WO2007012967A2

WO2007012967A2 - Combination of voriconazole and fluconazole

Info

Publication number: WO2007012967A2
Application number: PCT/IB2006/002148
Authority: WO
Inventors: Michael John Humphrey
Original assignee: Pfizer Limited
Priority date: 2005-07-27
Filing date: 2006-07-21
Publication date: 2007-02-01
Also published as: WO2007012967A3

Abstract

The invention provides a therapeutic combination comprising voriconazole and fluconazole in a 1:1 or 2:1 weight ratio. Pharmaceutical compositions, unit dosage forms and kits comprising voriconazole and fluconazole, and their use in the treatment of fungal infections, are also provided.

Description

Combination of voriconazole and fluconazole

Field of the Invention

This invention relates to a new combination therapy including voriconazole and fluconazole.

Background to the Invention

(2R,3S)-2-(2,4-Difluorophenyl)-3-(5-fluoro-4-pyrimidinyl)-1 -(1 H-1 ,2,4-triazol-1 -yl)-butan-2-ol, also known as voriconazole, is disclosed in EP-A-440372; see in particular Example 7. Voriconazole has the following structure:

and is useful in the treatment of fungal infections.

The pharmacokinetics of voriconazole are characterised by saturable metabolism resulting in non-linear increases in exposure with increasing dose levels. Furthermore, drug exposure varies to a significant extent between subjects. Voriconazole is metabolised by the cytochrome P450 isozymes CYP2C19, CYP2C9 and CYP3A4. The major circulating metabolite, the structure of which is given below, results from N-oxidation.

The metabolism of voriconazole is dependent to a large extent on the genotype of subjects being treated. One genotype metabolises voriconazole extensively, leading to rapid clearance of voriconazole from the body and, consequently, low plasma levels of voriconazole (ranging from about 0.6 to about 1.4 μg/ml). In this specification this genotype will be referred to as "extensive metabolisers". A second genotype can be characterised as a poor metaboliser of voriconazole: in this genotype, voriconazole is cleared much more slowly and therefore remains at higher levels in the body (ranging from about 3.5 to about 5.5μg/ml). In this specification this genotype will be referred to as "poor metabolisers". The genotyping is believed to be due to a recessive gene: homozygous extensive metabolisers make up about 73% of the Caucasian and 35% of the Japanese population; heterozygous extensive metabolisers make up about 25% of the Caucasian and 46% of the Japanese population. By contrast, poor metabolisers make up only about 2% of the Caucasian and about 19% of the Japanese population. It is now understood that the variability between genotypes with respect to the metabolism of voriconazole is dependent on the extent to which the enzyme cytochrome P450 2C19 (hereinafter referred to as CYP2C19) is present in the body: the enzyme CYP2C19 is present in extensive metabolisers, whereas poor metabolisers lack the functional enzyme. See, for example, M de Morals, G Wilkinson, J Blaisdell et al. J Biol Chem (1994), 269, 15419 -15422 (incorporated herein by reference).

In practice, voriconazole is administered to both extensive and poor metabolisers without dose adjustment. However, the need for voriconazole to be present in sufficient quantities in plasma to exert a therapeutic effect on extensive metabolisers requires a high dose of the drug: the usual recommended daily dose is 400mg (200mg twice a day). This dose in poor metabolisers results in higher systemic exposure which may lead to undesirable side effects. Moreover, the rapid clearance of the drug by extensive metabolisers requires the compound to be administered twice a day to enable it to maintain plasma levels throughout the day and exert a therapeutic effect. The need for twice-daily therapy raises compliance issues if voriconazole is to be self-administered by the patient. M. Ghannoum, N. Isham, M. Hossain and D. Sheehan, Int. J. Infect. Dis. (2002), Vol. 6 Supp. 2, 2S50 discloses in vitro combinations of voriconazole with antifungal agents including amphotericin B, Abelcet™, 5-fluorocytosine and fluconazole, and investigates their mechanistic synergy against a range of organisms. Combinations of voriconazole with fluconazole are stated to be 39% additive and 61% indifferent. M. Ghannoum, N. Isham and D. Sheehan, Abstracts of the lnterscience Conference on Antimicrobial Agents and Chemotherapy (2002) 42, 385 also discloses in vitro combinations of voriconazole with amphotericin B, Abelcet™, fluconazole, micafungin, ravuconazole and caspofungin. Combinations of voriconazole with fluconazole are stated to be 100% indifferent, i.e. no synergy of mechanism was observed. H.J. Scherpbier, M.I. Hilhorst and T.W. Kuijpers, Clin. Infect. Dis. (2003) 37, 828, discloses the treatment of an AIDS patient with a combination of antiretroviral drugs and report an interaction between protease inhibitors and voriconazole when the latter was added to a patient's therapy to treat oesophageal candidiasis. The interaction involved liver function impairment and elevated plasma concentrations of lopinavir, nevirapine and amprenavir. Plasma concentrations of voriconazole were not measured in the patients. N. Wood, K. Tan, L. Purkins, G. Layton, J. Hamlin, D. Kleinermans and D. Nichols, Br. J. Clin. Pharmacol. (2003) 56, 56 describes a study to determine the effects of the proton pump inhibitor omeprazole, a CYP2C19 inhibitor, on the steady state pharmacokinetics of voriconazole. The study concluded that omeprazole had no clinically relevant effect on voriconazole exposure, suggesting that no voriconazole dosage adjustment is necessary for patients in whom omeprazole therapy is initiated. Unpublished co-pending US patent application serial number 11/051 ,027 filed February 3, 2005 (incorporated herein by reference) discloses that co-administration of voriconazole and an antifungal CYP2C19 inhibitor, in particular fluconazole, markedly reduces metabolism in extensive metabolisers of voriconazole, causing the pharmacokinetic profile of such subjects to approximate to that of poor metabolisers. This results in markedly reduced intersubject variability and in therapeutic plasma levels being achieved at much lower doses of voriconazole. Voriconazole and the antifungal CYP2C19 inhibitor are administered in a weight ratio ranging from about 1 :4 to about 6:1 , preferably about 1 :2 to about 3:1 , and more preferably about 3:2 to about 5:2.

A lower dose of voriconazole will reduce overall exposure to the drug and likely reduce the incidence of side effects, especially in poor metabolisers. Ideally, the combination product of voriconazole and fluconazole should have linear kinetics between each dose level, thus enabling dose adjustments and drug-drug interactions to be managed more easily. The combination product should retain the efficacy of standard voriconazole therapy (400mg a day).

Surprisingly, the above mentioned objectives can be achieved by co-administration of specific amounts of voriconazole and fluconazole in a 1 :1 or 2:1 weight ratio. Summary of the Invention

The invention provides in a first aspect a therapeutic combination comprising voriconazole and fluconazole in specific amounts, defined in more detail hereinafter.

The invention also provides in a second aspect a pharmaceutical composition comprising a therapeutically effective amount of voriconazole and fluconazole, together with a pharmaceutically acceptable carrier or diluent.

The invention also provides in a third aspect a unit dosage form comprising a therapeutically effective amount of voriconazole and fluconazole.

The invention further provides in a fourth aspect a kit comprising a plurality of separate containers, wherein at least one container contains voriconazole and at least one different container contains fluconazole.

The invention additionally provides in a fifth aspect the use of the above combination, composition, kit or unit dosage form in the manufacture of a medicament for the treatment of a fungal infection in a mammal.

The invention also provides in a sixth aspect a method of treating a fungal infection in a mammal, comprising administering to a mammal in need of such treatment an effective amount of the above combination, composition, kit or unit dosage form.

Hereinafter the therapeutic combination, pharmaceutical composition, unit dosage form, kit, use and method of the present invention will be referred to jointly as "the combination of the present invention".

Brief Description of Drawings

Fig. 1 illustrates the pharmacokinetic profile of an extensive metaboliser of voriconazole when administered alone.

Fig. 2 illustrates the pharmacokinetic profile of a poor metaboliser of voriconazole when administered alone.

Fig. 3 illustrates the effect of fluconazole on N-oxide metabolite formation in vitro in human liver microsome HL-MIX 101 (control pool). Fig. 4 illustrates the effect of fluconazole on N-oxide metabolite formation in vitro in human liver microsome HH-92 (CYP2C19 poor metaboliser).

Fig. 5 illustrates the effect of fluconazole on N-oxide metabolite formation in vitro in human liver microsome HH-112 (CYP2C19 poor metaboliser).

Fig. 6 illustrates the effect of fluconazole on N-oxide metabolite formation in vitro in human liver microsome HH-100 (CYP2C19 extensive metaboliser).

Fig. 7 illustrates the effect of fluconazole on N-oxide metabolite formation in rCYP2C19. Fig. 8 illustrates the effect of fluconazole on N-oxide metabolite formation in rCYP3A4. Fig. 9 illustrates the effect of co-administered fluconazole on the steady state pharmacokinetics of voriconazole in young healthy volunteers. Detailed Description of Preferred Embodiments One part of the combination of the present invention is voriconazole. Voriconazole is disclosed in EP-A- 440372 (incorporated herein by reference); see in particular Example 7. As described in more detail below, voriconazole may be administered as the free base, or in the form of a salt, solvate or prodrug thereof. The other part of the combination of the present invention is fluconazole. Fluconazole is described in US 4,404,216, incorporated herein by reference. It has a Ki of 2μM - see L. C. Winkers, CJ. Wurden, E. Storch et al, Drug Metab Dispos (1996) 24, 610-4, incorporated herein by reference. Voriconazole and fluconazole are well-known antifungals and are approved for safe use in the same patient population. The overall antifungal effect depends, of course, on the specific infection being treated, the dose of voriconazole and fluconazole administered, and the age, sex, weight and condition of the patient being treated.

The combination of the present invention comprises voriconazole and fluconazole. Voriconazole and fluconazole may be administered as the free acid or base, or in the form of a pharmaceutically acceptable salt, solvate or prodrug thereof. Pharmaceutically acceptable salts of voriconazole and fluconazole include the acid addition and base salts thereof.

Suitable acid addition salts are formed from acids which form non-toxic salts. Examples include the acetate, adipate, aspartate, benzoate, besylate, bicarbonate/carbonate, bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate, hydrochloride/chloride, hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts. Suitable base salts are formed from bases which form non-toxic salts. Examples include the aluminium, arginine, benzathine, calcium, choline, diethylamine, diolamine, glycine, lysine, magnesium, meglumine, olamine, potassium, sodium, tromethamine and zinc salts.

For a review on suitable salts, see "Handbook of Pharmaceutical Salts: Properties, Selection, and Use" by Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002), incorporated herein by reference. A pharmaceutically acceptable salt of voriconazole or fluconazole may be readily prepared by mixing together solutions of voriconazole or fluconazole and the desired acid or base, as appropriate. The salt may precipitate from solution and be collected by filtration or may be recovered by evaporation of the solvent. The degree of ionisation in the salt may vary from completely ionised to almost non-ionised. Voriconazole and fluconazole may exist in both unsolvated and solvated forms. The term 'solvate' is used herein to describe a molecular complex comprising voriconazole and/or fluconazole and one or more pharmaceutically acceptable solvent molecules, for example, ethanol. The term 'hydrate' is employed when said solvent is water. Hereinafter all references to voriconazole and/or fluconazole include references to salts, solvates and solvates of salts thereof.

The combination of the invention includes voriconazole and fluconazole as hereinbefore defined, all polymorphs and crystal habits thereof, prodrugs and isomers thereof. As indicated, so-called 'prodrugs' of voriconazole and/or fluconazole are also within the scope of the invention. Thus certain derivatives of voriconazole and/or fluconazole which may have little or no pharmacological activity themselves can, when administered into or onto the body, be converted into compounds of formula (I) having the desired activity, for example, by hydrolytic cleavage. Such derivatives are referred to as 'prodrugs'. Further information on the use of prodrugs may be found in Pro- drugs as Novel Delivery Systems. Vol. 14, ACS Symposium Series (T. Higuchi and W. Stella) and

Bioreversible Carriers in Drug Design, Pergamon Press, 1987 (Ed. E. B. Roche, American

Pharmaceutical Association), both incorporated herein by reference.

Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in voriconazole and/or fluconazole with certain moieties known to those skilled in the art as 'pro-moieties' as described, for example, in "Design of Prodrugs" by H Bundgaard (Eisevier,

1985), incorporated herein by reference.

Voriconazole contains an alcohol functionality (-OH), therefore some examples of prodrugs in accordance with the invention may include an ester or ether thereof, for example, by replacement of the hydrogen by phosphorylation to provide (2R,3S)-2-(2,4-difluorophenyl)-3-(5-fluoro-4-pyrimtdinyl)-1-(1 H-1,2,4-triazol-1- yl)-2-butyl dihydrogen phosphate as described in WO 97/28169 (incorporated herein by reference); see in particular Example 3.

Similarly, fluconazole also contains an alcohol functionality (-OH), therefore some examples of prodrugs in accordance with the invention may include an ester or ether thereof, for example, by replacement of the hydrogen by phosphorylation to provide 2-(2,4-difluorophenyl)-1 ,3-bis(1 H-1 ,2,4-triazol-1-yl)-2-propyl dihydrogen phosphate as described in WO 97/28169; see in particular Example 1 , or a pharmaceutically acceptable salt thereof, especially the disodium salt (Prodif®). Further examples of replacement groups in accordance with the foregoing examples and examples of other prodrug types may be found in the aforementioned references.

The combination of the invention comprises specific amounts of fluconazole and voriconazole such that the weight ratio of voriconazole to fluconazole is about 1 :1 or 2:1.

In one embodiment, the combination of the invention comprises 60mg of fluconazole and 120mg of voriconazole.

In a further embodiment, the combination of the invention comprises 75mg of fluconazole and 150mg of voriconazole. In yet a further embodiment, the combination of the invention comprises 90mg of fluconazole and 180mg of voriconazole.

In yet a further embodiment, the combination of the invention comprises 105mg of fluconazole and

210mg of voriconazole.

In yet a further embodiment, the combination of the invention comprises 120mg of fluconazole and 240mg of voriconazole. In yet a further embodiment, the combination of the invention comprises 120mg of fluconazole and 120mg of voriconazole.

In one embodiment, the combination of the present invention can be administered to a patient once a day. This confers the particular advantage of better patient compliance. For example: a single dose of 120mg of fluconazole and a single dose of 240mg of voriconazole can be administered once or day. In another embodiment, the combination of the present invention can be administered to a patient twice a day. For example, doses of 60mg of fluconazole and of 120mg of voriconazole can be administered twice a day. Alternatively, doses of 120mg of fluconazole and of 120mg of voriconazole can be administered twice a day. Fungal infections which may be treated by the combination of the invention have been extensively described in the literature, including EP-A-440372, and include topical infections, mucosal infections, such as vaginal candidiasis, oesophageal and oropharyngeal candidiasis, and systemic infections. Furthermore, the combination of the invention may be used to treat allergic reactions, such as allergic rhinosinusitis. Fungal infections which may be treated by the combination of the invention include those caused by, inter alia, Candida spp, Trichophyton spp, Microsporum spp, Epidermophyton floccosum, Cryptococcus neoformans, Aspergillus spp, Fusarium spp, Scedosporium spp, Coccidioides immitis, Paracoccidioides brasiliensis, Histoplasma spp, Blastomyces dermatiditis, Alternaria spp, Exophiala spp, Fonsecaea pedrosoi, Penicillium marneffei, Phialophora spp or Paecilomyces lilacinus. It will be appreciated that reference to treatment is intended to include prophylaxis as well as the alleviation of established symptoms.

In the combination of the present invention, voriconazole and fluconazole may be administered, in terms of dosage forms, either separately or in conjunction with each other; and in terms of their time of administration, either simultaneously or sequentially. Thus, the administration of voriconazole may be prior to, concurrent with, or subsequent to the administration of fluconazole. The time in between administration may vary within a 24-hour dosing interval.

The unit dosage form of the invention is a dosage form in which both voriconazole and fluconazole are present. It may be a solid formulation for oral administration such as a tablet, a capsule containing a particulate, liquid, or powder, a lozenge (including liquid-filled), a chew, a gel, a solid solution, a liposome, a film (including muco-adhesive), an ovule, a spray or a liquid formulation, a parenteral formulation (typically an aqueous solution which may contain excipients as defined hereinbelow), or a formulation for topical administration to the skin or mucosa, (ie dermally or transdermal^) such as a hydrogel, a lotion, a solution, a cream, an ointment, a dusting powder, a dressing, a foam, a skin patch, a wafer, an implant, a sponge, a fibre, a bandage or a microemulsion. Preferably the unit dosage form of the invention is a tablet or capsule, especially a tablet, containing voriconazole and fluconazole. Compounds of the combination of the invention intended for pharmaceutical use may be administered as crystalline or amorphous products. They may be obtained, for example, as solid plugs, powders, or films by methods such as precipitation, crystallization, freeze drying, spray drying, or evaporative drying. Microwave or radio frequency drying may be used for this purpose. Generally, the combination of the invention will be administered as a formulation in association with one or more pharmaceutically acceptable excipients. The term "excipient" is used herein to describe any ingredient other than the compounds of the invention. The choice of excipient will to a large extent depend on factors such as the particular mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

Pharmaceutical compositions suitable for the delivery of compounds of the present invention and methods for their preparation will be readily apparent to those skilled in the art. Such compositions and methods for their preparation may be found, for example, in 'Remington's Pharmaceutical Sciences', 19th Edition (Mack Publishing Company, 1995), incorporated herein by reference.

The compounds of the combination of the invention may be administered orally. Oral administration may involve swallowing, so that the compounds enter the gastrointestinal tract, or buccal or sublingual administration may be employed by which the compounds enter the blood stream directly from the mouth. Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids, or powders, lozenges (including liquid-filled), chews, multi- and nano- particulates, gels, solid solution, liposome, films (including muco-adhesive), ovules, sprays and liquid formulations. Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet. The compounds of the combination of the invention may also be used in fast-dissolving, fast- disintegrating dosage forms such as those described in Expert Opinion in Therapeutic Patents, JM_, (6), 981-986 by Liang and Chen (2001), incorporated herein by reference.

For tablet dosage forms, depending on dose, the drug may make up from 1 wt% to 80 wt% of the dosage form, more typically from 5 wt% to 60 wt% of the dosage form. In addition to the drug, tablets generally contain a disintegrant. Examples of disintegrants include sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium alginate. Generally, the disintegrant will comprise from 1 wt% to 25 wt%, preferably from 5 wt% to 20 wt% of the dosage form. Binders are generally used to impart cohesive qualities to a tablet formulation. Suitable binders include microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, hydroxypropyl cellulose and hydroxypropyl methylcellulose. Tablets may also contain diluents, such as lactose (monohydrate, spray-dried monohydrate, anhydrous and the like), mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate. Tablets may also optionally include surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc. When present, surface active agents may comprise from 0.2 wt% to 5 wt% of the tablet, and glidants may comprise from 0.2 wt% to 1 wt% of the tablet. Tablets also generally contain lubricants such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants generally comprise from 0.25 wt% to 10 wt%, preferably from 0.5 wt% to 3 wt% of the tablet. Other possible ingredients include antioxidants, colourants, flavouring agents, preservatives and taste- masking agents.

Exemplary tablets contain up to about 80% drug, from about 10 wt% to about 90 wt% binder, from about 0 wt% to about 85 wt% diluent, from about 2 wt% to about 10 wt% disintegrant, and from about 0.25 wt% to about 10 wt% lubricant.

Tablet blends may be compressed directly or by roller to form tablets. Tablet blends or portions of blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tabletting. The final formulation may comprise one or more layers and may be coated or uncoated; it may even be encapsulated. The formulation of tablets is discussed in "Pharmaceutical Dosage Forms: Tablets, Vol. 1", by H. Lieberman and L. Lachman, Marcel Dekker, N.Y., N.Y., 1980 (ISBN 0-8247-6918-X), incorporated herein by reference.

Solid formulations for oral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

Suitable modified release formulations for the purposes of the invention are described in US Patent No. 6,106,864, incorporated herein by reference. Details of other suitable release technologies such as high energy dispersions and osmotic and coated particles are to be found in Verma et a/, Pharmaceutical Technology On-line, 25(2), 1-14 (2001), incorporated herein by reference. The use of chewing gum to achieve controlled release is described in WO 00/35298, incorporated herein by reference.

The compounds of the combination of the invention may also be administered directly into the blood stream, into muscle, or into an internal organ. Suitable means for parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrastemal, intracranial, intramuscular and subcutaneous. Suitable devices for parenteral administration include needle (including microneedle) injectors, needle-free injectors and infusion techniques. An example of a needle free injection is Powderject™.

Parenteral formulations are typically aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably, to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a powdered, dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.

The preparation of parenteral formulations under sterile conditions, for example, by lyophilisation, may readily be accomplished using standard pharmaceutical techniques well known to those skilled in the art. The solubility of voriconazole and fluconazole used in the preparation of parenteral solutions may be increased by the use of appropriate formulation techniques, such as the incorporation of solubility- enhancing agents. Formulations for use with needle-free injection administration comprise a compound of the invention in powdered form in conjunction with a suitable vehicle such as sterile, pyrogen-free water. For example, the aqueous solubility of voriconazole may be increased by formulating it with one or more poloxamers as described in WO 2005/051353 (incorporated herein by reference). Alternatively, the aqueous solubility of voriconazole may be increased by formulating it with a sulfobutyl ether cyclodextrin such as those disclosed in WO 91/11172 and WO 94/02518 (incorporated herein by reference). A formulation of voriconazole with a sulfobutyl ether cyclodextrin is described in WO 98/58677 (incorporated herein by reference).

Formulations for parenteral administration may be formulated to be immediate and/or modified/controlled release. Controlled/modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release. Thus compounds of the combination of the invention may be formulated as a solid, semi-solid, or thixotropic liquid for administration as an implanted depot providing modified release of the active compound. Examples of such formulations include drug-coated stents and

PGLA microspheres.

The compounds of the combination of the invention may also be administered topically to the skin or mucosa, that is, dermally or transdermally. Typical formulations for this purpose include gels, hydrogels, lotions, solutions, creams, ointments, dusting powders, dressings, foams, films, skin patches, wafers, implants, sponges, fibres, bandages and microemulsions. Liposomes may also be used. Typical carriers include alcohol, water, mineral oil, liquid petrolatum, white petrolatum, glycerin, polyethylene glycol and propylene glycol. Penetration enhancers may be incorporated - see, for example, J Pharm Sci, 88 (10), 955-958 by Finnin and Morgan (October 1999), incorporated herein by reference. Topical administration may also be achieved using a patch, such as a transdermal iontophoretic patch.

Other means of topical administration include delivery by electroporation, iontophoresis, phoπophoresis, sonophoresis and microneedle or needle-free (e.g. Powderject™, Bioject™, etc.) injection.

Formulations for topical administration may be formulated to be immediate and/or modified/controlled release. Controlled/modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

It is within the scope of the present invention that compositions containing voriconazole and fluconazole may conveniently be combined in the form of a kit suitable for coadministration of the compositions.

Thus the kit of the invention comprises two or more separate pharmaceutical compositions, at least one of which contains voriconazole and another contains fluconazole, and means for separately retaining said compositions, such as a container, divided bottle, or divided foil packet. An example of such a kit is the familiar blister pack used for the packaging of tablets, capsules and the like.

The kit of the invention is particularly suitable for administering different dosage forms, for example, oral and parenteral, for administering the separate compositions at different dosage intervals, or for titrating the separate compositions against one another. To assist compliance, the kit typically comprises directions for administration and may be provided with a so-called memory aid.

The invention is illustrated by the following examples.

Example 1: Inhibition of Cytochrome P450 activity in human liver microsomes by fluconazole The data in Table 1 below were determined according to the method described in Meier et al. (1985) Anal.Biochem. 151 , 286-291 , and in Funa and lmaoka (1987) Biochem. Biophys. Acta. 926, 349-358, both incorporated herein by reference.

TABLE 1

As demonstrated by the data above, fluconazole exhibits selective inhibition of the enzyme CYP2C19 over CYP3A4.

Example 2: In vitro metabolism of voriconazole in human liver microsomes and rCYP2C19 and rCYP3A4. Selectivity of enzyme inhibition by fluconazole

The following incubation mix (final concentrations) was used for all assays described in this study: 5OmM potassium phosphate buffer (pH 7.4), 5mM MgCI₂, 5mM isocitric acid, 1 U/ml_ isocitric acid dehydrogenase. Reducing equivalents required for P450 metabolism were provided by NADPH, which was regenerated using isocitric acid/isocitric acid dehydrogenase. Time Course Studies

Incubations were performed in the following human liver microsome preparations: control batch HL-MIX- 101 (prepared from a pool of 60 donors); two donors genotyped as CYP2C19 poor metabolisers (HH-92, HH-112) and a CYP2C19 extensive metaboliser with high CYP2C19 activity (HH-100). Relative CYP2C19 and CYP3A4 activities are shown in Table 2 below:

TABLE 2

Human liver microsome characteristics

CYP3A4 CYP2C19

CYP2C19 activity activity genotype Protein Total P450 (nmol/min/mg (nmol/min/mg

(mg/mL) (pmol/mL) protein) protein)

HL-MIX-101 Mixed pool 20.4 16 5.2 0.050

HH-92 PM 17.6 14 8.2 0.004

HH-112 PM 20.9 9 1.6 0.0012

HH-100 EM 18.7 7 3.8 0.18

(PM = poor metaboliser; EM = extensive metaboliser)

Additional incubations were performed with microsomes prepared from insect cells transfected with recombinant CYP2C19 and CYP3A4 (BDGENTEST® supersomes and Panvera baculosomes, respectively).

A series of preliminary studies were required to ensure the formation of the N-oxide metabolite was linear during the incubation period using the aforementioned reaction mixture. Here human liver microsomes

(1 mg/mL) were pre-incubated in the presence of the substrate voriconazole (25μM final concentration) prior to the addition of the NADPH. Note for studies using rCYP2C19 and rCYP3A4, pre-incubations were performed with NADPH and the reaction initiated by the addition of the substrate voriconazole. Aliquots (1mL) of the reaction mixture were collected over time (0-60mins) and added to 4mL of dichloromethane containing 50μl_ of internal standard (2-(2,4-difluorophenyl)-3-(4-pyrimidyl)-1-(1H-1,2,4- triazol-1-yl)-2-butanol; 15μg/mL). Samples were rotary mixed for 10 minutes, and then centrifuged at 3000 rpm for 5min (4°C). The upper aqueous layers were removed and discarded. The lower organic layer was evaporated to dryness at 37°C under a stream of N₂. Samples were subsequently reconstituted in 100μL mobile phase A (see below) and 25μL injected onto the HPLC.

Samples were analysed on an Agilent 1100 Series UV-HPLC. Chromatographic separation was performed using a Hichrom 100-5C18 column (150 x 4.6 mm) at a flow rate of 1mL/ min with UV- λ set at 254nm. The mobile phases used were: (A) 0.1 M ammonium phosphate in 70:30 H₂O:MeOH, (pH adjusted to 7.0, prior to the addition of MeOH) and (B) MeOH, using the following gradient: 0-2min (0% B), 2-20 (0-100% B), 20-23 (100% B), 23.1 (0% B). Column was allowed to re-equilibrate for 7min prior to next injection. Typical retention times were 7.9min for fluconazole; 11.7min for the N-oxide metabolite; 13.1min for the internal standard and 14.5min for voriconazole. The inhibitory potential of fluconazole against voriconazole N-oxidation was investigated in human liver microsome and rCYP2C19 and rCYP3A4 preparations. The final concentrations of fluconazole studied were 0, 0.1, 1 , 10, 100 and 1000μM.

Human liver microsomes were incubated at 1mg/mL for 60mins, rCYP2C19 at IOpmol CYP/mL for 60min and rCYP3A4 at IOOpmol CYP/mL for 20min. For each matrix, aliquots (4mL) of the reaction mixture were pre-warmed at 37°C in a waterbath followed by the addition of 200μL NADPH (2OmM) and 40μL of respective fluconazole solutions (0-10OmM). Reactions were initiated by the addition of 50μL voriconazole (2mM). At the end of the incubation time aliquots (n=3) were taken from each incubation mix and added into tubes containing 4mL of dichloromethane and 50μL of internal standard (2-(2,4-difluorophenyl)-3-(4- pyrimidyl)-1-(1H-1 ,2,4-triazol-1-yl)-2-butanol; 15μg/mL)_: The extraction and analysis procedures followed that described above.

At the concentrations reported, formation of the N-oxide metabolite was linear to 60min in human liver microsomes preparations and rCYP2C19. In rCYP3A4 linearity was measured to 20min. The inhibitory potential (IC₅₀) of fluconazole against metabolism of voriconazole to its N-oxide metabolite was studied in a pool of human liver microsomes (HL-MIX-101) as well as individual donors genotyped as being poor (HH-92 and HH-112) or extensive CYP2C19 metabolisers (HH-100). Furthermore the CYP2C19 PM's were subdivided as having either high and low CYP3A4 metabolic capacities. To better define the selectivity of fluconazole for inhibition of CYP3A4- or CYP2C19-mediated metabolism of voriconazole, rCYP3A4 or rCYP2C19 were also investigated. Results are summarised in Figures 3-8 and collated in Table 3, where concentrations of the N-oxide metabolite are given as values relative to the internal standard (IS). TABLE 3

ICgn determination for fluconazole in human liver microsomes (CYP2C19 PM & EM'S) and rCYP2C19 and

_/ rCYP —3 —A —4 -

HL-MIX-101 (mixed pool) 82

HH-92 (poor metaboliser) 214

HH-112 (poor metaboliser) 175

HH-100 (extensive metaboliser) 50 rCYP2C19 29 rCYP3A4 106

In CYP2C19 poor metaboliser human liver microsomes fluconazole is a weak inhibitor of voriconazole N- oxidation (IC₅₀~ 175μM and 214μM). More potent inhibitions were observed in the CYP2C19 extensive metaboliser microsomes (IC₅₀- 50μM). Experiments using rCYP2C19 and rCYP3A4 demonstrate fluconazole to be a more potent inhibitor of CYP2C19 mediated N-oxidation (IC₅₀~ 29μM) compared to CYP3A4 (IC₅₀- 106μM).

The overall finding from these experiments highlights a 3-4 fold selectivity for fluconazole towards inhibition of CYP2C19 mediated voriconazole N-oxidation over CYP3A4 mediated N-oxidation.

Example 3: Clinical trial

A study was conducted to investigate the effect of co-administered fluconazole on steady state pharmacokinetics of voriconazole in young healthy volunteers and to assess the safety and toleration of co-administered fluconazole and voriconazole. Ten healthy human subjects aged 21-55 were recruited to ensure at least 8 subjects, all extensive metaboliser phenotype, completed the study. The subjects received sequential treatment as follows:

1. dosing with voriconazole (120mg QD) to achieve steady-state by day 4,

2. addition of 60mg QD of fluconazole to the dosage regimen to achieve steady state by about day 9,

3. increasing the daily dose of fluconazole and voriconazole to 120 mg and 240 mg respectively to achieve steady state by day 14. Blood samples were taken on Days 4, 9 and 14 of the treatment schedule to provide plasma concentrations of voriconazole for the calculation of various pharmacokinetic parameters, including C_max, Tmax_> AUC and trough plasma levels for the end of each treatment period. These parameters were then used to compare voriconazole alone versus voriconazole plus fluconazole, and the effect of increasing dose on intersubject variability and the target levels for efficacy on a QD dosing regimen. The results of the clinical trial are shown in Figure 9 and Table 4 overpage. TABLE 4

A study to investigate the effect of co-administered fluconazole on steady state pharmacokinetics of voriconazole in young healthy volunteers

Parameter Voriconazole 120mg Voriconazole 120mg + Voriconazole 240mg + Fluconazole 60mg Fluconazole 120mg

AUCo-₂4 (ng.h/mL) 2851 7820 28396

C_max^a (ng/mL) 679.7 983.5 2572

C₂₄ ^a (ng/mL) 13.25 101.5 552.3

' Geometric mean; ^D Arithmetic mean

The results of the clinical trial unambiguously demonstrate that voriconazole exposure (AUC) is increased following co-administration of fluconazole. Indeed, comparing voriconazole 120mg to voriconazole 120mg + fluconazole 60mg indicates that voriconazole exposure is increased by approximately 275% when co-administered with fluconazole.

Significantly, the plasma concentrations of voriconazole during the voriconazole 240mg + fluconazole 120mg part of the study clearly indicate the potential for once a once a day (i.e. QD) voriconazole treatment, recognizing that the normal treatment for voriconazole, as mentioned at page 3, is a twice daily dose of 200mg (i.e. a total of 400mg). Thus administration of voriconazole 240mg + fluconazole 120mg has the potential to allow both a lower daily dose of voriconazole (i.e. a voriconazole sparing dose) together with the convenience of a once daily voriconazole treatment.

Finally, intersubject variability was significantly reduced. This would have been even more apparent if poor metabolisers had been included in the study.

Claims

1. A therapeutic combination comprising voriconazole and fluconazole, wherein voriconazole and fluconazole are present in the following amounts:

- 60mg of fluconazole and 120mg of voriconazole; or 75mg of fluconazole and 150mg of voriconazole; or 90mg of fluconazole and 180mg of voriconazole; or 105mg of fluconazole and 21 Omg of voriconazole; or - 120mg of fluconazole and 240mg of voriconazole; or

120mg of fluconazole and 120mg of voriconazole.

2. The therapeutic combination of claim 1 , wherein fluconazole is present in an amount of 60mg and voriconazole is present in an amount of 120mg.

3. The therapeutic combination of claim 1 , wherein fluconazole is present in an amount of 120mg and voriconazole is present in an amount of 240mg.

4. The therapeutic combination of claim 1 , wherein fluconazole is present in an amount of 120mg and voriconazole is present in an amount of 120mg.

5. A pharmaceutical composition comprising a therapeutically effective amount of voriconazole and fluconazole, together with a pharmaceutically acceptable carrier or diluent, wherein voriconazole and fluconazole are present in the amounts given in any of claims 1 to 4.

6. A kit comprising a plurality of separate containers, wherein at least one container contains voriconazole and at least one different container contains fluconazole, wherein voriconazole and fluconazole are present in the amounts given in any of claims 1 to 4.

7. A unit dosage form comprising a therapeutically effective amount of voriconazole and fluconazole, wherein voriconazole and fluconazole are present in the amounts given in any of claims 1 to 4.

8. The use of a combination according to any of claims 1 to 4, a composition according to claim 5, a kit according to claim 6 or a unit dosage form according to claim 7, in the manufacture of a medicament for the treatment of a fungal infection in a mammal.

9. A method of treating a fungal infection in a mammal, comprising administering to a mammal in need of such treatment a combination according to any of claims 1 to 4, a composition according to claim 5, a kit according to claim 6 or a unit dosage form according to claim 7.