M.-C. Coene

Effect of hypercholesterolemia on vascular reactivity in the rabbit. I. Endothelium-dependent and endothelium-independent contractions and relaxations in isolated arteries of control and hypercholesterolemic rabbits.

We studied the effects of hypercholesterolemia on vascular responsiveness in different arteries isolated from rabbits: control groups of rabbits and groups receiving the atherogenic diet consisted of eight animals each. In the arteries, 16 weeks of cholesterol-rich (0.3%) diet evoked intimal lesions which were more pronounced than those noted after 8 weeks of hypercholesterolemia; the aortic arch was affected significantly more by the lesions than the abdominal aorta and the pulmonary artery. Segments of the arteries were mounted in organ chambers for isometric tension recording or for measurement of the endothelium-derived relaxant factor. Contractions caused by acetylcholine and prostaglandin F2α were not altered by the hypercholesterolemia; those evoked by serotonin were moderately augmented only in the aortic arch of hypercholesterolemic rabbits. As the degree of intimal lesion formation increased, the contractions to norepinephrine and clonidine were progressively inhibited. The endothelium-independent relaxations to nitroglycerin were inhibited in only the most severely affected arteries; the endothelium-dependent relaxations to acetylcholine and adenosine triphosphate were progressively inhibited as the degree of fatty streak formation augmented. Thus, in the aortic arch, the relaxations to 3 ± 10–6 m acetylcholine, expressed as percent of the initial contraction, decreased from 86.7 ± 3.3% in control tissues to 16.3 ± 8.6% in the 16-week hypercholesterolemic vessels; in the abdominal aortas these relaxations averaged 93.5 ± 2.2% in control vessels and 72.0 ± 6.9% in the hypercholesterolemic tissues. The acetylcholine-induced release of endothelium-derived relaxant factor from the abdominal aorta was not significantly affected by the hypercholesterolemia. We conclude from these studies that in arteries obtained from hypercholesterolemic rabbits: (1) the contractions caused by serotonergic mechanisms tend to be augmented, while those to α-adrenergic activation are decreased, (2) the endothelium-independent relaxations are modified only in the more severely affected arteries, and (3) the endothelium-dependent relaxations are progressively inhibited as the degree of fatty streak formation augments, probably because a step subsequent to the release of endothelium-derived relaxant factor is altered.

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 strains 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.

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

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.

Biochemical Approaches to Selective Antifungal Activity. Focus on Azole Antifungals

Summary: Azole antifungals (e.g. the imida‐zoles: miconazole, clotrimazole, bifona‐zole, imazalil, ketoconazole, and the tria‐zoles: diniconazole, triadimenol, propico‐nazole, fluconazole and itraconazole) inhibit in fungal cells the 14α‐demethylation of lanosterol or 24–methylenedihydro‐lanosterol. The consequent inhibition of ergosterol synthesis originates from binding of the unsubstituted nitrogen (N‐3 or N‐4) of their imidazole or triazole moiety to the heme iron and from binding of their N‐1 substituent to the apoprotein of a cytochrome P‐450 (P‐45014DM) of the endo‐plasmic reticulum.

Effects of itraconazole on cytochrome P-450-dependent sterol 14 alpha-demethylation and reduction of 3-ketosteroids in Cryptococcus neoformans.

As in other pathogenic fungi, the major sterol synthesized by Cryptococcus neoformans var. neoformans is ergosterol. This yeast also shares with most pathogenic fungi a susceptibility of its cytochrome P-450-dependent ergosterol synthesis to nanomolar concentrations of itraconazole. Fifty percent inhibition of ergosterol synthesis was reached after 16 h of growth in the presence of 6.0 +/- 4.7 nM itraconazole, and complete inhibition was reached at approximately 100 nM itraconazole. This inhibition coincided with the accumulation of mainly eburicol and the 3-ketosteroid obtusifolione. The radioactivity incorporated from [14C]acetate in both compounds represents 64.2% +/- 12.9% of the radioactivity incorporated into the sterols plus squalene extracted from cells incubated in the presence of 10 nM itraconazole. The accumulation of obtusifolione as well as eburicol indicates that itraconazole inhibits not only the 14 alpha-demethylase but also (directly or indirectly) the NADPH-dependent 3-ketosteroid reductase, i.e., the enzyme catalyzing the last step in the demethylation at C-4. This latter inhibition obviates the synthesis of 4,4-demethylated 14 alpha-methylsterols that may function at least partly as surrogates of ergosterol. Eburicol and obtusifolione are unable to support cell growth, and the 3-ketosteroid has been shown to disturb membranes. The complete inhibition of ergosterol synthesis and the accumulation of the 4,4,14-trimethylsterol and of the 3-ketosteroid together with the absence of sterols, such as 14 alpha-methylfecosterol and lanosterol, which can partly fulfill some functions of ergosterol, are at the origin of the high activity of itraconazole against C. neoformans. Fifty percent inhibition of growth achieved after 16 h of incubation in the presence of 3.2 +/- 2.6 nM itraconazole.

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 11β‐hydroxylase, 20,22‐desmolase and 17α‐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, 17α‐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 11β‐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.

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.

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.

Effect of hypercholesterolemia on vascular reactivity in the rabbit. II. Influence of treatment with dipyridamole on endothelium-dependent and endothelium-independent responses in isolated aortas of control and hypercholesterolemic rabbits.

The effects of cholesterol-feeding in the presence of dipyridamole (0.60 g daily) on contractile responses and on endothelium-dependent and endothelium-independent relaxations in isolated rabbit aortas are described. The investigations were performed simultaneously with those described in Part I Cire Res 1986;58:552–564), where the effects of cholesterol feeding on vascular reactivity in rabbit arteries (n = 8 in each group) selected at random from the same group of animals was studied. In the hypercholesterolemic rabbits treated with dipyridamole for 8 or 16 weeks, both the increases in plasma cholesterol and the formation of fatty streaks were significantly less pronounced than in the hypercholesterolemic rabbits not receiving the drug. Segments of the isolated arteries were mounted in organ chambers for isometric tension recording. The contractions caused by acetylcholine, prosta-glandin F^, norepinephrine, clonidine, and serotonin and the endothelium-independent relaxations to nitroglycerin were not significantly altered by the hypercholesterolemia in rabbits treated with dipyridamole, even after 16 weeks of treatment. Thus, the decreased responses to norepinephrine, clonidine, and nitroglycerin and the augmented responses to serotonin noted in aortas of hypercholesterolemic rabbits in Part I were absent in the dipyridamole-treated hypercholesterolemic animals. The endothelium-dependent relaxations to ATP and acetylcholine were not affected after 8 weeks of hypercholesterolemia in presence of dipyridamole, while after 16 weeks the relaxations to ATP and acetylcholine were attenuated only in the more severely affected arteries. The effects of hypercholesterolemia + dipyridamole on endothelium-dependent relaxations were significantly less pronounced than those induced by hypercholesterolemia alone. We thus conclude from our studies that in the rabbit 1) dipyridamole can partially inhibit the augmentation in plasma cholesterol levels caused by a cholesterol-rich diet; 2) dipyridamole can slow down the process of formation of intimal aortic lesions caused by the cholesterol feeding; 3) dipyridamole inhibits the cholesterol-induced changes in vascular reactivity, and 4) the degree of formation of intimal aortic lesions determines the degree to which the vascular responses, especially the endothelium-dependent relaxations, are affected by the cholesterol-rich diet.

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

Summary. 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 μM itraconazole and 0.56–1.2 μM ketoconazole, whereas 91‐ > 100 μM 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 14α‐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.