.0 % liquid paraffin 30 % yellow εoft paraffin to 100 %

(12) polyhexanide 0.1 to 2.0 % polyethylene glycol 4000 to 100 %

Other ointments may be made by using different combinations and proportions of the substances listed in the three formulations given above.

Example 13 - Gel formulation Compositions are given as % w/v. polyhexanide 0.1 to 2.0 % carbomer 940 polymer 0.2 % mannitol 5.0 % hydrochloric acid/ qs pH 7.4 sodium hydroxide purified water to 100 % Example 14 - Liposomal formulation

Compositions are given as % w/v

(phospholipid formulation) polyhexanide 0.1 to 2.0 phoεphatidylglycerol 1.0 % phoεphatidylcholine 4.0 % cholesterol 5.0 % chloroform to 100 %

(See Surv. Ophthal. 1985;29;335-348. )

Bibliography

Larkin, D.F.P., Kilvington, S. & Dart, J.K.G., Ophthalmol¬ ogy; Vol. 99; No. 2 , 1992; 185-191, "Treatment of Acanthamoeba keratitis with polyhexanide biguanide".

Hay, John, Kirkness, Colin M. Seal, David V. & Wright, Peter Eye; Vol. 8; 1994; 555-563, "Drug resistance and Acanthamoeba keratitis; The queεt for alternative antiprotozoal chemotherapy",

Seal,- D.V. , BMJ, Vol. 308; 1994; 1116-1117, "Acanthamoeba keratitis"

Elder, M.J., Kilvington, S. & Dart., J.K.G., Invest.

Ophthal. & Vis. Sci.; Vol 35; 1994; 1059-1064,

"A Clinicopathologic Study of In Vitro Sensitivity Testing and Acanthamoeba keratitis"

Bacon, A.S., Frazer, D.G. , Dart, J.K.G., Mattheson, M. ,

Ficker, L.A. & Wright, P. Eye; Vol. 7; 1993; 719-725. "A

Review of 72 consecutive cases of Acanthamoeba keratitis. 1984-1992"

Masaki Imayasu, Takeshi Moriyama, Jun-ichi Ohashi, Hideji Ichijima, H. Dwight Cavanagh, Contact Lens Asεociation of Ophthalmologiεtε Journal; vol 18; October 1992; No. 4; 260- 266

Gilbert, Peter, et al., "Synergiεm within Polyhexa ethylene Biguanide biocide formulationε" ; Journal of Applied Bacteriology; 69; 1990; 93-8.

Iεenberg, H.D. "Clinical Microbiology Procedureε Handbook" American Society for Microbiology, Washington 1992,

(a) Section 5, pp 5.2.1-5.2.29 (MICε) &

(b) Section 5, pp 5.16.14-5.16.21 (Kill Timeε)

Claims

1. Use of a poly(hexamethylene biguanide) salt for the manufacture of a medicament for the topical treatment of microbial infection of the human or animal body, excluding amoebal infection of the eye.

2. Use of a poly(hexamethylene biguanide) salt for the manufacture of a medicament for the topical treatment of microbial infection of the eye excluding amoebal infection of the eye.

3. Use of a poly(hexamethylene biguanide) salt for the manufacture of a medicament for the topical treatment of microbial infection of the human or animal body, including infection of the eye, the salt being preεent in a concentration of at least 0.1 % w/v or w/w (w/v for solutions, w/w for other formulations) .

4. Use of a poly(hexamethylene biguanide) salt for the manufacture of an antiseptic for topical use in or on the human or animal body, including use on the eye.

5. A pharmaceutical preparation compriεing a physiologically acceptable amount of a poly(hexamethylene biguanide) salt in a form suitable for topical administration in or on.the human or animal body, excluding administration on the eye.

6. A pharmaceutical preparation comprising a physiologically acceptable amount of a poly(hexamethylene biguanide) salt in a form suitable for topical administration in or on the human or animal body, including on the eye, the salt being present in a concentration of at least 0.1 % w/v or w/w (w/v for solutionε, w/w for other formulationε) .

7. An antiεeptic preparation, comprising a physiologically acceptable amount of a poly(hexamethylene biguanide) salt in a form suitable for topical administration in or on the human or animal body, including on the eye.

8. An antiseptic preparation, comprising a phyεiologically acceptable amount of a poly(hexamethylene biguanide) salt in a form εuitable for topical administration in or on the human or animal body, including on the eye, the salt being present in a concentration of at 5 least 0.1 % w/v or w/w (w/v for solutions, w/w for other formulations) .

9. A method of treating microbial infection, other than amoebal infection on the eye, comprising administering a therapeutically effective amount of a poly(hexamethylene

10 biguanide) salt topically to the human or animal body.

10. - A method of treating microbial infection of the eye, other than an amoebal infection on the eye, comprising administering a therapeutically effective amount of a poly(hexamethylene biguanide) topically to the eye.

15 11. A method of treating a microbial infection comprising administering a therapeutically effective amount of a poly(hexamethylene biguanide) salt topically to the human or animal body, including to the eye, the salt being present in a concentration of at least 0.1% w/v or w/w (w/v

20 for solution, w/w for other formulations) .

12. A method of disinfecting a part of the human or animal body comprising administering an effective amount of a poly(hexamethylene biguanide) salt topically to the human or animal body, including to the eye.

25 13. Use of a poly(hexamethylene biguanide) salt as a preservative in liquid formulations for use in the eye, the salt being present in a concentration of greater than 0.01% w/v or w/w (w/v for solutions, w/w for other formulations) .

30 14. A use or a formulation as claimed in claim 13 wherein the εalt is present in a concentration of up to 2.0 % w/v or w/w (w/v for solutions, w/w for other formulations) .

15. A use or a formulation aε claimed in claim 14

35 wherein the εalt is present in a concentration of up to 0.02 % w/v or w/w (w/v for solutions, w/w for other formulationε) .

16. Uεe of a poly(hexamethylene biguanide) εalt as a disinfectant for surfaces.

17. A disinfectant for surfaces which comprises a poly 5 (hexamethylene biguanide) salt as an active agent.

18. A use, a composition or a method as claimed in any one of claims 1, 2, 5, 9 or 10 wherein the salt is present in a concentration of 0.02 % to 2.0 %, w/v or w/w (w/v for solutions, w/w for other formulations) .

10 19. A use, a composition or a method as claimed in any one of claims 1, 2, 3, 5, 6, 9, 10 or 11 wherein the salt is present in a concentration of 0.1 % to 1.0 %, w/v or w/w (w/v for solutions, w/w for other formulationε) .

20. The invention as claimed in any one of claims 1 to

15 19 wherein the poly(hexamethylene biguanide) salt iε poly (hexamethylene biguanide) hydrochloride.