Nizoral
Characterization of an azole-resistant Candida glabrata isolate.

vanden Bossche H, Marichal P, Odds FC, Le Jeune L, Coene MC.

Division of Medicinal Chemistry and Pharmacology, Janssen Research Foundation, Beerse, Belgium.

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.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1482129&dopt=Abstract ketoconazole Nizoral



Nizoral
Transcriptional regulation of rat microsomal epoxide hydrolase gene by imidazole antimycotic agents.

Kim SG.

Institute of Chemical Toxicology, Wayne State University, Detroit, Michigan 48201.

The effects of imidazole antifungal agents, including ketoconazole, clotrimazole, miconazole, and econazole, on the expression and regulation of microsomal epoxide hydrolase (mEH) were examined in rat hepatic tissue (doses of agents, 150 mg/kg of body weight/day, orally). Immunoblot analyses revealed that administration of either ketoconazole or clotrimazole caused a approximately 4-5-fold increase in mEH levels, whereas either miconazole or econazole resulted in a approximately 7-fold increase in mEH at day 3 after treatment. RNA hybridization analyses, probed with a 1.3-kilobase mEH cDNA, revealed that administration of these imidazole antifungal agents caused substantial elevation of hepatic mEH mRNA in total RNA. Hepatic mEH mRNA levels in total RNA were elevated approximately 11-, 15-, and 18-fold at 12, 24, and 72 hr, respectively, after ketoconazole treatment, whereas mEH mRNA levels were increased approximately 14-, 19-, and 22-fold, respectively, relative to control, at the same time points after clotrimazole treatment. The rate of increase of mEH mRNA caused by miconazole was more rapid than the rates observed for the other agents examined, with a maximal increase in mRNA being noted at 12 hr after treatment. The degree of mEH mRNA increase after 3 consecutive days of miconazole treatment was appreciably less than that observed at 12 hr after a single treatment. Econazole caused a maximal increase at 24 hr and subsequent decline in mEH mRNA levels after 3 consecutive days of treatment. Elevation of mEH mRNA levels by these antimycotic agents was confirmed in poly(A)+ RNA, as assessed by both Northern and slot blot hybridization analyses. Nuclear run-on analyses revealed that administration of ketoconazole, clotrimazole, or miconazole stimulated the rate of mEH gene transcription at 12 hr after treatment by 11-, 8.5-, and 9-fold, respectively, compared with control, whereas econazole resulted in a 4-fold increase in the rate of mEH gene transcription at the same time point. The transcription rates of mEH mRNA at 24 hr were significantly less than those observed at 12 hr after a single treatment with either ketoconazole, miconazole, or econazole, resulting in 6.5-, 2.5-, and 2-fold increases, respectively, relative to control. Clotrimazole, however, maintained the activated mEH transcription rate at 24 hr after treatment, exhibiting a 11-fold increase, compared with control. These results provide evidence that the imidazole antimycotic agents induce mEH and that the mEH induction involves large increases in mRNA, with transcriptional activation.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1513325&dopt=Abstract ketoconazole Nizoral



Nizoral
Ketoconazole binds to the human androgen receptor.

Eil C.

Department of Internal Medicine, Naval Hospital, Bethesda, Maryland.

Ketoconazole, an imidazole anti-fungal agent, has often produced features of androgen deficiency including decreased libido, gynecomastia, impotence, oligospermia, and decreased testosterone levels, in men being treated for chronic mycotic infections. Based on these potent effects on gonadal function in vivo as well as previous work in vitro demonstrating affinity of ketoconazole for receptor proteins for glucocorticoids and 1,25(OH)2 vitamin D3 and for sex steroid binding globulin (SSBG), the binding of ketoconazole to human androgen receptors (AR) in vitro was also examined. Ketoconazole competition with [3H]methyltrienolone (R1881) for androgen binding sites in dispersed, intact cultured human skin fibroblasts was determined at 22 degrees C. Fifty percent displacement of [3H]R1881 binding to AR was achieved by 6.4 +/- 1.8 (SE) x 10(-5) M ketoconazole. Additional binding studies performed with ketoconazole in the presence of increasing amounts of [3H]R1881 showed that the interaction of ketoconazole with AR was competitive when the data were analyzed by the Scatchard method. It should be noted, however, that the dose of ketoconazole required for 50% occupancy of the androgen receptor is not likely to be achieved in vivo, at least in plasma. Finally, androgen binding studies performed with other imidazoles, such as clotrimazole, miconazole, and fluconozole, revealed that in this class of compounds only ketoconazole appears to interact with the androgen receptor. Ketoconazole appears to be the first example of a non-steroidal compound which binds competitively to both SSBG and multiple steroid hormone receptors, suggesting that the ligand binding sites of these proteins share some features in common.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1526623&dopt=Abstract ketoconazole Nizoral



Nizoral
Uroporphyrinogen oxidation catalyzed by reconstituted cytochrome P450IA2.

Lambrecht RW, Sinclair PR, Gorman N, Sinclair JF.

Veterans Administration, White River Junction, Vermont 05009.

Previous work suggested that the oxidation of uroporphyrinogen to uroporphyrin is catalyzed by cytochrome P450IA2. Here we determined whether purified reconstituted mouse P450IA1 and IA2 oxidize uroporphyrinogen. Cytochromes P450IA1 and IA2 were purified from hepatic microsomes from 3-methylcholanthrene (MC)-treated C57BL/6 mice, using a combination of affinity chromatography and high performance liquid chromatography. Reconstituted P450IA1 was more active than P450IA2 in catalyzing ethoxyresorufin-O-deethylase (EROD) activity, whereas P450IA2 was more active than P450IA1 in catalyzing uroporphyrinogen oxidation (UROX). Both reactions required NADPH, NADPH-cytochrome P450 reductase, and either P450IA1 or IA2. Ketoconazole competitively inhibited both EROD and UROX activities, in microsomes from MC-treated mice. Ketoconazole also inhibited UROX catalyzed by reconstituted P450IA2. In contrast, ketoconazole did not inhibit UROX catalyzed by xanthine oxidase in the presence of iron-EDTA. Superoxide dismutase, catalase, and mannitol inhibited UROX catalyzed by xanthine oxidase/iron-EDTA, but did not affect UROX catalyzed by either microsomes or reconstituted P450IA2. These results suggest that UROX catalyzed by P450IA2 in microsomes and reconstituted systems does not involve free reactive oxygen species. Two known substrates of cytochrome P450IA2, 2-amino-3,4-dimethylimidazole[4,5-f]quinoline and phenacetin, were shown to inhibit the microsomal UROX reaction, suggesting that uroporphyrinogen binds to a substrate-binding site on the cytochrome P450.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1567206&dopt=Abstract ketoconazole Nizoral



Nizoral
The effect of ketoconazole on endocrine and reproductive parameters in male mice and rats.

Heckman WR, Kane BR, Pakyz RE, Cosentino MJ.

Department of Biology, Millersville University, Pennsylvania 17551.

Ketoconazole has been shown to reduce steroidogenesis by inhibiting the cytochrome P-450 enzymes in these pathways. This finding, along with the observation that the compound reduces sperm motility, led us to study the effectiveness of ketoconazole as a male contraceptive agent administered in acute and chronic studies of both rats and mice. Four hours after a single administration, male rats showed significant reductions in both serum testosterone and corticosterone levels that completely recovered (testosterone) or nearly recovered (corticosterone) 24 hours after administration. Chronic administration of ketoconazole to male rats and mice resulted in steroid levels comparable with those of control animals. Epididymal sperm motility was only slightly reduced in male mice 4 hours after administration of the drug. No effect on sperm motility was noted after chronic administration in either species studied. In vitro exposure of epididymal sperm to ketoconazole resulted in a significant reduction of sperm motility. Breeding trials after ketoconazole administration resulted in normal fertility and fecundity even at the highest dosage studied. The lack of correlation between steroid levels and sperm immobilization, along with rapid in vivo and in vitro effects on sperm motility, suggests that the reduction in sperm motility is not related to a decrease in steroid levels. From these data, the authors conclude that ketoconazole is probably not a viable approach to the development of a male contraceptive.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1601740&dopt=Abstract ketoconazole Nizoral



Nizoral
Inhibition mechanism of reconstituted cytochrome P-450scc-linked monooxygenase system by antimycotic reagents and other inhibitors.

Kurokohchi K, Nishioka M, Ichikawa Y.

Department of Biochemistry, Kagawa Medical School, Japan.

The effects of various antimycotic reagents and some other reagents on a cytochrome P-450-linked monooxygenase system were investigated with respect to the activities of NADPH-ferricyanide reductase. NADPH-cytochrome c reductase of NADPH-adreno-ferredoxin reductase from NADPH to cytochrome c via adreno-ferredoxin, NADPH-cytochrome P-450-phenylisocyanide complex reductase, and the cholesterol side chain cleavage of the cytochrome P-450scc-linked monooxygenase system. No reagents inhibited the NADPH-ferricyanide reductase activity. Only cloconazole inhibited about 50% of NADPH-cytochrome c reductase activity. Cloconazole, econazole, clotrimazole, etomidate and ketoconazole inhibited both NADPH-cytochrome P-450-phenylisocyanide complex reductase and the side chain cleavage activity of cholesterol of the cytochrome P-450scc-linked monooxygenase system. Cloconazole, econazole, etomidate and ketoconazole behaved like non-competitive inhibitors for NADPH-cytochrome P-450-phenylisocyanide reductase activities and their Ki values were 10(-4)-10(-6) M. Cloconazole was a non-competitive inhibitor of NADPH-cytochrome c reductase and its Ki value was 8.3 x 10(-4) M. Cloconazole, clotrimazole, econazole, etomidate, ketoconazole and mitotane completely inhibited the side chain cleavage activity of cholesterol.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1606041&dopt=Abstract ketoconazole Nizoral



Nizoral
Pharmacokinetics/pharmacodynamics of ketoconazole-prednisolone interaction.

Ulrich B, Frey FJ, Speck RF, Frey BM.

Division of Molecular Biology and Biochemistry, University of Berne, Switzerland.

Ketoconazole is commonly used in patients with fungal infections during immunosuppressive therapy with prednisolone. Ketoconazole inhibits mixed function oxidases, enzymes responsible for the catabolism of prednisolone, and might, by that mechanism, increase prednisolone concentrations and thus, the immunosuppressive effect of prednisolone. On the other hand, ketoconazole has been found to bind to the glucocorticoid receptor and, thereby, to function as a glucocorticoid antagonist in cultured cell preparations. In order to establish whether ketoconazole enhances or attenuates the immunosuppressive effect of prednisolone, the influence of ketoconazole on the kinetics of prednisolone and on the delayed hypersensitivity response was assessed in mice. Ketoconazole increased prednisolone concentrations, measured by high pressure liquid chromatography, in mice given a single dose of prednisolone or a continuous prednisolone treatment for 17 days. At four different doses of prednisolone administered for 17 days, the glucocorticoid therapy-associated inhibition of the delayed hypersensitivity response to keyhole limpet hemocyanin was enhanced by ketoconazole. Thus, coadministration of ketoconazole with prednisolone increases the exposure to the steroid and enhances the immunosuppressive effect.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1738098&dopt=Abstract ketoconazole Nizoral