Antimicrobial Agents and Chemotherapy, September 2004, p . 3272-3278, Vol . 48, No . 9

 
The antifungal activities of azole derivatives such as miconazole, clotrimazole, bifonazole, ketoconazole, itraconazole, fluconazole, and voriconazole arise from a complex multimechanistic process initiated by the inhibition of a cytochrome P450 (CYP) involved in the biosynthesis of ergosterol, namely, CYP51 (also called Erg11p, according to different gene-based nomenclatures) (6, 16, 20, 24) . CYP51 (the product of the CYP51 gene) catalyzes the oxidative removal of the 14-methyl group of lanosterol or eburicol .

Azole antifungal agents also affect other CYPs . At concentrations >100 nM, ketoconazole inhibits not only the mammalian CYP51 but also the 17-hydroxylase-17,20-lyase (CYP17), the cholesterol side chain cleavage enzyme (CYP11A1), and the 11ß-hydroxylase (CYP11B1) (18) . Itraconazole is almost devoid of effects on steroid metabolism (19) . However, like ketoconazole (10, 29), itraconazole inhibits CYP3A4, a major drug-metabolizing P450 isoform in the human liver (10, 29) . Lamb et al . (10) compared the inhibition by ketoconazole and itraconazole of human CYP3A4 and C . albicans CYP51 following heterologous expression in Saccharomyces cerevisiae . The 50% inhibitory concentrations (IC₅₀s) of ketoconazole and itraconazole for CYP3A4 inhibition were 250 and 200 nM, respectively . The IC₅₀s of ketoconazole and itraconazole for CYP51 inhibition were 8 and 7.6 nM, respectively . Fluconazole, miconazole (29), and voriconazole (6) are inhibitors of CYP2C9, which plays a major role in the metabolism of commonly prescribed drugs, such as phenytoin, S-warfarin, and a range of nonsteroidal anti-inflammatory drugs (11) . Voriconazole also inhibits CYP2C19 (S-mephenytoin hydroxylase) and CYP3A4 (6, 7) . The ideal azole antifungals are those that react strongly with fungal CYP51 and that have weak activities against mammalian CYPs .

Preclinical studies of new triazoles often consist of an in vitro test to help select congeners that will have the least drug-drug interactions in clinical trials .

In this study we describe the effects of R126638 and itraconazole on ergosterol synthesis in C . albicans, T . mentagrophytes, Trichophyton rubrum, and M . canis and on CYP-mediated reactions in mammalian cells, human liver and placental microsomes, rat testis microsomes, bovine adrenocortical and rat kidney mitochondria, and human skin epidermis .

Cells. he C . albicans (B2630), T . rubrum (B68183), T . mentagrophytes (B32663), and M . canis (B68128) isolates were from the Janssen Pharmaceutica collection . The mammalian cells used were human hepatoma cells (HepG2, ATCC HB 8065), human tongue squamous carcinoma cells (SCC25, ATCC CRL 1628), and HD-11 cells (v-myc-transformed chicken myelomonocytic cell line; a gift from Thomas Graf, European Molecular Biology Laboratory, Heidelberg, Germany) .

Inoculum preparation and culture conditions. C . albicans (2.5 x 10⁷ cells) was grown in 100 ml of CYG medium (casein hydrolysate [Merck, Darmstadt, Germany], yeast extract [Difco, Detroit, Mich.], and glucose, each at a concentration of 5 g liter^–1) in 500-ml Erlenmeyer flasks in a reciprocating shaker, set at 100 strokes per min, at 37°C (9) . T . rubrum, T . mentagrophytes, and M . canis were grown at 30°C on potato dextrose agar (39 g liter^–1; Difco) .

Ergosterol synthesis experiments. To study the effects of R126638 and itraconazole on ergosterol synthesis in C . albicans, 100 ml of CYG medium was supplemented with 5 µCi of sodium [¹⁴C]acetate (specific activity, 58 µCi mmol^–1; Radiochemical Center, Amersham, United Kingdom) and different concentrations of an azole antifungal and/or DMSO immediately before inoculation (12) . Inoculation (2.5 x 10⁵ cells ml^–1) and growth conditions were as described above . After 24 h of growth, cell concentrations were determined with a Coulter counter, as described earlier (21) . Cells were collected by centrifugation at 1,500 x g, and the cell pellet was washed twice with distilled water . The pellets were suspended in 8 ml of water and added to 5 g of acid-washed glass beads (diameter, 0.40 to 0.45 mm) in Packard Super polyethylene vials . Cells were shaken vigorously in a Retsch laboratory mixer mill 2000 set at maximum speed for 5 min . The homogenates were quantitatively separated from the glass beads, supplemented with 15% KOH dissolved in 90% ethanol, and saponified at 85°C . Nonsaponifiable lipids were extracted with 1 volume of n-heptane (spectrograde) (12) .

M . canis, T . mentagrophytes, and T . rubrum conidia were collected after 2 to 3 weeks of growth by pipetting a solution of 0.01% Tween 40 onto the agar surface, followed by agitation . The culture suspensions were transferred to 500-ml Erlenmeyer flasks containing three layers of sterile glass beads (diameter, 0.40 to 0.45 mm) . The flasks were shaken on a gyratory shaker at 250 rpm for 30 min, after which the suspensions were filtered through sterile glass wool and the filtrate was centrifuged aseptically for 20 min at 5,000 x g . The pellets were washed twice with physiological saline . To evaluate the effects of R126638 and itraconazole on ergosterol synthesis, T . mentagrophytes (2.5 x 10⁵ conidia), T . rubrum (5 x 10⁵ conidia), or M . canis (5 x 10⁵ conidia) was inoculated in 75 ml of PYG medium, which contained 10 g of polypeptone, 10 g of yeast extract, and 40 g of glucose per liter, and were grown in 175-cm² Falcon culture flasks at 30°C . Itraconazole or R126638 and/or DMSO was added to the medium immediately before inoculation . After 32 h (T . mentagrophytes), 40 h (T . rubrum), and 72 h (M . canis) of growth, 5 µCi of sodium [¹⁴C]acetate was added . T . mentagrophytes was grown for another 16 h, and T . rubrum and M . canis were grown for another 24 h . At the end of the incubation period, mycelium was collected by filtration on 8-µm-pore-size Millipore HAWP filters and washed with distilled water . The pellets were homogenized and saponified, and the nonsaponifiable lipids were extracted as described above for C . albicans . The total radioactivity in the extracts was determined by liquid scintillation counting (22) .

The heptane extracts were dried under a stream of nitrogen and dissolved in minimal volumes of methanol-water (95:5) . Sterols were separated by high-performance liquid chromatography (HPLC) on a Varian 9010 liquid chromatograph equipped with a Varian 9095 automatic injector, a Varian 9065 Polychrom detector, and a Berthold LB507A HPLC radioactivity monitor with Pico Aqua (Canberra Packard) as a scintillant and connected to a Compaq 386/33 computer (23) .

Cholesterol synthesis. The effects of R126638 and itraconazole on cholesterol synthesis from sodium [¹⁴C]acetate in human hepatoma cells (HepG2) were studied as described previously (22) . Cholesterol and its precursors were separated by thin-layer chromatography on precoated silica gel plates (no . 5554-60F254; Merck) and developed in a solvent system consisting of heptane-diisopropylether-acetic acid-ethyl acetate (60:40:4:34.7; vol/vol/vol/vol) . To visualize the radioactive fractions, the thin-layer chromatography plates were exposed to Kodak autoradiograph film for 3 days . The fractions were quantitated by digitizing the films with an RX5 video camera attached to a Macintosh IIfx computer . Integration of the density was done by means of Image (version 1.28) software .

Cholesterol side chain cleavage, 11ß-hydroxylase, and androgen and estrogen biosynthesis. Cholesterol side chain cleavage and 11ß-hydroxylase were studied by using extracts from sonicated bovine adrenocortical mitochondria (25) .

Androgen biosynthesis from [¹⁴C]pregnenolone in S 10,000 fractions (microsomes plus cytosol) of Wistar rat testes was studied as described previously (26) .

The conversion of [³H]androstenedione to estrone by aromatase in human placental microsomes was carried out as described previously (22) .

Retinoic acid metabolism. Human skin was obtained from patients undergoing plastic surgery at local hospitals and was immediately transported to the laboratory in sterile transport medium (T medium) containing phosphate-buffered saline (PBS; Gibco) with calcium (CaCl₂ · 2H₂O; 0.132 g liter^–1) and magnesium (MgCl₂ · 6H₂O; 0.10 g liter^–1) but without sodium bicarbonate . T medium also contained 10 µg of gentamicin ml of PBS^–1 . Upon arrival, the skin was washed three times with T medium, and the adipose tissue was scraped off . Punch specimens of 0.8 cm in diameter were obtained with a Keyes' skin puncher and placed in cold normal human keratinocyte medium (NHK medium) (27) . NHK medium contained 3 parts Dulbecco's modified Eagle's medium (DMEM; Gibco) and 1 part Ham's F-12 medium (Gibco) plus 10% fetal calf serum (FCS), 2 mM L-glutamine, 2 nM triiodothyronine (T3), 0.18 mM adenine, 0.1 nM cholera toxin, 5 µg of gentamicin, 0.4 µg of hydrocortisone, 5 µg of insulin, 5 µg of transferrin, and 10 ng of epidermal growth factor per ml of medium . Fifty skin punch specimens (diameter, 8 mm) were incubated overnight at 4°C in 15 ml of PBS (without calcium or magnesium) containing 2 ml of Dispase II (24 U ml^–1; Boehringer, Mannheim, Germany) . After incubation, the epidermis was separated from the dermis with sterile forceps and placed in a six-well plate (two epidermal sheets per well), dermal side down, on 2 ml of NHK medium containing 2 µl of drug and/or DMSO and 1 µCi of [11,12-³H(N)]all-trans retinoic acid (20 pmol; specific activity, 50.7 Ci mmol^–1; New England Nuclear, Boston, Mass.) . After 24 h of incubation at 37°C in a 5% CO₂-humidified atmosphere, the medium was extracted for 30 min with 5 ml of ethyl acetate containing 0.005% (wt/vol) butylated hydroxytoluene (BHT) . After evaporation of the organic solvent the samples were analyzed by HPLC as described by Van Wauwe et al . (28) . All operations were carried out in a darkened room illuminated with yellow light .

25-Hydroxyvitamin D₃-1-hydroxylase. v-myc-transformed chicken myelomonocytic (HD-11) cells (1) were grown on DMEM supplemented with 4 mM glutamine, 10% FCS, and 1% antibiotica-antimycotica mixture (Gibco) . Monolayers of HD-11 cells were preincubated for 16 h in serum-free medium, after which the medium was replaced by 2 ml of fresh medium, to which drugs and/or DMSO was added . The reaction was started by adding 100 pmol of 25-hydroxyvitamin D₃, of which 0.3 pmol was [26,27-³H]25-hydroxyvitamin D₃ (specific activity, 165 Ci.mmol^–1; New England Nuclear) . At the end of the 3-h incubation period, the medium was transferred into a brown test tube containing 3 ml of chloroform and 0.5 ml of 10% formic acid . The cells were trypsinized with 1 ml of 0.125% trypsin-0.01% EDTA, and after 10 to 15 min of incubation at room temperature, the trypsinized cells were combined with the medium-chloroform mixture . The six-well plates were washed twice with 1.5 ml of methanol . After 20 min of extraction, the chloroform layer was separated by centrifugation (10 min at 2,400 rpm [MSE-GF8 centrifuge]) and dried under a stream of nitrogen . 25-Hydroxyvitamin D₃ and its metabolites were separated by HPLC (Zorbax 5Si column; 250 by 4.5 mm; 5 µm; 30°C; flow rate, 1 ml min^–1) by a modification of the method described by Jones et al . (9) .

25-Hydroxyvitamin D₃-24-hydroxylase. Male Wistar rats (weight, 200 to 220 g) were pretreated (intraperitoneally) with 1,25-dihydroxyvitamin D₃ (1 µg kg of body weight^–1 per day) for three consecutive days . The animals were left for the last 24 h without food but with free access to water (8) . Kidney mitochondria were isolated as described by Vieth and Fraser (30) . They were suspended in ice-cold incubation medium, composed of 50 mM Tris-acetate buffer (pH 7.42), 10 mM MgCl₂ · 6H₂O, 12 mM isocitrate, and 2.5 µg of diphenyl-p-phenylenediamine . Incubations were carried out in brown tubes containing the following in a final volume of 1 ml of medium to which drugs and/or DMSO was added: 2 mg of mitochondrial protein, 500 pmol of 25-hydroxyvitamin D₃, and 0.05 µCi of [26,27-³H]25-hydroxyvitamin D₃ (specific activity, 165 Ci mmol^–1; New England Nuclear) . After incubation at 37°C for 30 min in a shaking water bath, the reaction was stopped by addition of 3.75 ml of methanol-chloroform (1:2; vol/vol) containing 0.01% BHT . Extraction and separation of the metabolites were performed as described above .

Catabolism of 1,25-dihydroxyvitamin D₃. Squamous carcinoma (SCC25) cells were seeded at a density of 2 x 10⁵ cells per 2 ml in six-well plates on a 1:1 mixture of Ham's F-12 medium and DMEM (Gibco) containing 0.36 µg of hydrocortisone ml^–1 and 10% FCS . The cells were grown for 4 days at 37°C in a humidified 5% CO₂ atmosphere . At the end of the incubation period, the medium was replaced by serum-free keratinocyte medium (Gibco), and the confluent cells were incubated for an additional 3 days .

Human hepatoma (HepG2) cells were seeded at a density of 10⁵ cells ml^–1 in six-well plates (2 ml of cell suspension per well) and grown for 1 week in MEM-Rega 3 medium (Gibco) containing 10% FCS .

Sixteen hours before and at the onset of the experiment, the media were replaced by 2 ml of serum-free keratinocyte medium (SCC25 cells) or serum-free MEM-Rega 3 medium (HepG2 cells) . The reaction was initiated by adding 0.1 µCi of 1,25-[26, 27-³H]dihydroxyvitamin D₃ (specific activity, 170 Ci mmol^–1) in 10 µl of ethanol and 2 µl of drug and/or DMSO . At the end of a 3-h incubation period, substrate and metabolites were extracted and separated as described above .

Effects of R126638 on oxidative drug metabolism by human liver microsomes. Microsomes were prepared from four human livers as described previously (3) . All inhibition experiments were performed in a pooled batch of microsomes, which was characterized for the different CYP activities (3) . Incubations were performed in triplicate at concentrations of 0, 1, 3, 10, and 30 mM R126638 for the CYP3A4 substrates cyclosporine, testosterone, and midazolam and the CYP2C19 substrate S-mephenytoin . Itraconazole was included as a reference compound . Inhibition of the other CYP forms was investigated at an R126638 concentration of 10 µM . The incubation conditions are summarized in Table 1 .

 
The percent inhibition of the metabolism of a probe substrate or the percent inhibition of metabolite formation after incubation with R126638 or itraconazole was determined as follows: 100 – [(C_{+ inhibitor}/C_control) x 100], where C_{+ inhibitor} and C_control represent the overall metabolism or the relative amounts of the metabolites in the presence and the absence of R126638, respectively .

IC₅₀s were determined from a plot of the percent inhibition against the logarithm of the R126638 or itraconazole concentration . The value was obtained by regression analysis of the linear part of the curve .

 
R126638 and itraconazole showed similar inhibitory activities against sterol synthesis in T . rubrum, with IC₅₀s of 33 and 18.5 nM, respectively . R126638 (IC₅₀ = 22 nM) was 3.7 times more active than itraconazole (IC₅₀ = 82 nM) against ergosterol biosynthesis in T . mentagrophytes . Compared to itraconazole, R126638 had a similar activity against sterol synthesis in M . canis, with IC₅₀s of 280 and 310 nM, respectively .

Inhibition of ergosterol synthesis in C . albicans coincided with the accumulation of 14-methyl-ergosta-8,24(28)-dien-3ß,6-diol (3,6-diol) (Fig . 2); small amounts of two other 14-methylated sterols, i.e., obtusifoliol and eburicol (24-methylenedihydrolanosterol) also accumulated .

In the presence of either R126638 or itraconazole, T . rubrum, T . mentagrophytes, and M . canis accumulated much more eburicol than 3,6-diol and obtusifoliol (Fig . 3) .

 
Effects on mammalian CYP-dependent reactions. In contrast to the potent inhibitory action of R126638 on ergosterol biosynthesis in intact C . albicans cells, a R126638 concentration 440 times higher was needed to inhibit mammalian cholesterol synthesis (Fig . 4) . Fifty percent inhibition of cholesterol synthesis from [¹⁴C]acetate was found when human hepatoma cells were incubated with 1.4 µM itraconazole or 3.1 µM R126638 .

 
The adrenal mitochondrial cholesterol side chain cleavage enzyme (CYP11A1) and 11ß-hydroxylase (CYP11B1), the 17-hydroxylase plus 17,20-lyase (CYP17) in the rat testis, the human placental aromatase (CYP19), and the 4-hydroxylation of all-trans retinoic acid (CYP26) in human skin epidermis were not affected at the concentrations of R126638 (10 µM) and itraconazole (5 µM) used .

As shown in Table 3, R126638 was also a poor inhibitor of the 1-hydroxylation of 25-hydroxyvitamin D₃ in chicken myelomonocytic cells; only 26% inhibition was achieved with 10 µM R126638 . The IC₅₀ of itraconazole for this CYP27B1-catalyzed reaction was 3.5 µM .

 
Both R126638 and itraconazole inhibited the 24-hydroxylation of 25-hydroxyvitamin D₃ at micromolar concentrations in rat kidney mitochondria . The catabolism of 1,25-dihydroxyvitamin D₃ by human squamous carcinoma (SCC25) cells and human hepatoma (HepG2) cells was inhibited at lower concentrations . The IC₅₀s of R126638 for the conversion of 1,25-dihydroxyvitamin D₃ into more polar metabolites by SCC25 and HepG2 cells were 0.17 and 1.82 µM, respectively, and those of itraconazole were 0.42 and 0.67 µM, respectively .

Effects on oxidative drug metabolism by human liver microsomes. The inhibitory effects of 10 µM R126638 on debrisoquine 4-hydroxylation (CYP2D6), coumarin 7-hydroxylation (CYP2A6), caffeine N₃-demethylation (CYP1A2), tolbutamide hydroxylation (CYP2C8, -9, and -10), phenytoin hydroxylation (CYP2C8, -9, and -10), S-mephenytoin 4-hydroxylation (CYP2C19), and chlorzoxazone hydroxylation (CYP2E1) were studied in human liver microsomes . For R126638, no clear inhibition of the metabolism of the CYP probe substrates could be observed .

The effects of R126638 on the metabolism of the CYP3A4 substrates testosterone, cyclosporine, and midazolam were investigated in more detail . The inhibitory potential of R126638 was compared to the inhibitory capacity of itraconazole in the same set of experiments . On the basis of the testosterone 6ß hydroxylation data and the overall metabolism data for cyclosporine, a lower intrinsic interaction potential with CYP3A4 substrates was observed for R126638 (Table 3) than for itraconazole . However, these triazole derivatives inhibited the formation of the 1'-hydroxy and 4-hydroxy metabolites from midazolam at similar concentrations (Table 3) .

R126638 and itraconazole are almost equipotent inhibitors of ergosterol biosynthesis in C . albicans and the dermatophytes studied . The decreased synthesis of ergosterol and the concomitant accumulation of 14-methylsterols provide indirect evidence that R126638 inhibits CYP51 in C . albicans, T . rubrum, T . mentagrophytes, and M . canis at nanomolar concentrations . The antifungal activity of R126638 in vivo was consistently superior to that of itraconazole (14) . Since R126638 is an almost equipotent inhibitor of the 14-demethylase as itraconazole, the higher degree of efficacy against guinea pig and mouse dermatophytoses (14) suggests that R126638 has a better pharmacokinetic profile in these particular animal models .

R126638 shares with other azole antifungal agents the capability to block mammalian cholesterol synthesis at CYP51 . However, as with the other azoles (e.g., itraconazole) the concentration required to cause the same degree of inhibition is much higher than that required to inhibit C . albicans and dermatophytes . Although the effects of R126638 on the in vivo 14-demethylation of lanosterol have not been studied, studies with itraconazole suggest that the interaction with mammalian cholesterol synthesis in vivo might be negligible . Eight days of treatment of female rats with itraconazole doses as high as 40 mg kg of body weight^–1 did not affect liver cholesterol synthesis (17) .

Micromolar concentrations of itraconazole are needed to inhibit the 25-hydroxyvitamin D₃-1-hydroxylase (CYP27B1) and 25-hydroxyvitamin D₃-24-hydroxylase (CYP24); R126638 is even less active . Both triazoles are, at least in the models used, more active against the further metabolism of 1,25-dihydroxyvitamin D₃ . In particular, the catabolism of 1,25-dihydroxyvitamin D₃ by human tongue squamous carcinoma cells is sensitive to inhibition . Again, however, R126638 affects 1,25-dihydroxyvitamin D₃ at concentrations much higher than those needed to inhibit ergosterol biosynthesis in C . albicans .

Contrary to ketoconazole (28), both itraconazole and R126638 do not interfere with the in vitro metabolism of retinoic acid . R126638 at concentrations up to 10 µM did not affect CYP1A2, CYP2A6, CYP2D6, CYP2C8, CYP2C9, CYP2C10, CYP2C19, or CYP2E1 activity .

Back and Tjia (2) studied ketoconazole, itraconazole, and fluconazole for their effects on the metabolism of cyclosporine by human liver microsomes . Cyclosporine is metabolized by the major drug-metabolizing CYP isoform in human liver, CYP3A4 . Ketoconazole caused marked inhibition of cyclosporine hydroxylase, with an IC₅₀ of 0.24 µM; itraconazole was 10 times less potent, and the fluconazole IC₅₀ was greater than 100 µM (2) . In the present study the IC₅₀ of itraconazole was similar; the inhibitory potency of R126638 was much lower . CYP3A4 also contributes to testosterone 6ß hydroxylation . Again, R126638 showed a lower interaction potential with testosterone 6ß-hydroxylase than itraconazole . Although the level of inhibition of these CYP3A4-catalyzed reactions by R126638 was much less than that by itraconazole, R126638 and itraconazole at similar concentrations inhibited the formation of the 1'-hydroxy and 4-hydroxy metabolites of midazolam, a well-known substrate of CYP3A4 . The clinical relevance of these effects observed in vitro will be further studied .

Our results suggest that R126638 has promising properties and is deserving of further clinical investigations as a treatment for dermatophyte and yeast infections of the skin, since R126638 showed high degrees of efficacy in animal models and has a low affinity for most mammalian P450s studied .

Present address: Steenweg op Gierle, 68, B2300 Turnhout, Belgium .

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