Hepatic effects of ketoconazole in the male Swiss Webster mouse: temporal changes in drug metabolic parameters

There have been conflicting observations regarding the effects of ketoconazole on hepatic metabolism. The objectives of these studies were to determine whether ketoconazole was an enzyme inducer or inhibitor in the mouse and then to establish the time frame of these ketoconazole-induced enzyme changes. Ketoconazole was administered (150 mg/kg p.o. X 4 days) to male Swiss Webster mice. Biochemical observations over a period of 6 days following treatment indicated that ketoconazole had a temporal biphasic effect on the liver. Although liver weight and microsomal protein were elevated, all other parameters monitored were lower at 2 h following ketoconazole treatment. At 24 h after the last dose of ketoconazole, hepatic biochemical parameters (liver wt., % liver wt./body wt., microsomal protein, and cytochrome P-450) were statistically elevated, while enzyme activities (benzphetamine N-demethylation, 6 beta- and 7 alpha-hydroxylation of testosterone, formation of androstenedione and UDP-glucuronyltransferase) were inhibited. At 72 h the ketoconazole-induced changes in the hepatic biochemical parameters were comparable to those observed at 24 h, and enzymatic parameters generally appeared to be induced by ketoconazole, with the exception of benzphetamine N-demethylase and UDP-glucuronyltransferase, which exhibited lower enzyme activities. Ethoxyresorufin O-deethylase, 7 alpha-hydroxylation of testosterone and glutathione S-transferase, on the other hand, were unaltered by ketoconazole treatment. The opposing effects of ketoconazole on benzphetamine N-demethylase and ethylmorphine N-demethylase at 72 h were further examined. Enzyme kinetics studies indicated that ketoconazole did not effect the Michaelis constants (Km) of the two substrates, but the maximum velocity (Vmax) of the reactions was altered.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antifungal activity of the essential oil of Agastache rugosa Kuntze and its synergism with ketoconazole

AIMS: To evaluate the fungitoxic activity of the essential oil of Agastache rugosa alone and to determine its combination effect with ketoconazole against Blastoschizomyces capitatus. METHODS AND RESULTS: The antifungal activities of the essential oil of A. rugosa and its main constituent estragole were investigated using the broth microdilution, disk diffusion methods and checkerboard microtitre assay. Both estragole and the essential oil exhibited strong activities against the tested fungi and showed synergism with ketoconazole against B. capitatus. CONCLUSIONS: Both estragole and the essential oil of A. rugosa have significant growth-inhibiting activities against B. capitatus showing strong synergistic effect with ketoconazole. SIGNIFICANCE AND IMPACT OF THE STUDY: The essential oil of A. rugosa, combined with ketoconazole, may be particularly useful against B. capitatus, a rare pathogenic fungus documented to cause severe and fatal mycoses in immunocompromised patients.     

In vitro inhibition by ketoconazole of human testicular steroid oxidoreductases

An oral antimycotic agent, ketoconazole has been demonstrated to be an inhibitor of cytochrome P-450-dependent monooxygenases. To investigate its effect on steroid oxidoreductases, in vitro studies were carried out using subcellular fractions of human testes. Ketoconazole competitively inhibited activities of 3 beta-hydroxy-5-ene-steroid oxidoreductase/isomerase and NADH-linked 20 alpha-hydroxysteroid oxidoreductase for steroid substrate and the Ki values were 2.9 and 0.9 microM, respectively. In contrast, ketoconazole inhibited neither 17 beta-hydroxysteroid oxidoreductase nor NADPH-linked 20 alpha-hydroxysteroid oxidoreductase, indicating that the two 20 alpha-hydroxysteroid oxidoreductases are distinct. Further, ketoconazole inhibited non-competitively the above enzyme activities for the corresponding cofactors of NAD and NADH. From the binding mode of ketoconazole to cytochrome P-450 and the present findings, it appears likely that the agent binds to a site which is different from that of steroids or pyridine nucleotides.     

Measurement of glucose-6-phosphate dehydrogenase and glutathione reductase activity in patients with paracoccidioidomycosis treated with ketoconazole

Hemoglobin rates, hematocrit and glucose-6-phosphate dehydrogenase (G6PD) and glutathione reductase activities were measured in 38 patients with paracoccidioidomycosis treated with ketoconazole or sulfadoxin, and in 13 normal individuals.Ketoconazole-treated patients showed reduced G6PD and glutathione reductase activities. One of these patients was found to be G6PD-deficient and suffered a hemolytic episode during treatment, which, however, did not require interruption of therapy.The authors suggest that patients showing an erythrocyte enzyme defect should be monitored hematologically during treatment with ketoconazole. They also suggest that ketoconazole is an oxidant drug in addition to being a possible inhibitor of antioxidant erythrocyte enzymes.     

Effect of antifungal azoles on the heme detoxification system of malarial parasite

The antimalarial activities of some antifungal azole agents (ketoconazole, miconazole, and clotrimazole) have been known for several years, however, their antimalarial mechanism remains equivocal. Our recent study showed that clotrimazole has a relative high affinity for heme, inhibits reduced glutathione-dependent heme catabolism, and enhances heme-induced hemolysis. In the present study, we have found that clotrimazole can remove heme from histidine rich peptide-heme complex, which initiates heme-polymerization in malaria. In addition, we show that two other azoles (ketoconazole and miconazole) behave similarly to clotrimazole in binding to heme: they bind to heme with similar affinities, remove heme from the histidine rich peptide-heme complex and from the reduced glutathione-heme complex to form stable heme-azole complexes with two nitrogenous ligands derived from the imidazole moieties of two azole molecules. We have also revealed that clotrimazole and miconazole have stronger promoting activities for heme-induced hemolysis than ketoconazole, implying that the stronger antimalarial activities of clotrimazole and miconazole might arise from their stronger ability to promote heme-induced hemolysis of clotrimazole and clotrimazole than that of ketoconazole. These results also suggest that ketoconazole and miconazole, like clotrimazole, might possess an antimalarial mechanism relating to their inhibition of heme polymerization and the degradation of reduced glutathione-dependent heme.     

Synthesis and preliminary biological profile of new NO-donor tolbutamide analogues

We describe a new class of NO-donor hypoglycemic products obtained by joining tolbutamide, a typical hypoglycemic sulfonylurea, with a NO-donor moiety through a hard link. As NO-donors we chose either furoxan (1,2,5-oxadiazole 2-oxide) derivatives or the classical nitrooxy function. A preliminary biological characterization of these compounds, including stimulation of insulin release from cultured rat pancreatic β-cells and in vitro vasodilator and anti-aggregatory activities, is reported.     

Response of plasma adrenal steroids to synthetic ACTH under ketoconazole in man

We have investigated the effect of a single oral administration of 600 mg ketoconazole, an imidazole derivative used in clinical practice as an antimycotic agent, on the response of plasma adrenocortical steroids to 1-24 ACTH in 5 normal male subjects pretreated with dexamethasone. The 2 mg of dexamethasone was administered orally at 2300 h on the preceding night, and then a rapid ACTH test was started at 0830 h. After a 1 week interval, the ACTH test was repeated in the same manner under the same dexamethasone pretreatment, but 600 mg of ketoconazole was given orally at 0500 h. The absolute plasma concentration and the increase over the basal level of each steroid after ACTH were evaluated and compared in both conditions with and without ketoconazole administration. A single ingestion of ketoconazole caused a decrease in both indices of the responsiveness of plasma dehydroepiandrosterone and a rise in the plasma level of 17 alpha-hydroxypregnenolone. The response of plasma aldosterone was clearly blunted by ketoconazole administration, whereas that of plasma corticosterone was clearly increased. Ketoconazole also blocked the response of plasma cortisol with concomitantly increased responses of plasma 11-deoxycortisol and 11-deoxycorticosterone. The increased response of plasma corticosterone seemed to be likely due to the severe inhibitory action of ketoconazole on the conversion of corticosterone to aldosterone. These results imply the inhibitory effect of ketoconazole on C17-20-lyase activity and on the conversion of corticosterone to aldosterone, and suggest an inhibitory action on 11 beta-hydroxylase activity. The effects of ketoconazole on the other enzyme activities in adrenal steroid biosynthesis were also discussed.     

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

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.     

Ketoconazole as a possible universal inhibitor of cytochrome P-450 dependent enzymes: its mode of inhibition

Modes of inhibition and binding of ketoconazole, an orally antimycotic agent, to NADPH-cytochrome P-450 dependent enzymes were investigated using subcellular fractions of human and rat testes, human adrenocortical adenoma tissue and rat adrenals and livers. Ketoconazole competitively inhibited the activities of steroid 17 alpha-hydroxylase and C17-20 lyase in rat and human testes, 16 alpha-hydroxylase in human testes and 21-hydroxylase in rat adrenal glands. Ki values were in the order of 10(-8)M for human testicular enzymes, while the order was 10(-7)-10(-6) M for rat adrenal and testicular enzymes. Kinetic studies indicated that ketoconazole bound to cytochrome P-450 and not to other components of monooxygenase systems. Spectrophotometric studies also revealed direct binding of ketoconazole to cytochrome P-450 component by inducing type II difference spectra in all tissue preparations examined, indicating that ketoconazole is possibly a universal inhibitor of NADPH-cytochrome P-450 dependent monooxygenases which are involved in metabolism of many substances including steroids, toxins, carcinogens and others.     

Effect of nitric oxide-donating agents on human monocyte cyclooxygenase-2

COX-2 is involved in inflammation and ischemic cardiovascular disease. As NO regulates COX activity in various cells, we investigated the effect of NO-donors and the novel NO-aspirin NC-4016 on human monocyte COX-2. Whole blood was incubated with LPS and PGE(2) was measured in plasma as an index of monocyte COX-2 activity. Serum TxB(2) was assessed as an index of platelet COX-1 activity. SNP, DetaNONOate, and NO-aspirin inhibited dose-dependently PGE(2) production while aspirin was ineffective. The guanylyl-cyclase inhibitor ODQ partially reversed the suppression of COX-2 activity by NO-aspirin, demonstrating a role of cGMP increase. NC-4016 and aspirin inhibited platelet COX-1 comparably while NO-donors were ineffective. COX-2 expression was not affected by NO-donors or NO-aspirin while aspirin or the selective COX-2-inhibitor DUP697 increased it. In conclusion, Nitroaspirin inhibits monocyte COX-2 activity by a cGMP-dependent mechanism. This might represent an advantage over aspirin, given the possible detrimental role of COX-2 in cardiovascular disease.     

Nitric oxide decreases intestinal haemorrhagic lesions in rat anaphylaxis independently of mast cell activation

The purpose of this study is to assess the role of nitric oxide (NO) in the intestinal lesions of passive anaphylaxis, since this experimental model resembles necrotizing enterocolitis. Sprague-Dawley rats were sensitized with IgE anti-dinitrophenol monoclonal antibody. Extravasation of protein-rich plasma and haemorrhagia were measured in the small intestine. Plasma histamine was measured to assess mast cell activation. The effect of exogenous NO on the lesions was assessed by using two structurally unrelated NO-donors: sodium nitroprusside and S-nitroso-Nacetyl-penicillamine (SNAP). An increased basal production of NO was observed in cells taken after anaphylaxis, associated with a reduced response to platelet-activating factor, interleukin 1beta, and IgE/DNP-bovine serum albumin complexes. The response to bacterial lipopolysaccharide and dibutyryl cyclic adenosine monophosphate (AMP) was enhanced 24 h after challenge, but at earlier times was not significantly different from that observed in controls. Treatment with either sodium nitroprusside or SNAP produced a significant reduction of the haemorrhagic lesions, which are a hallmark of rat anaphylaxis. The extravasation of protein-rich plasma was not influenced by NO-donors. The increase of plasma histamine elicited by the anaphylactic challenge was not influenced by SNAP treatment. NO-donors protect intestinal haemorrhagic lesions of rat anaphylaxis by a mechanism apparently independent of mast cell histamine release.     

Study of NO action on calcium-activated potassium channel of the rat artery smooth muscle cells

Nitric oxide (NO) released from the endothelium or from NO-donors is a powerful vasodilator. Its effect is mediated partly by vascular smooth muscle high conductance calcium-activated potassium (Kca) channels. Contradictory data exist as to whether NO activated the KCa channel directly or indirectly via protein kinase G (PKG). Thus the hypothesis that NO-donors can activate the KCa directly was investigated using the patch-clamp technique and freshly isolated smooth muscle cells from the rat tail artery. In inside-out experiments, the activity of KCa-channels was increased 1.61 +/- 0.20-fold (n = 10) by 10 microM SNP and 1.45 +/- 0.17-fold (n = 8) by 10 microM SNAP. However, the activity of KCa channels was also increased 1.46 +/- 0.20-fold (n = 8) by addition of the experimental bath solution. Thus these results suggest that NO released from NO-donors cannot activate KCa channel of the rat tail artery smooth muscle cells directly.     

Synthesis and antifungal activities of phenylenedithioureas

A total of 20 new phenylenedithiourea derivatives was synthesized by reaction of phenylenediisothiocyanates with aromatic amines as aminobenzoic, aminosalicylic acid and their derivatives. Their chemical structures were confirmed by elemental analysis, IR spectrometry and 1H NMR. The compounds were screened for in vitro antifungal, antibacterial activities and some of them have strong antifungal activities comparable to the activity observed for ketoconazole.     

In-vitro inhibitory activities of 2 new orally absorbable imidazole derivatives: BAY n 7133 and BAY 1 9139

The inhibitory activities of 2 new orally absorbed antifungal imidazole derivatives, BAY n 7133 and BAY 1 9139, were compared in vitro with those of ketoconazole and miconazole. Clinical isolates of pathogenic fungi tested included 35 yeasts, 62 dimorphic fungal pathogens, 37 filamentous fungi and 31 dermatophytes. LY 121019, a semisynthetic analog of echinocandin B, was included in tests with the pathogenic yeasts. Both BAY n 7133 and BAY 1 9139 were found to be broad spectrum antifungal agents. The spectra of these newer compounds were comparable to those of ketoconazole and miconazole; however only BAY n 7133 resembled these latter 2 imidazoles quantitatively in terms of the degree of antifungal activity as indicated by measurable MICs. In contrast, the spectrum of LY 121019 appeared to be confirmed only to isolates of Candida.     

Determination of susceptibility/resistance to antifungal drugs of Trichophyton mentagrophytes isolates by a macrodilution method

Onychomycosis is a common adult human mycosis, and dermatophytes of the Trichophyton genera are the most frequently isolated microorganism. Globally, from 3% to 10% of the human population is attacked by ony cho mycosis, and many cases involve toenails. The aim of this work was to determine the minimal inhibitory concentrations (MICs) of antifungal drugs (fluconazole, ketoconazole, itraconazole, terbinafine, and griseofulvin) often used for the treatment of ungueal dermatophytosis caused by Trichophyton mentagrophytes. The MICs were determined by the broth medium macrodilution method. The results showed that activities of terbinafine and itraconazole were significantly higher (MIC <0.007-0.015 microg.mL -1 and MIC = 0.062-1.000 microg.mL -1, respectively). All isolates had reduced susceptibility to fluconazole (MIC = 16 to >64 microg.mL -1). The MICs of ketoconazole and griseofulvin varied among strains, ranging from 0.125 to 2.000 microg.mL -1 for ketoconazole and from 0.25 to 2.00 microg.mL -1 for griseofulvin. These MICs were higher than those of other studies cited, possibly because of differences in culture medium used in the other studies.     

Propolis from the northwest of Argentina as a source of antifungal principles

AIMS: To determine the antimycotic and cytotoxic activities of partially purified propolis extract on yeasts, xylophagous and phytopathogenic fungi. To compare these activities with pinocembrin and galangin isolated from this propolis and with the synthetic drugs ketoconazole and clortrimazole. METHODS AND RESULTS: Ethanolic propolis extract was partially purified by cooling at -20 degrees C. Two of its components were isolated by HPLC and identified as pinocembrin and galangin. The antifungal activity was assayed by bioautography, hyphal radial growth, hyphal extent and microdilution in liquid medium. Cytotoxicity was studied with the lethality assay of Artemia salina. The obtained results were compared with the actions of ketoconazole and clortrimazole. The results showed that the antifungal potency of ketoconazole and clortrimazole is higher than pinocembrin, galangin and the partially purified propolis extract in this order. Otherwise, the cytotoxicity of the synthetic drugs is also the highest. CONCLUSIONS: Partially purified propolis extract inhibits fungal growth. The comparison of its relative biocide potency and cytotoxicity with synthetic drugs and two components of this propolis (pinocembrin and galangin) showed that the propolis from 'El Siambón', Tucumán, Argentina, is a suitable source of antifungal products. SIGNIFICANCE AND IMPACT OF THE STUDY: The partially purified propolis extract and its isolated compounds, pinocembrin and galangin, have the capacity of being used as antifungals without detriment to the equilibrium of agroecosystems. The impact of this study is that the preparation of agrochemicals with reduced economic costs using a partially purified preparation as the active principle is possible.     

Effect of the monooxygenase activity inhibitor ketoconazole on cholesterol esterification in mouse peritoneal macrophages

In order to determine the feasible role of monooxygenases in regulation of the macrophage acyl-CoA: cholesterol acyltransferase (ACAT) activity, the effects of ketoconazole on the activities of benz(a)pyrene hydroxylase and ACAT as well as on the [14C]oleate incorporation into cholesterol esters in cultured mouse peritoneal macrophages (MPM) were studied. Ketoconazole (0.5-50 M) inhibited the benz(a)pyrene hydroxylase activity but increased the free cholesterol (FC) level in MPM cultured with an acetylated low density lipoprotein (acetyl-LDL). An addition of ketoconazole (1-50 M) eliminated the increase in the rate of FC esterification after incubation of MPM with acetyl-LDL (but not with 25-hydroxycholesterol). In contrast, progesterone, an ACAT activity inhibitor, used at 5-30 M diminished the rate of FC esterification, when MPM were incubated with acetyl-LDL of 25-hydroxycholesterol. Ketoconazole provoked a dose-dependent decrease of the [3H]FC incorporation into macrophage polar oxysteroids. The data obtained suggest that the ketoconazole (1-30 M) effect on FC esterification in MPM cultured with acetyl-LDL is determined by its inhibiting monooxygenases, which produce oxidized forms of FC that are potential activators of ACAT.     

Nitric oxide decreases cell surface expression of aquaporin-5 and membrane water permeability in lung epithelial cells

Nitric oxide (NO) is implicated in the pathogenesis of lung inflammation and edema. In this study, the effects of nitric oxide (NO)-donors on membrane water permeability and cell surface expression of aquaporin-5 (AQP5) in mouse lung epithelial cells were examined. NO-donors, GSNO and NOC-18 decreased cell surface expression of AQP5, concentration- and time-dependently, whereas they did not affect the amount of AQP5 in whole cell lysates. The membrane water permeability of cells was also decreased by treatment with NO-donors. The decrease in cell surface AQP5 by NO was abolished by simultaneous treatment with methyl-beta-cyclodextrin, but not with ODQ, an inhibitor of the cGMP-dependent pathway. In addition, immunocytochemistry with anti-AQP5 indicated that NO changed AQP5 localization from the plasma membrane to the intracellular fraction. These data indicate that NO stimulates AQP5 internalization from the plasma membrane through a cGMP-independent mechanism, and decreases membrane water permeability.     

Studies on the effect of ketoconazole on the fusion of L6 myoblasts

Summary The effect of ketoconazole on the fusion of L6 myoblasts was studied. Ketoconazole was a potent inhibitor of myoblast fusion at concentrations as low as 0.1 M, but fusion was restored when the inhibitor was removed. The inhibitor resulted in decreased binding of conA and WGA to cell surface oligosaccharides showing that it was inhibiting N-linked cell surface glycoproteins. Inhibition of fusion by ketoconazole was accompanied by reduced creatine phosphokinase activities showing that it is affecting biochemical differentiation. Incorporation of labelled mannose from GDP-mannose into lipid-sugar and lipid-oligosaccharide complexes involved in the synthesis of N-linked oligosaccharides was also inhibited by ketoconazole, but the inhibition was reversed by addition of exogenous dolichol phosphate to the incorporation mixture. The main conclusion from these studies was that ketoconazole inhibited fusion of L6 myoblasts by affecting the synthesis of dolichol-phosphate required for the synthesis of lipid-oligosaccharides needed for the synthesis of fusogenic cell surface N-linked glycoproteins.Abbreviations HMG-CoA 3-hydroxy-3-methylglutaryl Coenzyme A - Dol-P Dolichol Phosphate - Man Mannose - GlcNAc N-acetylglucosamine - Glc Glucose - conA concanavalin A - WGA Wheat Germ Agglutinin - CPK Creatine Phosphokinase