NADPH-Independent Inactivation of CYP2B6 and NADPH-Dependent Inactivation of CYP3A4/5 by PBD: Potential Implication for Assessing Covalent Modulators for Time-Dependent Inhibition

Abstract

Pyrrolo [2,1-c][1,4]benzodiazepine dimer (PBD) has shown broad antitumor properties and potential as a therapeutic agent for cancers. During a routine drug-drug interaction assessment, it was found that PBD is a reversible inhibitor of CYP2C8 (IC 50 = 1.1 mM) but not CYP1A2, 2B6, 2C9, 2C19, 2D6, or 3A4/5. Additionally, PBD is a classic time-dependent inhibition (TDI) of CYP3A4/5, with >30-fold shift in IC 50 after a preincubation with NADPH. All other CYPs tested did not show evidence for TDI, but potent inhibition of CYP2B6 (IC 50 = 1.5 mM) was observed after a preincubation with or without (w/wo) NADPH, which was an unexpected observation given the fact that no inhibition was observed in the direct inhibition assay. No other CYP isoforms were susceptible to this apparent non-NADPH-dependent inhibition, suggesting that PBD may selectively inactivate CYP2B6 without metabolic activation. The washing of the human liver microsome pellet after incubation with PBD did not fully recover CYP2B6 activity, indicating that PBD is covalently bound to CYP2B6, leading to inactivation of the enzyme. To further investigate the mechanism of NADPH-independent inhibition, the IC 50 shift was determined for several PBD analogs, and it was found that the compounds without both reactive imines did not show NADPHindependent inhibition of CYP2B6, implying that NADPHindependent inactivation was likely caused by direct covalent binding of PBD to the enzyme in a highly structure-specific manner. These data clearly highlight the need to assess direct and time-dependent inhibition w/wo NADPH to adequately characterize the in vitro CYP inhibitory properties of drug candidates with reactive moieties. SIGNIFICANCE STATEMENTWe described a very unique in vitro CYP inhibition profile of pyrrolo [2,1-c][1,4]benzodiazepine dimer as a potent reversible CYP2C8 inhibitor, an NADPH-dependent CYP3A4/5 time-dependent inhibition (TDI) inhibitor, and an NADPH-independent CYP2B6 TDI inhibitor, and inhibition of CYPs occurs through three distinct mechanisms: reversible drug-enzyme binding, enzyme inactivation via bioactivation, and enzyme inactivation by covalent binding via chemical reactions. Our results suggest that, for compounds with reactive functional moieties, false positives can be reported when the conventional TDI assay is utilized.

Risk‐Based Pharmacokinetic and Drug–Drug Interaction Characterization of Antibody–Drug Conjugates in Oncology Clinical Development: An International Consortium for Innovation and Quality in Pharmaceutical Development Perspective

“…In case of pyrrolobenzodiazepine derivatives, the ADC database showed that one out of three pyrrolobenzodiazepine derivatives is not an inhibitor or inducer of CYP3A and P‐gp while the other two are not reported. A recent publication showed that in vitro CYP inhibitory profiles depend on the structure of pyrrolobenzodiazepine derivatives 29 . Additionally, the ADC database showed that the unconjugated pyrrolobenzodiazepine was not detectable in the systemic circulation for two out of three pyrrolobenzodiazepine‐containing ADCs, while for the third ADC, it was not reported.…”

“…A recent publication showed that in vitro CYP inhibitory profiles depend on the structure of pyrrolobenzodiazepine derivatives. 29 Additionally, the ADC database showed that the unconjugated pyrrolobenzodiazepine was not detectable in the systemic circulation for two out of three pyrrolobenzodiazepine-containing ADCs, while for the third ADC, it was not reported. Consistently, phase I studies with three pyrrolobenzodiazepine-containing ADCs, namely rovalpituzumab tesirine, vadastuximab talirine, and SGN-CD70A, showed that unconjugated pyrrolobenzodiazepine was generally not detectable in the systemic circulation at tested clinical doses, even though the unconjugated pyrrolobenzodiazepine assays were sensitive in the range of 10-60 pg/mL.…”

Inhibitory effects of Triphala on CYP isoforms in vitro and its pharmacokinetic interactions with phenacetin and midazolam in rats

“…Briefly, a portion of HLMs in the final protein concentration of 0.28 mg/mL in 100 mM potassium phosphate buffer (pH 7.4) was pre-incubated with Triphala extract (12.5–125 μg/mL), gallic acid, ellagic acid or one of the CYP 1A2, 2C9, 2D6 and 3A4 inhibitors (as positive control), i.e., α-naphthoflavone (0.025–0.1 μM), sulfaphenazole (0.01–1 μM), quinidine (0.5–2 μM) and ketoconazole (0.002–0.2 μM), at 37 °C for 5 min. Then, the reaction was initiated by the addition of an NADPH (1.3 mM) in 100 mM potassium phosphate buffer pH 7.4 and each probe substrate for CYP isoform ( Kosaka et al., 2020 ). The mixture was further incubated at 37 °C for various intervals (30, 10, 20 and 15 min for the determination of CYP 1A2, 2C9, 2D6 and 3A4, respectively) and terminated by adding ice-cooled acetonitrile.…”

Inhibition of drug-metabolizing enzymes by Jingyin granules: implications of herb–drug interactions in antiviral therapy

“…CYP2C19 and CYP3A inactivation kinetic experiments were performed as previously reported [ 34 – 36 ], with an inactivation group and an activity evaluation group. The final concentration of the organic solvent was <1% (v/v).…”