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Low dose ketoconazole attenuates serum androgen levels in patients with polycystic ovary syndrome and inhibits ovarian steroidogenesis in vitro.

Gal M, Orly J, Barr I, Algur N, Boldes R, Diamant YZ.

Department of Obstetrics and Gynecology, Shaare Zedek Medical Center, Jerusalem, Israel.

OBJECTIVE: To investigate the effects of a low-dose ketoconazole on ovarian steroidogenesis and on serum androgen levels in polycystic ovary syndrome (PCOS). DESIGN: In vitro, human granulosa-luteal cells were incubated with ketoconazole and radiolabeled steroid substrates, to follow their metabolic fate by thin-layer chromatography analysis. In vivo, normally cycling women (n = 7) in their luteal phase were administered one tablet of 200 mg ketoconazole at 8 A.M. Serum steroid levels, sampled basally and at 12 P.M., 4 P.M., and 8 A.M. the next morning, were compared with untreated control group (n = 7) values. Polycystic ovary syndrome women (n = 11) were similarly administered ketoconazole 6 to 10 days after occurrence of spontaneous menses. Adrenal origin of hyperandrogenemia was excluded by stimulation with ACTH and a normal basal DHEAS. The steroid diurnal variation was determined in the same patients a day before treatment. RESULTS: In vitro, ketoconazole selectively inhibited the key steroidogenic cytochromes, namely P450scc, P45017 alpha, and P450arom (IC50 = 0.5 to 1.0 microgram/mL). In vivo, in the luteal phase, ketoconazole transiently decreased serum values (mean +/- SE) of E2 (19.2% +/- 2.1%) and P (38.3% +/- 8.5%) within 4 to 8 hours. The same low-dose ketoconazole, administered to PCOS women, decreased serum values of androstenedione (17.6% +/- 4.7%), T (24.6% +/- 7.6%), and free T (30.7% +/- 7.7%). In contrast, 17 alpha-hydroxyprogesterone increased concomitantly (78.5% +/- 10.8%), suggesting a greater suppressibility of the P45017 alpha lyase activity. The E2 levels in PCOS patients were slightly elevated (29.1% +/- 5.6%), resulting in a 1.7- to 2.3-fold increase of the E2:T ratio. CONCLUSIONS: These findings suggest that a low-dose ketoconazole may facilitate a decreased intraovarian T:E2 ratio, which may prove favorable for follicular maturation in PCOS.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8174717&dopt=Abstract ketoconazole Nizoral



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Effect of dexamethasone and cytochrome P450 inhibitors on the formation of 7 alpha-hydroxydehydroepiandrosterone by human adipose stromal cells.

Khalil MW, Strutt B, Vachon D, Killinger DW.

Department of Medicine, University of Western Ontario, London, Canada.

7 alpha-Hydroxydehydroepiandrosterone (7 alpha-OHDHA) is a major metabolite of dehydroepiandrosterone (DHA) using adipose stromal cells. To gain a better understanding of the factors regulating DHA metabolism, we examined the effect of dexamethasone and cytochrome P450 inhibitors on the formation of 7 alpha-OHDHA. Dexamethasone (10(-9) to 10(-7) M) stimulated 7 alpha-OHDHA formation in a dose-dependent manner with a 2- to 5-fold stimulation at 10(-7) M. The dexamethasone stimulated 7 alpha-OHDHA formation was inhibited by RU486 in a dose-dependent manner with suppression to basal levels at 10(-6) M. Progesterone (10(-7) M) had no effect on 7 alpha-OHDHA formation suggesting that the dexamethasone stimulation was acting through the glucocorticoid receptor. Conversion of DHA to 7 alpha-OHDHA was inhibited by ketoconazole and metyrapone. An inhibition of 70-80% was obtained with ketoconazole and 25-60% with metyrapone at concentrations of 10(-5) M. Aminoglutethimide phosphate was less effective than either ketoconazole or metyrapone in inhibiting 7 alpha-OHDHA formation with < 30% inhibition at 10(-5) M. These studies indicate that 7-hydroxylation provides an alternative pathway for the metabolism of DHA in peripheral tissues. This pathway, which is regulated by glucocorticoids, may influence the amount of DHA available for conversion to androstenedione and its subsequent aromatization to estrone. The biological role of the 7-oxygenated metabolites and their effects on other steroidogenic pathways have not been established.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=8180117&dopt=Abstract ketoconazole Nizoral



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Development and characterization of an open tubular column containing immobilized P-glycoprotein for rapid on-line screening for P-glycoprotein substrates.

Moaddel R, Bullock PL, Wainer IW.

Laboratory of Clinical Investigation, Intramural Research Program, Bioanalytical and Drug Discovery Unit, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825, USA.

Cellular membranes from a cell line expressing P-glycoprotein (Pgp(+)) and from a cell line that does not express Pgp (Pgp(-)) were immobilized on the surface of glass capillaries (25 cm x 100 microm i.d.) by non-covalent interactions using the avidin-biotin coupling system to create two open tubular columns, Pgp(+)-OT and Pgp(-)-OT. Frontal displacement chromatography on the Pgp(+)-OT demonstrated that the immobilized Pgp retained its ability to specifically bind the known Pgp substrates vinblastin and ketoconazole. The calculated affinities, expressed as K(d), for vinblastin and ketoconazole were 97 nM and 12.1 microM, which were comparable with previously reported K(d) values of 37 nM and 8.6 microM, respectively. The results confirm that the Pgp(+)-OT can be used to quantitatively estimate binding affinities for the Pgp. Frontal displacement chromatography on the Pgp(-)-OT demonstrated that the immobilized membranes retained the ability to bind some Pgp substrates, but that the binding was not due to specific binding to Pgp. A cohort of compounds containing high affinity Pgp substrates (vinblastin, prazosin) and moderate-low affinity Pgp substrates (doxorubicin, verapamil, ketoconazole) and a non-substrate (nicotine) were chromatographed on the Pgp(+)-OT and Pgp(-)-OT using fast frontal analysis and mass spectrometric detection. The results demonstrated that when the retention on the Pgp(+)-OT was corrected by subtraction of the retention on the Pgp(-)-OT, the test compounds could be accurately sorted into high, moderate-low and non-substrate categories. The data from the study indicates that a single 30-min parallel chromatographic experiment can be used to rank a compound based upon its relative affinity for the immobilized Pgp.

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Influence of serum protein binding on the in vitro activity of anti-fungal agents.

Schafer-Korting M, Korting HC, Rittler W, Obermuller W.

Fachbereich Pharmazie der Freien Universitat Berlin, Germany.

Historically it has been assumed that the pharmacological effect is related to the free drug concentration. In exposing Candida albicans to itraconazole and ketoconazole serum concentration-time profiles, however, antifungal activity was not diminished despite intense albumin binding. The relevance of serum protein binding was further investigated, by in vitro susceptibility testing of C. albicans (40 clinical isolates) and Trichophyton rubrum (ten strains) against antifungal agents using microdilution tests allowing the determination of IC30- and MIC-values. The range of serum protein binding ranges from 11% with fluconazole to > 99% with itraconazole and terbinafine. The ratios of IC30- and MIC-values with and without serum protein (albumin, alpha- and gamma-globulin, human plasma) were related to the loss of susceptibility expected according to the free-drug hypothesis. A difference in the albumin effect with the test strains was not observed. With most antifungals including terbinafine, the activity declined as expected. IC30- and MIC-ratios for miconazole were 7 and 13 (observed) vs. 12-20 (expected), for fluconazole 1.5 and 3.5 vs. 1.1, for amphotericin B 10 vs. 11-20, for griseofulvin 3.6 vs. 4, and for terbinafine 61 vs. 100. Itraconazole activity, however, was not diminished by albumin (expected ratio 286), and ketoconazole effects decreased less than expected (ratio 5-15, expected about 100). alpha-globulin, but not gamma-globulin induced a major loss in anti-Candida activity of itraconazole and ketoconazole, which is paralleled by a decline in ketoconazole (but not itraconazole) activity due to plasma. With the other antifungals (except for ciclopiroxolamine) IC30-values for C. albicans increased, too. Due to the complete inhibition of T. rubrum growth by gamma-globulin, this species proved unsuitable for studying the gamma-globulin effects. The present study demonstrates that the effects of intense protein binding on drug activity are only partly predictable from binding studies in vitro.

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The anti-Malassezia furfur activity in vitro and in experimental dermatitis of six imidazole antifungal agents: bifonazole, clotrimazole, flutrimazole, ketoconazole, miconazole and sertaconazole.

Van Gerven F, Odds FC.

Department of Bacteriology and Mycology, Janssen Research Foundation, Beerse, Belgium.

Bifonazole, clotrimazole, flutrimazole, ketoconazole, miconazole and sertaconazole were tested for their activity against 23 isolates of Malassezia furfur by agar dilution in vitro. Topical formulations of the same agents were evaluated for efficacy against M. furfur skin infections in guinea pigs in vivo. The most potent inhibitor in vitro was ketoconazole (geometric mean minimum inhibitory concentration 0.51 microgram ml-1), followed by bifonazole (8.1 micrograms ml-1), then miconazole (14 micrograms ml-1), clotrimazole (15 micrograms ml-1) and flutrimazole (16 micrograms ml-1), with sertaconazole the least active (52 micrograms ml-1). In animal experiments involving three consecutive days of topical treatments, bifonazole 1% cream, clotrimazole 1% cream, flutrimazole 1% and 2% creams, ketoconazole 2% cream and shampoo and miconazole 2% cream all reduced M. furfur dermatitis lesion severity below that of untreated control animals; however, sertaconazole 2% gel and cream showed no reduction in lesion severity below control. The results confirm that ketoconazole is a more potent inhibitor of M. furfur in vitro than other topical antifungal agents of its class and suggest that sertaconazole is the least effective of such agents among those tested.

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Modification of the sterol composition of Trypanosoma (Schizotrypanum) cruzi epimastigotes by delta 24(25)-sterol methyl transferase inhibitors and their combinations with ketoconazole.

Urbina JA, Vivas J, Visbal G, Contreras LM.

Laboratorio de Quimica Biologica, Instituto Venezolano de Investigaciones Cientificas, Caracas, Venezuela.

We report a detailed analysis of the sterol composition of Trypanosoma cruzi epimastigotes grown in the absence or presence of two sterol analogs previously reported as inhibitors of delta 24(25) sterol methyltransferase (24(25)-SMT,E.C.2.1.1.43) in yeast and fungi, a cholestanol analog with a 6-membered aza ring as side chain (22,26-azasterol) and 24-(R,S),25-epiminolanosterol, as well as combinations of these compounds with the C14 demethylase inhibitor ketoconazole. Both sterol analogs produced a dose-dependent reduction in the incorporation of radioactivity from [methyl-14C]methionine with IC50 values of 640 nM and 70 nM for 22,26-azasterol and 24,25-(R,S)-epiminolanosterol, respectively, indicating a specific inhibition of 24(25)-SMT. Correspondingly, it was found that the sterols present in control cells (ergosterol, 24-ethylcholesta-5,7,22-trien-3 beta-ol and precursors) were almost completely replaced by zymosterol (cholesta-8,24-dien-3 beta-ol) or a mixture of zymosterol, cholesta-7,24-dien-3 beta-ol and cholesta-5,7,24-trien-3 beta-ol when the parasites were exposed to the minimal growth inhibitory concentrations of 22,26-azasterol and 24-(R,S),25-epiminolanosterol, respectively. At sub-optimal concentrations of the inhibitors a complete disappearance of the 24-ethyl sterols was observed and a concomitant increase in the proportion of 24-methyl sterols, particularly delta 24(24') sterols. This showed that in T. cruzi the second methenylation step (catalyzed by delta 24(24') sterol methyl transferase) was significantly more sensitive to these inhibitors than the first and that the sterol analogs were also powerful inhibitors of the delta 24(24') sterol reductase. In growth-arrested epimastigotes resulting from their treatment with low (1-3 microM) concentrations of either sterol analog combined with sub optimal (100-300 nM) levels of ketoconazole the main sterol was lanosterol with no evidence 24-methylenedihydrolanosterol, the main sterol found in cells treated with growth inhibitory concentrations of the azole alone. Taken together, these results indicated that 24-alkyl sterols are essential growth factors for T. cruzi and that the preferred substrate of the delta 24(25) sterol methyl transferase in this organism is zymosterol.

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Occurrence of dermatomycoses and in-vitro therapeutic efficacy of three antifungal drugs on the growth of Epidermophyton floccosum.

Awoderu VA, Nebo GC, Otudero VO.

Department of Biological Sciences, Olabisi Onabanjo University, P. M. B. 2002, Ago-Iwoye, Nigeria. awoderu ibadan.skannet.com.ng

The occurrence of dermatomycoses and the in-vitro therapeutic efficacy of some antifungal agents on dermatomycotic organisms were investigated. Of the 550 primary school children screened, the incidence was one hundred (18%), 70 were males (representing 20% of the males screened) and 30 females (15% of the females sampled). The differences between male and female prevalence were insignificant. Three species of dermatophytes were isolated and identified. These were Microsporum canis, Trichophyton tonsurans and Epidermophyton floccosum. The antifungal agents tested on E. floccosum were griseofulvin, terbinafine and ketoconazole. They produced different sized zones of inhibition against the growth of E. floccosum. Griseofulvin exhibited a 50% inhibition of growth on E. floccosum at 63.00 mg/L. Terbinafine on the other hand exhibited varying levels of inhibition of growth at varying concentrations, at 0.07 mg/L, terbinafine achieved 46% inhibition of growth on E. floccosum. The drug achieved 100% inhibition of growth on the isolate at 61.81 mg/L. In the case of ketoconazole, 50% inhibition of growth was achieved at 100 mg/L while 100% inhibition of growth was achieved at 200 mg/L. The antifungal effects of the three drugs were confirmed by broth dilution tests where terbinafine was found to be fungistatic on the growth of E. floccosum at concentrations ranging from 0.013-1.700 mg/L and was fungicidal at concentrations ranging from 0.027-1.700 mg/L. Ketoconazole was found to inhibit the growth of E. floccosum at 0.003-1.700 mg/L and was fungicidal at concentrations ranging from 0.027-1.700 mg/L. It however did not succeed in killing the isolate under the same range of concentrations. Griseofulvin exhibited fungistatic effects on the growth of E. floccusum at 0.013-1.700 mg/L. In conclusion, ketoconazole and griseofulvin were found to be fungistatic and not fungicidal while terbinafine was both fungistatic and fungicidal on the pathogen. Terbinafine was found to be the most effective drug in inhibiting E. floccosum.

Online source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14682454&dopt=Abstract ketoconazole Nizoral