Potent inhibition of the cytochrome P-450 3A-mediated human liver microsomal metabolism of a novel HIV protease inhibitor by ritonavir: A positive drug-drug interaction.

ABT-378 is a potent in vitro inhibitor of the HIV protease and is currently being developed for coadministration with another HIV protease inhibitor, ritonavir, as an oral therapeutic treatment for HIV infection. In the present study, the effect of ritonavir, a potent inhibitor of cytochrome P-450 (CYP) 3A, on the in vitro metabolism of ABT-378 was examined. Furthermore, the effect of ABT-378-ritonavir combinations on several CYP-dependent monooxygenase activities in human liver microsomes was also examined. ABT-378 was found to undergo NADPH- and CYP3A4/5-dependent metabolism to three major metabolites, M-1 (4-oxo) and M-3/M-4 (4-hydroxy epimers), as well as several minor oxidative metabolites in human liver microsomes. The mean apparent K(m) and V(max) values for the metabolism of ABT-378 by human liver microsomes were 6.8 +/- 3.6 microM and 9.4 +/- 5.5 nmol of ABT-378 metabolized/mg protein/min, respectively. Ritonavir inhibited human liver microsomal metabolism of ABT-378 potently (K(i) = 0.013 microM). The combination of ABT-378 and ritonavir was much weaker in inhibiting CYP-mediated biotransformations than ritonavir alone, and the inhibitory effect appears to be primarily due to the ritonavir component of the combination. The ABT-378-ritonavir combinations (at 3:1 and 29:1 ratios) inhibited CYP3A (IC(50) = 1.1 and 4.6 microM), albeit less potently than ritonavir (IC(50) = 0.14 microM). Metabolic reactions mediated by CYP1A2, CYP2A6, and CYP2E1 were not affected by the ABT-378-ritonavir combinations. The inhibitory effects of ABT-378-ritonavir combinations on CYP2B6 (IC(50) = >30 microM), CYP2C9 (IC(50) = 13.7 and 23.0 microM), CYP2C19 (IC(50) = 28.7 and 38.0 microM), and CYP2D6 (IC(50) = 13.5 and 29.0 microM) were marginal and are not likely to produce clinically significant drug-drug interactions.

In vitro metabolism of the HIV-1 protease inhibitor ABT-378: species comparison and metabolite identification.

HIV protease inhibitor ABT-378 (ABT-378) was metabolized very extensively and rapidly by liver microsomes from mouse, rat, dog, monkey, and humans. The rates of NADPH-dependent metabolism of ABT-378 ranged from 2.39 to 9.80 nmol.mg microsomal protein-1.min-1, with monkey liver microsomes exhibiting the highest rates of metabolism. ABT-378 was metabolized to 12 metabolites (M-1 to M-12), which were characterized by mass and NMR spectroscopy. The metabolite profile of ABT-378 in liver microsomes from all five species was similar, except that the mouse liver microsomes did not form M-9, a minor secondary metabolite. The predominant site of metabolism was the cyclic urea moiety of ABT-378. In all five species, the major metabolites were M-1 (4-oxo-ABT-378) and M-3 and M-4 (4-hydroxy-ABT-378). Metabolite M-2 (6-hydroxy-ABT-378) was formed by rodents at a faster rate than by dog, monkey, and human liver microsomes. Metabolites M-5 to M-8 were identified as monohydroxylated derivatives of ABT-378. Metabolites M-9 and M-10 were identified as hydroxylated products of M-1. Metabolites M-11 and M-12 were identified as dihydroxylated derivatives of ABT-378. The metabolite profile in human hepatocytes and liver slices was similar to that of human liver microsomes. The results of the current study indicate that ABT-378 is highly susceptible to oxidative metabolism in vitro, and possibly in vivo, in humans.

The orally active renin inhibitor A-74273. In vivo and in vitro morpholine ring metabolism in rats, dogs, and humans.

The metabolism and disposition of [14C]A-74273--a potent, orally active renin inhibitor--were investigated in beagle dogs and Sprague-Dawley rats. Two male and two female dogs received a single 10 mg/kg oral or 1 mg/kg intravenous dose in a cross-over experiment and urine and feces were collected for 5 days. After both intravenous and oral dosing, > 92% of the dose was recovered in the feces and < 3% was recovered in the urine. The predominance of hepatobiliary elimination in the clearance of A-74273 was verified in a bile-exteriorized dog, where 79.8% of a 1 mg/kg intravenous dose was excreted in the bile within 6 hr after administration. Similarly, administration of a 1 mg/kg intravenous dose to a bile-exteriorized rat resulted in biliary excretion of 60.5% of the dose within 6 hr. Radio-HPLC analysis of bile and feces from both species indicated extensive metabolism of A-74273 to three major morpholine ring-opened metabolites; the ethanolamine A-78242, the amine A-78030, and the carboxylic acid A-81307. These three metabolites each contributed 12.0-20.2% of the biliary radioactivity after intravenous dosing, while unchanged A-74273 contributed 5-17%. Incubation of [14C]A-74273 with rat, dog, and human liver microsomes afforded nearly equal amounts of the three in vivo metabolites for all three species, suggesting that the in vitro system was representative of A-74273 in vivo metabolism and that humans should also convert A-74273 to the morpholine ring-opened metabolites in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of liver microsomal cytochrome p450 (CYP2D6) activity using [O-methyl-14C]dextromethorphan.

The activity of human liver microsomal cytochrome P4502D6 (CYP2D6) is readily estimated by following the O-demethylation of [O-methyl-14C]dextromethorphan. The basis of the assay is the quantitative measurement of [14C]formaldehyde (0.05-4.0 microM) after addition of NaOH to the microsomal incubates and extraction with methylene chloride. The assay is relatively simple, sensitive (limit of detection is approximately 5.0 pmol HCHO/h/mg microsomal protein) and does not require the use of HPLC or an internal standard. Formation of radiolabeled formaldehyde in human liver microsomes is linear for 20 min, up to a final protein concentration of 1.0 mg/ml. Furthermore, the O-demethylase activity in a panel of microsomes prepared from a series of human livers was significantly correlated with the immunochemically determined levels of CYP2D6 protein (r = 0.925, p < 0.001), and was inhibited (> 89%) by quinidine and lobeline. In addition, [O-methyl-14C]-dextromethorphan O-demethylation was exclusively catalyzed by cDNA-expressed CYP2D6 in microsomes prepared from human B-lymphoblast cells. The method is suitable for rapid screening of compounds as potential CYP2D6 cosubstrates and/or inhibitors.

Combined enzymatic and chemical approaches to the synthesis of unique polyribonucleotides.

The enzymatic polymerization by polynucleotide phosphorylase of 6-chloro-9-(beta-D-ribofuranosyl)purine 5'-diphosphate to poly(6-chloropurinylic acid) and its conversion to poly(6-thioninosinic acid) is described. The sulfur isostere of poly(I) was found not to form a complex with poly(C), but to form a self-association complex with a Tm around 295 degrees K. The sedimentation velocities, pKa and Tm values of the polymer have been examined under various conditions. A two (or more) stranded helical array is suggested as the most probable structure. Thermal loss of the thione chromophore was noted for poly- (S6I), S6IMP and S6I; the degradation product from S6I was shown to be inosine.

The use of s-2-cyanoethyl phosphorothioate in the preparation of oligo 5'-deoxy-5'-thiothymidylates.

An improvement of our strategy for the stepwise synthesis of oligo 5'-deoxy-5'-thiodeoxyribonucleotides [Chladek and Nagyvary (1972) J. Amer. Chem. Soc. 94, 2079] involves the use of 5'-O-tosylthymidine 3'-S-2-cyanoethyl phosphorothioate. The displacement of the tosylate by thymidine 3'-phosphorothioate and subsequent alkaline deblocking afforded the dinucleotide (Tps)2. The process of displacement and deblocking was repeated three more times at an average yield of 30 percent per step. The corresponding bifunctional derivative of deoxyadenosine was found much less reactive and practically unsuitable for repeated chain elongation. The ORD and CD spectra of the analogs are similar to those of the natural oligonucleotides.