Augmentation of near infrared diffuse reflectance and transmittance spectral data for the development of robust PLSBC models for classifying double blind clinical trial tablets.

The water content of clinical trial tablets can be different between and within different tablet batches, depending on the relative humidity conditions during their production, packaging, storage and analysis. These water variations lead to important spectral variations in the near infrared spectral region which can lead to a wrong identification if the classification model was based on unrepresentative data towards the water content. As model development for clinical trial studies needs to be extremely fast - within one working day - with generally only one batch available, the principle of data augmentation has to be applied to render more robust classification models. Therefore, tablets available for constructing the model are being processed in order to increase or decrease their water content and to make them more representative for tablets to be tested in the future. The inclusion of a deliberate water variation is the most efficient way to develop a model, for which no additional model redevelopment will be required to pass the system suitability tests and to obtain a correct identification.

Effects of liarozole, a new antitumoral compound, on retinoic acid-induced inhibition of cell growth and on retinoic acid metabolism in MCF-7 human breast cancer cells.

Liarozole is a new imidazole derivative with antitumoral properties. Effects of the compound alone and in combination with all-trans-retinoic acid on proliferation of MCF-7 human breast cancer cells were examined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. Following 9 days of drug exposure, MCF-7 cell growth was concentration dependently inhibited by all-trans-retinoic acid (drug concentration resulting in 50% growth inhibition, 2 x 10(-8) M), while liarozole at 10(-5) M inhibited cell growth by only 35%. When MCF-7 cells were incubated with a combination of all-trans-retinoic acid and liarozole, the antiproliferative effect of all-trans-retinoic acid was clearly enhanced. This enhancement was dependent on the liarozole concentration and was more than 10-fold. A combination of 10(-8) M all-trans-retinoic acid and 10(-6) M liarozole resulted in a greater antiproliferative effect than that obtained with 10(-7) M all-trans-retinoic acid alone. When MCF-7 cells were incubated for 4 h with [3H]all-trans-retinoic acid, the radioactivity in the supernatant consisted of unaltered retinoid. However, when cells had been pretreated with 10(-6) M all-trans-retinoic acid overnight, they were able to substantially metabolize [3H]all-trans-retinoic acid during a subsequent 4-h incubation. High-performance liquid chromatography analysis of the supernatants revealed that the reaction products consisted mainly of very polar metabolites. Liarozole inhibited the metabolism of all-trans-retinoic acid in MCF-7 cells with 10(-5) M liarozole reducing the amount of polar metabolites by 87%. It is concluded that the enhancement by liarozole of the antiproliferative effects of retinoic acid on MCF-7 human breast cancer cells is probably due to inhibition of retinoic acid metabolism. Further research into these effects in MCF-7 cells as well as in other cancer cell lines will provide more information concerning the exact mechanism of action of liarozole and the use of inhibitors of retinoid metabolism in cancer treatment.

Arachidonic acid metabolism by cultured mesothelial cells. Different transformations of exogenously added and endogenously.

The capacity of cultured mesothelial cells to produce prostaglandins from both exogenous an endogenous arachidonic acid has been investigated. Incubations with labelled [1-14C]arachidonic acid and [1-14C]prostaglandin endoperoxide H2 indicated the formation of prostacyclin and prostaglandin E2. Evaluation of the transformation of endogenously released arachidonic acid, however, could only confirm the production of prostacyclin.

Characterization of monohydroxylated lipoxygenase metabolites of arachidonic and linoleic acid in rabbit peritoneal tissue.

Rabbit peritoneal tissue contains a lipoxygenase which converts arachidonic acid preferentially into 15-hydroxy-5,8,11,13-eicosatetraenoic acid. Stereochemical analysis of the menthyloxycarbonyl derivative of this metabolite by means of a high-pressure liquid chromatography method, involving the use of a Ag+ -loaded cation-exchange column, indicated that it has mainly the 15-Ls-hydroxy configuration. The biosynthesis of 15-hydroxy-5,8,11,13-eicosatetraenoic acid could be confirmed during examination of the monohydroxy acids obtained without addition of fatty acids, thus formed from endogenously released substrate. However, the 9-and 13-hydroxy derivatives of linoleic acid were also formed and in quantities exceeding those of 15-hydroxy-5,8,11,13-eicosatetraenoic acid.

The influence of cisapride and clarithromycin on QT intervals in healthy volunteers.

OBJECTIVE: Recently a few cases of long QT syndrome were reported during treatment with cisapride. In most of these cases, risk factors for cardiac arrhythmias or pharmacologic interactions might have been involved, and the role of cisapride remained unclear. Macrolides such as clarithromycin potentially interact with the metabolic elimination of cisapride and have overlapping indication areas. We therefore studied whether combined treatment with clarithromycin and cisapride leads to pharmacokinetic changes and increased QT intervals. METHODS: The study was an open, randomized, 2-way crossover study with washout periods of 1 week. Twelve healthy volunteers were recruited. Treatments were cisapride (10 mg 4 times a day) for 10 days with concomitant clarithromycin (500 mg twice a day) from days 6 through 10, or clarithromycin (500 mg twice a day) for 10 days combined with cisapride (10 mg 4 times a day) from days 6 through 10. Frequent ECG recordings were performed for 24 hours before drug treatment (baseline). After 5 days of monotherapy and combination therapy, frequent ECG recordings and assessments of plasma drug levels were performed for 24 hours. RESULTS: Clarithromycin alone was associated with a minimal increase in QTc intervals. Monotherapy with 10 mg cisapride 4 times a day led to a concentration-dependent QTc elevation, amounting to 6 ms during steady state. Combination of cisapride and clarithromycin caused an average QTc increase of 25 ms above pretreatment values and 3-fold increases in cisapride concentrations. CONCLUSIONS: QTc elevations after cisapride or clarithromycin alone remained within the normal range of diurnal variation. Coadministration of cisapride and clarithromycin produced a substantial QT prolongation. The data support the recently purported interaction between cisapride and clarithromycin and thus the filed contraindication to combine these drugs.

Ketoconazole inhibits the biosynthesis of leukotrienes in vitro and in vivo.

Ketoconazole inhibits in vitro (IC50:2.6 X 10(-5) M) the formation of 5-HETE and LTB4 by isolated, carrageenin-elicited rat peritoneal PMN leukocytes, challenged with the Ca2+-ionophore A23187 in the presence of [14C]-arachidonic acid ([14C]-AA). The relative potency of various compounds tested in this respect is NDGA greater than nafazatrom greater than phenidone greater than ketoconazole greater than BW 755C. In contrast to the other compounds studies, ketoconazole in vitro, up to 1 X 10(-4) M, has no effect on the fatty acid cyclo-oxygenase or the 12-lipoxygenase-mediated metabolism of [14C]-AA by isolated human platelets; however, it stimulates the 15-lipoxygenase activity in phenylhydrazine-induced rabbit reticulocytes. After oral administration (10-40 mg/kg, -2 hr), ketoconazole inhibits in a dose-dependent way, the leukotriene-mediated anaphylactic bronchoconstriction in guinea pigs. This study demonstrates that ketoconazole is a comparatively specific and orally active inhibitor of the 5-lipoxygenase activity bearing on the production of leukotrienes derived from arachidonic acid.

R 76713 and enantiomers: selective, nonsteroidal inhibitors of the cytochrome P450-dependent oestrogen synthesis.

The triazole derivative, R 76713 and its enantiomers R 83839(-) and R 83842(+) are effective inhibitors of the aromatization of androstenedione. For human placental microsomes, the (+) enantiomer (R 83824) is about 1.9- and 32-times more active than the racemate (IC50 2.6 nM) and the (-) enantiomer, respectively. R 83842 is about 30- and 1029-times more active than 4-hydroxyandrostene-3,17-dione and aminoglutethimide. This potency might originate from its high affinity for the microsomal cytochrome P450 (P450). Indeed, R 83842, compared to R 76713 and R 83839, forms a more stable P450-drug complex. Difference spectral measurements indicate that the triazole nitrogen N-4 coordinates to the haem iron. The reversed type 1 spectral changes suggest that R 76713 is able to displace the substrate from its binding place and the stable complex formed in particular with the (+) enantiomer suggests that its N-1-substituent occupies a lipophilic region of the apoprotein moiety. Kinetic analysis implies that there is a competitive part in the inhibition of the human placental aromatase by R 76713. The Ki values for R 76713, R 83842 and R 83839 are 1.3 nM, 0.7 nM and 18 nM, respectively. These results are indicative of stereospecificity for binding. Up to 10 microM, R 76713 and its enantiomers have no statistically significant effect on the regio- and stereoselective oxidations of testosterone in male rat liver microsomes. All three compounds have no effect on the P450-dependent cholesterol synthesis, cholesterol side-chain cleavage and 7 alpha-hydroxylation and 21-hydroxylase. At 10 microM, R 76713 has a slight effect on the bovine adrenal 11 beta-hydroxylase. This effect originates mainly from R 83839, the less potent aromatase inhibitor. On the other hand, the inhibition of the 17,20-lyase of rat testis observed at concentrations greater than or equal to 0.5 microM, originates rather from R 83842. However, 50% inhibition is only achieved at 1.8 microM R 83842, i.e. at a concentration about 1300-times higher than that needed to reach 50% inhibition of the human placental aromatase.

Liarozole fumarate inhibits the metabolism of 4-keto-all-trans-retinoic acid.

The metabolism of 4-keto-all-trans-retinoic-acid (4-keto-RA), a biologically active oxygenated metabolite of all-trans-retinoic (RA), has been examined. In vitro, incubation of [14C]4-keto-RA with hamster liver microsomes in the presence of NADPH produced two major radioactive metabolites which were more polar than the parent compound. Following isolation, appropriate derivatization and analysis by GC-MS, these compounds were tentatively identified as 2-hydroxy- and 3-hydroxy-4-ketoretinoic acid. Formation of both hydroxy-keto derivatives was suppressed by the imidazole-containing P450 inhibitor liarozole fumarate (IC50, 1.3 microM). In vitro, an i.v. injection of 4-keto-RA (20 micrograms) into rats was followed by rapid disappearance of the retinoid from plasma with a half-life of 7 min. Pretreatment with liarozole fumarate (40 mg/kg, -60 min) reduced the elimination rate of 4-keto-RA: it prolonged the plasma half-life of the retinoid to 12 min, without affecting its distribution volume. These results indicate the important role of the P450 enzyme system in the metabolism of 4-keto-RA both in vitro and in vivo. The inhibitory effect of liarozole fumarate on this metabolic process may contribute to the reported retinoid-mimetic activity of this drug.

Experimental studies with liarozole (R 75,251): an antitumoral agent which inhibits retinoic acid breakdown.

Liarozole reduced tumor growth in the androgen-dependent Dunning-G and the androgen-independent Dunning MatLu rat prostate carcinoma models as well as in patients with metastatic prostate cancer who had relapsed after orchiectomy. In vitro, liarozole did not have cytostatic properties, as measured by cell proliferation in breast MCF-7 and prostate DU145 and LNCaP carcinoma cell lines. It did not alter the metabolism of labeled testosterone i.e. the 5 alpha-reductase in cultured rat prostatic cells. In mouse F9 teratocarcinoma cells liarozole did not show any retinoid-like properties but enhanced the plasminogen activator production induced by retinoic acid. Furthermore, liarozole and retinoic acid similarly reduced the growth of the androgen-dependent Dunning-G tumor in nude mice and inhibited tumor promotion elicited by phorbol ester in mouse skin. These data have raised the hypothesis that the antitumoral properties of liarozole may be related to inhibition of retinoic acid degradation, catalyzed by a P-450-dependent enzyme that is blocked by the drug.

Inhibition of rabbit platelet activation by lipoxygenase products of arachidonic and linoleic acid.

The hydroperoxy fatty acids, 15-hydroperoxyeicosatetraenoic acid (15-HPETE), 13-hydroperoxy and 9-hydroperoxyoctadecadienoic acid (13- and 9-HPODE) and the corresponding hydroxy compounds (15-HETE and 13-HODE) were synthesized and purified. Washed rabbit platelets were incubated with these fatty acid derivatives before aggregation was induced. Arachidonic acid-induced aggregation, as well as the secretion of ATP and the formation of thromboxane B2 (TXB2) were dose-dependently inhibited by these compounds. Low thrombin-, collagen- and ADP-induced aggregations were also suppressed by 15-HPETE. Platelet activation induced by the calcium ionophore A23187 and by high thrombin concentrations were not affected by 15-HPETE. In addition, doses of 15-HPETE which were inactive by themselves, potentiated the anti-aggregating activity of prostacyclin (PGI2). It is suggested that the hydroperoxy and hydroxy compounds suppress platelet activation by interference with the rise in cytoplasmic calcium in addition to the inhibition of cyclo-oxygenase.

The metabolism of arachidonic and linoleic acid in rabbit peritoneal tissue: a short review.

The results obtained in the biotransformation studies demonstrate that lipoxygenase and PG-cyclo-oxygenase reactions represent major pathways in the metabolism of both arachidonic and linoleic acid in rabbit peritoneal tissue. Although the physiological significance of the hydroxy derivatives of arachidonic acid and linoleic acid is still unclear, it is tempting to assume that in tissues containing lipoxygenase activity, some of the effects ascribed to hydroxy arachidonates and their hydroperoxy precursors, e.g. inhibition of leukotriene and of PGI2 biosynthesis (16, 17) could for a great part be invoked by linoleic acid derivatives, as these products can be formed in larger quantities than the corresponding arachidonate derivatives.

Effects of high dose ketoconazole therapy on the main plasma testicular and adrenal steroids in previously untreated prostatic cancer patients.

In vitro, ketoconazole has been shown to block testicular and adrenal 17,20-lyase, which converts progestins to androgens. At higher concentrations, it also inhibits 11 beta-hydroxylase, 20,22-desmolase and 17 alpha-hydroxylase. To determine the differential hormonal effects of a 2-week ketoconazole high-dose therapy, the plasma levels of 10 major androgens, gluco- and mineralocorticoids were measured in 14 previously untreated patients with metastatic prostate cancer. Within 24 h, plasma testosterone fell from 14.6 +/- 1.4 nmol/l (mean +/- SEM) to 3.7 +/- 0.7 nmol/l. Thereafter, it decreased to about 2.5 nmol/l and remained at that level. Plasma androstenedione and dehydroepiandrosterone decreased more gradually, respectively from 3.1 +/- 0.4 nmol/l to 0.64 +/- 0.17 nmol/l and from 6.6 +/- 1.0 nmol/l to 2.82 +/- 0.55 nmol/l (on day 14). In contrast, 17 alpha-hydroxyprogesterone and progesterone rose respectively 2- and 5-fold. Plasma cortisol and aldosterone levels remained unchanged whereas 11-deoxycorticosterone and 11-deoxycortisol rose by factors of 14 and 6.7 respectively. Plasma corticosterone also increased, but to a much lesser extent (3-fold). These results demonstrate that ketoconazole high dose therapy blocks mainly the 17,20-lyase of both adrenal and testis. In addition it inhibits mitochondrial 11 beta-hydroxylase to a lesser extent. The inhibition of 20,22-desmolase also seems to be of little clinical relevance. However, since clinical or laboratory symptoms suggestive of hypo-adrenalism have been reported in a small minority of patients, replacement therapy should be considered in such cases.

Leukotriene B4 production by stimulated whole blood: comparative studies with isolated polymorphonuclear cells.

A new method was developed to study leukotriene B4 (LTB4) production by stimulated whole blood. The calcium ionophore A23187 and serum-treated zymosan induced LTB4 production, measured by radioimmunoassay, in a dose- and time-dependent manner. The pattern of LTB4 production by whole blood differed markedly from that observed with isolated, purified polymorphonuclear leukocytes. Higher levels of LTB4 were reached and maintained in whole blood. The system allowed to detect drug effects on LTB4 synthesis in vitro. This new method to study the synthesis of LTB4 takes into account the complex interactions between different cell types which can modulate LTB4 metabolism.

Vitamin C increases the prostacyclin production and decreases the vascular lesions in experimental atherosclerosis in rabbits.

Feeding a cholesterol-rich diet (0.3%) to rabbits resulted in an intimal thickening and lipid infiltration of the aorta. The prostacyclin production by the vascular endothelium was significantly decreased, after a transient increase after 2 weeks of diet. The arachidonic acid metabolism in platelets was hardly changed. Addition of a low dose vitamin C (150 mg/day) to the cholesterol rich diet resulted in decreased lipid infiltration and intimal thickening and the transient increase of the prostacyclin production was postponed to the 4th week. Although this dose of vitamin C could not restore the decreased prostacyclin production observed after 6 weeks diet, a higher dose of vitamin C (600 mg/day), besides its beneficial effect on the lipid infiltration and the intimal thickening in the thoracic aorta, kept the intimal prostacyclin production at normal levels for at least 8 weeks.

Biphasic response of intimal prostacyclin production during the development of experimental atherosclerosis.

Feeding a cholesterol rich diet (0.3%) to rabbits for up to 10 weeks resulted in morphological changes of the vascular wall. Microscopic evaluation of the aorta revealed a lipid infiltration and an intimal thickening containing foam cells, which both became more pronounced as the cholesterol feeding was more prolonged. The intimal prostacyclin production showed a transient increase after 2 weeks, but was significantly decreased after 6 weeks of diet and remained at this low level during the rest of the experiment. No significant changes in formation of thromboxane B2 by the platelets could be observed, whereas the production of 12-HETE was enhanced.

Effects of cytochrome P-450 inhibitors on the in vivo metabolism of all-trans-retinoic acid in rats.

This study examines the effects of ketoconazole, R 75 251 and some other cytochrome P-450 inhibitors on the in vivo metabolism of all-trans-retinoic acid (RA) in normal rats. Oral treatment with ketoconazole or R 75 251 (40 mg/kg, -1 hr) reduced the elimination rate of i.v. injected RA from plasma: the half-life of RA increased from 27 min in control-treated animals to 43 min and 76 min after dosing with ketoconazole and R 75 251, respectively. However, neither drug had an effect on the distribution volume of the retinoid. Two hours after i.v. injection of RA, residual plasma levels of the retinoid were 11.2 ng/ml in ketoconazole and 22.7 ng/ml in R 75 251-treated rats. The other P-450 inhibitors, aminoglutethimide, cimetidine, itraconazole, metyrapone and saperconazole, showed no sparing effect on RA elimination: plasma levels of the acid were below 1 ng/ml, as in control-treated animals. Administration of ketoconazole or R 75 251 (40 mg/kg, -2 hr) to rats also enhanced endogenous plasma concentrations of RA. Levels of the retinoid were raised from mostly undetectable values (less than 0.5 ng/ml) to 1.3 +/- 0.1 and 2.5 0.1 ng/ml after treatment with ketoconazole and R 75 251, respectively. These data are indicative of the important contribution of the cytochrome P-450 enzyme system to the in vivo metabolic process of RA. In vivo inhibition of the P-450 pathway not only increased the biological half-life of exogenously administered RA, but also enhanced the endogenous plasma level of this vitamin A derivative.

Characterization of an azole-resistant Candida glabrata isolate.

A Candida (Torulopsis) glabrata strain (B57149) became resistant to fluconazole after a patient carrying the organism was treated with the drug at 400 mg once daily for 9 days. Growth of the pretreatment isolate (B57148) was inhibited by 50% with 0.67 microM ketoconazole, 1.0 microM itraconazole, and 43 microM fluconazole, whereas growth of B57149 was inhibited slightly by 10 microM ketoconazole but was unaffected by 10 microM itraconazole or 100 microM fluconazole. This indicates cross-resistance to all three azole antifungal agents. The cellular fluconazole content of B57149 was from 1.5- to 3-fold lower than that of B57148, suggesting a difference in drug uptake between the strains. However, this difference was smaller than the measured difference in susceptibility and, therefore, cannot fully explain the fluconazole resistance of B57149. Moreover, the intracellular contents of ketoconazole and itraconazole differed by less than twofold between the strains, so that uptake differences did not account for the azole cross-resistance of B57149. The microsomal cytochrome P-450 content of B57149 was about twice that of B57148, a difference quantitatively similar to the increased subcellular ergosterol synthesis from mevalonate or lanosterol. These results indicate that the level of P-450-dependent 14 alpha-demethylation of lanosterol is higher in B57149. Increased ergosterol synthesis was also seen in intact B57149 cells, and this coincided with a decreased susceptibility of B57149 toward all three azoles and amphotericin B. B57149 also had higher squalene epoxidase activity, and thus, more terbinafine was needed to inhibit the synthesis of 2,3-oxidosqualene from squalene. P-450 content and ergosterol synthesis both decreased when isolate B57149 was subcultured repeatedly on drug-free medium. This repeated subculture also fully restored the strain's itraconazole susceptibility, but only partly increased its susceptibility to fluconazole. The results suggest that both lower fluconazole uptake and increased P-450-dependent ergosterol synthesis are involved in the mechanism of fluconazole resistance but that only the increased ergosterol synthesis contributes to itraconazole cross-resistance.

Effects of high-dose ketoconazole treatment on adrenal mineralocorticoid biosynthesis in dogs and rats.

At high doses, ketoconazole blocks both testicular and adrenal androgen biosyntheses and partially inhibits the glucocorticoid production. To investigate the effects of this imidazole derivative on the mineralocorticoid biosynthesis, 7 male mongrel dogs received a single oral dose of 15 mg/kg of ketoconazole or placebo, in a cross-over way. From 2 to 4 h after treatment, an iv infusion of angiotensin II (10 ng/kg per min) was performed. Ketoconazole treatment significantly blunted the aldosterone and cortisol increment, whereas 18-hydroxycorticosterone, corticosterone, 11-deoxycorticosterone (DOC), progesterone, and 17 alpha-hydroxyprogesterone rose to peak concentrations, respectively 2.5-, 6-, 8-, 2.5- and 1.5-fold higher than those observed after placebo administration. Plasma 11-deoxycortisol and renin activity levels remained similar in both groups. On the other hand, 2 X 2 groups of 10 male adult rats each were fed with a normal or a sodium-depleted diet. Of the two sets of groups, one was treated ip with ketoconazole (20 mg/kg twice a day), the other with vehicle solution. In animals on either diet, ketoconazole lowered 18-hydroxycorticosterone and aldosterone concentrations. Plasma DOC rose up to 25-fold in the salt-deprived animals. Serum Na+, Cl-, corticosterone and plasma renin activity remained unaffected by the treatment. These results show that high-dose ketoconazole treatment partially inhibits the biosynthesis of aldosterone by affecting the cytochrome P-45011 beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical basis for the activity and selectivity of oral antifungal drugs.

The ergosterol biosynthesis-inhibiting (EBI) antifungals constitute the most important group of compounds developed for the control of fungal diseases in man. Currently, representatives of two classes of EBI antifungals are available: the squalene epoxidase inhibitors and those that interfere with cytochrome P450-dependent ergosterol synthesis. The allylamines (eg, terbinafine) inhibit squalene epoxidase in sensitive fungi, Trichophyton mentagrophytes being the most sensitive species. The most important developments have come from the introduction of the N-substituted imidazoles and triazoles, the so-called azole antifungals. Most of the currently available imidazoles (eg, miconazole, clotrimazole, econazole) and the triazole derivative terconazole are mainly for topical treatment. Ketoconazole was the first azole derivative orally active against yeasts, dermatophytes and dimorphic fungi. The new triazole, itraconazole, appears to be among the most promising orally active systemic agents. All the azole antifungals inhibit the cytochrome P450-dependent, 14 alpha-demethylase, a key enzyme in the synthesis of ergosterol, the main sterol in most fungal cells. Of all the azoles tested, itraconazole shows the highest affinity for the cytochrome P450 involved. It is about three and ten times more active in vitro than miconazole and the bis-triazole, fluconazole, respectively. Itraconazole's high affinity for the fungal P450 originates from its triazole group as well as from the nonligating lipophilic tail.

Origin of differences in susceptibility of Candida krusei to azole antifungal agents.

Two Candida krusei isolates were used to compare the effects of fluconazole, ketoconazole and itraconazole on growth and ergosterol synthesis, and to measure intracellular drug contents. Fifty per cent inhibition (IC50) of growth was achieved at 0.05-0.08 microM itraconazole and 0.56-1.2 microM ketoconazole, whereas 91-->100 microM fluconazole was needed to reach the IC50 value. Similar differences in sensitivity to these azole antifungal agents were seen when their effects on ergosterol synthesis from [14C]acetate were measured after 4 h and 24 h of growth. However, when the effects of the azoles on ergosterol synthesis from [14C]mevalonate by subcellular fractions were measured, fluconazole was only 2.3-6.1 times less active than itraconazole, and the IC50 values for ketoconazole were almost similar to those obtained with itraconazole. These results indicate that differences in susceptibility to itraconazole and ketoconazole are unrelated to differences in affinity for the C. krusei cytochrome P450. The much lower growth-inhibitory effects of fluconazole can also be explained partly only by a lower affinity for the P450-dependent 14 alpha-demethylase. The differences in sensitivity of both C. krusei isolates appeared to arise from differences in the intracellular itraconazole, ketoconazole and fluconazole contents. Depending on the experimental conditions, these isolates accumulated 6-41 times more itraconazole than ketoconazole and the intracellular ketoconazole content was 3.0-19.0 times higher than that of fluconazole.