Identification of a new metabolite of macrolide immunosuppressant, like rapamycin and SDZ RAD, using high performance liquid chromatography and electrospray tandem mass spectrometry.

Previously unknown metabolites from the two macrolide immunosuppressants rapamycin (sirolimus) and SDZ RAD [40-O-(2-hydroxyethyl)rapamycin] obtained after in vitro incubation with human liver microsomes have been purified. Structure elucidation was performed by nanoelectrospray ionization tandem mass spectrometry applying low energy collision activated dissociation. This ionization method is, as shown here, a powerful tool to determine metabolic pathways by analysis of even low abundance products. Product ion spectra of the isolated metabolites indicate a new kind of biotransformation reaction for rapamycin and SDZ RAD. The proposed metabolic pathway starts with an ester hydrolysis which leads to a ring-opened structure. A dehydration on C33-C34 and a supplementary hydrogenation at C33-C34 result in a structure similar to the ring-opened isomer with an single bond at C33-C34.

Small intestinal metabolism of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor lovastatin and comparison with pravastatin.

We compared the intestinal metabolism of the structurally related 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors lovastatin and pravastatin in vitro. Human small intestinal microsomes metabolized lovastatin to its major metabolites 6'beta-hydroxy (apparent K(m) = 11.2 +/- 3.3 microM) and 6'-exomethylene (apparent K(m) = 22.7 +/- 9.0 microM) lovastatin. The apparent K(m) values were similar for lovastatin metabolism by human liver microsomes. 6'beta-Hydroxylovastatin formation by pig small intestinal microsomes was inhibited with the following inhibition K(i) values: cyclosporine, 3.3 +/- 1.2 microM; ketoconazole, 0.4 +/- 0.1 microM; and troleandomycin, 0.8 +/- 0.9 microM. K(i) values for 6'-exomethylene lovastatin were similar. Incubation of pravastatin with human small intestinal microsomes resulted in the generation of 3'alpha,5'beta, 6'beta-trihydroxypravastatin (apparent K(m) = 4560 +/- 1410 microM) and hydroxypravastatin (apparent K(m) = 5290 +/- 1740 microM). In addition, as in the liver, pravastatin was metabolized in the small intestine by sulfation and subsequent degradation to its main metabolite 3'alpha-iso-pravastatin. It was concluded that lovastatin is metabolized by cytochrome P-450 3A enzymes in the small intestine. Compared with lovastatin, the cytochrome P-450-dependent intestinal intrinsic clearance of pravastatin was >5000-fold lower and cannot be expected to significantly affect its oral bioavailability or to be a significant site of drug interactions.

Simultaneous on-line extraction and analysis of sirolimus (rapamycin) and ciclosporin in blood by liquid chromatography-electrospray mass spectrometry.

We developed a sensitive and specific semi-automated liquid chromatography-electrospray mass spectrometric (HPLC-ESI-MS) assay for the simultaneous quantification of sirolimus and ciclosporin in blood. Following a simple protein precipitation step, the supernatants were injected into the HPLC system and extracted on-line. After column switching, the analytes were backflushed from the extraction column onto the analytical narrow-bore column and eluted into the ESI-MS system. The assay was linear from 0.4 to 100 microg/l sirolimus and from 2 to 1500 microg/l ciclosporin. The mean recoveries of sirolimus and ciclosporin were 98 and 96%, respectively. The mean interday precision/accuracy was 8.6%/-4.8% for sirolimus and 9.3%/-2.9% for ciclosporin.

Comparison of cytochrome P-450-dependent metabolism and drug interactions of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors lovastatin and pravastatin in the liver.

In an in vitro study, the cytochrome P-450 3A (CYP3A)-dependent metabolism and drug interactions of the 3-hydroxy-3-methylglutaryl-Co A reductase inhibitors lovastatin and pravastatin were compared. Lovastatin was metabolized by human liver microsomes to two major metabolites: 6'beta-hydroxy [Michaelis-Menten constant (Km): 7.8 +/- 2.7 microM] and 6'-exomethylene lovastatin (Km,10.3 +/- 2.6 microM). 6'beta-Hydroxylovastatin formation in the liver was inhibited by the specific CYP3A inhibitors cyclosporine (Ki, 7.6 +/- 2.3 microM), ketoconazole (Ki, 0.25 +/- 0.2 microM), and troleandomycin (Ki, 26.6 +/- 18.5 microM). Incubation of pravastatin with human liver microsomes resulted in the generation of 3'alpha,5'beta, 6'beta-trihydroxy pravastatin (Km, 4,887 +/- 2,185 microM) and hydroxy pravastatin (Km, 20,987 +/- 9,389 microM). The formation rates of 3'alpha,5'beta,6'beta-trihydroxy pravastatin by reconstituted CYP3A enzymes were (1,000 microM pravastatin) 1.9 +/- 0.6 pmol.min-1.pmol CYP3A4 and 0.06 +/- 0.04 pmol.min-1.pmol CYP3A5, and the formation rates of hydroxy pravastatin were 0.12 +/- 0.02 pmol.min-1.pmol CYP3A4 and 0.02 +/- 0.004 pmol.min-1.pmol CYP3A5. The specific CYP3A inhibitors cyclosporine, ketoconazole, and troleandomycin significantly inhibited hydroxy pravastatin formation by human liver microsomes, but only ketoconazole inhibited 3'alpha, 5'beta,6'beta-trihydroxy pravastatin formation, suggesting that other CYP enzymes are involved in its formation. It is concluded that, compared with lovastatin [CLint formation 6'beta-hydroxylovastatin (microl.min-1.mg-1): 199 +/- 248, 6'-exomethylene lovastatin: 138 +/- 104)], CYP3A-dependent metabolism of pravastatin [CLint formation 3'alpha,5'beta, 6'beta-trihydroxy pravastatin (microl.min-1.mg-1): 0.03 +/- 0.03 and hydroxy pravastatin: 0.02 +/- 0.02] is a minor elimination pathway. In contrast to lovastatin, drug interactions with pravastatin CYP3A-catalyzed metabolism cannot be expected to have a clinically significant effect on its pharmacokinetics.