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Dimethocaine, a synthetic cocaine derivative: Studies on its in vitro metabolism catalyzed by P450s and NAT2
Toxicology Letters 225 (2014) 139–146
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Dimethocaine, a synthetic cocaine derivative: Studies on its in vitro metabolism catalyzed by P450s and NAT2 Markus R. Meyer ∗ , Carina Lindauer, Hans H. Maurer Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, Homburg, Saar, Germany
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• 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.
a r t i c l e
i n f o
Article history: Received 18 October 2013 Received in revised form 23 November 2013 Accepted 26 November 2013 Available online 3 December 2013 Keywords: Designer drug DMC Cocaine Metabolism P450 NAT
a b s t r a c t 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 M. 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. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Dimethocaine (DMC, larocaine, 3-diethylamino-2,2-dimethylpropyl)-4-aminobenzoate) was marketed as local anesthetic in
∗ Corresponding author. Tel.: +49 6841 16 26430; fax: +49 6841 16 26051. E-mail address: [email protected] (M.R. Meyer). 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.11.033
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, 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
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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). 2. Materials and methods 2.1. 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 of human cDNA-expressed P450 1A2, P450 2A6, P450 2B6, P450 2C8, P450 2C9, P450 2C19, P450 2D6, P450 2E1 (2 nmol/mL), P450 3A4, or P450 3A5 (2 nmol/mL) and pooled human liver microsomes (pHLM, 20 mg microsomal protein/mL, 400 pmol total P450/mg protein). After delivery, the microsomes were thawed at 37 ◦ C, aliquoted, snap-frozen in liquid nitrogen, and stored at −80 ◦ C until use. Baculovirus-insect cell-expressed NAT1 (human arylamine N-acetyltransferase 1*4, wild-type allele) and NAT2 (human arylamine N-acetyltransferase 2*4, wild-type allele) as well as control cell cytosol were from BD Biosciences (Heidelberg, Germany). After delivery, the cytosols were thawed at 37 ◦ C, aliquoted, snap-frozen in liquid nitrogen, and stored at −80 ◦ C until use.
microsomes and stopped with 50 L of an ice-cold mixture of acetonitrile with 0.1% formic acid, containing the internal standard (diphenhydramine, 5 M). The solution was centrifuged for 2 min at 14,000 × g, 50 L of the supernatant was transferred to an autosampler vial, and injected onto the LC apparatus for analysis. For initial screening studies, incubations were performed at final concentration of 25 M DMC and 50 pmol/mL of P450 1A2, P450 2A6, P450 2B6, P450 2C8, P450 2C9, P450 2C19, P450 2D6, P450 2E1, P450 3A4, or P450 3A5 for 30 min. For kinetic studies, incubations were performed for 5 min with 32 pmol/mL protein concentration of P450 1A2, P450 2C19, P450 2D6, P450 3A4. The described incubation conditions (incubation time and protein concentration) were chosen to be within a linear range of metabolite formation. DMC was incubated using substrate concentrations ranging from 0.1 to 2000 M. Enzyme kinetic constants were estimated by non-linear curvefitting using GraphPad Prism 5.00 software (San Diego, CA). The Michaelis–Menten equation (Eq. (1)) was used to calculate apparent Km values and Vmax values for single-enzyme systems. V=
Vmax × [S] Km + [S]
(1)
The relative activity factor (RAF) approach was used to account for differences in functional levels of redox partners between the two enzyme sources (Crespi and Miller, 1999; Venkatakrishnan et al., 2000). The turnover rates (TR) of P450 1A2 (probe substrate (PS) phenacetin), P450 2C19 (PS (S)-mephenytoin), P450 2D6 (PS bufuralol) and P450 3A4 (PS testosterone) in insect cell microsomes (ICM) and human liver microsomes (HLM) were taken from the supplier’s data sheets. The RAFs were calculated according to Eq. (2).
RAFenzyme =
TRPS in HLM TRPS in ICM
(2)
The intrinsic clearances Clint (see Eq. (3)) for the respective metabolic reactions were calculated and then multiplied with the corresponding RAF leading to a value, which is defined as ‘contribution’. The Vmax and the Km values (Eq. (1)) were obtained from the incubations with cDNA-expressed P450s. Clint,enzyme =
Vmax Km
(3)
Clenzyme = RAFenzyme × Clint,enzyme
(4)
From these corrected activities (Clenzyme ), the net clearance by a particular P450 at a certain substrate concentration can be calculated based on Eq. (5) using individual activities. net clearance, enzyme(%) =
Clenzyme × 100 ˙(Clenzymes )
(5)
2.2. Microsomal incubations with P450 isoforms
2.3. Chemical inhibition studies
Microsomal incubations were conducted according to previously published protocols (Meyer et al., 2012). Incubations were performed at 37 ◦ C using HLM, P450 1A2, P450 2A6, P450 2B6, P450 2C8, P450 2C9, P450 2C19, P450 2D6, P450 2E1, P450 3A4, or P450 3A5. Besides enzymes and substrate, incubation mixtures (final volume: 50 L) consisted of 90 mM phosphate buffer (pH 7.4), 5 mM Mg2+ , 5 mM isocitrate, 1.2 mM NADP+, 0.5 U/mL isocitrate dehydrogenase, and 200 U/mL superoxide dismutase. For incubations with P450 2A6 or P450 2C9, phosphate buffer was replaced with 45 mM or 90 mM Tris-buffer, respectively, according to the Gentest manual. Reactions were started by addition of pre-warmed
The effect of 1 M quinidine (P4502D6 inhibitor), 10 M omeprazole (P450 2C19 inhibitor), 1 M ␣-naphthoflavone (P450 1A2 inhibitor), 1 M ketoconazole (P450 3A4 inhibitor), and a mixture of them in the given concentrations on metabolite formation was assessed. The incubation (n = 6 each) was performed with 0.5 mg HLM protein/mL and 5 M of DMC for 20 min and the metabolite formation was determined as described below. The metabolite formation in control incubations (n = 6) without inhibitors were set to 100% for calculation of the percentage of inhibition. Significance of inhibition was tested by one-tailed unpaired Student’ t-test using GraphPad Prism software.
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Fig. 1. Mass chromatogram of the protonated molecules of DMC (m/z 279), hydroxyl DMC (m/z 295), and N-deethyl DMC (m/z 251) and their respective chemical structures, product ion spectra (MS2), and postulated main MS fragmentations.
2.4. Incubations with NAT1 and NAT2 Incubations were performed at 37 ◦ C with 50 M DMC or the PS sulfamethazine using human NAT1, human NAT2, and control cytosol for 20 min. Besides enzymes and substrate, incubation mixtures (final volume: 100 L) consisted of TEA-Buffer pH 7.5 (triethanolamine 100 mM, ethylenediaminetetraacetic acid 500 mM, and dithiotreitol 50 mM) and CoA-system (acetyl-CoA 1 mM, acetyl-carnitine 23 mM, and carnitin-acetyltransferase 0.08 U/L). The CoA-System was preincubated for 10 min and TEA-Buffer for 5 min at 37 ◦ C. Reactions were started by addition of the pre-warmed cytosol and stopped with 100 L of an ice-cold mixture of methanol, containing the internal standard (diphenhydramine, 5 M). The solution was centrifuged for 2 min at
14,000 × g, 50 L of the supernatant phase was transferred to an autosampler vial, and injected onto the LC apparatus for analysis. For initial screening studies, incubations were performed with 50 M of DMC or sulfamethazine and 0.05 mg/mL NAT1, NAT2, or control cytosol for 20 min. Kinetic data of N-acetylation were deduced from incubations with an incubation time of 10 min and 0.05 mg/mL (NATs) protein concentration. Incubation time and enzyme concentration were chosen to be within a linear range of metabolite formation. Sulfamethazine and DMC were incubated using substrate concentrations ranging from 0.5 to 2000 M. Values were estimated by non-linear curve-fitting using GraphPad Prism 5.00 software (San Diego, CA). The Michaelis–Menten equation (Eq. (1)) was used to
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Fig. 2. (a) Michaelis–Menten fitted plots for deethylation of DMC catalyzed by P450 1A2 (top left), P450 2C19 (top right), P450 2D6 (bottom left), and P450 3A4 (bottom right). Data points represent means of duplicate incubations. V given as dimensionless PAR per pmol protein and min. Curves were calculated by nonlinear curve fit (one-site binding model). (b) Michaelis–Menten fitted plots for hydroxylation of DMC catalyzed by P450 1A2 (top left), P450 2C19 (top right), P450 2D6 (bottom left), and P450 3A4 (bottom right). Data points represent means of duplicate incubations. V given as dimensionless PAR per pmol protein and min. Curves were calculated by nonlinear curve fit (one-site binding model).
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calculate apparent Km and Vmax values for single-enzyme systems as described above. Additionally a modified equation was used (Eqs. (5) and (6)) to calculate estimated Ki representing the inhibition constant considering the total substrate concentration range.
V=
Vmax × [S] × Km + [S]
1 + S Ki
(6)
The best kinetic model was selected, considering the randomness of the residuals, the standard errors of the estimates and the correlation coefficients.
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Table 1 Km (M) and Vmax values (dimensionless PAR/min and mg protein.) for deethylation, hydroxylation, and acetylation of DMC and acetylation of sulfamethazin. Isozyme
P450 1A2 P450 2C19 P450 2D6 P450 3A4
NAT2
Deethylation of DMC
Hydroxylation of DMC
Km
Km
220 25 195 26
Vmax ± ± ± ±
30 3 34 5
0.5 0.7 0.3 1.2
± ± ± ±
0.02 0.02 0.01 0.06
190 26 3.6 58
Vmax ± ± ± ±
25 3 0.3 10
1.9 0.8 1.1 0.1
± ± ± ±
0.07 0.02 0.02 0.01
Acetylation of DMC
Acetylation of sulfamethazin
102 ± 19
588 ± 40
1.1 ± 0.06
24 ± 1.6
2.5. LC–MSn apparatus for analysis of P450 and NAT incubations and chemical inhibition studies All samples were analyzed using a ThermoFisher Scientific (TF, Dreieich, Germany) LXQ linear ion trap mass spectrometer equipped with a heated electrospray ionization source and coupled to a TF Accela ultra UHPLC system consisting of a degasser, a quaternary pump, and an autosampler. LC conditions were as follows. Gradient elution was performed on a TF Hypersil GOLD C18 column (100 mm × 2.1 mm, 1.9 m) guarded by a TF Hypersil GOLD C18 Drop-in guard cartridge and a TF Javelin column filter with 10 mM aqueous ammonium formate plus 0.1% formic acid pH 3.4 (eluent A) and acetonitrile plus 0.1% formic acid (eluent B). The flow rate was set to 0.5 mL/min, and the gradient was programmed as follows: 0–1.0 min 98% A, 1.0–3.0 min to 90% A, 3.0–5.0 min to 85% A, 5.0–7.5 min to 80% A, 7.5–10.0 min to 75% A, 10.0–11.5 min to 70% A, 11.5–13.0 min to 65% A, 13.0–14.5 min to 50% A, 14.5–16.0 min to 40% A, 16.0–19.0 min to 0% A, and 19.0–21.0 hold 0% A. The injection volume for all samples was 10 L each. The MS was operated in positive electrospray ionization mode; sheath gas, nitrogen at flow rate of 34 arbitrary units (AU); auxiliary gas, nitrogen at flow rate of 11 AU; vaporizer temperature, 250 ◦ C; source voltage, 3.00 kV; ion transfer capillary temperature, 300 ◦ C; capillary voltage, 31 V; and tube lens voltage, 80 V. Automatic gain control was set to 15,000 ions for full scan and 5000 ions for MSn. The maximum injection time for full scan (MS1 stage) was set to 100 ms. Collision-induced dissociation (CID)-MSn experiments were performed on precursor ions selected from MS1 using information-dependent acquisition (IDA): MS1 was performed in the full scan mode (m/z 100–800). For identification of metabolites, MS2 and MS3 were performed in the IDA mode.
Fig. 3. RAF corrected, calculated total net clearances of P450 1A2, P450 2C19, P450 CY2D6, and P450 3A4 at a DMC concentration of 1 M for the deethylation (upper part) and hydroxylation (lower part).
2.6. LC–MS apparatus for analysis of NAT incubations The NAT incubation extracts were separated and quantified using an Agilent Technologies (AT, Waldbronn, Germany) AT 1100 series atmospheric pressure chemical ionization (APCI) electrospray LC-MSD, SL version and a LC-MSD ChemStation using the A.08.03 software. The general conditions were according to Peters et al. (2008). Briefly, gradient elution was achieved on a Merck
Fig. 4. Metabolite formed without inhibitor (control) and with the following P450 inhibitors measured in HLM with 1 M DMC: ␣-naphthoflavone (P450 1A2), omeprazole (P450 2C19), quinidine (P450 2D6), and ketoconazole (P450 3A4). Each bar represents the mean metabolite formation of six incubations ± SEM. The metabolite concentrations formed with inhibitors were compared with those without inhibitors (set to 100%) and tested for significance (*, p < 0.5; **, p < 0.05; ***, p < 0.005).
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Fig. 5. Mass chromatogram of the protonated molecules of DMC (m/z 279) and N-acetyl DMC (m/z 321) of incubations of NAT1 or NAT2 and their respective chemical structures, product ion spectra (MS2), and postulated main MS fragmentations.
Fig. 6. Michaelis–Menten fitted plots for N-acetylation of DMC (left) and substrate inhibition fitted plots for sulfamethazine (right) catalyzed by NAT2. Data points represent means of duplicate incubations. V given as dimensionless PAR per mg protein and min. Curves were calculated by nonlinear curve fit.
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LiChroCART column (125 mm × 2 mm I.D.) with Superspher 60 RP Select B as stationary phase and a LiChroCART 10-2 Superspher 60 RP Select B guard column. The mobile phase consisted of 50 mM ammonium formate adjusted to pH 3 with formic acid (eluent A) and acetonitrile containing 1 mL/L formic acid (eluent B). The MS operated in full-scan mode (m/z 100–600). For quantification, the peak area ratios of the respective target ions [M+H]+ of DMC, acetylated DMC, and the internal standard diphenhydramine were used.
3. Results and discussion
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3.2. Chemical inhibition studies In order to further confirm the contribution of the different P450s to the two reactions, the impact of the corresponding specific inhibitors (Topletz et al., 2013) on the metabolite formation was tested in HLM (Fig. 4). Inhibition of P450 3A4 in HLM, mainly catalyzing the N-deethyation according to the kinetic studies and RAF approach, lead to a decrease of metabolite formation of 90%. Inhibition of other P450s had no significant impact on N-deethyl formation. For hydroxylation, inhibition of P450 1A2, 2D6, and 3A4 lead to a significant decrease in metabolite formation, which was also in accordance to the predictions based on the kinetic studies and the subsequent RAF calculation.
3.1. 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 structure of both in vitro metabolites and the unchanged parent compound are shown in Fig. 1. Their MS2 mass spectra together with their analytical separation are shown in Fig. 1. After identification of catalyzing enzymes, kinetic parameters could be determined for the isozymes P450 1A2, 2C19, 2D6, and 3A4 for each reaction respectively. In case of P450s 2B6, 2C8, 2C9, and 3A5 activities were too low for calculation of kinetic parameters. Unfortunately, no reference substances of the metabolites were available. Therefore, only relative estimations of Vmax values, expressed as dimensionless peak area ratios (PAR) per pmol protein and minute could be obtained. However, this should have no negative impact on the estimation of the in vivo contribution of P450s as this was previously shown to be possible using PARs (Meyer et al., 2013a). All kinetic profiles best fit into Michaelis–Menten kinetics, as shown in Fig. 2a and b for deethylation and hydroxylation, respectively. The resulting Km , representing the in vitro affinity, and Vmax values are listed in Table 1. Using a modified approach (Meyer et al., 2008, 2009a,b; Venkatakrishnan et al., 2001) for calculating the in vivo clearances of individual P450 enzymes at a defined substrate concentration, the net clearances (illustrated in Fig. 3) for deethylation of DMC were calculated to be 3% for P450 1A2, 2% for P450 2C19, <1% for P450 2D6, and 95% for P450 3A4. The net clearances for hydroxylation of DMC were calculated to be 21% for P450 1A2, 4% for P450 2C19, 67% for P450 2D6, and 7% for P450 3A4. P450 3A4 should be the most relevant isozyme for deethylation and P450 2D6 for hydroxylation of DMC (Fig. 3). Similar results were obtained for the chemically related compound lidocaine. Wang et al. (2000) reported that P450 3A4 but also 1A2 were the major P450 isoforms involved in lidocaine Ndeethylation. Gantenbein et al. (2000) reported P450 3A4 to most efficiently N-dealkylate bupivacaine. Even the formation of norcocaine was mainly catalyzed by P450 3A4 (8488174) (LeDuc et al., 1993). These findings are all in line with the reported results. It should also be taken into account, that interactions might be possible between DMC and P450 3A4 and/or P450 2D6 inhibitors such as macrolide antibiotics as inhibitors of P450 3A4 and SSRIs as inhibitors of P450 2D6 but as DMC was metabolized via two CYP catalyzed steps and different isoforms were involved in the hepatic clearance, a clinically relevant interaction with single inhibitors should not be expected.
3.3. NAT 1 and 2 kinetic studies with DMC and sulfamethazine It seemed likely that isozyme NAT1 should be of importance for DMC N-acetylation because of its p-aminobenzoic acid structure, which was described to be a substrate of this enzyme (Butcher et al., 2002). However, initial activity screening with conditions adequate to make a statement on the general involvement of the NAT isozymes revealed that only NAT2 was capable catalyzing the N-acetylation of DMC (chemical structure in Fig. 5). This was further supported by enzyme kinetic studies, where data could only be acquired for NAT2 due to a very low product formation in NAT1 incubations. The MS2 mass spectrum of acetylated DMC together with its analytical separation is shown in Fig. 5. The kinetic profile of DMC acetylation by NAT2 best fits into Michaelis–Menten kinetic as shown in Fig. 6. To check proper incubation conditions, sulfamethazine was used as suitable test substrate with Km values available for both NAT enzymes (Grant et al., 1991; Hein et al., 1993). The Km value for DMC acetylation was determined to be 102 M, the Vmax value to be 1.1 units/min/pmol. The kinetic profile of sulfamethazine N-acetylation best followed a substrate inhibition equation with Km = 588 M, Vmax = 24 units/min/pmol, and Ki = 12 (Fig. 6), which fit well to previously published data (Delomenie et al., 1997; Grant et al., 1991; Hein et al., 1993). Endogenous N-acetylation of aromatic or heterocyclic amines by NAT1 or NAT2 is an important metabolic detoxification step of many drugs. As both enzymes, NAT1 and NAT2, are highly polymorphic, individual variations in the biotransformation of aromatic amines may occur (Yalin et al., 2007). It is obvious that there might be individual pharmacokinetic differences of the endogenous N-acetylation of DMC, which may lead to increased negative side effects such as carcinogenicity (Vineis and Pirastu, 1997). Such interactions may be much more important for people with a slow acetylation phenotype, in fact 50% of all Europeans have the slow or intermediary acetylation phenotype (Crettol et al., 2010). To what extent these points affect the plasma concentrations of DMC and also the concentration of acetylated DMC metabolites in urine should be target of further studies. In conclusion, the P450 isozymes involved in the initial steps of human metabolism could be identified to be P450 1A2, P450 2C19, P450 2D6, and P450 3A4. The endogenous N-acetylation as main part of DMC metabolism was catalyzed by the NAT2 isozyme. 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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Meyer, M.R., Peters, F.T., Maurer, H.H., 2009a. Stereoselective differences in the cytochrome P450-dependent dealkylation and demethylenation of N-methylbenzodioxolyl-butanamine (MBDB, Eden) enantiomers. Biochem. Pharmacol. 77, 1725–1734. Meyer, M.R., Peters, F.T., Maurer, H.H., 2009b. The role of human hepatic cytochrome P450 isozymes in the metabolism of racemic 3,4methylenedioxyethylamphetamine and its single enantiomers. Drug Metab. Dispos. 37, 1152–1156. Meyer, M.R., Vollmar, C., Schwaninger, A.E., Maurer, H.H., 2012. New cathinonederived designer drugs 3-bromomethcathinone and 3-fluoromethcathinone: studies on their metabolism in rat urine and human liver microsomes using GC–MS and LC-high-resolution MS and their detectability in urine. J. Mass Spectrom. 47, 253–262. Peters, F.T., Meyer, M.R., Theobald, D.S., Maurer, H.H., 2008. Identification of cytochrome P450 enzymes involved in the metabolism of 4 -methylpyrrolidino-butyrophenone (MPBP), a new designer drug, in human liver microsomes. Drug Metab. Dispos. 36, 163–168. Topletz, A.R., Dennison, J.B., Barbuch, R.J., Hadden, C.E., Hall, S.D., Renbarger, J.L., 2013. The relative contributions of CYP3A4 and CYP3A5 to the metabolism of vinorelbine. Drug Metab. Dispos. 41, 1651–1661. Venkatakrishnan, K., von Moltke, L.L., Court, M.H., Harmatz, J.S., Crespi, C.L., Greenblatt, D.J., 2000. Comparison between cytochrome P450 (CYP) content and relative activity approaches to scaling from cDNA-expressed CYPs to human liver microsomes: ratios of accessory proteins as sources of discrepancies between the approaches. Drug Metab. Dispos. 28, 1493–1504. Venkatakrishnan, K., von Moltke, L.L., Greenblatt, D.J., 2001. Application of the relative activity factor approach in scaling from heterologously expressed cytochromes p450 to human liver microsomes: studies on amitriptyline as a model substrate. J. Pharmacol. Exp. Ther. 297, 326–337. Vineis, P., Pirastu, R., 1997. Aromatic amines and cancer. Cancer Causes Control 8, 346–355. Wang, J.S., Backman, J.T., Taavitsainen, P., Neuvonen, P.J., Kivisto, K.T., 2000. Involvement of CYP1A2 and CYP3A4 in lidocaine N-deethylation and 3-hydroxylation in humans. Drug Metab. Dispos. 28, 959–965. Wilcox, K.M., Kimmel, H.L., Lindsey, K.P., Votaw, J.R., Goodman, M.M., Howell, L.L., 2005. In vivo comparison of the reinforcing and dopamine transporter effects of local anesthetics in rhesus monkeys. Synapse 58, 220–228. Wilcox, K.M., Rowlett, J.K., Paul, I.A., Ordway, G.A., Woolverton, W.L., 2000. On the relationship between the dopamine transporter and the reinforcing effects of local anesthetics in rhesus monkeys: practical and theoretical concerns. Psychopharmacology (Berl.) 153, 139–147. Woodward, J.J., Compton, D.M., Balster, R.L., Martin, B.R., 1995. In vitro and in vivo effects of cocaine and selected local anesthetics on the dopamine transporter. Eur. J. Pharmacol. 277, 7–13. Yalin, S., Hatungil, R., Tamer, L., Ates, N.A., Dogruer, N., Yildirim, H., Karakas, S., Atik, U., 2007. N-acetyltransferase 2 polymorphism in patients with diabetes mellitus. Cell Biochem. Funct. 25, 407–411.
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Toxicology Letters journal homepage: www.elsevier.com/locate/toxlet
Dimethocaine, a synthetic cocaine derivative: Studies on its in vitro metabolism catalyzed by P450s and NAT2 Markus R. Meyer ∗ , Carina Lindauer, Hans H. Maurer Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, Homburg, Saar, Germany
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
• 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.
a r t i c l e
i n f o
Article history: Received 18 October 2013 Received in revised form 23 November 2013 Accepted 26 November 2013 Available online 3 December 2013 Keywords: Designer drug DMC Cocaine Metabolism P450 NAT
a b s t r a c t 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 M. 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. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Dimethocaine (DMC, larocaine, 3-diethylamino-2,2-dimethylpropyl)-4-aminobenzoate) was marketed as local anesthetic in
∗ Corresponding author. Tel.: +49 6841 16 26430; fax: +49 6841 16 26051. E-mail address: [email protected] (M.R. Meyer). 0378-4274/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.toxlet.2013.11.033
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, 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
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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). 2. Materials and methods 2.1. 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 of human cDNA-expressed P450 1A2, P450 2A6, P450 2B6, P450 2C8, P450 2C9, P450 2C19, P450 2D6, P450 2E1 (2 nmol/mL), P450 3A4, or P450 3A5 (2 nmol/mL) and pooled human liver microsomes (pHLM, 20 mg microsomal protein/mL, 400 pmol total P450/mg protein). After delivery, the microsomes were thawed at 37 ◦ C, aliquoted, snap-frozen in liquid nitrogen, and stored at −80 ◦ C until use. Baculovirus-insect cell-expressed NAT1 (human arylamine N-acetyltransferase 1*4, wild-type allele) and NAT2 (human arylamine N-acetyltransferase 2*4, wild-type allele) as well as control cell cytosol were from BD Biosciences (Heidelberg, Germany). After delivery, the cytosols were thawed at 37 ◦ C, aliquoted, snap-frozen in liquid nitrogen, and stored at −80 ◦ C until use.
microsomes and stopped with 50 L of an ice-cold mixture of acetonitrile with 0.1% formic acid, containing the internal standard (diphenhydramine, 5 M). The solution was centrifuged for 2 min at 14,000 × g, 50 L of the supernatant was transferred to an autosampler vial, and injected onto the LC apparatus for analysis. For initial screening studies, incubations were performed at final concentration of 25 M DMC and 50 pmol/mL of P450 1A2, P450 2A6, P450 2B6, P450 2C8, P450 2C9, P450 2C19, P450 2D6, P450 2E1, P450 3A4, or P450 3A5 for 30 min. For kinetic studies, incubations were performed for 5 min with 32 pmol/mL protein concentration of P450 1A2, P450 2C19, P450 2D6, P450 3A4. The described incubation conditions (incubation time and protein concentration) were chosen to be within a linear range of metabolite formation. DMC was incubated using substrate concentrations ranging from 0.1 to 2000 M. Enzyme kinetic constants were estimated by non-linear curvefitting using GraphPad Prism 5.00 software (San Diego, CA). The Michaelis–Menten equation (Eq. (1)) was used to calculate apparent Km values and Vmax values for single-enzyme systems. V=
Vmax × [S] Km + [S]
(1)
The relative activity factor (RAF) approach was used to account for differences in functional levels of redox partners between the two enzyme sources (Crespi and Miller, 1999; Venkatakrishnan et al., 2000). The turnover rates (TR) of P450 1A2 (probe substrate (PS) phenacetin), P450 2C19 (PS (S)-mephenytoin), P450 2D6 (PS bufuralol) and P450 3A4 (PS testosterone) in insect cell microsomes (ICM) and human liver microsomes (HLM) were taken from the supplier’s data sheets. The RAFs were calculated according to Eq. (2).
RAFenzyme =
TRPS in HLM TRPS in ICM
(2)
The intrinsic clearances Clint (see Eq. (3)) for the respective metabolic reactions were calculated and then multiplied with the corresponding RAF leading to a value, which is defined as ‘contribution’. The Vmax and the Km values (Eq. (1)) were obtained from the incubations with cDNA-expressed P450s. Clint,enzyme =
Vmax Km
(3)
Clenzyme = RAFenzyme × Clint,enzyme
(4)
From these corrected activities (Clenzyme ), the net clearance by a particular P450 at a certain substrate concentration can be calculated based on Eq. (5) using individual activities. net clearance, enzyme(%) =
Clenzyme × 100 ˙(Clenzymes )
(5)
2.2. Microsomal incubations with P450 isoforms
2.3. Chemical inhibition studies
Microsomal incubations were conducted according to previously published protocols (Meyer et al., 2012). Incubations were performed at 37 ◦ C using HLM, P450 1A2, P450 2A6, P450 2B6, P450 2C8, P450 2C9, P450 2C19, P450 2D6, P450 2E1, P450 3A4, or P450 3A5. Besides enzymes and substrate, incubation mixtures (final volume: 50 L) consisted of 90 mM phosphate buffer (pH 7.4), 5 mM Mg2+ , 5 mM isocitrate, 1.2 mM NADP+, 0.5 U/mL isocitrate dehydrogenase, and 200 U/mL superoxide dismutase. For incubations with P450 2A6 or P450 2C9, phosphate buffer was replaced with 45 mM or 90 mM Tris-buffer, respectively, according to the Gentest manual. Reactions were started by addition of pre-warmed
The effect of 1 M quinidine (P4502D6 inhibitor), 10 M omeprazole (P450 2C19 inhibitor), 1 M ␣-naphthoflavone (P450 1A2 inhibitor), 1 M ketoconazole (P450 3A4 inhibitor), and a mixture of them in the given concentrations on metabolite formation was assessed. The incubation (n = 6 each) was performed with 0.5 mg HLM protein/mL and 5 M of DMC for 20 min and the metabolite formation was determined as described below. The metabolite formation in control incubations (n = 6) without inhibitors were set to 100% for calculation of the percentage of inhibition. Significance of inhibition was tested by one-tailed unpaired Student’ t-test using GraphPad Prism software.
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Fig. 1. Mass chromatogram of the protonated molecules of DMC (m/z 279), hydroxyl DMC (m/z 295), and N-deethyl DMC (m/z 251) and their respective chemical structures, product ion spectra (MS2), and postulated main MS fragmentations.
2.4. Incubations with NAT1 and NAT2 Incubations were performed at 37 ◦ C with 50 M DMC or the PS sulfamethazine using human NAT1, human NAT2, and control cytosol for 20 min. Besides enzymes and substrate, incubation mixtures (final volume: 100 L) consisted of TEA-Buffer pH 7.5 (triethanolamine 100 mM, ethylenediaminetetraacetic acid 500 mM, and dithiotreitol 50 mM) and CoA-system (acetyl-CoA 1 mM, acetyl-carnitine 23 mM, and carnitin-acetyltransferase 0.08 U/L). The CoA-System was preincubated for 10 min and TEA-Buffer for 5 min at 37 ◦ C. Reactions were started by addition of the pre-warmed cytosol and stopped with 100 L of an ice-cold mixture of methanol, containing the internal standard (diphenhydramine, 5 M). The solution was centrifuged for 2 min at
14,000 × g, 50 L of the supernatant phase was transferred to an autosampler vial, and injected onto the LC apparatus for analysis. For initial screening studies, incubations were performed with 50 M of DMC or sulfamethazine and 0.05 mg/mL NAT1, NAT2, or control cytosol for 20 min. Kinetic data of N-acetylation were deduced from incubations with an incubation time of 10 min and 0.05 mg/mL (NATs) protein concentration. Incubation time and enzyme concentration were chosen to be within a linear range of metabolite formation. Sulfamethazine and DMC were incubated using substrate concentrations ranging from 0.5 to 2000 M. Values were estimated by non-linear curve-fitting using GraphPad Prism 5.00 software (San Diego, CA). The Michaelis–Menten equation (Eq. (1)) was used to
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Fig. 2. (a) Michaelis–Menten fitted plots for deethylation of DMC catalyzed by P450 1A2 (top left), P450 2C19 (top right), P450 2D6 (bottom left), and P450 3A4 (bottom right). Data points represent means of duplicate incubations. V given as dimensionless PAR per pmol protein and min. Curves were calculated by nonlinear curve fit (one-site binding model). (b) Michaelis–Menten fitted plots for hydroxylation of DMC catalyzed by P450 1A2 (top left), P450 2C19 (top right), P450 2D6 (bottom left), and P450 3A4 (bottom right). Data points represent means of duplicate incubations. V given as dimensionless PAR per pmol protein and min. Curves were calculated by nonlinear curve fit (one-site binding model).
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calculate apparent Km and Vmax values for single-enzyme systems as described above. Additionally a modified equation was used (Eqs. (5) and (6)) to calculate estimated Ki representing the inhibition constant considering the total substrate concentration range.
V=
Vmax × [S] × Km + [S]
1 + S Ki
(6)
The best kinetic model was selected, considering the randomness of the residuals, the standard errors of the estimates and the correlation coefficients.
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Table 1 Km (M) and Vmax values (dimensionless PAR/min and mg protein.) for deethylation, hydroxylation, and acetylation of DMC and acetylation of sulfamethazin. Isozyme
P450 1A2 P450 2C19 P450 2D6 P450 3A4
NAT2
Deethylation of DMC
Hydroxylation of DMC
Km
Km
220 25 195 26
Vmax ± ± ± ±
30 3 34 5
0.5 0.7 0.3 1.2
± ± ± ±
0.02 0.02 0.01 0.06
190 26 3.6 58
Vmax ± ± ± ±
25 3 0.3 10
1.9 0.8 1.1 0.1
± ± ± ±
0.07 0.02 0.02 0.01
Acetylation of DMC
Acetylation of sulfamethazin
102 ± 19
588 ± 40
1.1 ± 0.06
24 ± 1.6
2.5. LC–MSn apparatus for analysis of P450 and NAT incubations and chemical inhibition studies All samples were analyzed using a ThermoFisher Scientific (TF, Dreieich, Germany) LXQ linear ion trap mass spectrometer equipped with a heated electrospray ionization source and coupled to a TF Accela ultra UHPLC system consisting of a degasser, a quaternary pump, and an autosampler. LC conditions were as follows. Gradient elution was performed on a TF Hypersil GOLD C18 column (100 mm × 2.1 mm, 1.9 m) guarded by a TF Hypersil GOLD C18 Drop-in guard cartridge and a TF Javelin column filter with 10 mM aqueous ammonium formate plus 0.1% formic acid pH 3.4 (eluent A) and acetonitrile plus 0.1% formic acid (eluent B). The flow rate was set to 0.5 mL/min, and the gradient was programmed as follows: 0–1.0 min 98% A, 1.0–3.0 min to 90% A, 3.0–5.0 min to 85% A, 5.0–7.5 min to 80% A, 7.5–10.0 min to 75% A, 10.0–11.5 min to 70% A, 11.5–13.0 min to 65% A, 13.0–14.5 min to 50% A, 14.5–16.0 min to 40% A, 16.0–19.0 min to 0% A, and 19.0–21.0 hold 0% A. The injection volume for all samples was 10 L each. The MS was operated in positive electrospray ionization mode; sheath gas, nitrogen at flow rate of 34 arbitrary units (AU); auxiliary gas, nitrogen at flow rate of 11 AU; vaporizer temperature, 250 ◦ C; source voltage, 3.00 kV; ion transfer capillary temperature, 300 ◦ C; capillary voltage, 31 V; and tube lens voltage, 80 V. Automatic gain control was set to 15,000 ions for full scan and 5000 ions for MSn. The maximum injection time for full scan (MS1 stage) was set to 100 ms. Collision-induced dissociation (CID)-MSn experiments were performed on precursor ions selected from MS1 using information-dependent acquisition (IDA): MS1 was performed in the full scan mode (m/z 100–800). For identification of metabolites, MS2 and MS3 were performed in the IDA mode.
Fig. 3. RAF corrected, calculated total net clearances of P450 1A2, P450 2C19, P450 CY2D6, and P450 3A4 at a DMC concentration of 1 M for the deethylation (upper part) and hydroxylation (lower part).
2.6. LC–MS apparatus for analysis of NAT incubations The NAT incubation extracts were separated and quantified using an Agilent Technologies (AT, Waldbronn, Germany) AT 1100 series atmospheric pressure chemical ionization (APCI) electrospray LC-MSD, SL version and a LC-MSD ChemStation using the A.08.03 software. The general conditions were according to Peters et al. (2008). Briefly, gradient elution was achieved on a Merck
Fig. 4. Metabolite formed without inhibitor (control) and with the following P450 inhibitors measured in HLM with 1 M DMC: ␣-naphthoflavone (P450 1A2), omeprazole (P450 2C19), quinidine (P450 2D6), and ketoconazole (P450 3A4). Each bar represents the mean metabolite formation of six incubations ± SEM. The metabolite concentrations formed with inhibitors were compared with those without inhibitors (set to 100%) and tested for significance (*, p < 0.5; **, p < 0.05; ***, p < 0.005).
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Fig. 5. Mass chromatogram of the protonated molecules of DMC (m/z 279) and N-acetyl DMC (m/z 321) of incubations of NAT1 or NAT2 and their respective chemical structures, product ion spectra (MS2), and postulated main MS fragmentations.
Fig. 6. Michaelis–Menten fitted plots for N-acetylation of DMC (left) and substrate inhibition fitted plots for sulfamethazine (right) catalyzed by NAT2. Data points represent means of duplicate incubations. V given as dimensionless PAR per mg protein and min. Curves were calculated by nonlinear curve fit.
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LiChroCART column (125 mm × 2 mm I.D.) with Superspher 60 RP Select B as stationary phase and a LiChroCART 10-2 Superspher 60 RP Select B guard column. The mobile phase consisted of 50 mM ammonium formate adjusted to pH 3 with formic acid (eluent A) and acetonitrile containing 1 mL/L formic acid (eluent B). The MS operated in full-scan mode (m/z 100–600). For quantification, the peak area ratios of the respective target ions [M+H]+ of DMC, acetylated DMC, and the internal standard diphenhydramine were used.
3. Results and discussion
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3.2. Chemical inhibition studies In order to further confirm the contribution of the different P450s to the two reactions, the impact of the corresponding specific inhibitors (Topletz et al., 2013) on the metabolite formation was tested in HLM (Fig. 4). Inhibition of P450 3A4 in HLM, mainly catalyzing the N-deethyation according to the kinetic studies and RAF approach, lead to a decrease of metabolite formation of 90%. Inhibition of other P450s had no significant impact on N-deethyl formation. For hydroxylation, inhibition of P450 1A2, 2D6, and 3A4 lead to a significant decrease in metabolite formation, which was also in accordance to the predictions based on the kinetic studies and the subsequent RAF calculation.
3.1. 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 structure of both in vitro metabolites and the unchanged parent compound are shown in Fig. 1. Their MS2 mass spectra together with their analytical separation are shown in Fig. 1. After identification of catalyzing enzymes, kinetic parameters could be determined for the isozymes P450 1A2, 2C19, 2D6, and 3A4 for each reaction respectively. In case of P450s 2B6, 2C8, 2C9, and 3A5 activities were too low for calculation of kinetic parameters. Unfortunately, no reference substances of the metabolites were available. Therefore, only relative estimations of Vmax values, expressed as dimensionless peak area ratios (PAR) per pmol protein and minute could be obtained. However, this should have no negative impact on the estimation of the in vivo contribution of P450s as this was previously shown to be possible using PARs (Meyer et al., 2013a). All kinetic profiles best fit into Michaelis–Menten kinetics, as shown in Fig. 2a and b for deethylation and hydroxylation, respectively. The resulting Km , representing the in vitro affinity, and Vmax values are listed in Table 1. Using a modified approach (Meyer et al., 2008, 2009a,b; Venkatakrishnan et al., 2001) for calculating the in vivo clearances of individual P450 enzymes at a defined substrate concentration, the net clearances (illustrated in Fig. 3) for deethylation of DMC were calculated to be 3% for P450 1A2, 2% for P450 2C19, <1% for P450 2D6, and 95% for P450 3A4. The net clearances for hydroxylation of DMC were calculated to be 21% for P450 1A2, 4% for P450 2C19, 67% for P450 2D6, and 7% for P450 3A4. P450 3A4 should be the most relevant isozyme for deethylation and P450 2D6 for hydroxylation of DMC (Fig. 3). Similar results were obtained for the chemically related compound lidocaine. Wang et al. (2000) reported that P450 3A4 but also 1A2 were the major P450 isoforms involved in lidocaine Ndeethylation. Gantenbein et al. (2000) reported P450 3A4 to most efficiently N-dealkylate bupivacaine. Even the formation of norcocaine was mainly catalyzed by P450 3A4 (8488174) (LeDuc et al., 1993). These findings are all in line with the reported results. It should also be taken into account, that interactions might be possible between DMC and P450 3A4 and/or P450 2D6 inhibitors such as macrolide antibiotics as inhibitors of P450 3A4 and SSRIs as inhibitors of P450 2D6 but as DMC was metabolized via two CYP catalyzed steps and different isoforms were involved in the hepatic clearance, a clinically relevant interaction with single inhibitors should not be expected.
3.3. NAT 1 and 2 kinetic studies with DMC and sulfamethazine It seemed likely that isozyme NAT1 should be of importance for DMC N-acetylation because of its p-aminobenzoic acid structure, which was described to be a substrate of this enzyme (Butcher et al., 2002). However, initial activity screening with conditions adequate to make a statement on the general involvement of the NAT isozymes revealed that only NAT2 was capable catalyzing the N-acetylation of DMC (chemical structure in Fig. 5). This was further supported by enzyme kinetic studies, where data could only be acquired for NAT2 due to a very low product formation in NAT1 incubations. The MS2 mass spectrum of acetylated DMC together with its analytical separation is shown in Fig. 5. The kinetic profile of DMC acetylation by NAT2 best fits into Michaelis–Menten kinetic as shown in Fig. 6. To check proper incubation conditions, sulfamethazine was used as suitable test substrate with Km values available for both NAT enzymes (Grant et al., 1991; Hein et al., 1993). The Km value for DMC acetylation was determined to be 102 M, the Vmax value to be 1.1 units/min/pmol. The kinetic profile of sulfamethazine N-acetylation best followed a substrate inhibition equation with Km = 588 M, Vmax = 24 units/min/pmol, and Ki = 12 (Fig. 6), which fit well to previously published data (Delomenie et al., 1997; Grant et al., 1991; Hein et al., 1993). Endogenous N-acetylation of aromatic or heterocyclic amines by NAT1 or NAT2 is an important metabolic detoxification step of many drugs. As both enzymes, NAT1 and NAT2, are highly polymorphic, individual variations in the biotransformation of aromatic amines may occur (Yalin et al., 2007). It is obvious that there might be individual pharmacokinetic differences of the endogenous N-acetylation of DMC, which may lead to increased negative side effects such as carcinogenicity (Vineis and Pirastu, 1997). Such interactions may be much more important for people with a slow acetylation phenotype, in fact 50% of all Europeans have the slow or intermediary acetylation phenotype (Crettol et al., 2010). To what extent these points affect the plasma concentrations of DMC and also the concentration of acetylated DMC metabolites in urine should be target of further studies. In conclusion, the P450 isozymes involved in the initial steps of human metabolism could be identified to be P450 1A2, P450 2C19, P450 2D6, and P450 3A4. The endogenous N-acetylation as main part of DMC metabolism was catalyzed by the NAT2 isozyme. 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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