Mechanism-Based Inactivation of Cytochrome P450 3A4 and 3A5 by the Fibroblast Growth Factor Receptor Inhibitor Erdafitinib
Lloyd Wei Tat Tang, Jian Wei Teng, Siew Kwan Koh, Lei Zhou, Mei Lin Go, and Eric Chun Yong Chan*
ABSTRACT:
Erdafitinib (ERD) is a first-in-class pan inhibitor of fibroblast growth factor receptor 1−4 that has garnered global regulatory approval for the treatment of advanced or metastatic urothelial carcinoma. Although it has been previously reported that ERD elicits time-dependent inhibition (TDI) of cytochrome P450 (P450) 3A4 (CYP3A4), the exact biochemical nature under- pinning this observation remains obfuscated. Moreover, it is also uninterrogated if CYP3A5 its highly homologous counterpart could be susceptible to such interactions. Mechanism-based inactivation (MBI) of P450 is a unique subset of TDI that hinges on prior bioactivation of the drug to a reactive intermediate and possesses profound clinical and toXicological implications due to its irreversible nature. Here, we investigated and confirmed that ERD inactivated both CYP3A isoforms in a time-, concentration-, and NADPH-dependent manner with KI, kinact, and partition ratio of 4.01 and 10.04 μM, 0.120 and 0.045 min−1, and 32 and 55 for both CYP3A4 and CYP3A5, respectively, when rivaroXaban was employed as the probe substrate. Co-incubation with an alternative substrate or direct inhibitor of CYP3A attenuated the rate of inactivation, whereas the addition of glutathione or catalase did not induce such protection. The lack of enzyme activity recovery following dialysis for 4 h and oXidation with potassium ferricyanide combined with the lack of a Soret peak in spectral scans collectively substantiated that ERD is an irreversible covalent MBI of CYP3A. Finally, glutathione trapping and high-resolution mass spectrometry experiments illuminated a plausible bioactivation mechanism of ERD by CYP3A arising from metabolic epoXidation of its quinoXaline ring.
1. INTRODUCTION
The fibroblast growth factor receptors (FGFR) are a family of four transmembrane receptor tyrosine kinases that are involved in a wide range of physiological processes such as proliferation, differentiation, migration, and survival.1 Recent evidence has demonstrated that deregulations in the FGF/FGFR signaling axis may serve to independently promote oncogenesis and drive resistance to existing anticancer therapies.2,3 In fact, a recent study reported that genomic aberrations in FGFR (i.e., gene amplifications or chromosomal translocations) were present in 7.1% of all malignancies and may be as prevalent is also under clinical investigation for other FGFR-driven cancers including cholangiocarcinoma and hepatocellular carcinoma among many others.8
Early metabolic studies have revealed that ERD undergoes phase I metabolism primarily via O-demethylation at its dimethoXybenzene ring moiety by cytochrome P450 (P450) enzymes.9,10 Apart from CYP2C9, it is also known to be a major substrate of CYP3A4, which is the most abundant hepatic P450 isoform in humans.9 Due to its wide substrate and catalytic promiscuity, CYP3A4along with its highly homologous counterpart CYP3A5is known to participate in as 32% in certain tumor types (i.e., urothelial carcinoma).4 Consequently, pharmacological ablation of FGFR signaling pathways is readily gaining traction as a promising modality in cancer therapeutics.5,6 Erdafitinib (ERD) formerly desig- nated as JNJ-42756493 (1) (Scheme 1)is a highly potent, first-in-class pan-selective inhibitor of FGFR1−4 that has recently been granted accelerated approval by the U.S Food and Drug Administration (FDA) in 2019 for the treatment of advanced or metastatic urothelial carcinoma.7,8 Additionally, it
Scheme 1. Proposed Bioactivation Pathway of ERD by CYP3A
Mechanism-based inactivation (MBI) is a unique subset of time-dependent inhibition which hinges on prior metabolic activation (termed bioactivation) of the drug to a reactive electrophilic intermediate that subsequently primes itself for irreversible covalent adduction to nucleophilic residues within the apoprotein and/or heme porphyrin ring or through coordination with the heme catalytic iron to form a quasi- irreversible metabolite−intermediate (MI) complex.16 As the loss of P450 activity is irrevocable and persists in vivo even after the perpetrator has been systemically cleared from the body and only restored upon biosynthesis of new enzymes,
the extent of pharmacokinetic-pharmacodynamic DDIs elicited tends to be exaggerated as compared to that elicited by a reversible inhibitor.17 Furthermore, such covalent modification of the P450 protein may constitute neoantigens and trigger adaptive immune responses which can culminate in immune- mediated idiosyncratic toXicities.18 Apart from the character- istic time-dependent nature of MBI, an archetypal MBI of P450 also exhibits these following features: cofactor-depend- ency of inactivation, saturable kinetics of inactivation, protection against inactivation by a competing substrate, lack of protection by exogenous nucleophiles or scavengers of reactive oXygen species (ROS), irreversibility of inactivation, and a 1:1 binding stoichiometry.19
While the propensity for ERD to cause time-dependent inhibition of CYP3A4 has been recognized, some seeding questions remain unanswered. It is not known if ERD elicits MBI of CYP3A4. Moreover, it remains unelucidated if ERD could similarly inactivate CYP3A5. In that regard, previous studies interrogating MBI in CYP3A4 and CYP3A5 have unraveled vastly disparate susceptibilities and/or potencies to MBI between both CYP3A isoforms.20,21 Furthermore, given that the active site of CYP3A possesses considerable bulk and plasticity, probe substrate-dependent inactivation has also been observed.22 Taken together, it is imperative to investigate the MBI susceptibilities and/or potencies of CYP3A4 and CYP3A5 to ERD separately using an array of probe substrates to discern if it elicits any MBI due to its profound clinical and toXicological ramifications.
In this study, the nature of interactions between ERD and CYP3A was investigated using the two structurally unrelated FDA-recommended CYP3A probe substrates (i.e., testosterone and midazolam) and a clinically relevant substrate of CYP3A levels of CYP3A5 are highly polymorphic and vary between different ethnic groups due to the presence of the defective *3 allelic variant.13,14 Conversely, in wild-type *1 carrier, CYP3A5 may further augment CYP3A-mediated metabolism.15 Con- sequently, inhibition of this important P450 subfamily can potentially precipitate drug−drug interactions (DDI) and lead to deleterious clinical consequences in pharmaceutical agents with narrow therapeutic indices. At this point, ERD has been reported to cause time-dependent inhibition of CYP3A4.9
CYP3A4 and CYP3A5 in a time-, concentration-, and cofactor- dependent manner albeit with varying potencies. We then demonstrated that ERD fulfilled all of the other aforemen- tioned criteria for an MBI of P450 and elucidated the structure of the putative reactive metabolite implicated in the irreversible covalent modification of CYP3A through glutathione (GSH) trapping experiments.
2. MATERIALS AND METHODS
2.1. Chemicals and Reagents. ERD and infigratinib were acquired from MedChem EXpress (Monmouth Junction, NJ). Catalase, dexamethasone, GSH, ketoconazole, prednisolone, and rivaroXaban were purchased from Sigma-Aldrich (St. Louis, MO). Midazolam was procured from Tocris Bioscience (Bristol, UK). Testosterone was acquired from Tokyo Chemical Industries (Tokyo, Japan). Potassium ferricyanide was procured from VWR International (Leuven, Belgium). The NADPH regenerating system comprised NADP+ and glucose-6-phosphate (G6P) (NADPH A) and glucose-6- phosphate dehydrogenase (G6PDH) (NADPH B) and recombinant human P450 3A4 (rhCYP3A4) and 3A5 (rhCYP3A5) Supersomes coexpressing Cytochrome b5 and NADPH P450 reductase and was commercially purchased from Corning Gentest (Woburn, MA). High- performance liquid chromatography (HPLC)-grade acetonitrile was (1)procured from Tedia Company Inc. (Fairfield, OH). Ultrapure water (type I) was obtained using a Milli-Q water purification system (Millipore Corporation, Bedford, MA). All other commercially available chemicals were of analytical or HPLC grade.
2.2. Substrate Depletion of ERD in CYP3A4 and CYP3A5. All incubations described in this work were performed in 96-well plates. Incubation miXtures containing 20 pmol/mL rhCYP3A4 or rhCYP3A5, 1 μM ERD, G6PDH, and 100 mM potassium phosphate buffer (pH 7.4) were prepared in triplicate. The reaction was initiated via the addition of NADP+/G6P after preincubating for 5 min at 37°C. The final primary incubation miXture (100 μL) contained <1% v/ v organic solvent. Following this, at specific predefined time intervals (0, 5, 10, 15, 30, 45, 60, 80, 100, and 120 min), 80 μL aliquots of each incubation miXture were withdrawn and quenched with equal volumes of ice-cold acetonitrile spiked with 100 nM infigratinib (internal standard). The quenched samples were then centrifuged at 4000g at 4°C for 30 min, following which aliquots of the supernatant were sampled to quantify the amount of ERD remaining using LC/MS/ MS.
2.3. Time-, Concentration-, and NADPH-Dependent Inacti- vation of CYP3A. Enzyme inactivation kinetic assays were performed as previously described using three structurally distinct probe substrate of CYP3A (i.e., testosterone, midazolam, and rivaroXaban).23 Briefly, primary incubation miXtures comprising 20− 40 pmol/mL rhCYP3A4 or rhCYP3A5, ERD (0, 1, 2.5, 5, 15, 25, and 50 μM), G6PDH, and 100 mM potassium phosphate buffer (pH 7.4) were prepared in triplicate. After preincubating at 37 °C for 5 min, the reaction was initiated via the addition of NADP+/G6P. The final primary incubation miXture (100 μL) contained <1% v/v organic solvent. At several preincubation intervals (0, 3, 8, 15, 22, and 30 min), a 5 μL aliquot of each primary incubation miXture was withdrawn and transferred to 95 μL of prewarmed secondary incubation miXture consisting of a CYP3A-specific probe substrate at concentrations more than ∼4× their respective Km (i.e., 200 μM testosterone, 25 μM midazolam, and 50 μM rivaroXaban), an NADPH regenerating system (1 mM) and 100 mM potassium phosphate buffer (pH 7.4). This yielded a 20-fold dilution. The secondary incubation miXtures were further incubated at 37 °C for 10 min (for assays involving testosterone or midazolam) or 2 h (for experiments involving rivaroXaban). Thereafter, an 80 μL aliquot was immediately withdrawn and quenched with equal volumes of ice-cold acetonitrile spiked with either 1 μM prednisolone (internal standard for quantification of 6β-hydroXytestosterone and 1′-hydroXymidazolam) where kinact represents the maximal inactivation rate constant; KI is the concentration of the inactivator at half-maximum inactivation rate constant, and [I] is the in vitro concentration of the inactivator (ERD). Equation 1 assumes that there is negligible change of [I] during the incubation period and that the loss of enzyme activity is purely attributed to inactivation by ERD. The ratio of kinact to KI was determined by dividing the mean values of kinact by KI. Lastly, the time required for half of the enzyme molecules to be inactivated (t1/2) was determined by eq 2. t1/2 = ln2 kinact (2)
2.5. Partition Ratio. RivaroXaban was employed as the probe substrate in the secondary incubation miXture in all subsequent CYP3A MBI assays described in this work. Primary incubation miXtures consisting of 100 pmol/mL rhCYP3A4 or rhCYP3A5, ERD (0, 1, 2.5, 5, 15, 25, and 50 μM), G6PDH, and 100 mM potassium phosphate buffer (pH 7.4) were prepared in triplicate. After prewarming the miXture at 37 °C for 5 min, the reaction was initiated via the addition of NADP+/G6P and incubated for an additional 45 min to allow inactivation to go to completion. The final primary incubation miXture (50 μL) contained <1% v/v organic solvent. Thereafter, aliquots of the primary incubation miXture were withdrawn and transferred to a prewarmed secondary incubation miXture (similar to that prepared for the inactivation experiments) and incubated at 37 °C for another 2 h. Samples were then quenched and assayed for residual enzyme activity as described above. The partition ratio was estimated as outlined in our previous study.23 Briefly, the percentage of residual CYP3A activity was plotted against the molar ratio of ERD to CYP3A4 or CYP3A5 concentration. The turnover number (partition ratio +1) was obtained by extrapolating the intercept of the linear regression line plotted at lower ratios with the straight line plotted at higher ratios to the abscissa. Finally, the partition ratio was back-calculated by subtracting the turnover number by a numerical value of 1.
2.6. Substrate Protection. To investigate if enzyme inactivation could be amendable to substrate protection, an alternative CYP3A substrate testosterone, at a concentration of 100 and 200 μM (corresponding to 1:4 and 1:8 the molar ratio of ERD in this assay), and a potent direct inhibitor of CYP3A ketoconazole, at a concentration of 0.1 and 1 μM (approXimately 1× and 10× its Ki value), were coincubated separately in triplicate to the primary incubation miXture consisting of 40 pmol/mL rhCYP3A4 or 4 μM dexamethasone (internal standard for quantification of hydroXylated rivaroXaban). The quenched samples were centrifuged at 4000g at 4 °C for 30 min to obtain the supernatant for LC/MS/MS analysis. Negative control experiments were performed by substituting NADP+/G6P with 100 mM potassium phosphate buffer (pH 7.4).
2.4. Calculation of MBI Kinetic Parameters (KI and kinact). To derive the inactivation kinetic parameters (KI and kinact), the mean of triplicate peak area ratios was used to calculate the natural logarithm of percentage residual P450 enzyme activity normalized to vehicle, which was then plotted against preincubation time for each concentration of ERD. The resulting data points were fitted to linear regression, and the observed first-order inactivation rate constant (kobs) was derived from the slope of the linear decline in CYP3A activity for each ERD concentration. Specifically, in MBI assays comprising CYP3A4, points from preincubation time points 0 to 15 min were utilized in the derivation of the kobs, whereas in assays involving CYP3A5, points throughout 0 to 30 min were used as the decline in enzymatic activity was determined to be linear even after 30 min of preincubation. Thereafter, a plot of kobs against ERD concentrations [I] allowed the fitting of inactivation kinetic parameters (KI and kinact) to nonlinear least-squares regression based on eq 1 in GraphPad 8.0.2 (San Diego, CA) rhCYP3A5, 25 μM ERD, G6PDH, and 100 mM potassium phosphate buffer (pH 7.4). After prewarming at 37 °C for 5 min, the enzymatic reaction was initiated by the addition of NADP+/G6P. Aliquots were then withdrawn at different preincubation time points (0, 3, 8, and 15 min) and transferred to the secondary incubation miXture and subsequently assayed for residual CYP3A enzymatic activity as described above. Primary incubation miXtures that excluded the addition of either testosterone or ketoconazole or both ERD and testosterone or ketoconazole served as the negative controls.
2.7. Effect of Exogenous Nucleophile and Scavenger of ROS on Inactivation. To interrogate if an exogenous nucleophile or scavenger of ROS could attenuate enzyme inactivation, GSH (2 mM) and catalase (800 U/mL) were incorporated individually into to the primary incubation miXture comprising 40 pmol/mL rhCYP3A4 or rhCYP3A5, 25 μM ERD, G6PDH, and 100 mM potassium phosphate buffer (pH 7.4). After preincubating at 37 °C for 5 min, the enzymatic reaction was initiated via the addition of NADP+/G6P. At specific preincubation time points (0, 3, 8, and 15 min), aliquots were transferred to the secondary incubation miXtures and subsequently assayed for residual CYP3A enzymatic activity as previously described. Negative controls were prepared without both ERD and GSH or catalase or only without GSH or catalase in the primary incubation miXture.
2.8. Reversibility of Inactivation. The reversibility of CYP3A inactivation by ERD was interrogated by two different approaches, namely, equilibrium dialysis and oXidation by potassium ferricyanide, as expounded upon in our previous works.23−25 In the dialysis experiments, triplicate primary incubation miXtures comprising 40 pmol/mL rhCYP3A4 or rhCYP3A5, 25 μM ERD, G6PDH, and 100 mM potassium phosphate buffer (pH 7.4) were prewarmed at 37 °C for 5 min. Enzymatic reaction was initiated by the addition of NADP +/G6P and allowed to proceed for 30 min. After this, a 5 μL aliquot was transferred to 95 μL of the secondary incubation miXture yielding a 20-fold dilution. Concurrently, 90 μL of the remaining primary incubation miXture was transferred to a Slide-A-Lyzer mini dialysis device (0.1 mL, molecular weight cutoff of 10,000; Pierce Chemical Co., Rockford, IL) and placed in a glass beaker filled with 500 mL of ice-cold 100 mM potassium phosphate buffer (pH 7.4). The buffer system was maintained on ice (4 °C) with constant gentle stirring and accompanied by one fresh buffer change at the second hour. After 4 h, 5 μL of the dialyzed miXture was transferred to each prewarmed secondary incubation well. All secondary miXtures were further incubated at 37 °C for 2 h and subsequently assayed for residual CYP3A enzymatic activity as previously described.
On the other hand, in the experiments involving oXidation with potassium ferricyanide, a series of three separate incubations were performed sequentially. Briefly, the primary incubation comprised 40 pmol/mL rhCYP3A4 or rhCYP3A5, G6PDH, and 100 mM potassium phosphate buffer (pH 7.4) in the presence or absence of 25 μM ERD. Following initiation of the reaction with the addition of NADP+/G6P and incubation at 37 °C for either 0 or 30 min, 20 μL of the primary incubation miXture was aliquoted into an equal volume of secondary incubation miXture containing 100 mM potassium phosphate buffer (pH 7.4) with or without 2 mM potassium ferricyanide. The secondary miXtures were then allowed to incubate at 37 °C for another 10 min. Thereafter, 10 μL of the miXture was withdrawn and diluted 10-fold into a tertiary incubation miXture containing 50 μM rivaroXaban (probe substrate), an NADPH regenerating system (1 mM), and 100 mM potassium phosphate buffer (pH 7.4). The reaction miXture was further incubated at 37 °C for another 2 h and subsequently assayed for residual CYP3A activity as previously described. The percentage of CYP3A metabolic activity remaining after 0 or 30 min incubation with ERD compared to the corresponding controls in the absence of ERD was calculated using eqs 3 and 4, respectively.
GSH, and 100 mM potassium phosphate buffer (pH 7.4) were prepared and prewarmed at 37 °C for 5 min. The reaction was then initiated via the addition of NADP+/G6P and incubated at 37 °C for 1 h. Thereafter, an equal volume of ice-cold acetonitrile was added to quench the reaction. The resulting miXture was centrifuged at 14,000g at 4 °C for 15 min. Following this, the supernatant was transferred to a new microcentrifuge tube and dried under a gentle stream of nitrogen gas (TurboVap LV; Caliper Life Science, Hopkinton, MA). The residue was then reconstituted with 60 μL of ACN−water miXture (3:7), vortexed, and centrifuged at 14,000g at 4 °C for 15 min. The supernatant was then carefully removed for LC/MS/MS analysis. Samples which omitted the inclusion of ERD in the incubation miXture served as the negative controls.
2.11. Measurement of ERD and Residual P450 Activity by LC/MS/MS. All samples were analyzed using the liquid chromatog- raphy tandem mass spectrometry (LC/MS/MS) system consisting of an Agilent 1290 Infinity ultrahigh pressure liquid chromatography (Agilent Technologies Inc., Santa Clara, CA) interfaced with AB SCIEX QTRAP 3500 tandem mass spectrometry (MS/MS) (AB SCIEX, Framingham, MA). Chromatographic separation of ERD and infigratinib (internal standard) in the substrate depletion assay was achieved with an ACQUITY UPLC ethylene bridged hybrid (BEH) C18,1.7 μM, 2.2 × 100 mm column (Waters, Milford, MA), whereas the ACQUITY UPLC BEH C18, 1.7 μM, 2.1 × 50 mm column (Waters, Milford, MA) was utilized for the chromatographic separation of the rest of the analytes and internal standards described in this work. The aqueous mobile phase (A) was 0.1% formic acid in water, whereas the organic mobile phase (B) was 0.1% formic acid in acetonitrile. Mobile phases were delivered at a flow rate of 0.5 mL/ min. The column and autosampler temperatures were set to 45 and 4
°C, respectively. The gradient elution conditions were as follows: linear gradient from 20% to 80% B (0−1.20 min), isocratic at 100% B (1.21−2.00 min), and isocratic at 20% B (2.01−2.50 min). All analytes were detected in positive electrospray ionization (ESI) mode. The source-dependent MS parameters were as follows: ion spray voltage = 5500 V; source temperature = 500 °C; curtain gas (CUR) = 25 psi; ion source gas 1 (sheath gas) = 30 psi; ion source gas 2 (drying gas) = 30 psi. The MRM transitions and compound-dependent MS parameters of the analytes are summarized in Table 1. Chromato-
2.9. Spectral Difference Scanning. Incubation miXtures (200 μL) containing 200 pmol/mL rhCYP3A4 or rhCYP3A5, 25 μM ERD, aDP: declustering potential, EP: entrance potential, CE: collision energy, CXP: collision exit potential. G6PDH, and 100 mM potassium phosphate buffer (pH 7.4) were prepared and prewarmed at 37 °C for 5 min. Thereafter, the enzymatic reaction was initiated via the addition of NADP+/G6P and immediately scanned from 400 to 500 nm at 5 min intervals over a 1 h duration using an Hidex Sense microplate reader (Hidex, Turku, Finland) maintained at 37 °C. The spectral differences were obtained by comparing the UV absorbances between the sample and reference wells, which consisted of vehicle in place of ERD. Finally, the extent of metabolite-intermediate (MI) complex formation was also quantita- tively assessed by measuring the absorbance difference between 454 and 490 nm with time. 2.10. GSH Trapping. GSH trapping experiments were performed as described in our previous work.25,26 Incubation miXtures (500 μL) containing 50 pmol/mL rhCYP3A4, 25 μM ERD, G6PDH, 50 mM graphic peak integration was performed using Analyst software version 1.6.2 (Applied Biosystems). For all LC/MS/MS analyses, the peak area of the analyte was expressed as a ratio to the peak area of the internal standard. 2.12. Detection of GSH Adducts. GSH adducts of putative reactive electrophilic intermediate(s) of ERD were analyzed using the LC/MS/MS system consisting of an Agilent 1290 Infinity ultrahigh pressure liquid chromatography (Agilent Technologies Inc., Santa Clara, CA) coupled to an AB SCIEX QTRAP 5500 MS/MS (AB SCIEX, Framingham, MA). Chromatographic separation was performed on an ACQUITY UPLC BEH C18,1.7 μM, 2.2 × 100 mm column (Waters, Milford, MA). The aqueous (A) and organic
Figure 1. Substrate depletion of ERD by CYP3A. Percentage of ERD remaining against time in the presence of (A) CYP3A4 and (B) CYP3A5 plotted on a linear scale and the corresponding substrate depletion graphs of ERD in the presence of (C) CYP3A4 and (D) CYP3A5 as plotted on a seminatural logarithmic scale. Each point in (A to D) represents the mean and SD of triplicate experiments. (B) mobile phases were 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. Mobile phases were delivered at a flow rate of 0.45 mL/min. The column and autosampler temperatures were maintained at 45 and 4 °C, respectively. The gradient elution conditions were as follows: linear gradient 5 to 60% B (0−6.25 min), isocratic at 95% B (6.26−7.00 min), and isocratic at 5% B (7.01−8.00 min). An information-dependent acquisition experiment was conducted to detect ERD-derived GSH conjugates including precursor ion scan (PIS) of m/z 272 in negative ESI mode and neutral loss scan (NL) of 129 Da in positive ESI mode. Enhanced product ion (EPI) scan was subsequently performed for all potential GSH adducts identified. The source-dependent MS parameters utilized were as follows ion spray voltage = 5000 V; source temperature = 650 °C; curtain gas (CUR) = 20 psi; ion source gas 1 (sheath gas) = 45 psi; ion source gas 2 (drying gas) = 60 psi.
Accurate mass measurement of the prospective ERD-derived GSH adduct identified in the GSH trapping experiments was performed using an ACQUITY UPLC system (Waters, Milford, MA) interfaced with an AB SCIEX TripleTOF 5600 quadrupole time-of-flight (QTOF) high-resolution MS (AB SCIEX, Framingham, MA) equipped with a DuoSpray ion source (AB SCIEX, Framingham, MA). Chromatographic separation was carried out on an ACQUITY = 5500 V; source temperature = 500 °C; CUR = 30 psi; ion source gas 1 (sheath gas) = 55 psi; ion source gas 2 (drying gas) = 60 psi. The collision energy was set to 31 V with a collision energy spread of 5 V. The QTOF-MS data were extracted and analyzed using the PeakView software version 2.2 (AB SCIEX, Framingham, MA).
3. RESULTS
3.1. ERD Substrate Depletion by CYP3A4 and CYP3A5. As a cardinal feature of MBI hinges on the prior bioactivation of the substrate to a reactive intermediate, we conducted substrate depletion studies to ascertain if ERD is a substrate of CYP3A. Monitoring the depletion of ERD over time by CYP3A4 and CYP3A5 revealed that both CYP3A isoforms were indeed capable of metabolizing ERD albeit with slight differences in efficiencies. Specifically, the percentage of ERD remaining after 2 h was 36.60 ± 6.47% in CYP3A4 incubations and 51.73 ± 1.24% in CYP3A5 incubations (Figure 1A and B). Remarkably, analysis of the log-transformed substrate depletion profiles revealed that ERD metabolism by CYP3A4 appears to follow a biphasic decline (Figure 1C). Consequently, two different elimination rate slow,CYP3A4 , respectively) were conditions were identical to those employed in the LC/MS/MS experiments described earlier in this section. EXternal mass calibration was performed at the start and after every five samples with the APCI Positive Calibration Solution (AB SCIEX, Framingham, MA) to ensure high mass accuracy and reproducibility. Other source- dependent MS parameters are as follows: ion spray voltage floating calculated for the substrate depletion of ERD by CYP3A4. In contrast, a monophasic decline was observed for CYP3A5 instead (Figure 1D). A comparison of the elimination rate constants in the initial phases of incubation revealed that ERDnwas metabolized ∼4.8 times faster by CYP3A4 as compared to CYP3A5 (i.e., kfast,CYP3A4 = 0.026 ± 0.002 min−1 compared with
Figure 2. Time- and concentration-dependent inactivation of CYP3A4 by ERD using (A) testosterone, (B) midazolam, and (C) rivaroXaban as probe substrates. Nonlinear regression of observed first-order inactivation rate constants (kobs) versus ERD concentration yielded KI and kinact values of 1.14 ± 0.35 μM and 0.039 ± 0.002 min−1 when (D) testosterone was utilized as the probe substrate, 6.35 ± 0.57 μM and 0.150 ± 0.004 min−1 when (E) midazolam was harnessed as the probe substrate, and 4.01 ± 0.42 μM and 0.120 ± 0.003 min−1 when (F) rivaroXaban was employed as probe substrates, respectively. Each point in (A to C) represents the mean and SD of triplicate experiments.
Figure 3. Time- and concentration-dependent inactivation of CYP3A5 by ERD using (A) testosterone, (B) midazolam, and (C) rivaroXaban as probe substrates. Nonlinear regression of observed first-order inactivation rate constants (kobs) versus ERD concentration yielded KI and kinact values of 2.87 ± 0.73 μM and 0.019 ± 0.001 min−1 when (D) testosterone was utilized as the probe substrate, 26.80 ± 2.40 μM and 0.052 ± 0.002 min−1 when (E) midazolam was harnessed as the probe substrate, and 10.04 ± 1.91 μM and 0.045 ± 0.003 min−1 when (F) rivaroXaban was employed as probe substrates, respectively. Each point in (A to C) represents the mean and SD of triplicate experiments. kCYP3A5 = 0.005 ± 0.001 min−1). However, after around 30 min of incubation, its rate of metabolism by CYP3A4 was markedly reduced (i.e., kslow,CYP3A4 = 0.004 ± 0.001 min−1), thereby quantitative characterization of the inactivation kinetics of ERD against CYP3A4 and CYP3A5 using the US FDA- prototypical CYP3A substrates testosterone and midazolam. suggesting possible time-dependent inhibition of the enzyme. We also adopted rivaroxaban as a clinically relevant probe
3.2. Time-, Concentration-, and NADPH-Dependent Inactivation of CYP3A. We then proceeded with a deeper substrate of CYP3A. Our findings demonstrated that ERD inactivated both CYP3A4 and CYP3A5 in a time- and
Figure 4. Cofactor NADPH-dependent inactivation of CYP3A4 and CYP3A5 by ERD using (A and B) testosterone and (C and D) midazolam as probe substrates. Each point in (A to D) represents the mean and SD of triplicate experiments.
Table 2. CYP3A Inactivation Kinetic Parameters for ERD Derived Using Either Testosterone 6β-Hydroxylation, Midazolam 1′-Hydroxylation or Morpholinone Hydroxylation of Rivaroxaban As Surrogate Markers of Residual CYP3A Activitya
P450 isoform probe substrate KI (μM) kinact (min−1) kinact/KI (min−1 mM−1) t1/2 (min) partition ratio
CYP3A4 Testosterone 1.14 ± 0.35 0.039 ± 0.002 34.2 17.8 N.Db
Midazolam 6.35 ± 0.57 0.150 ± 0.004 23.6 4.6 N.D
RivaroXaban 4.01 ± 0.42 0.120 ± 0.003 29.9 5.8 32
CYP3A5 Testosterone 2.87 ± 0.73 0.019 ± 0.001 6.6 36.5 N.D
Midazolam 26.80 ± 2.40 0.052 ± 0.002 1.9 13.3 N.D
RivaroXaban 10.04 ± 1.91 0.045 ± 0.003 4.9 15.4 55
aData are presented as means ± S.D. bN.D: not determined. concentration-dependent manner in all three probe substrates of CYP3A (Figure 2A−C and Figure 3A−C), with the greatest loss of enzyme activity achieved when 50 μM ERD was preincubated with CYP3A for 30 min. The intentional omission of cofactor NADPH from the primary incubation miXtures also nullified the loss of CYP3A-mediated testoster- one 6β-hydroXylase and 1′-midazolam hydroXylase activity when preincubated with 50 μM ERD for up to 30 min (Figure 4A−D). This dependence on NADPH implied that prior metabolic activation of ERD was a key molecular-initiating event leading to its eventual inactivation. Moreover, as the kobs determined from the slopes of each of the concentrations of ERD approached a maximum inactivation rate constant (kinact) (Figure 2D−F and Figure 3D−F), it denoted that the loss of CYP3A activity elicited by ERD exhibited saturable kinetics. At this outset, the inactivation kinetic parameters (KI and kinact) of ERD for CYP3A4 derived from the Kitz-Wilson plot27 were 1.14 ± 0.35 μM and 0.039 ± 0.002 min−1, respectively, when testosterone was utilized as the probe substrate, 6.35 ± 0.57 μM and 0.150 ± 0.004 min−1, respectively, when midazolam was harnessed as the probe substrate, and 4.01 ± 0.42 μM and 0.120 ± 0.003 min−1 when rivaroXaban was employed as the probe substrate. This in turn yielded respective kinact/KI ratios of 34.2, 23.6, and 29.9 min−1 mM−1 and inactivation t1/2 of 17.8, 4.6, and 5.8 min; whereas the corresponding inactivation kinetic parameters (KI and kinact) of ERD for CYP3A5 were 2.87 ± 0.73 μM and 0.019 ± 0.001 min−1, respectively, when testosterone was utilized as the probe substrate, 26.80 ± 2.40 μM and 0.052 ± 0.002 min−1, respectively, when midazolam was harnessed as the probe substrate, and 10.04 ± 1.91 μM and 0.045 ± 0.003 min−1 when rivaroXaban was employed as the probe substrate. This in turn corresponded to kinact/KI ratios of 6.6, 1.9, and 4.9 and inactivation t1/2 of 36.5, 13.3, andn15.4 min, respectively. Taken together, our results obtained
Figure 5. Partition ratio for the inactivation of (A) CYP3A4 and (B) CYP3A5 by ERD determined by extrapolating the intercept of the linear regression line at lower ratios and the straight line for the high ratios to the x-axis was estimated to be 32 and 55, respectively. Each point in (A and B) represents the mean and SD of triplicate experiments.
Figure 6. Inactivation of CYP3A4 and CYP3A5 by ERD was attenuated in the presence of (A and D) an alternative CYP3A substrate testosterone (TES) and (B and E) a direct CYP3A inhibitor ketoconazole (KTC). (C and F) Conversely, the presence of either GSH or catalase did not protect against enzymatic inactivation. Each point in A−F represents the mean and SD of triplicate experiments. with three different probe substrates of CYP3Aconsistently demonstrated that ERD was a more potent MBI of CYP3A4 as compared to CYP3A5. All reported values of KI, kinact, kinact/KI ratio, and t1/2 are summarized in Table 2.
3.3. Partition Ratio. A titration method previously described28 was adopted, which determined the turnover number for the inactivation of CYP3A4 to be ∼33 (Figure 5A) and CYP3A5 to be ∼56 (Figure 5B). This in turn corresponded to an estimated partition ratio of 32 and 55 for CYP3A4 and CYP3A5, respectively (Table 2).
3.4. Substrate Protection. Inactivation of CYP3A by ERD was protected in the presence of both an alternative substrate and a direct inhibitor of CYP3A. Coincubation with either testosterone (Figure 6A and D) or ketoconazole (Figure 6B and E) attenuated the rate of inactivation of CYP3A by ERD as evident by the diminished rate of enzyme inactivation with time. Furthermore, the extent of substrate protection conferred appeared to be dose-dependentwith inactivation being completely abolished when 1 μM ketoconazole was coincubated with ERD and CYP3A in the primary incubation miXture.
3.5. Effect of Exogenous Nucleophile and Scavenger of ROS on Inactivation. The presence of an exogenous nucleophile or a scavenger of ROS had no appreciable protective effect on the rate of enzyme inactivation elicited by ERD. As illustrated in Figure 6C and F, CYP3A4 and CYP3A5 were inactivated to a similar extent in incubation miXtures comprising ERD with GSH or catalase compared to ERD alone.
3.6. Reversibility of Inactivation. To establish whether the inactivation of CYP3A by ERD is quasi-irreversible or irreversible, the nature of inactivation was interrogated via two distinct approaches dialysis and oXidation with potassium ferricyanide. Our findings revealed that the magnitude of both CYP3A4 and CYP3A5 activities was not restored after dialysis (Figure 7A and B). Rather, the marginal decrease in residual enzyme activity observed post-dialysis could be ascribed to enzymatic degradation that might have occurred during dialysis as confirmed in vehicle control experiments. On the other
Figure 7. Percentage activity of (A) CYP3A4 and (B) CYP3A5 remaining did not increase after extensive dialysis at 4 °C for 4 h. Potassium ferricyanide (KFC) only restored the metabolic activity of (C) CYP3A4 by 11.60 ± 1.83% and (D) CYP3A5 by 8.43 ± 1.86% after a 30 min incubation with 25 μM ERD. Results from all four graphs show the mean and SD of two independent experiments conducted in triplicate. hand, oXidation with potassium ferricyanide after a 30 min pre- incubation with 25 μM ERD only restored CYP3A activity marginally. Specifically, CYP3A4 and CYP3A5 activities were restored by 11.60 ± 1.83% (Figure 7C) and 8.43 ± 1.86% (Figure 7D), respectively.
3.7. Spectral Difference Scanning. There was a lack of an observable Soret peak in the region of 448−458 nm associated with the formation of quasi-irreversible MI complexes when incubation miXtures containing ERD and CYP3A4 or CYP3A5 were scanned from 400 to 500 nm at 5 min intervals for 1 h (Figure 8A and B). Moreover, tracking the increase in absorbance between 454 nm and the isosbestic point at 490 nm further substantiated the lack of MI complex formation with ERD (Figure 8C and D).
3.8. GSH Trapping. As our previous experiments offered corroboratory evidence that the inactivation of CYP3A arose due to the formation of irreversible covalent adducts as opposed to quasi-irreversible MI complexes, a GSH trapping assay was subsequently conducted to confirm the generation of electrophilic reactive intermediates of ERD. One peak that was suggestive of an ERD-derived GSH adduct with an MH+ ion at m/z of 752 (retention time: 3.00 min) was detected in both the PIS at m/z 272 in ESI negative mode and NL of 129 Da in positive ESI mode when ERD and CYP3A4 were enriched with GSH but was absent in negative control samples lacking ERD (data not shown). The resultant EPI scan performed at m/z 752 yielded a spectrum that was characteristic of collision- induced dissociation fragmentation of a GSH conjugate due to the neutral mass loss of 129 Da corresponding to cleavage of the pyroglutamic acid moiety in GSH. To further confirm the identity of the potential GSH adduct, accurate mass measure- ments were performed. As expected, we were able to recapitulate the nominal mass patterns generated using the QTRAP-MS when the GSH adduct was subjected to accurate mass measurements using the QTOF-MS. The proposed elemental composition, theoretical and experimental exact m/ z, and mass accuracy (in both Da and ppm) of the GSH adduct are summarized in Table 3. Additionally, the accurate mass MS/MS spectrum and proposed fragmentation of the ERD- derived GSH adduct are shown in Figure 9.
4. DISCUSSION
ERD is the first pan-FGFR inhibitor that has garnered global regulatory approval for the treatment of advanced or metastatic urothelial carcinoma. While early metabolic studies have revealed that it elicits time-dependent inhibition of CYP3A4, the specific biochemical nature underpinning this observation remains uninterrogated. Moreover, it is also unclear if CYP3A5, which possesses considerable sequence homology with CYP3A4, could be susceptible to such interactions. Here, our findings confirmed the time-, concentration-, and NADPH-dependent inhibition of both CYP3A isoforms by ERD and suggested irreversible MBI of these enzymes.
The phenomenon of probe substrate-dependent modulation of P450 catalytic activity by xenobiotics is well described in the literature.29−31 These atypical interactions complicate safety evaluation of clinical drug combinations, as the biochemical behavior observed with one probe substrate may not accurately
Figure 8. Spectral difference measured over 60 min failed to elicit a Soret peak in the absorbance ranges of 448−458 nm for (A) CYP3A4 and (B) CYP3A5 incubated with 25 μM ERD. Similarly, a comparison of the absorbance at the reference of 454 nm against the isosbestic point at 490 nm failed to demonstrate an increase in the extent of MI complex formation over time in (C) CYP3A4 and (D) CYP3A5 incubation miXtures.
Table 3. Accurate Mass Measurement of the Parent and Product Ions of the ERD-Derived GSH Adduct (m/z 752) Using a Mass Tolerance of 5 ppm
increasingly being coadministered in urothelial carcinoma patients for prophylaxis and treatment of cancer-associated venous thromboembolism, its adoption as a probe substrate here bears clinical relevance.36 Although our findings revealed a lack of site-specific effects on inactivation by ERD for both CYP3A isoforms, there were marked differences in the inactivation kinetics yielded for each isoform depending on reflect those obtained with another.32 Although the exact molecular mechanisms underpinning these observations remain nebulous, it has been reported more frequently in CYP3A isoforms which are known to possess larger active sites with a greater degree of conformational flexibility.33 In that regard, docking studies and molecular dynamic simulations of CYP3A have since demonstrated that these cavities can simultaneously accommodate multiple substrates with different binding modes and affinities.34,35 Consequently, it is crucial to employ multiple structurally distinct substrates of CYP3A to investigate if there are any site-specific effects in enzymatic inactivation. Here, in addition to both FDA-recommended substrates (i.e., testosterone and midazolam), we also included rivaroXaban as a probe substrate in our assays. As rivaroXaban is midazolam for both CYP3A4 and CYP3A5. These salient differences in inactivation potencies reiterate the importance of using a clinically relevant probe substrate to derive accurate inhibition/inactivation kinetic constants which are paramount in pharmacokinetic DDI studies.
A further comparison of the kinact/KI ratio yielded with all three probe substrates consistently demonstrated that ERD elicits more potent inactivation of CYP3A4 as compared to CYP3A5. This is further substantiated by our substrate depletion experiments involving CYP3A4 in which ERD concentration was found to decline in a biphasic manner and is suggestive of possible time-dependent inhibition of the enzyme. In contrast, we did not manage to elucidate such a trend in our parallel substrate depletion experiments comprising CYP3A5, thereby indicating that at the common concentration of 1 μM, ERD only elicits appreciable time- dependent inhibition in CYP3A4 but not CYP3A5. Our observations here are corroborated by other reports in which
Figure 9. Proposed accurate mass fragmentation pattern of the ERD-derived GSH adduct. The MS/MS spectrum depicts the experimental m/z values, whereas the chemical structure illustrates the theoretical m/z values of the parent and product ions of the adduct as outlined in Table 3 using a mass tolerance of 5 ppm.
the potency of MBI elicited by a common inactivator in CYP3A4 and CYP3A5 can differ substantially despite the close similarity between both isoforms.37,38 At this outset, due to the overlapping substrate specificities of both CYP3A isoforms, it is plausible that the metabolism of ERD and other concomitant CYP3A substrates may be preserved due to the less potent inactivation of CYP3A5. However, as the expression levels of CYP3A5 are highly polymorphic among different ethnic groups, deeper mechanistic studies are warranted to better understand the pharmacogenomic and clinical implications of our findings and how they may contribute to inter-individual variability in ERD interactions.
The partition ratio, which is defined as the number of inactivator molecules metabolized for every molecule of enzyme inactivated, is commonly employed as an indicator of the overall efficiency of MBI. Partition ratios ranging from near-zero to several thousands have been reported,39 with highly efficient inactivators possessing ratios <50.40 As such, our data demonstrated that ERD is a relatively efficient MBI of CYP3A and further suggested that the inactivation of CYP3A4 is likely more efficient than that of CYP3A5 due to the lower partition ratio obtained (Table 2). Additionally, our findings also revealed that the rate of inactivation evoked by ERD could be attenuated in the presence of an alternative substrate or direct inhibitor of CYP3A (i.e., testosterone and ketoconazole, respectively), thereby implying that enzymatic inactivation by ERD occurred within its active site. This was starkly contrasted by the lack of protection conferred by GSH or catalase which confirmed that the putative reactive metabolite(s) of ERD inactivates the enzyme before it is released from the active site and dismisses the role of ROS in enzyme inactivation.
While our experimental findings to date collectively substantiated the MBI of CYP3A by ERD, the exact nature of inactivation still remains obfuscated. Enzymatic inactivation is known to occur via two distinct modes namely, via the formation of MI complexes or through covalent modification of the heme moiety and/or apoprotein.41 MI complexes, which are derived from the coordination of the reactive intermediate to the catalytic heme ferrous, are pseudoirreversible and can be dissociated in vitro by dialysis or with a strong oXidizing agent (e.g., potassium ferricyanide), thereby reviving enzymatic activity. Conversely, the formation of covalent adducts to the apoprotein and/or porphyrin ring of the prosthetic heme moiety is irreversible and cannot be mitigated by both of the aforementioned experimental approaches. Furthermore, de- spite the close homology between both CYP3A isoforms, diverging inactivation modalities have been reported with a common inactivator. For instance, our group has previously demonstrated that the dual kinase inhibitor lapatinib exhibits a dichotomous inactivation pattern in CYP3A4 and CYP3A5,26,42 wherein it exhibited MI complex formation and covalent binding to the apoprotein in an isoform-selective manner.16 Consequently, it is important to delineate the nature of inactivation of both CYP3A isoforms by ERD independ- ently. Our findings demonstrated that the loss of CYP3A activity is irrevocable and could not be recovered after dialysis, whereas the addition of potassium ferricyanide only restored CYP3A4 and CYP3A5 activity by 11.6% and 8.43%, respectively, which, when interpreted under the context of a published criterion by Watanabe et al.,43 further substantiates the absence of MI complex formation, since enzymatic activity was not restored by more than 20%. Finally, the lack of the characteristic Soret peak signature in our spectral analyses further reinforced our postulations that ERD did not form any MI complex with CYP3A.44 Taken together, our findings suggest that ERD inactivates both CYP3A isoforms irreversibly via covalent modification.
To elucidate electrophilic reactive intermediate of ERD implicated in MBI, reaction miXtures were fortified with the nucleophilic trapping agent GSH. We employed a de novo screening method comprising two survey scans namely PIS at m/z 272 in negative mode and NL at 129 Da in positive mode,45,46 which triggered the subsequent acquisition of the EPI spectrum of a single prospective ERD-derived GSH adduct (m/z 752). These two well-established survey scans, which monitor the loss of the deprotonated γ-glutamyl-dehydroa- lanyl-glycine and pyroglutamic acid moiety from GSH, respectively, are frequently harnessed during drug discovery to identify drug candidates that pose potential metabolic liabilities. Successful recapitulation of the nominal mass EPI spectrum in our high-resolution QTOF-MS (Figure 9) coupled together with mass accuracies well below the mass tolerance threshold of 5 ppm (Table 3) gave us confidence in the predicted elemental compositions of the parent and product ions for structural elucidation of the ERD-derived GSH adduct. Notably, as the fragment ion at m/z 447.2496 as well as 388.1751 and 362.1608 corresponded to the parent and product ions of ERD,47 it indicated that the glutathionyl moiety was alkylated directly to the ERD core structure. Consequently, we proposed that the bioactivation of ERD resulting in MBI of CYP3A might involve epoXidation of its quinoXaline ring (Scheme 1). Thereafter, nucleophilic attack of the reactive epoXide intermediate (2) by GSH produces a glutathionyl conjugate (3), which can further undergo dehydration to the detected ERD-derived GSH adduct (m/z 752) (4). At this outset, epoXidation of drugs by P450 is commonly regarded as the rate-determining step in the overall metabolic pathway leading to enzymatic inactivation.48 Notably, a similar mechanism of CYP3A-mediated bioactiva- tion to reactive epoXide intermediates has been reported by Wang et al.49 for the uricosuric agent benzbromarone, which was later posited to be implicated in its MBI of CYP3A4.23 Therefore, it is plausible that the epoXide intermediate of ERD could also covalently adduct to nucleophilic residues within the apoprotein and/or heme porphyrin ring of CYP3A4 and elicit MBI. However, further studies will be necessary to definitively elucidate the exact mechanism of covalent modification by ERD against CYP3A.
In conclusion, our findings demonstrated that ERD fulfills all of the established criteria as an archetypal MBI of CYP3A4 and CYP3A5. Moreover, we also determined the nature of inactivation to be irreversible and proposed the identity of the putative reactive intermediate implicated in enzyme inactivation. Future physiologically based pharmacokinetic modeling DDI studies will help ascertain the clinical significance of CYP3A inactivation by ERD.
■ AUTHOR INFORMATION
Corresponding Author
Eric Chun Yong Chan − Department of Pharmacy, Faculty of Science, National University of Singapore, 169856, Singapore; orcid.org/0000-0001-6107-9072;
Phone: +65-6516 6137; Email: [email protected];
Fax: +65-6779 1554
Authors
Lloyd Wei Tat Tang − Department of Pharmacy, Faculty of Science, National University of Singapore, 169856, Singapore; orcid.org/0000-0001-7113-4397
Jian Wei Teng − Department of Pharmacy, Faculty of Science, National University of Singapore, 169856, Singapore
Siew Kwan Koh − Singapore Eye Research Institute (SERI), Singapore
Lei Zhou − Singapore Eye Research Institute (SERI), Singapore; Department of Ophthalmology, Yong Loo Lin School of Medicine, National University of Singapore, 117597, Singapore; Ophthalmology and Visual Sciences
Academia Clinical Program, Duke-National University of Singapore Medical School, 169857, Singapore
Mei Lin Go − Department of Pharmacy, Faculty of Science, National University of Singapore, 169856, Singapore
Complete contact information is available at: https://pubs.acs.org/10.1021/acs.chemrestoX.1c00178
Author Contributions
Participated in research design: Tang, Chan. Conducted experiments: Tang, Teng, Koh. Contributed new reagents or analytical tools: Zhou. Performed data analysis: Tang, Teng, Go, Chan. Wrote or contributed to the writing of the manuscript: Tang, Teng, Chan.
Funding
This work was supported by the Agency for Science, Technology and Research (A*STAR) Industry Alignment Fund − Pre-Positioning (IAF-PP) [Grant H18/01/a0/C14] and the Joseph Lim Boon Tiong Urology Cancer Research Initiative [Grant: R-148-000-302-720] to E.C.Y.C. L.W.T.T is supported by the National University of Singapore (NUS) President’s Graduate Fellowship (PGF).
Notes
The authors declare no competing financial interest.
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