engleski

KETOCONAZOLE AND VERAPAMIL CAUSE THE REDUCTION OF HEME

Ketoconazole is an antiandrogen and antifungal medicine used to treat certain serious fungal infections in the body. Some of the uses of ketoconazole include tinea, cutaneous candidiasis, pityriasis versicolor, dandruff and seborrheic dermatitis. Verapamil is a calcium channel blocker medicine used to treat patients with high blood pressure, angina and supraventricular tachycardia. Cytochrome P450 3A4 (CYP3A4) enzymes are a part of a large group of enzymes which are essential for the metabolism of many medicines and endogenous compounds. CYP3A4 is responsible for the metabolism of more than 50% of medicines. CYP3A4 is subject to reversible and mechanism-based (irreversible) inhibition. The latter involves the inactivation of the enzyme via the formation of metabolic intermediates that bind irreversibly to the enzyme and then inactivate it. The clinical effects of a mechanistic inactivator are more prominent after multiple dosing and last longer than those of a reversible inhibitor. Ketoconazole and verapamil are known as strong inhibitors of the CYP3A4 enzyme with proven in vitro and in vivo inhibitions of the CYP3A4 activity. CYP3A4 is a hemoprotein, which means that it has a heme iron in its active center. The aim of this study was to examine the reduction of the heme, which is believed to be caused by the inhibition mechanism of ketoconazole and verapamil. The examination of the change in heme concentration due to binding to the reactive intermediate to heme was performed according to the method described earlier in the literature. To perform the test, the calibration curve of the hemin solution in dimethyl sulfoxide (0.6 to 0.1 μM) was first prepared, and a spectrum at a wavelength of 500 to 600 nm was recorded on a spectrophotometer. The incubation mixture was prepared to a volume of 1.5 mL and consisted of ultrapure water, appropriate hemin solution, 0.83 M pyridine (final concentration) and 0.06 M sodium hydroxide (final concentration). The prepared calibration curve was used to calculate the heme concentration in the samples. The incubation mixture was prepared according to the described method, where the volume of the incubation mixture was increased to 200 μL, and the concentration of inhibitor was 25 μM. The tests were performed in triplicate. The reaction is started by the addition of a generating system, with incubation for 30 min. Ultrapure water is then added to the mixture. The mixture was transferred to spectrophotometer cuvettes and pyridine and sodium hydroxide were added. Samples were recorded on a spectrophotometer within 1 min from the addition of alkaline solution, due to the instability of pyridine hemochromogen under basic conditions. The results were confirmed by re-incubation, adding 5 international units of catalase and superoxide dismutase. An 83.72% and a 69.92% reduction of heme was observed using ketoconazole and verapamil as substrates. The second testing with superoxide dismutase and catalase showed similar results with 77.34% and 58.23% of heme reduction, respectively. These results indicate the irreversible nature of the CYP3A4 inhibition by ketoconazole and verapamil and are useful for future clinical practices and drug regulations.

ketoconazole ; verapamil ; heme ; reduction

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