Elsevier

Toxicology Letters

Volume 225, Issue 1, 10 February 2014, Pages 139-146
Toxicology Letters

Dimethocaine, a synthetic cocaine derivative: Studies on its in vitro metabolism catalyzed by P450s and NAT2

https://doi.org/10.1016/j.toxlet.2013.11.033Get rights and content

Highlights

  • Elucidation of human NATs and P450s involved in DMC major metabolic steps.

  • Kinetic profiles of NAT and P450 catalyzed reactions were presented.

  • Net clearance data for involved P450 were presented.

  • Results compared to chemical inhibition in human liver microsomes.

  • Clinically relevant interaction with single P450 inhibitors should not be expected.

Abstract

Dimethocaine (DMC), a synthetic derivative of cocaine, is distributed and consumed as “new psychoactive substance” (NPS) without any safety testing at the forefront. It is mainly metabolized by N-acetylation, N-deethylation or hydroxylation. Therefore, the aim of the presented study was to determine the human NAT and P450 isozymes involved in this major metabolic steps, to measure the kinetics of the reactions, and to estimate the contribution on in vivo hepatic clearance. For these studies, cDNA-expressed NATs and P450s were used and formation of metabolites after incubation was measured using LC–MS or LC–MSn. For N-acetylation, NAT2 could be shown to be the only isoform catalyzing the reaction in vitro hence assuming to be the only relevant enzyme for in vivo acetylation. Kinetic profiles of all P450 catalyzed metabolite formations followed classic Michaelis–Menten behavior with enzyme affinities (Km values) between 3.6 and 220 μM. Using the relative activity factor approach, the net clearances for deethylation of DMC were calculated to be 3% for P450 1A2, 1% for 2C19, <1% for 2D6, and 96% for 3A4. The net clearances for hydroxylation of DMC were calculated to be 32% for P450 1A2, 5% for 2C19, 51% for 2D6, and 12% for 3A4. Furthermore, these data were confirmed by chemical inhibition tests in human liver microsomes. As DMC is metabolized via two main steps and different P450 isoforms were involved in the hepatic clearance of DMC, a clinically relevant interaction with single P450 inhibitors should not be expected. However, a slow acetylation phenotype or inhibition of NAT2 could lead to decreased N-acetylation and hence leading to an increased risk of side effects caused by this arylamine.

Introduction

Dimethocaine (DMC, larocaine, 3-diethylamino-2,2-dimethylpropyl)-4-aminobenzoate) was marketed as local anesthetic in the 1930s and used in dentistry and ophthalmology. Besides local anesthetic effects, DMC has also effects on the central nervous system acting as dopamine-reuptake-inhibitor (Graham et al., 1995, Wilcox et al., 2000, Wilcox et al., 2005, Woodward et al., 1995). It is listed by the EMCDDA under the category “synthetic cocaine derivatives” and offered in numerous online shops (European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), 2013). The abuse is described to produce feelings of euphoria and sometimes relaxed feeling accompanied with side effect such as a strong hangover and fatigue. The in vivo pharmacological properties of DMC were compared to cocaine after intraperitoneal injection into rats (Graham et al., 1995). DMC was shown to have high affinity for the DA transporter mainly in the nucleus accumbens stimulating the reward system. Furthermore, Woodward et al. (1995) have shown that DMC is nearly as potent as cocaine concerning the DA-reuptake-inhibitor efficiency.

So far, nothing is known about its toxicokinetics such as the metabolic fate and involved cytochrome P450 (P450) isozymes. However, studies on the toxicokinetics of “new psychoactive substances” (NPSs) play a major role in clinical and forensic toxicology for developing new drug testing strategies and for assessing toxic risks e.g. based on drug–drug or drug–food interactions. Therefore, the authors investigated in previous studies the affinity of DMC to human ABC transporter P-glycoproteine (P-gp) (Meyer et al., 2013c) and the metabolic pathway of DMC in rat (Meyer et al., 2013b). Main observed metabolic steps were the N-acetylation, hydroxylation, and N-deethylation. Therefore, the aim of the presented study was to elucidate in vivo contribution of human N-acetyltransferases (NATs) and cytochrome P450s (P450 s) to the hepatic metabolism of DMC using the relative activity factor (RAF) approach (Venkatakrishnan et al., 2001). These data should be confirmed by selective P450 inhibition tests in pooled human liver microsomes (HLM).

Section snippets

Chemicals and reagents

DMC was obtained from LGC (Teddington, UK), NADP+ from Biomol (Hamburg, Germany), and isocitrate, isocitrate dehydrogenase, carnitin-acetyl-transferase (from pigeon breast muscle), and acetyl-d,l-carnitine from Sigma–Aldrich (Taufkirchen, Germany). All other chemicals and reagents were from VWR (Darmstadt, Germany) and were of analytical grade. The following microsomes were from BD Biosciences (Heidelberg, Germany): baculovirus-infected insect cell microsomes (Supersomes), containing 1 nmol/mL

Initial P450 activity screening and P450 kinetic studies

The initial activity screening studies with the ten most abundant human hepatic P450s and HLM were performed to identify their ability for catalyzing the formation of the initial metabolites in vitro. According to the supplier's advice, the initial incubation conditions chosen were adequate to make a statement on the general involvement of a particular P450 enzyme. The two main metabolic steps observed were the N-deethylation and the hydroxylation at the p-aminobenzoic acid part. The chemical

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgements

The authors would like to thank Jessica Welter, Carina S.D. Wink, Julia Dinger, Golo M.J. Meyer, Carsten Schröder, Armin Weber, and Gabriele Ulrich for their support.

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