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Research ArticleArticle

Metabolic Switching of BILR 355 in the Presence of Ritonavir. II. Uncovering Novel Contributions by Gut Bacteria and Aldehyde Oxidase

Yongmei Li, Jun Xu, W. George Lai, Andrea Whitcher-Johnstone and Donald J. Tweedie
Drug Metabolism and Disposition June 2012, 40 (6) 1130-1137; DOI: https://doi.org/10.1124/dmd.111.044362

Abstract

Ritonavir (RTV) was used as a boosting agent to increase the clinical exposure of 11-ethyl-5,11-dihydro-5-methyl-8-[2-[(1-oxido-4-quinolinyl)oxy]ethyl]-6H-dipyrido[3,2-b:2′,3′-e][1,4]diazepin-6-one (BILR 355), an inhibitor of the human immunodeficiency virus, by inhibiting the CYP3A-mediated metabolism of BILR 355. However, although the levels of BILR 355 increased upon concomitant administration of RTV, a metabolite of BILR 355, BILR 516, which was not detected previously in humans dosed with BILR 355 alone, became a disproportionate human metabolite with levels exceeding the parent levels at steady state. This was an unusual finding based on the in vitro and in vivo metabolic profiles of BILR 355 available at that time. Our studies reveal that BILR 355 is reduced to an intermediate, BILR 402, by gut bacteria and the reduced metabolite (BILR 402) is then oxidized by aldehyde oxidase to form BILR 516, the disproportionate human metabolite. The role of aldehyde oxidase helped to explain the somewhat unique formation of BILR 516 in humans compared with preclinical animal species. This article underlines the increasing importance of two individually atypical enzymes in drug development, gut bacterial biotransformation and aldehyde oxidase, which in combination provided a unique metabolic pathway. In addition, this article clearly elucidates an example of novel metabolic switching and, it is hoped, raises awareness of the potential for metabolic switching in combination drug therapies.

Introduction

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have played an important role in antiretroviral combination regimens for the treatment of human immunodeficiency virus (HIV)-1 infection, along with nucleoside reverse transcriptase inhibitors, protease inhibitors, fusion inhibitors, CCR5 antagonists, and integrase inhibitors (Klimas et al., 2008). However, first-generation NNRTIs have a low genetic barrier to resistance (Thompson et al., 2010; de Béthune, 2010). 11-Ethyl-5,11-dihydro-5-methyl-8-[2-[(1-oxido-4-quinolinyl) oxy]ethyl]-6H-dipyrido[3,2-b:2′,3′-e][1,4]diazepin-6-one (BILR 355) (Fig. 1) is a second-generation NNRTI that was developed for the treatment of HIV-1 infection in adults and children (Bonneau et al., 2005; Boone, 2006). BILR 355 is highly specific toward HIV-1 reverse transcriptase and exhibits an attractive resistance profile against a broad spectrum of NNRTI-resistant viruses (Bonneau et al., 2005).

Fig. 1.
Fig. 1.

Chemical structures of BILR 355, BILR 402, and BILR 516.

BILR 355 has a very short half-life (2–5 h) and low exposure after oral administration to humans (Huang et al., 2008). In vitro metabolism studies suggested that cytochrome P450 (P450) 3A4 was playing a significant role in limiting systemic exposure to BILR 355 (Li et al., 2012). Ritonavir (RTV) is a potent inhibitor of CYP3A that has been used as a boosting agent to increase the exposure of a number of drugs, such as atazanavir and darunavir (Hull and Montaner, 2011). After concomitant administration of BILR 355 with RTV, BILR 355 exposure improved dramatically with AUC increasing 16- to 30-fold (Huang et al., 2008). However, an unexpected consequence of this boosting strategy was that a metabolite, BILR 516 (Fig. 1), which was not detected previously in humans given BILR 355 alone, emerged as a disproportionate human metabolite (DHM) with plasma levels exceeding those of the parent at steady-state (Li et al., 2012).

The appearance of BILR 516 as a DHM provided challenges in achieving adequate plasma exposure in preclinical species to cover the human systemic levels. It was therefore of interest to explore how this metabolite was formed. This article delineates the metabolic pathways for the conversion of BILR 355 to the DHM, BILR 516, and discusses the importance of two individually atypical enzymes in drug development, which in combination provided a unique metabolic pathway. In addition, this article clearly elucidates the complicated mechanisms of the metabolic switching of BILR 355 upon concomitant administration of the boosting agent, RTV.

Materials and Methods

Chemicals, Reagents, and Other Materials.

BILR 355, BILR 402, and BILR 516 were synthesized at Boehringer Ingelheim Pharmaceuticals, Inc. (Ridgefield, CT) (Fig. 1). BILR 483 (D3-BILR 355 with three deuterium atoms on the methyl group) and nevirapine were also synthesized at Boehringer Ingelheim Pharmaceuticals, Inc. NADPH was purchased from Sigma-Aldrich (St. Louis, MO). All other reagents and solvents were of analytical grade or higher purity and were obtained from commercial suppliers. Pooled human liver microsomes (HLMs), human liver (HL) cytosol, and HL S9 were obtained from BD Biosciences (Woburn, MA). Individual lots of HLMs (lots HL081696 and HL082396B) were prepared in-house. All recombinant P450s (produced in baculovirus-infected insect cells) and control insect cell microsomes were obtained from BD Biosciences. Cytosolic extract of Escherichia coli expressing recombinant human aldehyde oxidase (AO) and control cytosolic extract of E. coli without recombinant human AO were purchased from Cypex Ltd. (Dundee, Scotland, UK). All enzymes were stored at −80°C until used.

In Vitro Incubations.

Incubations were performed in 0.05 M potassium phosphate buffer, pH 7.4. For incubations with recombinant P450s, HLMs, and HL S9 using NADPH as the cofactor, substrates, inhibitors (when needed), and enzymes were preincubated in buffer for 5 min at 37°C, and each reaction was initiated with the addition of NADPH at a final concentration of 2.5 mM. For incubations with HL cytosol and S9 in the absence of NADPH, enzymes and inhibitors (when needed) were preincubated for 5 min at 37°C, and each reaction was initiated with the addition of substrate. The final concentrations of enzymes varied and are specified in each study as described below. The final incubation volume was 0.5 or 1 ml and the final organic solvent concentration in each reaction did not exceed 1%. Reactions were performed for 60 min, and at the specified time points, a 50-μl aliquot was transferred to a glass filter plate assembly containing 100 μl of quench solution (0.1 μM nevirapine in acetonitrile or 0.2 μM nevirapine in 40% acetonitrile, 59.9% water, and 0.1% acetic acid). The filter plate assembly was centrifuged at 1600g for 10 min at 4°C. Filtrates from the centrifugation step were analyzed by LC-MS/MS. Each experiment was performed in duplicate. The general in vitro incubation conditions were applied to all assays described below unless specifically stated otherwise.

Incubation of BILR 355 with Hepatic Enzymes.

BILR 355 at a final concentration of 5 μM was incubated with HLMs, HL cytosol, or HL S9. Protein concentration was 1 mg/ml. The formation of BILR 516 or BILR 402 was monitored at 0, 10, 20, 30, and 60 min by LC-MS/MS.

In Vitro Gut Bacteria Incubation.

Individual fecal samples from two healthy male volunteers were collected and transferred to an anaerobic chamber (Coy Laboratory Products, Inc., Grass Lake, MI). The Coy anaerobic chamber provides a strict anaerobic condition of 0 to 5 ppm oxygen atmosphere through a hydrogen gas (5% in nitrogen gas) reacting with a palladium catalyst to remove oxygen. A heavy-duty vacuum airlock is connected to the chamber, which allows sample transfer without changes to the internal atmosphere. Dulbecco's phosphate-buffered saline (DPBS) was stirred in the anaerobic chamber overnight to remove oxygen. The fecal samples were mixed with DPBS to obtain a concentration of 0.1 g/ml (weight of fecal sample/volume of DPBS). The samples were then homogenized and centrifuged at 20 g for 5 min at 4°C to remove debris. The supernatant was diluted to 0.05 g/ml. The samples were transferred to individual wells and preincubated for 5 min at 37°C. Reactions were initiated by the addition of BILR 355 at a final concentration of 100 μM to each sample. The incubations were performed in duplicate at 37°C. Reactions were terminated at 0, 2.5, 5, 7.5, 10, 15, 20, 30, 40, 60, 90, and 120 min, and the formation of BILR 402 was measured by LC-MS/MS. All of the in vitro steps up to reaction termination were performed under anaerobic condition.

Incubation of BILR 402 with Hepatic Enzymes.

BILR 402, at a final concentration of 10 μM, was incubated with HL S9 or cytosol. The final protein concentration used in this study was 1 mg/ml. The amount of BILR 516 generated from BILR 402 was measured at 10, 20, 30, and 60 min by LC-MS/MS.

Inhibition of the Biotransformation of BILR 402 to BILR 516 in HL S9 and Cytosol by Enzyme-Selective Inhibitors.

To investigate the role of cytosolic enzymes involved in the formation of BILR 516 from BILR 402, BILR 402 (5 μM) was incubated with HL S9 or cytosol at a final protein concentration of 0.5 mg/ml in the presence of various selective inhibitors. The selective chemical inhibitors used in this study were as follows: 50 μM menadione and 50 μM hydralazine for AO, 50 μM allopurinol for xanthine oxidase, 50 μM disulfiram for aldehyde dehydrogenase, and 100 μM pyrazole for alcohol dehydrogenase (Rochat et al., 1998). Controls were performed in the absence of the inhibitors. The incubations were performed for 60 min, and the formation of BILR 516 was measured at 5, 10, 20, 30, and 60 min by LC-MS/MS.

18O Incorporation Assay.

BILR 402 was incubated at a final concentration of 5 μM with HL cytosol in 0.05 M potassium phosphate buffer containing 100% H216O or a mixture of H216O and H218O (82:18, v/v). The final protein concentration was 1 mg/ml. The reactions were terminated at 10, 20, 30, and 60 min, and the samples were analyzed by LC-MS/MS. The formation of both BILR 516-16O (BILR 516 containing 16O) and BILR 516-18O (BILR 516-containing 18O) were monitored on the basis of LC-MS/MS peak areas.

Incubation with Recombinant AO.

BILR 402 was incubated at a final concentration of 5 μM with recombinant human AO or the control cytosol without recombinant human AO. The final protein concentration was 0.1 mg/ml. The formation of BILR 516 at 0, 2, 5, 10, 15, 20, 30, 45, and 60 min was measured by LC-MS/MS.

Apparent Km and Vmax Determination of the Biotransformation of BILR 402 to BILR 516 in HL Cytosol.

To determine kinetic parameters for the biotransformation of BILR 402 to BILR 516 in HL cytosol, BILR 402 was incubated at 12 different concentrations (0.061, 0.122, 0.244, 0.488, 0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, and 125 μM). The incubations were performed for 10 min at a cytosolic protein concentration of 0.1 mg/ml. These reaction conditions were within the linear range with respect to enzyme content and incubation time. The formation of BILR 516 was monitored by LC-MS/MS.

Apparent Km and Vmax Determination of BILR 402 Metabolism: Depletion of BILR 402 and Formation of BILR 355 by HLMs.

The apparent Km and Vmax values for both BILR 402 depletion and BILR 355 formation in two lots of individual HLMs were determined. Incubations were performed in 0.1 M potassium phosphate buffer with 5 mM MgCl2, pH 7.4. The final concentration of HLMs was 0.05 mg/ml, and reactions were conducted for 30 min. These reaction conditions were within the linear range with respect to enzyme concentration and incubation time. The initial rates of metabolism were measured at various BILR 402 concentrations (0.05, 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, and 20.0 μM). Aliquots (100 μl) were transferred from the incubation mixtures at various time intervals (0.5, 1, 2, 4, 10, 15, 20, and 30 min) and quenched with an equal volume of acetonitrile. Then, 100 μl of BILR 483 (0.2 μM in acetonitrile) was added to each well as the internal standard. All samples were centrifuged at 1600g for 10 min, and both the depletion of BILR 402 and the formation of BILR 355 were monitored by LC-MS/MS.

Metabolism of BILR 402 by Recombinant P450 Isoforms.

BILR 402 (1 or 10 μM) was incubated with recombinant P450 isoforms, including rCYP1A2, rCYP2A6, rCYP2B6, rCYP2C9, rCYP2C19, rCYP2D6, and rCYP3A4, in a 0.1 M potassium phosphate buffer with 5 mM MgCl2, pH 7.4. The total incubation volume was 900 μl. Control samples were prepared identically, except that microsomal protein from nontransfected insect cells was substituted for individual recombinant P450 isoforms. Aliquots of incubation mixtures were transferred at 0.5, 1, 2, 4, 10, 15, 20, and 30 min and quenched with an equal volume of acetonitrile. Then, 100 μl of BILR 483 (0.2 μM in acetonitrile) was added to each well as the internal standard. The plates were centrifuged. The depletion of BILR 402 and the formation of BILR 355 were monitored by LC-MS/MS.

Inhibition of BILR 402 Metabolism in HLMs by Isoform-Selective Inhibitors.

BILR 402 (1 μM) was incubated in the presence and absence of isoform-selective chemical inhibitors using the same procedure as described in Li et al. (2012) with the exception that ticlopidine was added as another inhibitor of CYP2C19 at a final concentration of 20 μM, and ketoconazole was used as an inhibitor of CYP3A4 only at a final concentration of 3 μM. The final protein concentration was 0.05 mg/ml, and the incubation time was 30 min. Both the depletion of BILR 402 and the formation of BILR 355 were monitored by LC-MS/MS.

Equipment and Chromatographic Conditions for In Vitro Assays.

For apparent Km and Vmax determination of BILR 402 metabolism by HLMs and P450 phenotyping studies of BILR 402, concentrations of BILR 355 and BILR 402 were quantitated by an LC-MS/MS system, consisting of a Gilson 215 liquid handler autosampler (Gilson, Inc., Middleton, WI), two series 200 micro pumps (PerkinElmer Life and Analytical Sciences, Waltham, MA), and an Applied Biosystems MDS Sciex API4000 triple quadrupole mass spectrometer (Applied Biosystems/Sciex, Thornhill, ON, Canada). The column used was a Waters Symmetry C18 (2 × 50 mm, 3.5-μm particle size; Waters, Milford, MA). Mobile phase compositions were as follows: mobile phase A, water-acetonitrile-acetic acid (95:5:0.05, v/v/v); and mobile phase B, water-acetonitrile-acetic acid (5:95:0.05, v/v/v). A 5-min gradient was used, which started at 0% B for 0.2 min, then increased to 100% B in 4.5 min, and was maintained at 100% B for 0.2 min before returning to 0% B at a flow rate of 0.25 ml/min. The retention times for BILR 355, BILR 402, and the isotope-labeled internal standard, BILR 483, were 2.7, 2.8, and 2.7 min, respectively. The mass spectrometer was optimized with an IonSpray voltage of 5.5 kV, an ion source temperature of 650°C, a declustering potential at 80 V, and collision energy of 45 V for BILR 402 and 30 V for BILR 355 and BILR 483, respectively. The multiple reaction monitoring transitions requested for BILR 402, BILR 355, and BILR 483 were m/z 426 → 281, m/z 442 → 281, and m/z 445 → 284, respectively.

Different LC-MS/MS instrumentation was used for other experiments. The system consisted of an SIL-5000 autosampler and two LC-10AD vp pumps (Shimadzu Scientific Instruments, Norwell, MA) connected with an Applied Biosystems MDS Sciex 4000 QTrap mass spectrometer (Applied Biosystems/Sciex, Thornhill, ON, Canada). An Atlantis dC18 column (3.9 × 150 mm, 3-μm particle size; Waters) was used. Mobile phases were similar to those described previously except that the concentration of acetic acid was 0.1%. A 5-min gradient with mobile phase B increasing from 45 to 65% was used at a flow rate of 0.7 ml/min. The multiple reaction monitoring transitions requested for BILR 402, BILR 355, BILR 516, BILR 516-18O, and the internal standard nevirapine were m/z 426 → 281, m/z 442 → 281, m/z 442 → 281, m/z 444 → 281, and m/z 267 → 226, respectively, and their retention times were 1.9, 2.7, 3.7, 3.7, and 2.4 min, respectively.

Data Analysis.

The formation rates of BILR 516 from the incubations of BILR 402 with HL cytosol were calculated. The kinetic parameters were determined by nonlinear regression analysis using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA) and fit to a biphasic equation (Tracy, 2006) as shown below: Embedded Image where [S] is substrate concentration, v is reaction velocity in picomoles per minute per milligram of HL cytosolic protein, Vmax1 is the normalized maximum reaction velocity in picomoles per minute per milligram of HL cytosolic protein, and Km1 is the Michaelis-Menten constant for the high-affinity, low-capacity site (i.e., low Km and low Vmax), CLint2 is the in vitro intrinsic clearance in milliliters per minute per milligram of HL cytosolic protein for the second low-affinity, high-capacity site (i.e., high Km and high Vmax).

CLint1, in vitro of BILR 402 metabolism to BILR 516 in cytosol was calculated by Vmax1/Km1. The following constants were used to estimate CLint1,in vivo from CLint1,in vitro: a value of 80.7 mg of cytosolic protein/g liver (Houston and Galetin, 2008), 1800 g human liver weight, and average 70 kg human body weight (Davies and Morris, 1993). To calculate the hepatic clearance, the well stirred model was applied, and CLh1 (milliliters per minute per kilogram) was calculated using the following equation (Pang and Rowland, 1977): Embedded Image where Qh is hepatic blood flow in humans (20.7 ml · min−1 · kg−1) (Davies and Morris, 1993) and fu is fraction of unbound drug (assume that fu = 1).

Enzyme kinetics (Km and Vmax) of the depletion of BILR 402 and the formation of BILR 355 by HLMs were measured and the clearance (CLint, in vitro and CLh) of both processes was calculated using the same procedure as described in Li et al. (2012).

Results

The DHM BILR 516 Is Not Generated Directly from BILR 355.

BILR 355 was incubated with HLMs, HL S9, or HL cytosol over 60 min at a concentration of 5 μM, which was close to the maximal plasma concentration of BILR 355 in humans (1500 ng/ml, ∼3.40 μM) at steady state after oral administration of 150 mg BILR 355 and 100 mg RTV twice a day (Huang et al., 2009). No formation of the DHM was observed, suggesting that the DHM is not formed directly from BILR 355.

Formation of the Reduced Metabolite BILR 402 from BILR 355.

On the basis of the structures of BILR 355 and the DHM, it was hypothesized that BILR 355 could go through a two-step metabolic pathway to form the DHM, including reduction of the N-oxide and subsequent oxidation on the carbon adjacent to the heterocyclic nitrogen.

The first step, reduction of N-oxide of BILR 355 to form the reduced metabolite BILR 402 (Fig. 1), can be mediated by liver enzymes or gut bacteria. The formation of the reduced metabolite was then monitored in the incubations of BILR 355 with HLMs, HL S9, and HL cytosol. There was no formation of the reduced metabolite from the parent in these matrices. The possibility of the involvement of gut bacteria in the biotransformation of the parent to the reduced metabolite was tested. Human fecal samples, containing gut bacteria, were obtained from two healthy volunteers and then incubated with BILR 355. The total incubation time was 120 min. Loss of linearity and saturation were observed beyond 15 min. The linear ranges of the formation of the reduced metabolite were plotted (r2 > 0.85) for both subjects and are shown in Fig. 2.

Fig. 2.
Fig. 2.

Formation of BILR 402 from the incubations of BILR 355 with human feces (n = 2).

The second step involved oxidation of the reduced metabolite to the DHM. This possibility was explored in hepatic enzyme systems, including HL S9 and cytosol, and the results are shown in Fig. 3. The initial concentration of the reduced metabolite in the incubations was 10 μM. In the absence of NADPH, 59 and 81% of the reduced metabolite was converted to the DHM after 60-min incubations with HL S9 and cytosol, respectively. Of interest, compared with what was observed in the absence of NADPH, significantly less of the DHM was generated by HL S9 in the presence of NADPH. The level of the DHM reached approximately 20% of the starting material at 10 min and leveled off thereafter. Furthermore, on the basis of LC-MS/MS analysis, BILR 355 was generated from the reduced metabolite in the S9 incubations in the presence of NADPH, whereas BILR 355 was not detected in S9 incubations without NADPH or in the cytosol incubations.

Fig. 3.
Fig. 3.

Formation of BILR 516 in incubations of BILR 402 with (A) human liver cytosol in the absence of NADPH and (B) human liver S9 in the absence and presence of NADPH (n = 2).

A role for AO in the formation of the DHM from the reduced metabolite was supported by studies using specific chemical inhibitors of major cytosolic enzymes, including menadione and hydralazine for AO, allopurinol for xanthine oxidase, disulfiram for aldehyde dehydrogenase, and pyrazole for alcohol dehydrogenase. The results are shown in Table 1. In the presence of menadione or hydralazine, two specific inhibitors of AO, the formation of the DHM from the reduced metabolite was almost completely abolished. The inhibition effects by other chemical inhibitors were negligible, except that disulfiram, a specific inhibitor of aldehyde dehydrogenase, inhibited the reaction more than 50%. However, it has been reported that disulfiram can also inhibit AO (Kitamura et al., 2001). In addition, aldehyde dehydrogenase is known to oxidize only aldehyde to acid, whereas aldehyde oxidase can catalyze nucleophilic oxidation at an electrodeficient carbon atom in N-heterocycles (e.g., quinoline) (Beedham et al., 2003). On the basis of the chemical inhibition results, it is likely that AO is involved in the formation of the DHM from the reduced metabolite.

View this table:
TABLE 1

Effect of inhibitors on the biotransformation of BILR 402 to BILR 516 in HL S9 and cytosol (n = 2)

Water is the source of oxygen in reactions mediated by AO. Water labeled with 18O was added to the incubations of the reduced metabolite with HL cytosol. The LC-MS/MS peaks of BILR 516-16O and BILR 516-18O were monitored, and representative LC-MS/MS chromatograms from incubations containing either the mixture of H216O and H218O (82:18, v/v) or 100% H216O (control sample) are presented in Fig. 4. In the LC-MS/MS chromatograms generated from control samples, the peak of BILR 516-16O was predominant, with a very small peak (0.8% of BILR 516-16O based on peak area comparison) shown in the LC-MS/MS transition for BILR 516-18O, which corresponded to the M+2 isotope of BILR 516-16O (Fig. 4A). The LC-MS/MS chromatograms generated from incubations containing the mixture of H216O and H218O were different from this control. The BILR 516-18O peak increased significantly compared with the control (Fig. 4B). The peak area ratios of BILR 516-16O to BILR 516-18O were 82 to 18 after the contribution of the M+2 isotope of BILR 516-16O was subtracted from the peak area of BILR 516-18O across all time points, which was consistent with the ratio of 16O to 18O (82:18) in the starting incubation material. This result confirmed that water was the source for the oxygen incorporated into the DHM, BILR 516, in the incubations of the reduced metabolite with HL cytosol and corroborated the involvement of AO.

Fig. 4.
Fig. 4.

Incorporation of 18O into BILR 516 in incubations of BILR 402 with HL cytosol in (A) 50 mM phosphate buffer (no H218O) and (B) 50 mM phosphate buffer containing H218O (final concentration 18%) (n = 2). XIC, extracted ion chromatogram; amu, atomic mass units.

Furthermore, the reduced metabolite was incubated with recombinant human AO, and formation of the DHM was clearly observed (Fig. 5). The DHM was not generated in the control cytosol without recombinant human AO.

Fig. 5.
Fig. 5.

Formation of BILR 516 in incubations of BILR 402 with recombinant human AO (n = 2).

Kinetic Analysis of the Biotransformation of the Reduced Metabolite BILR 402 to the DHM BILR 516.

Apparent Km and Vmax parameters for the metabolism of the reduced metabolite to the DHM were determined in HL cytosol. The Eadie-Hofstee plot of the reaction is shown in Fig. 6A and indicated that the kinetics followed a biphasic model. The apparent Km1 and Vmax1 values were obtained on the basis of the biphasic model (Fig. 6B). The intrinsic clearance of the high-affinity, low-capacity site, CLint1, in vitro, was calculated to be 437 μl · min−1 · mg−1 cytosolic protein on the basis of Vmax1/Km1. Subsequently, CLint1, in vivo was estimated as 907 ml · min−1 · kg−1 and CLh1 was calculated as 20.2 ml · min−1 · kg−1.

Fig. 6.
Fig. 6.

Eadie-Hofstee plot (A) and enzyme kinetic curve (B) of the metabolism of BILR 402 to BILR 516 in HL cytosol (n = 2).

Kinetic Analysis of the Metabolism of the Reduced Metabolite BILR 402 by HLMs.

Two lots of precharacterized HLMs (HL082396B and HL081696) were selected for the incubations with the reduced metabolite, BILR 402, on the basis of their testosterone 6β-hydroxylase activities (1600 and 3400 pmol · min−1 · mg−1 microsomal protein, respectively) representing a range of low to average CYP3A4 activities from available individual lots of human liver microsomes. The formation of BILR 355 from the reduced metabolite was observed in the incubations. The apparent Km and Vmax values for the depletion of the reduced metabolite and the formation of BILR 355 in each lot of HLMs were calculated using nonlinear regression analysis based on the Michaelis-Menten equation, and the corresponding intrinsic clearance values were derived. The results are summarized in Table 2. The intrinsic clearance of the reduced metabolite ranged from 1.09 to 5.16 ml · min−1 · mg−1 HLM protein and the intrinsic clearance for the formation of BILR 355 ranged from 0.24 to 0.55 ml · min−1 · mg−1 HLM protein. The hepatic clearance values for both processes were close to 20 ml · min−1 · kg−1.

View this table:
TABLE 2

Michaelis-Menten kinetic parameters for BILR 402 metabolism and BILR 355 formation in human liver microsomes (n = 2)

Identification of P450 Isoforms Involved in the Metabolism of the Reduced Metabolite BILR 402.

In vitro metabolic rates of the depletion of the reduced metabolite and the formation of BILR 355 were measured in incubations of the reduced metabolite (1 and 10 μM) with recombinant CYP1A2, -2A6, -2B6, -2C9, -2C19, -2D6, and -3A4. Time-dependent depletion of the reduced metabolite was only observed in the incubations with rCYP3A4 in the presence of NADPH. The substrate was depleted from the 1 μM starting concentration to 0.25 μM and from 10 μM starting concentration to 5 μM in the first 4 min. In addition, NADPH-dependent formation of BILR 355 from the reduced metabolite was only observed in the incubations with rCYP3A4. The level of BILR 355 initially increased with the incubation time (0.15 μM BILR 355 was formed by 4 min when the substrate concentration was 1 μM, and 0.9 μM BILR 355 was formed at the same time when the substrate concentration was 10 μM) and then started to decrease. This observation is consistent with the fact that BILR 355 is also a good substrate of CYP3A (Li et al., 2012) and would be further metabolized by CYP3A as the incubation proceeded. The amount of each P450 enzyme used in the incubations was adjusted on the basis of the relative level of each P450 isoform in an average set of human liver microsomes (Shimada et al., 1994). The final concentrations of each P450 isoform were as follows: 1A2, 2 pmol/ml; 2A6, 1 pmol/ml; 2B6, 0.1 pmol/ml; 2C9,1.5 pmol/ml; 2C19, 1.5 pmol/ml; 2D6, 0.3 pmol/ml; and 3A4, 5 pmol/ml (calculated on the basis of 0.05 mg/ml microsomal protein). To bolster this conclusion, the reduced metabolite was also incubated with recombinant P450 isoforms (CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, or CYP2D6) at 20-fold higher enzyme levels. No depletion of the reduced metabolite was observed (data not shown). The results confirmed that these P450 isoforms are not responsible for the overall P450-mediated metabolism of the reduced metabolite.

In addition, isoform-selective chemical inhibitors of the six major drug-metabolizing P450 isoforms were used to investigate the relative contributions of these isoforms to the metabolism of the reduced metabolite. Metabolism of the reduced metabolite by HLMs was monitored as both the depletion of the reduced metabolite and the formation of BILR 355. The results showed that, in the presence of ketoconazole, a selective inhibitor of CYP3A4, 70% of the overall metabolism of the reduced metabolite and 94% of the formation of BILR 355 in HLMs were inhibited. Furafylline, sulfaphenazole, and quinidine, as selective inhibitors of CYP1A2, CYP2C9, and CYP2D6, respectively, had no inhibitory effect on the depletion of the reduced metabolite or the formation of BILR 355. Tranylcypromine, at a concentration of 100 μM, inhibited approximately 30% of the overall metabolism of the reduced metabolite but had no effect on the formation of BILR 355. Tranylcypromine is an inhibitor of CYP2C19 and CYP2A6 (Dierks et al., 2001). However, another more potent CYP2C19 inhibitor, ticlopidine, did not inhibit the metabolism of the reduced metabolite and as shown above, neither recombinant CYP2C19 nor CYP2A6 catalyzed the metabolism of the reduced metabolite. Although the mechanism of this inhibition is not clear, the data do not support the role of CYP2C19 or CYP2A6 in the metabolism of the reduced metabolite. The current data clearly demonstrate that CYP3A4 is the major enzyme responsible for the P450-mediated metabolism of the reduced metabolite and the regeneration of BILR 355 by HLMs.

Discussion

Previous studies with BILR 355 had shown that it was extensively metabolized by CYP3A (Li et al., 2012). Concomitant administration of RTV with BILR 355 to inhibit CYP3A and boost the exposure of BILR 355 had the desired effect (Huang et al., 2008). However, a high-level DHM, BILR 516, was observed (Li et al., 2012). Because the DHM had not been previously observed in humans administered BILR 355 alone, it was important to elucidate the metabolic pathway and identify enzymes responsible for the conversion of BILR 355 to the DHM. In this article, two atypical enzymatic processes were identified, which in combination, provided a unique metabolic pathway. In addition, the metabolic switching of BILR 355 with concomitant administration of RTV was also elucidated.

We first explored potential metabolic mechanisms for the biotransformation of BILR 355 to the DHM. This biotransformation involves reduction of the N-oxide and oxidation on the carbon next to the N-oxide. The likelihood of this biotransformation as a single step reaction is low, which was confirmed by the in vitro incubations of BILR 355 with HLMs, HL S9, and HL cytosol, for which no formation of the DHM was observed.

Our hypothesis was that this biotransformation is a two-step process with the reduced metabolite, BILR 402, as the intermediate. The first step would be reduction of BILR 355 to BILR 402. Reduction of N-oxide has been shown to occur presystemically or by hepatic enzymes (Kitamura and Tatsumi, 1984a,b; Guengerich, 2001; Sousa et al., 2008). The second step is the oxidation of BILR 402 at the carbon next to the nitrogen in the quinoline moiety, which is a typical reaction mediated by AO (Pryde et al., 2010).

The first step was confirmed to be mediated by gut bacteria, because no BILR 402 was formed after incubations of BILR 355 with HLMs, HL S9, and HL cytosol, whereas extensive formation of BILR 402 was observed in in vitro anaerobic incubations with human feces containing gut bacteria. As shown in Fig. 2, approximately 8% of BILR 355 was metabolized to the reduced metabolite within 15 min by human feces. This in vitro finding is consistent with observations from the 14C absorption, distribution, metabolism, and excretion studies for BILR 355 in animals, which indicated that unchanged BILR 355 was present in only trace amounts in feces of mice, rats, and dogs (0–3.3% of the total radioactivity), and the majority of fecal radioactivity could be accounted for by the reduced metabolite and its downstream metabolites (72.5–90.4% of the total radioactivity) (Boehringer Ingelheim Pharmaceuticals, Inc., data on file).

The second step is mediated by AO as confirmed with selective chemical inhibitors (Table 1), incorporation of oxygen from H2O (Fig. 4), and metabolism by recombinant AO (Fig. 5). In addition, the activity was found in the cytosolic subcellular fraction (more than 80% conversion from the reduced metabolite to the DHM by 60 min) and did not require NADPH (Fig. 3A).

The biotransformation of the reduced metabolite to form the DHM followed biphasic kinetics with a low Km1 value of 0.772 μM (Fig. 6). The turnover of the reduced metabolite to the DHM is predicted to be high, with a calculated in vivo hepatic clearance of 20.2 ml · min−1 · kg−1, which approximates hepatic blood flow in humans (20.7 ml · min−1 · kg−1), particularly because in vitro data generally underpredicts in vivo clearance of AO-mediated metabolism (Zientek et al., 2010).

With the combination of two highly efficient metabolic reactions (BILR 355 forms the reduced metabolite, which is then further metabolized to the DHM) and the long in vivo half-life of the DHM (54.5 h) (Li et al., 2012), it is not surprising to see the high exposure of the DHM at steady state in humans.

The reduced metabolite, BILR 402, is the intermediate of the biotransformation from BILR 355 to the DHM. Therefore, the metabolism of the reduced metabolite was studied. The reduced metabolite was extensively metabolized by P450s to other products, as demonstrated by high hepatic clearance (>20 ml · min−1 · kg−1) of the reduced metabolite (Table 2). When the reduced metabolite was incubated with HL S9, there was substantially less of the DHM formed in the presence of NADPH compared with that formed in its absence (Fig. 3B). This could be attributable to extensive metabolism of the reduced metabolite by P450s in the presence of NADPH; thus, minimal substrate was available for AO metabolism at later time points (i.e., beyond 10 min). In addition, it is also possible that the DHM can be further metabolized by P450s, which could offset the formation of the DHM by AO and result in nearly zero net change of the DHM level after 10 min. The reduced metabolite can also be oxidized back to the parent BILR 355 by P450s as shown in HLMs (11% for HL081696 and 22% for HL082396B of the overall intrinsic clearance of the reduced metabolite) (Table 2). CYP3A4 was identified as the major P450 isoform responsible for the metabolism of the reduced metabolite and specifically the formation of BILR 355 on the basis of studies using recombinant P450 isoforms and isoform-selective inhibitors. Although not tested in these studies, CYP3A5 may also be involved in the metabolism of BILR 402 and the formation of BILR 355, considering the overlap in substrate specificity between CYP3A4 and CYP3A5. Of note, ketoconazole only inhibited approximately 70% of HLM-catalyzed metabolism of the reduced metabolite. Thus, it is possible that other liver microsomal enzymes might play a minor role in the metabolism of the reduced metabolite.

The DHM was not detected in the plasma of human subjects administered BILR 355 alone. So why is there such a difference upon addition of RTV? When administered alone, BILR 355 is significantly metabolized by CYP3A to multiple metabolites as illustrated in Fig. 7A. Reduction of BILR 355 by gut bacteria results in the formation of the reduced metabolite BILR 402. The reduced metabolite is also a very good substrate of CYP3A, and the absorbed fraction could be extensively metabolized, including oxidation back to BILR 355. Without concomitant administration of RTV, CYP3A-mediated metabolism of BILR 355 and metabolism of the reduced metabolite are the predominant metabolic pathways and the formation of the DHM, BILR 516, by AO is negligible. The concomitant administration of RTV with BILR 355 results in the shutdown of the extensive CYP3A-mediated clearance of BILR 355 and of the reduced metabolite (Fig. 7B). BILR 355 levels now increase and, in addition, the absorbed fraction of the reduced metabolite is no longer subject to CYP3A metabolism but is mainly cleared by AO to form the DHM, which is now the predominant pathway. In addition, it is possible that RTV may inhibit the metabolism of BILR 516, which could also cause an increase in BILR 516 exposure.

Fig. 7.
Fig. 7.

A, summary of key metabolic pathways of BILR 355 clearance and the formation of BILR 516. B, metabolic switching in the presence of RTV.

These studies have clearly elucidated the unusual metabolic pathways of BILR 355 with the involvement of gut bacteria and AO and the novel metabolic switching upon concomitant administration of RTV.

There are 100 trillion microbes resident in a normal human intestine with a higher density in the lower gastrointestinal tract (Ley et al., 2006). Because of the complexity of drug development, more and more drugs are developed with low solubility or low permeability or are administered with extended release formulations. Thus, these drugs will be more likely to reach the lower gastrointestinal tract, presenting themselves to the host gut bacteria (Sousa et al., 2008). The possible involvement of gut bacteria in the metabolism of drugs needs to be seriously considered.

In addition, AO-mediated metabolism is drawing more and more attention in drug development. Two key areas of concern are the potential for low oral bioavailability of development compounds in humans, resulting from high turnover by AO, and also the marked species differences in AO activity leading to gross species differences in exposure of metabolites formed by AO (Pryde et al., 2010). In general, high AO activity is observed in human and monkey, followed by hamster, rabbit, guinea pig, rat, and mouse, and AO is deficient in dog (Beedham et al., 1987, 1995; Kitamura et al., 2001, 2006). Because AO activity is generally low or deficient in the standard toxicology species, a metabolite generated by AO that is major in humans (>10% of the total drug-related materials), has a higher chance of being a DHM. BILR 355 metabolism provides a clear example of this issue.

Although it was expected from in vitro studies (Li et al., 2012) that CYP3A would be a major clearance pathway for BILR 355, the extent of metabolic switching upon concomitant administration of RTV was a surprise. Metabolic switching should certainly be considered as more drugs are developed as a combination therapy either to boost the exposure (e.g., HIV protease inhibitors and RTV) or to maximize efficacy with complementary treatment mechanisms (e.g., combinations of antiretroviral agents with different targets). In addition, this could be more of an issue as a result of potential drug-drug interactions.

This article supports the increasing importance of non-P450 drug-metabolizing enzymes (e.g., AO and gut bacteria in this case) and, it is hoped, raises awareness of potential metabolic switching associated with combination drug therapies.

Authorship Contributions

Participated in research design: Li, Lai, and Tweedie.

Conducted experiments: Li, Xu, Lai, and Whitcher-Johnstone.

Performed data analysis: Li, Xu, and Lai.

Wrote or contributed to the writing of the manuscript: Li and Tweedie.

Acknowledgments

We thank Yanping Mao, Elsy Philip, and Dr. Lin-Zhi Chen for providing the results of the 14C absorption, distribution, metabolism, and excretion studies in animals and thank Dr. Timothy S. Tracy for scientific advice and review of the manuscript.

Footnotes

  • This research was funded by Boehringer Ingelheim Pharmaceuticals, Inc.

  • Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

    http://dx.doi.org/10.1124/dmd.111.044362.

  • ABBREVIATIONS:

    NNRTI
    non-nucleoside reverse transcriptase inhibitor
    HIV
    human immunodeficiency virus
    BILR 355
    11-ethyl-5,11-dihydro-5-methyl-8-[2-[(1-oxido-4-quinolinyl)oxy]ethyl]-6H-dipyrido[3,2-b:2′,3′-e][1,4]diazepin-6-one
    P450
    cytochrome P450
    RTV
    ritonavir
    DHM
    disproportionate human metabolite
    HLMs
    human liver microsomes
    HL
    human liver
    AO
    aldehyde oxidase
    r
    recombinant
    LC
    liquid chromatography
    MS/MS
    tandem mass spectrometry
    DPBS
    Dulbecco's phosphate-buffered saline.

  • Received December 16, 2011.
  • Accepted March 5, 2012.

References

  1. ↵
    1. Beedham C,
    2. Bruce SE,
    3. Critchley DJ,
    4. al-Tayib Y,
    5. Rance DJ
    (1987) Species variation in hepatic aldehyde oxidase activity. Eur J Drug Metab Pharmacokinet 12:307–310.
    OpenUrlPubMed
  2. ↵
    1. Beedham C,
    2. Miceli JJ,
    3. Obach RS
    (2003) Ziprasidone metabolism, aldehyde oxidase, and clinical implications. J Clin Psychopharmacol 23:229–232.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Beedham C,
    2. Peet CF,
    3. Panoutsopoulos GI,
    4. Carter H,
    5. Smith JA
    (1995) Role of aldehyde oxidase in biogenic amine metabolism. Prog Brain Res 106:345–353.
    OpenUrlPubMed
  4. ↵
    1. Bonneau P,
    2. Robinson PA,
    3. Duan J,
    4. Doyon L,
    5. Simoneau B,
    6. Yoakim C,
    7. Garneau M,
    8. Bos M,
    9. Cordingley M,
    10. Brenner B,
    11. Spira B,
    12. Wainberg M,
    13. Huang F,
    14. Drda K,
    15. Ballow C,
    16. Koenen-Bergmann M,
    17. Mayers D
    (2005) Antiviral characterization and human experience with BILR 355 BS, a novel next-generation non-nucleoside reverse transcriptase inhibitor (NNRTI) with a broad anti HIV-1 profile. Proceedings of the 12th Conference on Retroviruses and Opportunistic Infections; 2005 Feb 22–25; Boston, MA. Poster 558.
  5. ↵
    1. Boone LR
    (2006) Next-generation HIV-1 non-nucleoside reverse transcriptase inhibitors. Curr Opin Investig Drugs 7:128–135.
    OpenUrlPubMed
  6. ↵
    1. Davies B,
    2. Morris T
    (1993) Physiological parameters in laboratory animals and humans. Pharm Res 10:1093–1095.
    OpenUrlCrossRefPubMed
  7. ↵
    1. de Béthune MP
    (2010) Non-nucleoside reverse transcriptase inhibitors (NNRTIs), their discovery, development, and use in the treatment of HIV-1 infection: a review of the last 20 years (1989–2009). Antiviral Res 85:75–90.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Dierks EA,
    2. Stams KR,
    3. Lim HK,
    4. Cornelius G,
    5. Zhang H,
    6. Ball SE
    (2001) A method for the simultaneous evaluation of the activities of seven major human drug-metabolizing cytochrome P450s using an in vitro cocktail of probe substrates and fast gradient liquid chromatography tandem mass spectrometry. Drug Metab Dispos 29:23–29.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Guengerich FP
    (2001) Common and uncommon cytochrome P450 reactions related to metabolism and chemical toxicity. Chem Res Toxicol 14:611–650.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Houston JB,
    2. Galetin A
    (2008) Methods for predicting in vivo pharmacokinetics using data from in vitro assays. Curr Drug Metab 9:940–951.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Huang F,
    2. Drda K,
    3. MacGregor TR,
    4. Scherer J,
    5. Rowland L,
    6. Nguyen T,
    7. Ballow C,
    8. Castles M,
    9. Robinson P
    (2009) Pharmacokinetics of BILR 355 after multiple oral doses coadministered with a low dose of ritonavir. Antimicrob Agents Chemother 53:95–103.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Huang F,
    2. Koenen-Bergmann M,
    3. Macgregor TR,
    4. Ring A,
    5. Hattox S,
    6. Robinson P
    (2008) Pharmacokinetic and safety evaluation of BILR 355, a second-generation nonnucleoside reverse transcriptase inhibitor, in healthy volunteers. Antimicrob Agents Chemother 52:4300–4307.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Hull MW,
    2. Montaner JS
    (2011) Ritonavir-boosted protease inhibitors in HIV therapy. Ann Med 43:375–388.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Kitamura S,
    2. Ohashi KNK,
    3. Sugihara K,
    4. Hosokawa R,
    5. Akagawa Y,
    6. Ohta S
    (2001) Extremely high drug-reductase activity based on aldehyde oxidase in monkey liver. Biol Pharm Bull 24:856–859.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Kitamura S,
    2. Sugihara K,
    3. Ohta S
    (2006) Drug-metabolizing ability of molybdenum hydroxylases. Drug Metab Pharmacokinet 21:83–98.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Kitamura S,
    2. Tatsumi K
    (1984a) Involvement of liver aldehyde oxidase in the reduction of nicotinamide N-oxide. Biochem Biophys Res Commun 120:602–606.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Kitamura S,
    2. Tatsumi K
    (1984b) Reduction of tertiary amine N-oxides by liver preparations: function of aldehyde oxidase as a major N-oxide reductase. Biochem Biophys Res Commun 121:749–754.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Klimas N,
    2. Koneru AO,
    3. Fletcher MA
    (2008) Overview of HIV. Psychosom Med 70:523–530.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Ley RE,
    2. Peterson DA,
    3. Gordon JI
    (2006) Ecological and evolutionary forces shaping microbial diversity in the human intestine. Cell 124:837–848.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Li Y,
    2. Lai WG,
    3. Whitcher-Johnstone A,
    4. Busacca CA,
    5. Eriksson MC,
    6. Lorenz JC,
    7. Tweedie DJ
    (2012) Metabolic switching of BILR 355 in the presence of ritonavir. I. Identifying an unexpected disproportionate human metabolite. Drug Metab Dispos 40:1122–1129.
    OpenUrlAbstract/FREE Full Text
  21. ↵
    1. Pang KS,
    2. Rowland M
    (1977) Hepatic clearance of drugs. I. Theoretical considerations of a “well-stirred” model and a “parallel tube” model. Influence of hepatic blood flow, plasma and blood cell binding, and the hepatocellular enzymatic activity on hepatic drug clearance. J Pharmacokinet Biopharm 5:625–653.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Pryde DC,
    2. Dalvie D,
    3. Hu Q,
    4. Jones P,
    5. Obach RS,
    6. Tran TD
    (2010) Aldehyde oxidase: an enzyme of emerging importance in drug discovery. J Med Chem 53:8441–8460.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Rochat B,
    2. Kosel M,
    3. Boss G,
    4. Testa B,
    5. Gillet M,
    6. Baumann P
    (1998) Stereoselective biotransformation of the selective serotonin reuptake inhibitor citalopram and its demethylated metabolites by monoamine oxidases in human liver. Biochem Pharmacol 56:15–23.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Shimada T,
    2. Yamazaki H,
    3. Mimura M,
    4. Inui Y,
    5. Guengerich FP
    (1994) Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270:414–423.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Sousa T,
    2. Paterson R,
    3. Moore V,
    4. Carlsson A,
    5. Abrahamsson B,
    6. Basit AW
    (2008) The gastrointestinal microbiota as a site for the biotransformation of drugs. Int J Pharm 363:1–25.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Thompson MA,
    2. Aberg JA,
    3. Cahn P,
    4. Montaner JS,
    5. Rizzardini G,
    6. Telenti A,
    7. Gatell JM,
    8. Günthard HF,
    9. Hammer SM,
    10. Hirsch MS,
    11. et al
    . (2010) Antiretroviral treatment of adult HIV infection: 2010 recommendations of the International AIDS Society-USA panel. JAMA 304:321–333.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Tracy TS
    (2006) Atypical cytochrome P450 kinetics: implications for drug discovery. Drugs R D 7:349–363.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Zientek M,
    2. Jiang Y,
    3. Youdim K,
    4. Obach RS
    (2010) In vitro-in vivo correlation for intrinsic clearance for drugs metabolized by human aldehyde oxidase. Drug Metab Dispos 38:1322–1327.
    OpenUrlAbstract/FREE Full Text
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METABOLIC SWITCHING OF BILR 355 IN THE PRESENCE OF RTV. II

Yongmei Li, Jun Xu, W. George Lai, Andrea Whitcher-Johnstone and Donald J. Tweedie
Drug Metabolism and Disposition June 1, 2012, 40 (6) 1130-1137; DOI: https://doi.org/10.1124/dmd.111.044362
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