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Research ArticleArticle

The Potentially Significant Role of CYP3A-Mediated Oxidative Metabolism of Dabigatran Etexilate and Its Intermediate Metabolites in Drug-Drug Interaction Assessments Using Microdose Dabigatran Etexilate

Udomsak Udomnilobol, Suree Jianmongkol and Thomayant Prueksaritanont
Drug Metabolism and Disposition September 2023, 51 (9) 1216-1226; DOI: https://doi.org/10.1124/dmd.123.001353

Abstract

Dabigatran etexilate (DABE), a double ester prodrug of dabigatran, is a probe substrate of intestinal P-glycoprotein (P-gp) commonly used in clinical drug-drug interaction (DDI) studies. When compared with its therapeutic dose at 150 mg, microdose DABE (375 µg) showed approximately 2-fold higher in DDI magnitudes with CYP3A/P-gp inhibitors. In this study, we conducted several in vitro metabolism studies to demonstrate that DABE, at a theoretical gut concentration after microdosing, significantly underwent NADPH-dependent oxidation (~40%–50%) in parallel to carboxylesterase-mediated hydrolysis in human intestinal microsomes. Furthermore, NADPH-dependent metabolism of its intermediate monoester, BIBR0951, was also observed in both human intestinal and liver microsomes, accounting for 100% and 50% of total metabolism, respectively. Metabolite profiling using high resolution mass spectrometry confirmed the presence of several novel oxidative metabolites of DABE and of BIBR0951 in the NADPH-fortified incubations. CYP3A was identified as the major enzyme catalyzing the oxidation of both compounds. The metabolism of DABE and BIBR0951 was well described by Michaelis-Menten kinetics, with Km ranging 1–3 µM, significantly below the expected concentrations following the therapeutic dose of DABE. Overall, the present results suggested that CYP3A played a significant role in the presystemic metabolism of DABE and BIBR0951 following microdose DABE administration, thus attributing partly to the apparent overestimation in the DDI magnitude observed with the CYP3A/P-gp inhibitors. Therefore, DABE at the microdose, unlike the therapeutic dose, would likely be a less predictive tool and should be considered as a clinical dual substrate for P-gp and CYP3A when assessing potential P-gp–mediated impacts by dual CYP3A/P-gp inhibitors.

SIGNIFICANT STATEMENT This is the first study demonstrating a potentially significant role of cytochrome P450–mediated metabolism of the prodrug DABE following a microdose but not a therapeutic dose. This additional pathway, coupled with its susceptibility to P-glycoprotein (P-gp), may make DABE a clinical dual substrate for both P-gp and CYP3A at a microdose. The study also highlights the need for better characterization of the pharmacokinetics and metabolism of a clinical drug-drug interaction probe substrate over the intended study dose range for proper result interpretations.

Introduction

Dabigatran etexilate (DABE) is an orally bioavailable direct thrombin inhibitor used at 75–300 mg for treatments of deep vein thrombosis and pulmonary edema. It is designed as a double ester prodrug because of the poor oral bioavailability of its active moiety, dabigatran (DAB) (FDA, 2010a). After absorption, DABE has been shown to undergo two sequential hydrolysis by carboxylesterase (CES) enzymes to liberate DAB into the systemic circulation (Fig. 1) (Laizure et al., 2014). DABE is known to be primarily hydrolyzed by the intestinal CES2 enzyme to form an intermediate monoester metabolite called dabigatran ethylester (BIBR0951), which is further converted to DAB by the hepatic CES1 enzyme. To a much lesser extent, DAB can also be formed through the hydrolysis of another intermediate monoester metabolite, desethyl dabigatran etexilate (BIBR1087). The prodrug DABE, but not the active drug DAB, was shown in vitro and through several clinical drug-drug interaction (DDI) studies following the therapeutic dose (150–300 mg) of DABE to be a substrate of the human intestinal efflux transporter P-glycoprotein (P-gp) (Härtter et al., 2013; Ishiguro et al., 2014; Hodin et al., 2018). Citing its sensitivity and selectivity to intestinal P-gp, worldwide regulatory agencies have recommended DABE as a clinical probe substrate for investigating the perpetrator’s potential on intestinal P-gp inhibition (EMA, 2012; FDA, 2020a).

Fig. 1.
Fig. 1.

Metabolic conversion from DABE to DAB in human. The bold and dashed arrows indicate the major and minor pathways of DAB formation, respectively.

Recently, a microdose DABE (375–750 µg) has been used for studying DDI as well as phenotyping intestinal P-gp function in several populations (Prueksaritanont et al., 2017; Rattanacheeworn et al., 2021; Tatosian et al., 2021). Interestingly, the systemic exposure following a microdose DABE was approximately 2-fold lower than expected from its therapeutic dose, suggesting a nonlinear pharmacokinetic (PK) (Prueksaritanont et al., 2017). Furthermore, DDI magnitudes after the microdose DABE and P-gp inhibitors, i.e., clarithromycin (CTC) and itraconazole (ITZ), were higher than those observed using the therapeutic dose. In the microdose study, CTC increased the area under the plasma concentration-time curve of DAB by fourfold (Prueksaritanont et al., 2017) as compared with less than twofold under the therapeutic dose condition (FDA, 2010a; Delavenne et al., 2013). Two hypotheses have been proposed for the dissimilar interaction magnitudes following two different doses of DABE (Prueksaritanont et al., 2017). First, the intestinal P-gp is partially saturated at high doses of DABE but fully active when the intestinal concentration of DABE is relatively low following the microdose. The DDI magnitudes are thus increased through the maximal inhibition of intestinal P-gp following microdose DABE administration (Prueksaritanont et al., 2017). A recent published literature using physiologically based pharmacokinetic (PBPK) modeling suggested that regional differences in P-gp inhibition could possibly explain the different magnitudes of DABE-CTC interaction between microdose and standard dose of DABE (Lang et al., 2021). However, Prueksaritanont et al. (2017) hypothesized that CYP3A could be involved and contribute to the nonlinearity and the disparity in DDI magnitudes in the presence of either CTC or ITZ after the microdose DABE administration based on the following two reasons: 1) apart from being P-gp inhibitors, CTC and ITZ are also potent inhibitors of CYP3A enzymes (Ke et al., 2014) and 2) using a recombinant enzyme system, CYP3A4-mediated DABE oxidation was reported, albeit as an insignificant metabolic pathway when studied at a relatively high concentration (Blech et al., 2008; FDA, 2010a). Nevertheless, this hypothesis has not been thoroughly investigated elsewhere.

In this study, the oxidative metabolism of DABE and its intermediate metabolites (BIBR0951 and BIBR1087) was investigated using an in vitro approach to explore whether CYP3A-mediated metabolism is a potential underlying mechanism for the disparity in DDI magnitudes of DABE-P-gp inhibitor (CTC and ITZ) following administration of the microdose and the therapeutic dose of DABE.

Materials and Methods

Materials

All materials used in this study were the highest grade available commercially. DABE, BIBR1087, BIBR0951, and DAB were purchased from Toronto Research Chemical Inc. (Ontario, Canada). Acetonitrile was purchased from Honeywell Burdick and Jackson (Fischer Scientific, MI). NADPH, ketoconazole (KTZ), labetalol hydrochloride, and formic acid were purchased from Tokyo Chemical Industry Co., Ltd. (Chuo-ku, Tokyo, Japan). Bis(4-nitrophenyl) phosphate (BNPP) was purchased from Sigma Aldrich (St. Louis, MO). Pooled human liver microsomes (HLM) were purchased from Gibco Life Technologies (Thermo Fischer Scientific Inc., MA). Recombinant human CYP3A4 and CYP3A5 with oxidoreductase and cytochrome b5 (Corning Supersome) and pooled human intestinal microsome (HIM) were purchased from Corning Incorporated (NY).

In Vitro Metabolic Stability

The metabolism of DABE and its monoester metabolites (BIBR1087 and BIBR0951) was investigated in HLM, HIM, and recombinant human (rh)-CYP3A4 (rhCYP3A4) and CYP3A5 (rhCYP3A5) enzymes. Briefly, the 100-µL reaction mixtures were prepared to obtain a final concentration of 0.1 M potassium phosphate buffer (pH 7.4), 3.3 mM MgCl2, and specific protein concentration in the presence or absence of 1.3 mM NADPH. The HLM concentration was at 0.025, 0.2, and 0.5 mg/mL for DABE, BIBR0951, and BIBR1087, respectively. For HIM incubation, the protein concentration was at 0.05 mg/mL for DABE and at 0.1 mg/mL for both BIBR0951 and BIBR1087. The final rhCYP3A4 concentration was 5 pmol/mL for both DABE and BIBR0951, whereas the rhCYP3A5 concentration was at 20 and 5 pmol/mL for DABE and BIBR0951, respectively. After prewarming at 37°C for 10 minutes, the reactions were initiated by addition of compound. The organic solvent (acetonitrile) in the reactions was kept constant at 1%. After incubation, the reactions were quenched with one volume of ice-cold acetonitrile plus 0.1% formic acid containing labetalol as an internal standard. Then, the supernatants of reaction mixtures were collected by centrifugation and diluted with an appropriate volume of water plus 0.1% formic acid prior to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The natural logarithm of peak area ratios of the analytes to the internal standard were plotted against incubation time points. The first-order depletion rate constant (kdep) was estimated from the slope of the graph using linear regression analysis. The in vitro intrinsic clearance (CLint) of the compounds was calculated from the formula kdep × (incubation volume)/(mg of microsomal protein or pmol of recombinant enzyme).

In some experiments, the reactions were coincubated either with 100 μM BNPP (nonspecific CES inhibitor) or 1 μM KTZ (selective CYP3A inhibitor) to investigate the involvement of CES and CYP3A enzymes to overall microsomal metabolism.

In Vitro Enzyme Kinetic Studies

Kinetic parameters of CES-mediated hydrolysis of DABE and BIBR0951 were determined in HIM and HLM incubations without NADPH under the linear conditions for metabolite formation. The experiments were conducted as described under the section of in vitro metabolic stability by varying substrate concentrations from 1–400 µM. The Michaelis-Menten constants (Km) and maximum enzyme velocity (Vmax) were estimated from a graph plotted between rates of metabolite formation versus the corresponding substrate concentrations using a two-parameter hyperbolic equation in the SigmaPlot software version 13.0 (SyStat Software, Inc., CA).

Kinetic parameters of CYP3A-mediated oxidative metabolism of DABE and BIBR0951 were determined in the NADPH-fortified rhCYP3A4/5 incubations by a substrate depletion method as described previously (Obach and Reed-Hagen, 2002). Briefly, the experiments were performed using the procedure described in In Vitro Metabolic Stability, except that the substrate concentrations were varied from 0.1–30 µM. The first-order depletion rate constants (kdep) were plotted against the corresponding substrate concentrations. The Km was estimated from the inflection points of nonlinear curve fitting from a four-parameter Hill equation using the SigmaPlot software. The Vmax was calculated from multiplying Km to the CLint value obtained from the incubation at the lowest substrate concentration tested (0.1 µM).

Quantitative LC-MS/MS Bioanalysis

DABE and its metabolites in the samples were quantified by AB SCIEX QTRAP6500+ LC-MS/MS system. The chromatographic separation was performed by an Exion LC AD100 system coupled with a Phenomenex Kinetex C18, 1.3 µm, 50 × 2.1 mm analytical column. For mobile phases, the flow rate was set at 0.3 mL/min using a binary system of water and acetonitrile (both were fortified with 0.1% formic acid) as phase A and B, respectively. The gradient program was set as equilibration at 5% B for 0.5 minutes, linear gradient 5%–50% B for 3.5 minutes, ramping to 95% B for 1 minute, holding at 95% B for 1 minute, column reconditioning to 5% B for 0.2 minutes, and postanalysis equilibration at 5% B for 0.8 minutes. The Turbo V ion source was operated in the electrospray positive ionization mode under 40 psi of curtain gas, 4500 V of ion spray voltage, 450°C of source temperature, and 40 psi for both heating and nebulizing gases. The peak area of analytes was acquired by a multiple reaction monitoring mode using ion transitions and compound-specific parameters (i.e., declustering potential, entrance potential, collision energy, and collision exit potential) obtained from automatic compound optimization following direct infusion of analytical standards. For commercially unavailable metabolites, the quantification was relative based on multiple reaction monitoring transitions of their parents. Data were processed by Analyst version 1.6 and MultiQuant version 3.0 softwares (AB SCIEX, MA).

Metabolite Identification

Oxidative metabolites in the samples at the longest incubation time were identified using an AB SCIEX X500B QTOF LC-MS/MS system. The chromatographic separation was performed by an Exion LC AD100 system coupled with a Phenomenex Kinetex C18, 1.7 µm, 50 × 2.1 mm analytical column. For mobile phases, the flow rate was set at 0.4 mL/min using a binary system of water and acetonitrile (both were fortified with 0.1% formic acid) as phase A and B, respectively. The gradient program was similar to that described in Quantitative LC-MS/MS Bioanalysis. The Turbo V ion source was operated in the electrospray positive ionization mode under 40 psi of curtain gas, 4500 V of ion spray voltage, 7 psi of collision gas, 450°C in temperature, and 40 psi for both heating and nebulizing gases. An information-dependent data acquisition method was applied for metabolite identification. A full scan TOF MS ranging from m/z 50–1000 was used as a survey scan using declustering potential and a declustering potential spread of 60 and 20 V, respectively. The product ion spectra were acquired for the m/z at a range of 50–1000 using collision energy and a collision energy spread at 35 and 15 V, respectively. The data were processed and interpreted using SCIEX OS version 1.6 and MetabolitePilot version 2.0 softwares (AB SCIEX, MA).

Statistical Analysis

Data were expressed as mean ± S.D. from at least three independent experiments (n ≥ 3). The statistical analysis was performed by IBM SPSS Statistics 22 (Armonk, NY) using the independent t test or one-way analysis of variance (ANOVA) followed by a post-hoc Bonferroni test, where appropriate. Statistical significance was considered at p-value <0.05.

Results

NADPH-Dependent Metabolism of DABE and Its Intermediate Metabolites in HIM and HLM

To investigate the involvement of NADPH-dependent metabolic pathways in metabolism of DABE and its intermediate metabolites (BIBR1087 and BIBR0951), the metabolic stability assay was first conducted by separately incubating each compound at 1 μM, a concentration falling within the intestinal concentration range estimated following the microdose (see Discussion) with HIM and HLM in the presence or absence of NADPH. For all three compounds, enzyme-mediated reactions were confirmed based on minimal parent disappearance and no detectable metabolite formation was observed in the incubations without microsomal protein (Supplemental Fig. 1).

DABE Metabolism

In HIM incubations, DABE disappearance was faster in the presence of NADPH than in the absence of NADPH, with the CLint value differing by ~1.6-fold (Fig. 2A; Table 1). However, the formation of its major metabolite, BIBR0951, was comparable under these two conditions. Another intermediate metabolite BIBR1087 was also observed, albeit at a much lower extent (<3% of DABE) (Fig. 2A). The results suggested that in HIM, DABE underwent both NADPH-independent, primarily to BIBR0951 and to a much lesser extent to BIBR1087, as well as NADPH-dependent metabolism, to others yet to be identified metabolites. In HIM, the relative contribution of NADPH-dependent metabolic pathways constituted ~40% of the overall DABE metabolism (Table 1).

Fig. 2.
Fig. 2.

Metabolic stability of DABE (A), BIBR0951 (B), and BIBR1087 (C) following incubation of 1 μM in HIM (left panel) and HLM (right panel). The compounds are represented by the symbols as follows: DABE (circle), BIBR0951 (diamond), BIBR1087 (square), and DAB (triangle). Black symbols and solid lines represent the incubations with NADPH, whereas white symbols and dashed lines represent the incubations without NADPH. Data are expressed as percent of compounds presented in the incubations compared with their parents at 0 minutes (mean ± S.D. from n ≥ 3). w/, with; w/o, without.

View this table:
TABLE 1

CLint of DABE, BIBR0951, and BIBR1087 in HIM and HLM incubations with or without NADPH and in the presence or absence of inhibitors

Data are expressed as mean ± S.D. from n ≥ 3.

In HLM incubations, DABE was comparably metabolized in the presence and absence of NADPH to form BIBR1087 as a major metabolite, whereas BIBR0951 and DAB were barely detected (Fig. 2A). There was no significant difference between the two incubation conditions in either DABE disappearance or formation of its metabolites BIBR0951 and BIBR1087 (Fig. 2A; Table 1), suggesting that unlike HIM, the metabolism of DABE in HLM was mediated primarily by non–NADPH-dependent enzymes and mainly to BIBR1087.

BIBR0951 Metabolism

In HIM incubations, BIBR0951 disappearance was pronounced (~40% consumption in 10 minutes) in the incubation fortified with NADPH but very minimal in the absence of NADPH (Fig. 2B; Table 1). DAB formation in both incubations with and without NADPH was very low (Fig. 2B). In HLM incubations with BIBR0951, there was a more rapid consumption of BIBR0951, resulting in 2-fold higher in the CLint in the presence than that in the absence of NADPH (Fig. 2B; Table 1). However, its hydrolytic metabolite (DAB) was formed at a similar level in the HLM incubations with and without NADPH (Fig. 2B). These results suggested that in both HIM and HLM incubations, in addition to CES-mediated hydrolysis, NADPH-dependent enzymes were likely involved in the metabolism of BIBR0951 to some unidentified metabolites. Additionally, the formation of DAB in both HIM and HLM was mediated primarily by hydrolytic enzymes.

BIBR1087 Metabolism

In HIM incubations, the disappearance of BIBR1087 was 68% higher with NADPH than that without NADPH, whereas DAB formation was comparable between the two incubation conditions (Fig. 2C; Table 1). Unlike HIM incubations, there was no difference in both BIBR1087 disappearance and DAB formation (Fig. 2C; Table 1) in the HLM incubations with and without NADPH. These results indicated that BIBR1087 underwent metabolism by NADPH-dependent enzymes only in the HIM but not in the HLM. DAB, the major metabolite of BIBR1087, was likely generated via hydrolytic enzymes in both the HIM and the HLM.

Identification of Oxidative Metabolites of DABE and BIBR0951 in HIM and HLM

Oxidative Metabolites in the Incubation with BIBR0951

We first identified the NADPH-dependent metabolites of BIBR0951 generated in HIM, which included M324, M488, M400, M516 (1), M416, M516 (2), M498 (1), M498 (2), and M514 (Fig. 3A). The key product ions of BIBR0951 and its metabolites are listed in Supplemental Table 1. A fragmentation pattern of the BIBR0951 showed the intense daughter ions of its core structure at the m/z 289.10 and 306.11 amu and a weak ion of ethyl 3-(pyridin-2-ylamino)propanoate ion at the m/z 195.11 amu. All oxidative metabolites found also showed the product ion at the m/z 289.10, suggesting that the core structure was intact and that the oxidation occurred on the surrounding functional groups. The most abundant oxidative metabolite, M400, was an N-dealkylated metabolite with a loss of 100 amu, equivalent to N-(ethyl propionate) group (C5H8O2). The M516 (1) and M516 (2) shared the common mass shift of +16 amu or hydroxylation. For M516 (1), the presence of product ions at the m/z 289.10 and 306.13 amu suggested that the hydroxylation could occur on either N-(ethyl propionate) or pyridine moiety. In the case of M516 (2), its product ion at the m/z 193.0978 amu indicated the hydroxylation on an aliphatic carbon of the N-(ethyl propionate) moiety, which possibly dehydrated during MS fragmentation. Both M498 (1) and M498 (2) showed a mass shift of −2 amu, which could result from dehydrogenation or oxidation followed by dehydration. The presence of product ions at the m/z 289.10 and 306.13 amu pointed to the metabolic site on the N-(ethyl propionate) moiety. For other oxidative metabolites (M324, M488, M416, and M514), the product ions indicated that they were the secondary metabolites of BIBR0951. A mass shift of −176 amu of M324 corresponded to a dual loss of pyridine and N-(ethyl propionate) moieties, whereas M488 and M416 were the hydroxylated versions (+16 amu) of DAB and M400, respectively. For M514, a mass shift of +14 indicated an addition of one atomic oxygen plus a loss of two hydrogen atoms, possibly generated from either M516 or M498.

Fig. 3.
Fig. 3.

Profiling of BIBR0951 metabolites. The extracted ion chromatograms of BIBR0951 and its metabolites detected in the HIM (A), HLM (B), rhCYP3A4 (C), and rhCYP3A5 (D) incubations with (left panel) or without (right panel) NADPH.

In the HLM incubation, all primary oxidative metabolites of BIBR0951 [including M400, M516 (1), M516 (2), M498 (1), and M498 (2)] were detected only in the presence of NADPH (Fig. 3B). The M400 was still the most abundant oxidative metabolite detected in the HLM incubation. In addition, none of the secondary oxidative metabolites of BIBR0951 (M324, M488, and M416) were observed in the HLM incubation except the M514 (Fig. 3B).

Oxidative Metabolites in the Incubation with DABE

In HIM, there were five oxidative metabolites, including M400, M644 (1), M644 (2), M264, and M528, with M400 and M264 being major products (Fig. 4A). After fragmentation, the parent DABE showed three dominated daughter ions at the m/z 289.11, 434.22, and 526.22 amu (Supplemental Table 2). Based on both product ion spectra and retention time, M400 was the same oxidative metabolite found in the microsomal incubation with BIBR0951, suggesting that M400 was a secondary metabolite of DABE generated subsequently from BIBR0951. Although a small peak of M264 was also detected in all HIM incubations with DABE, an increase of its peak intensity over time in the incubation with NADPH supported that M264 was an oxidative metabolite of DABE. The molecular ion at the m/z 264.1707 amu corresponded to a structure of hexyl ((4-aminophenyl)(imino)methyl)carbamate, possibly formed by an oxidative N-dealkylation on the aliphatic carbon adjacent to the 1-methyl-1H-benzo[d]imidazole ring (Supplemental Table 2). The M644 (1) and M644 (2) shared a common mass shift of +16 amu or addition of an atomic oxygen (hydroxylation). The common product ion at the m/z 526.22 suggested that hydroxylation could be assigned on two different aliphatic carbons of the hexyl carbamate moiety (Supplemental Table 2). For M528, a neutral loss of 100 amu from both mother and daughter m/z indicated a cleavage of N-(ethyl propionate) group (C5H8O2) through an oxidative N-dealkylation reaction (Supplemental Table 2).

Fig. 4.
Fig. 4.

Profiling of DABE metabolites. The extracted ion chromatograms of DABE and its metabolites detected in the HIM (A), HLM (B), rhCYP3A4 (C), and rhCYP3A5 (D) incubations with (left panel) or without (right panel) NADPH.

In HLM, all oxidative metabolites found were detected in the HIM, with M264 showing the highest intensity (Fig. 4B).

Investigation of CYP3A-Mediated Oxidative Metabolism of DABE and BIBR0951 Using Chemical Inhibitors

Effects of Chemical Inhibitors on DABE Metabolism

Given that the oxidative metabolism of DABE occurred primarily in the HIM, we next investigated the potential involvement of CYP3A in the HIM metabolism of DABE. In the presence of KTZ, a known CYP3A inhibitor, DABE disappearance in the HIM incubation with NADPH was inhibited by 53% when compared with the solvent control (Fig. 5A; Table 1). KTZ drastically inhibited the formation of BIBR1087 but exerted little effect on BIBR0951 formation (Supplemental Fig. 2, A and D). In addition, KTZ completely inhibited the formation of all primary oxidative metabolites of DABE (Supplemental Fig. 2, B, C, and E). As expected, BNPP, a known CES inhibitor, had no effect on the formation of the primary oxidative metabolites of DABE [i.e., M644 (1), M644 (2), M264, and M528] (Supplemental Fig. 2, B, C, and E). These results suggested that CYP3A was the predominant NADPH-dependent enzyme responsible for the oxidative metabolism of DABE to form BIBR1087 and other oxidative metabolites [M644 (1), M644 (2), M264, and M528].

Fig. 5.
Fig. 5.

Effects of KTZ (1 µM) and BNPP (100 µM) on the disappearance of DABE in HIM (A), BIBR0951 in HIM (B), and BIBR0951 in HLM (C) in the presence (left panel) or absence (right panel) of NADPH. Data are expressed as percent of compounds at 0 minutes (mean ± S.D. from n = 3).

Effects of Chemical Inhibitors on BIBR0951 Metabolism

In the NADPH-fortified HIM incubation, KTZ inhibited BIBR0951 metabolism by 88% (Fig. 5B; Table 1) and completely abolished the formation of all primary oxidative metabolites of BIBR0951 [i.e., M400, M516 (1), M516 (2), M498 (1), and M498 (2)] (Supplemental Fig. 3, B–F) but with minimal effect on the formation of DAB (Supplemental Fig. 3A). In contrast, BNPP had modest effects on both BIBR0951 disappearance (Fig. 5B; Table 1) and DAB formation (Supplemental Fig. 3A). Interestingly, both KTZ and BNPP were able to inhibit the formation of DAB in the incubation without NADPH (Supplemental Fig. 3A).

In NADPH-fortified HLM incubation, KTZ significantly decreased BIBR0951 disappearance by ∼58% but had no effect on DAB formation (Fig. 5C; Table 1; Supplemental Fig. 4A). In addition, KTZ completely inhibited the formation of the primary oxidative metabolites of BIBR0951 (Supplemental Fig. 4B–F). In this system, BNPP significantly reduced BIBR0951 disappearance by 40% and dramatically decreased DAB formation (Fig. 5C; Table 1; Supplemental Fig. 4A). As expected, BNPP had no effect on the formation of the primary oxidative metabolites of BIBR0951 (Supplemental Fig. 4B–F). In HLM incubation without NADPH, BNPP significantly inhibited CES-mediated BIBR0951 hydrolysis by decreasing both BIBR0951 disappearance and DAB formation by more than 80%. Under the same incubation condition, KTZ had minimal effects on both BIBR0951 disappearance and DAB formation (Fig. 5C; Table 1; Supplemental Fig. 4A). Notably, the CLint of BIBR0951 calculated from the HLM incubation without NADPH was comparable to that obtained from the HLM incubation with NADPH and KTZ (Table 1).

Therefore, these results indicated that the oxidative metabolism of BIBR0951 observed earlier in HIM and HLM incubations was mediated through CYP3A enzymes. In addition, CYP3A also partly metabolized BIBR0951 to DAB.

Investigation of CYP3A-Mediated Oxidative Metabolism of DABE and BIBR0951 Using Recombinant Enzymes

To further confirm CYP3A-mediated DABE and BIBR0951 oxidation, DABE and BIBR0951 at 1 µM were incubated with rhCYP3A4 and rhCYP3A5 enzymes.

NADPH-dependent disappearance of DABE and BIBR0951 was clearly observed in both rhCYP3A4 and rhCYP3A5 incubations (data not shown), confirming the roles of both CYP3A isoforms in the oxidative metabolism of DABE and BIBR0951. When the intersystem extrapolation factor and enzyme abundance were considered for comparison the relevance of two isoforms (Bohnert et al., 2016), DABE and BIBR0951 were oxidative metabolized mainly by CYP3A4 but with a lesser extent by CYP3A5.

As shown in Fig. 4, C and D, all primary oxidative metabolites of DABE [including M644 (1), M644 (2), M264, and M528] were detected in both rhCYP3A4 and rhCYP3A5 systems, with M264 and BIBR1087 being the major metabolites. Interestingly, the peaks of BIBR0951 and BIBR1087 in the NADPH-fortified rhCYP3A4/5 incubations were higher than those in the NADPH-free incubations, suggesting that BIBR0951 and BIBR1087 were formed, at least partly, by CYP3A4/5-mediated ester cleavage of DABE.

As shown in Fig. 3, C and D, both rhCYP3A4 and rhCYP3A5 could catalyze the formation of all primary oxidative metabolites of BIBR0951 detected in HIM and HLM, with M400 being the most abundant metabolite. Interestingly, the metabolites M516 (1) and M498 (2) were formed by only rhCYP3A4 but not rhCYP3A5. Furthermore, the intensity of DAB in NADPH-fortified rhCYP3A4/5 incubations was higher than that in NADPH-free incubations, indicating that DAB was formed minorly through CYP3A4/5-mediated ester cleavage of BIBR0951.

Determination of Kinetic Parameters of CES- and CYP3A4/5-Mediated Metabolism of DABE and BIBR0951

We next determined the enzyme kinetic parameters, especially the Km of CYP3A4/5-mediated oxidation (determined based on substrate depletion as well as metabolite formation) and compared them to those of CES-mediated hydrolysis of DABE and BIBR0951 (determined based on metabolite formation).

For DABE, the Km values for rhCYP3A4- and rhCYP3A5-mediated DABE depletion as determined were 1.4 and 1.1 µM, respectively (Table 2; Supplemental Fig. 5A), comparable to or lower than the Km values for both intestinal and hepatic CES-mediated DABE hydrolysis (1–3 and 8 to 9 µM for BIBR0951 and BIBR1087 formation, respectively) (Table 2; Supplemental Fig. 6, A and B). Of note, the Km value for DABE hydrolysis in HIM of this study (1.2 µM) was about 7-fold lower than reported previously (8.6 µM) (Laizure et al., 2014), possibly due to the batch-to-batch variation in HIM quality and preparation (Hatley et al., 2017).

View this table:
TABLE 2

Enzyme kinetic parameters following incubation of either DABE or BIBR0951 in HIM, HLM, rhCYP3A4, and rhCYP3A5

All incubations with HIM and HLM were performed in the absence of NADPH, whereas those with rhCYP3A4/5 were performed in the presence of NADPH. Data are expressed as mean ± S.D. from n = 3.

For BIBR0951, the Km values for rhCYP3A4- and rhCYP3A5-mediated BIBR0951 depletion were at 2.8 and 0.6 µM (Table 2; Supplemental Fig. 5B), respectively. The values were 185- and 863-fold lower than that for CES-mediated DAB formation in HLM (Table 2; Supplemental Fig. 6C). The Km for CES-mediated DAB formation in HIM was not determined given the very low formation of DAB in this system.

In the rhCYP3A4/5 systems, the Km values based on metabolite formation following incubation with DABE and BIBR0951 (<5 µM; Supplemental Table 3; Supplemental Figs. 7 and 8) were in line with those observed based on substrate depletion. Overall, the findings suggested that CYP3A4/5-mediated metabolism in HIM and HLM could play an important role at relatively low concentrations of DABE and BIBR0951, whereas CES-mediated hydrolysis could become dominant at higher concentrations of both compounds.

Investigation of Saturation of CYP3A4/5-Mediated Oxidative Metabolism of DABE and BIBR0951

We next quantified the relative contribution of CYP3A to overall microsomal metabolism at various concentrations of DABE and BIBR0951 (1, 10, and 100 µM). As illustrated in Table 3, DABE at the concentration of 1 µM was metabolized in HIM by both CES and CYP3A, with the relative contribution of CYP3A at 38%. By increasing the concentration of DABE from 1 to 10 µM, the involvement of CYP3A was decreased from 38% to 19%. Due to a solubility limit of DABE, the CLint of DABE disappearance and the relative contribution of CYP3A were not evaluated at the concentration of 100 µM.

View this table:
TABLE 3

CLint and relative contribution of CYP3A to overall microsomal metabolism at various concentrations of DABE and BIBR0951 in HIM and/or HLM incubations

Data are expressed as mean ± S.D. from n = 3. The contribution of CYP3A was calculated by 100 × (CLint with NADPH − CLint without NADPH)/(CLint with NADPH).

In HIM incubations, BIBR0951 was entirely metabolized by CYP3A (100% contribution) at all concentrations tested (1, 10, and 100 µM) (Table 3). In HLM, the relative contribution of CYP3A was about 52% when BIBR0951 was incubated at the concentration of 1 µM but significantly reduced to 15% at the concentration of 10 µM. At the high concentration of BIBR0951 (100 µM), CYP3A-mediated BIBR0951 oxidation was completely abolished and BIBR0951 was solely metabolized by NADPH-independent hydrolysis (Table 3).

These results supported that at relatively low substrate concentrations, both CES and CYP3A enzymes contributed significantly to the metabolism of DABE (in HIM) and BIBR0951 (in HLM). However, when the concentrations were increased beyond 10 μM, the CYP3A-mediated oxidation was saturated, and the metabolism of DABE and BIBR0951 in HIM and of DABE in HLM was mediated almost exclusively by CES enzymes.

Discussion

In this study, we used several in vitro techniques to demonstrate that DABE and its intermediate metabolites (primarily BIBR0951, the major gut metabolite) underwent substantial CYP3A-mediated metabolism in HIM. Additionally, a significant extent of BIBR0951 oxidation was also observed in HLM. The oxidative metabolism was saturable at a concentration range similar to the estimated intestinal concentrations following microdosing at 375 ug, supporting the earlier postulation that CYP3A might be involved in and contribute to the nonlinearity of DAB PK and the greater increase of DAB exposure by CTC and ITZ with the microdose versus with the therapeutic dose of DABE (Prueksaritanont et al., 2017).

Shown in Fig. 6 is a comprehensive metabolic scheme proposed based on the present in vitro findings. Overall, DABE is metabolized in the intestine by both CYP3A4/5 and hydrolysis (presumably the CES2-mediated pathway), with CYP3A4/5 accounting for approximately 40%–50% of the overall gut metabolism. The CYP3A4/5 enzymes catalyze the formation of several oxidative metabolites of DABE [M644 (1), M644 (2), M264, and M528]; these findings have never been reported previously, either in vitro (FDA, 2010b; Hu et al., 2013) or in vivo (Blech et al., 2008; FDA, 2010a,b). As demonstrated previously (Laizure et al., 2014), the hydrolysis of DABE by intestinal CES2 enzymes yields BIBR0951, which was subsequently metabolized exclusively by CYP3A4/5 in the small intestine to form several novel oxidative metabolites [M400, M516 (1), M516 (2), M498 (1), and M498 (2)], along with DAB. The formation of another intermediate monoester, BIBR1087, from DABE in the gut is minimal, and the reaction was mediated by both CES and CYP3A enzymes. In the liver, DABE is hydrolyzed mainly by the CES1 enzyme to form BIBR1087, which further liberates the active DAB via CES2-mediated hydrolysis (Laizure et al., 2014). CYP3A4/5 involvement in BIBR1087 metabolism in the liver is very limited. Unlike DABE, BIBR0951 is metabolized in the liver to a similar extent by both CES1 and CYP3A4/5 enzymes. The DAB formation pathway through BIBR0951 by CES1 is more pronounced than that via CYP3A4/5 enzymes. In addition, the metabolism of BIBR0951 by hepatic CYP3A4/5 yields the same oxidative products as those formed in the gut epithelia. Notably, the CYP3A4/5-mediated oxidative metabolism of both DABE and BIBR0951 in the small intestine and of BIBR0951 in the liver was shown to be saturable, especially when the concentrations of both compounds exceeded 10 µM (exceeding the Km values of CYP3A4/5).

Fig. 6.
Fig. 6.

Proposed scheme of metabolic conversion from DABE to DAB following oral administration of microdose DABE in human. The bold and dashed arrows indicate the major and minor disposition pathways of microdose DABE, respectively. The plain arrow (nondashed, nonbold) indicates the potentially minor pathway.

Among the oxidative metabolites identified in this study, the M400 (N-dealkylated metabolite of BIBR0951) was the most abundant metabolite. It is noteworthy that the previous literature proposed that M400 was formed by direct DAB oxidation in vivo (Blech et al., 2008). However, our in vitro results demonstrated that DAB was metabolically stable in HLM (Supplemental Fig. 9), with no metabolite detected. In addition, DAB was also reportedly not being metabolized by any cytochrome P450 isoform (FDA, 2010a). A human mass balance study following intravenous administration of DAB also confirmed that DAB is excreted mainly unchanged (>90% of dose), with less than 4% recovered as oxidative (but not M400) metabolites (Blech et al., 2008). Therefore, M400 is likely originated from CYP3A4/5-mediated BIBR0951 oxidation rather than from the direct oxidation of DAB. It is noteworthy that M400 was also found following oral administration of radiolabeled DABE at 200 mg, with recoveries of 0.2% and 5.8% of dose in the urine and feces, respectively (Blech et al., 2008). Although the in vivo formation of M400 has never been reported under the microdose condition, we expect that it could be formed more significantly when compared with the therapeutic dose condition.

The in vitro metabolism data obtained in this study (Fig. 6) may shed some light on the disparity in the DDI magnitude by CTC and ITZ following microdose versus following the therapeutic dose of DABE. After oral administration of microdose DABE at 375 μg, the theoretical gut concentration of DABE and BIBR0951 is approximately at 2.4 μM (FDA, 2020b) or even lower when considering an intestinal P-gp mediated DABE efflux. Under this condition, the pathways of CYP3A4/5-mediated DABE and BIBR0951 oxidation may compete with the typical DAB formation pathway, possibly resulting in the lower systemic exposure of DAB following microdose DABE than expected from the therapeutic dose DABE. In the presence of either CTC or ITZ, the DDI magnitude following microdose DABE would be magnified from their inhibitory effects on CYP3A4/5-mediated oxidative metabolism of DABE and BIBR0951, in addition to P-gp. On the contrary, following oral administration of a therapeutic dose DABE at 150 mg (the expected gut concentrations >100 μM), the relative contribution of CYP3A4/5 to overall disposition is expected to be insignificant, and therefore, the P-gp–mediated DAB efflux is the major mechanism producing the DDI magnitude.

Using a semimechanistic PBPK model, a previous study proposed that the higher magnitudes of microdose DABE-CTC DDI could arise from the regional difference in P-gp inhibition (Lang et al., 2021). In the present study, a comprehensive in vitro data set supported another possibility that the disposition of microdose DABE may not rely solely on the intestinal P-gp–mediated DABE efflux but also depends on the CYP3A-mediated oxidative metabolism of DABE and its intermediate, BIBR0951. Furthermore, an interplay between CYP3A4/5 and P-gp in the gut may possibly occur for DABE, thus increasing the complexity of its disposition following the microdose administration. However, the relative significance of each pathway (P-gp and CYP3A4/5) to the overall disposition of microdose DABE remains to be investigated, via either a clinical study or PBPK modeling or both.

Although the microdosing approach provides some advantages in early drug development in terms of safe assessment in first-in-human PK and DDI evaluations, there are several previous cases showing that their PK following microdose were not in line with those following therapeutic dose (Burt et al., 2020). According to the previous meta-analysis of 46 drugs administered orally, the microdose PK of 13 drugs (32%) could not be linearly extrapolated from the PK at therapeutic dose, mostly caused by saturation of enzymes and/or transporters at the therapeutic dose (van Nuland et al., 2019). In addition to those known cases, our in vitro findings for DABE and its metabolites exemplified another case that the saturation of CYP3A and/or P-gp could be the underlying mechanisms of nonlinearity and a disparity in DDI magnitude following microdose versus following therapeutic dose. It is worth pointing out that dual P-gp and CYP3A inhibitors, like dual P-gp and CYP3A substrates, are commonly found (Yasuda et al., 2002; Vermeer et al., 2016), and thus, the microdose DABE remains a valuable clinical tool for DDI assessments, although with different interpretations from when using the therapeutic dose DABE. No substantial differences in result interpretations are anticipated when using either the microdose or therapeutic doses of DABE with selective P-gp inhibitors.

In conclusion, DABE and its gut intermediate monoester, BIBR0951, were significantly metabolized by the saturable CYP3A-mediated oxidation, in parallel to the previously known CES-mediated hydrolysis. The findings offer an alternative explanation to the well established intestinal P-gp–mediated efflux for the difference in DDI magnitudes of DABE-P-gp inhibitor (CTC and ITZ) following microdose versus therapeutic doses of DABE. The present results also imply that the microdose, unlike the therapeutic dose, of DABE should be used as a dual P-gp and CYP3A, not just P-gp, marker substrate when assessing the DDI potential of P-gp and CYP3A inhibitors. Finally, the present study highlights another example of different result interpretations following the microdose versus the therapeutic dose and suggests the need to establish linearity in pharmacokinetics and characterize associated disposition determinants of a compound over its intended study dose range.

Acknowledgments

The authors would like to thank Cuyue Tang and Dan Cui for their suggestions and critical reviews of this article.

Data Availability

The authors declare that all the data supporting the findings of this study are available within the paper and its Supplemental Materials.

Authorship Contributions

Participated in research design: Udomnilobol, Jianmongkol, Prueksaritanont.

Conducted experiments: Udomnilobol.

Performed data analysis: Udomnilobol.

Wrote or contributed to the writing of the manuscript: Udomnilobol, Jianmongkol, Prueksaritanont.

Footnotes

    • Received April 5, 2023.
    • Accepted May 15, 2023.

  • This research was supported by the Health System Research Institute [Grant 60-094], Thailand.

  • All authors declare that they have no competing interests.

  • dx.doi.org/10.1124/dmd.123.001353.

  • ↵Embedded ImageThis article has supplemental material available at dmd.aspetjournals.org.

Abbreviations

BIBR0951
dabigatran ethylester
BIBR1087
desethyl dabigatran etexilate
BNPP
bis(4-nitrophenyl) phosphate
CES
carboxylesterase
CLint
intrinsic clearance
CTC
clarithromycin
DAB
dabigatran
DABE
dabigatran etexilate
DDI
drug-drug interaction
HIM
human intestinal microsome
HLM
human liver microsome
ITZ
itraconazole
kdep
first-order depletion rate constant
Km
Michaelis-Menten constants
KTZ
ketoconazole
LC-MS/MS
liquid chromatography-tandem mass spectrometry
PBPK
physiologically based pharmacokinetics
P-gp
P-glycoprotein
PK
Pharmacokinetics
rh
recombinant human
Vmax
maximum enzyme velocity

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CYP3A-Mediated Oxidation of DABE and BIBR0951

Udomsak Udomnilobol, Suree Jianmongkol and Thomayant Prueksaritanont
Drug Metabolism and Disposition September 1, 2023, 51 (9) 1216-1226; DOI: https://doi.org/10.1124/dmd.123.001353
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