Numerical Analysis of Time-Dependent Inhibition by MDMA

Abstract

Methylenedioxymethamphetamine (MDMA) is a known drug of abuse and schedule 1 narcotic under the Controlled Substances Act. Previous pharmacokinetic work on MDMA used classic linearization techniques to conclude irreversible mechanism-based inhibition of CYP2D6. The current work challenges this outcome by assessing the possibility of two alternative reversible kinetic inhibition mechanisms known as the quasi-irreversible (QI) model and equilibrium model (EM). In addition, progress curve experiments were used to investigate the residual metabolism of MDMA by liver microsomes and CYP2D6 baculosomes over incubation periods up to 30 minutes. These experiments revealed activity in a terminal linear phase at the fractional rates with respect to initial turnover of 0.0354 6 0.0089 in human liver microsomes and 0.0114 6 0.0025 in baculosomes. Numerical model fits to percentage of remaining activity (PRA) data were consistent with progress curve modeling results, wherein an irreversible inhibition pathway was found unnecessary for good fit scoring. Both QI and EM kinetic mechanisms fit the PRA data well, although in CYP2D6 baculosomes the inclusion of an irreversible inactivation pathway did not allow for convergence to a reasonable fit. The kinetic complexity accessible to numerical modeling has been used to determine that MDMA is not an irreversible inactivator of CYP2D6, and instead follows what can be generally referred to as slowly reversible inhibition. SIGNIFICANCE STATEMENTThe work herein describes the usage of computational models to delineate between irreversible and slowly reversible time-dependent inhibition. Such models are used in the paper to analyze MDMA and classify it as a reversible time-dependent inhibitor.

3D Kinetic Analysis: A Rapid, Model Independent, Method for Enzyme Kinetics

“…(2019), featuring a reversible ES-complex inactivation pathway as well as an irreversible death pathway for unbound protein. Upon tting this kinetic scheme to the 3D plot, an inhibition rate of k 4 = 0.44± 0.05 min -1 was obtained which was consistent with earlier reports of k inact =0.309±0.008 min -1 for this interaction 29 . Here, we have demonstrated the inaccuracy of the steady state assumption for enzymes with nonlinear kinetics such as AO using ES simulations.…”

“…A kinetic scheme was constructed in congruence with that reported by Rodgers et al(2019), featuring a reversible ES-complex inactivation pathway as well as an irreversible death pathway for unbound protein. Upon tting this kinetic scheme to the 3D plot, an inhibition rate of k 4 = 0.44± 0.05 min -1 was obtained which was consistent with earlier reports of k inact =0.309±0.008 min -1 for this interaction29 .…”

“…The quantitation was conducted on an API 4000 Q-Trap MS system (Applied Biosystems/MDS Sciex, Foster City, CA) with turbo spray electrospray ionization operating in positive ion mode. The program gradient timing of the mobile phases and the MS tuning parameters were similar to a method previously published [29][30][31] were among the best performing models used for different case studies (Fig. 2).…”

“…Therefore, the reversible TDI model in this study (Fig. 8) has been adopted from the previously developed numerical model for MDMA and 2D6 interaction 29 with minor adjustments to remove the unnecessary use of a probe substrate for 3D Kinetics analysis (RevTDI). In this model, enzyme inactivation rate is represented by k 4 = 0.44± 0.05 which is consistent with what was previously reported as k inact = 0.309±0.008 min -1 using a probe substrate and the traditional replot method 29 (Table 2).…”

“…The quantitation was conducted on an API 4000 Q-Trap MS system (Applied Biosystems/MDS Sciex, Foster City, CA) with turbo spray electrospray ionization operating in positive ion mode. The program gradient timing of the mobile phases and the MS tuning parameters were similar to a method previously published [29][30][31] only using different Q1 and Q3 for various substrates. The ow rate was 0.25 mL/min and the metabolites and internal standard were detected using multiple reaction monitoring mode following these m/z transitions: DACA-acridone, 310.2 Menten equation for each time point, ending up with a total of 8 saturation curves with each providing k cat and K m values.…”