Metabolism-mediated drug interactions associated with ritonavir-boosted tipranavir in mice

Abstract

Tipranavir (TPV) is the first nonpeptidic protease inhibitor used for the treatment of drug-resistant HIV infection. Clinically, TPV is coadministered with ritonavir (RTV) to boost blood concentrations and increase therapeutic efficacy. The mechanism of metabolism-mediated drug interactions associated with RTV-boosted TPV is not fully understood. In the current study, TPV metabolism was investigated in mice using a metabolomic approach. TPV and its metabolites were found in the feces of mice but not in the urine. Principal component analysis of the feces metabolome uncovered eight TPV metabolites, including three monohydroxylated, three desaturated, one dealkylated, and one dihydroxylated. In vitro study using human liver microsomes recapitulated five TPV metabolites, all of which were suppressed by RTV. CYP3A4 was identified as the primary enzyme contributing to the formation of four TPV metabolites (metabolites II, IV, V, and VI), including an unusual dealkylated product arising from carbon-carbon bond cleavage. Multiple cytochromes P450 (2C19, 2D6, and 3A4) contributed to the formation of a monohydroxylated metabolite (metabolite III). In vivo, RTV cotreatment significantly inhibited eight TPV metabolic pathways. In summary, metabolomic analysis revealed two known and six novel TPV metabolites in mice, all of which were suppressed by RTV. The current study provides solid evidence that the RTV-mediated boosting of TPV is due to the modulation of P450-dependent metabolism.

Fig. 1.

Metabolomic analysis of control and TPV-treated mouse feces. Wild-type mice (n = 3) were treated with 40 mg/kg TPV p.o., and 18-h urine and feces samples were collected for analysis. A, separation of control and TPV-treated mouse feces in PCA scores plot. The t[1] and t[2] values represent the scores of each sample in principal component 1 and 2, respectively. B, loading S-plot generated by OPLS-DA analysis. The x-axis is a measure of the relative abundance of ions, and the y-axis is a measure of the correlation of each ion to the model. These loading plots represent the relationship between variables (ions) in relation to the first and second components present in A. Top ranking ions are marked. *, sodium adduct of original ion. The number of ions (metabolite identification) was accordant with that in Figs. 2 and 6. C, the chromatograms of TPV and its metabolites: I (TPV); II, III, and IV (monohydroxylated metabolite); V (dealkylated metabolite); VI, VII, and VIII (dehydrogenated metabolites); and IX (dihydroxylated metabolite).

Fig. 2.

MS/MS structural elucidation of TPV metabolites in mouse feces. Feces samples from mice were collected for 18 h after oral administration of 40 mg/kg TPV. Screening and identification of major metabolites were performed by using MarkerLynx software based on accurate mass measurement (mass errors less than 10 ppm). MS/MS fragmentation was conducted with collision energy ramping from 10 to 30 eV. Major daughter ions from fragmentation were interpreted in the inlaid structural diagrams. A, TPV I (m/z 603⁺), retention time at 6.62 min. B, monohydroxylated metabolite II (m/z 619⁺), retention time at 6.30 min. C, monohydroxylated metabolite III (m/z 619⁺), retention time at 5.87 min. D, monohydroxylated metabolite IV (m/z 619⁺), retention time at 6.58 min. E, depropylated metabolite V (m/z 561⁺), retention time at 6.23 min. F, dehydrogenated metabolite VI (m/z 601⁺), retention time at 7.45 min. G, dehydrogenated metabolite VII (m/z 601⁺), retention time at 6.01 min. H, dehydrogenated metabolite VIII (m/z 601⁺), retention time at 6.11 min. I, dihydroxylated metabolite IX (m/z 635⁺), retention time at 5.75 min.

Fig. 3.

TPV metabolism in vitro and inhibition by RTV. Duplicate incubations were conducted in 1× PBS (pH 7.4) containing TPV (50 μM), NADPH (1.0 mM), HLM (0.5 g protein/l) or MLM (0.5 g protein/l), or cDNA-expressed CYP3A4 (10 nM), CYP2D6 (10 nM), and CYP2C19 (10 nM), with coincubation of RTV (RTV co), or preincubation of RTV (RTV pre) respectively. Metabolites II, III, IV, V, and VI were analyzed by UPLC-TOFMS. A, effect of RTV (0–100 μM) on TPV metabolism in HLM. B, effect of preincubation of RTV and coincubation of RTV (1 μM) on TPV metabolism in HLM. C, effect of preincubation of RTV and coincubation of RTV (1 μM) on TPV metabolism in cDNA-expressed CYP3A4. D, effect of coincubation of 1 and 50 μM RTV on TPV metabolism in cDNA-expressed CYP2C19 and CYP2D6. E, effect of preincubation of RTV and coincubation of RTV (50 μM) on TPV metabolism in cDNA-expressed CYP2C19 and CYP2D6 (10 nM). F, comparison of TPV metabolism in HLM and MLM. The overall abundance of TPV metabolites was set as 100% in the incubation of HLM and MLM. The relative abundance of each metabolite was compared and is presented as relative ratio (MLM versus HLM). G, effect of preincubation of RTV and coincubation of RTV (20 μM) on TPV metabolism in HLM. H, effect of preincubation of RTV and coincubation of RTV (20 μM) on TPV metabolism in MLM (10 nM). The data are expressed as means. For each metabolic pathway, the incubation without RTV was set as 100%. N.D., not detected. The metabolite identification was accordant with that in Figs. 2 and 6.

Fig. 4.

TPV and RTV tissue distribution in mice. Mice were treated orally with TPV (100 mg/kg), RTV (40 mg/kg), or TPV/r (100/40 mg/kg), respectively. Tissues including liver, brain, lung, kidney, spleen, and eyes were collected 30 min after treatment. TPV and RTV were extracted and analyzed by UPLC-TOFMS. A, TPV tissue distribution in TPV-treated and TPV/r-cotreated mice. B, RTV tissue distribution in TPV/r-cotreated mice. The data are expressed as means ± S.D. (n = 4). *, P < 0.05 versus TPV-treated mice.

Fig. 5.

Relative quantification of TPV and its metabolites in liver and serum from TPV-treated and TPV/r-cotreated mice. TPV and its metabolites were analyzed by UPLC-TOFMS. The overall abundance of TPV and its metabolites was set as 100% in each tissue. The data are expressed as means (n = 4). The metabolite identification is accordant with that in Figs. 2 and 6. A, TPV and its metabolites in the serum of TPV-treated mice. B, TPV and its metabolites in the liver of TPV-treated mice. C, TPV and its metabolites in the serum of TPV/r-cotreated mice. D, TPV and its metabolites in the liver of TPV/r-cotreated mice.

Fig. 6.

TPV metabolism and inhibition by RTV in mice. By using a metabolomic approach, the TPV metabolic map was extended to include two known and six novel pathways. CYP3A was identified as the primary enzyme contributing to the formation of four TPV metabolites (II, IV, V, and VI). CYP2C, CYP2D, and CYP3A have collaboratively contributed to the formation of a monohydroxylated metabolite (III). All TPV metabolic pathways were significantly inhibited by RTV in vivo.