Article pubs.acs.org/Langmuir
Nanocomposites of Graphene and Cytochrome P450 2D6 Isozyme for Electrochemical-Driven Tramadol Metabolism Dongmei Cui, Li Mi, Xuan Xu, Jusheng Lu, Jing Qian, and Songqin Liu* Jiangsu Province Hi-Tech Key Laboratory for Bio-medical Research, School of Chemistry and Chemical Engineering, Southeast University, Jiangning District 211189, Nanjing, Jiangsu Province, People’s Republic of China S Supporting Information *
ABSTRACT: Cytochrome P450 enzymes (cyt P450s) with an active center of iron protoheme are involved in most clinical drugs metabolism process. Herein, an electrochemical platform for the investigation of drug metabolism in vitro was constructed by immobilizing cytochrome P450 2D6 (CYP2D6) with cyt P450 reductase (CPR) on graphene modified glass carbon electrode. Direct and reversible electron transfer of the immobilized CYP2D6 with the direct electron transfer constant of 0.47 s−1 and midpoint potential of -0.483 V was obtained. In the presence of substrate tramadol, the electrochemical-driven CYP2D6 mediated catalytic behavior toward the conversion of tramadol to o-demethyl-tramadol was confirmed. The Michaelis−Menten constant (Kmapp) and heterogeneous reaction rate constant during the metabolism of tramadol were calculated to be 23.85 μM and 1.96 cm s−1, respectively. The inhibition effect of quinidine on CYP2D6 catalyze-cycle was also investigated. Furthermore, this system was applied to studying the metabolism of other drugs. nate11 and poly(dimethyldiallylammonium chloride)12 modified electrodes. Besides the chemicals and polyelectrolyte, nanomaterials were widely used for the immobilization of enzymes. Niwa and coworkers used carbon nanofibers as the matrix for the immobilization of CYP3A4 on the electrode. Based on this platform, the drug metabolism and inhibition reactions were achieved.13 Our group has reported the successful immobilization and drug metabolism of cyt P450s on nanocomposites modified electrodes, where the nanocomposites were composed of zirconium dioxide nanoparticles and platinum,14 colloidal gold nanoparticles and chitosan,15 indium tin oxide nanoparticles and chitosan,16 colloidal gold and graphene.17 Graphene has attracted a great deal of attention with its excellent thermal, unique mechanical, and superior electrical properties.18 It is often derived from oxide graphene (GO) by a reducing procedure. However, the obtained reduced oxide graphene (RGO) sheets tend to form irreversible agglomerates through π−π stacking and van der Waals attractive interactions.19 Polyethylenimine (PEI) is a kind of cationic polymer with high density of amino and has excellent filmforming property, and thus it has received wide applications in the fields of biology and medicine.20,21 Through electrostatic interaction, PEI can be coated on graphene oxide to form homogeneous suspension, and further convert GO to RGO. Herein, the nanocomposite of PEI modified graphene was constructed for the immobilization of the model enzyme,
1. INTRODUCTION Cytochrome P450 enzymes (cyt P450s) are a superfamily of heme proteins, which are responsible for the oxidative metabolism of ∼75% clinical drugs. The metabolism reactions normally include hydroxylation, oxygen dealkylation, and nitrogen oxidation.1−4 Drug metabolism via the cyt P450s can cause drug−drug or drug−food interactions that result in toxicities and undesired consequences. Effective methods to monitor the drug metabolism via cyt P450s in vitro are very important, especially for the discovery and development of new drugs.5 Cyt P450s catalyze drugs or chemicals by two electrons delivery from NADPH to cyt P450 reductase (CPR) for subsequent delivery to cyt P450 heme.1,6 Great efforts have been made for initiation of cyt P450s catalysis by replacing the electron donation from expensive NADPH with electrodes. Since the first electrochemistry of cyt P450cam was achieved in 1996,7 researchers devoted themselves to investigating the electrochemical methods to study the cyt P450s’ role in drug metabolism in vitro. Some polymer materials have been used as matrix for enzyme immobilization to improve the electron transfer. Rusling and coworkers used poly(diallyldimethylammonium chloride) and poly(sodium 4styrenesulfonate) as the matrix for layer by layer immobilizing CYP1A2 or CYP2E1, and accurately mimicked the natural cyt P450s catalytic cycle.8 Gilardi and coworkers successfully linked P450 BMP on the gold surface by using spacers of cystamineN-succinimidyl 3-maleimidopropionate and dithio-bismaleimidoethane, the electrochemical activity of the immobilized P450 BMP was achieved.9 The direct electrochemistry and catalysis of P450 BM3, CYP1A2 and CYP3A4 were also monitored at didodecyldimethylammonium bromide,10 polystyrenesulfo© XXXX American Chemical Society
Received: April 22, 2014 Revised: September 7, 2014
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2.2. Instrumentation. Raman spectra were collected with a DXR-laser micro Raman spectrophotometer (Thermo Fisher Scientific Company, USA) with an excitation wavelength of 532 nm. UV−vis absorption spectra were gathered on a UV-2450 spectrometer (Shimadzu, Japan). The morphology of the asprepared materials was analyzed by a Tecnai G2 transmission electron microscope (TEM, FEI, Netherland) with an acceleration voltage of 200 kV. Elemental analysis of graphene before and after reduction was carried out on the energy dispersive spectroscopy with an X-MAX electric refrigeration detector (EDS, Oxford Instruments). All the electrochemical measurements were performed on a CHI 750C or 660C electrochemical workstation. The traditional three-electrode system was consisted of a modified glassy carbon electrode as a working electrode, a Pt wire as a counter electrode and a saturated calomel electrode (SCE) as a reference electrode. Electrocatalytic drug metabolism was conducted by a rotating ring-disk electrode (RDE). The catalytic products were analyzed by high performance liquid chromatography (HPLC, Thermo Fisher Scientific Company, USA) and quadrupole electrostatic field orbit trap high-resolution mass spectrometry (MS). A solution of 3 μL sample was injected into Thermo Hypersil Gold C18 column (100 mm × 2.1 mm, 3 μm) and the high performance liquid chromatography separation was carried out using the method of gradient elution with a mobile phase contained 0.1 wt % formic acid in water (C) and acetonitrile (D) at a flow rate of 0.3 mL min−1. The ratio of C to D was 90:10 for the first 2 min, then it was turned to 80:20 from 2 to 24 min, 90:10 from 24 to 30 min. Before the liquid flowed into the MS, the switch valve was used to separate PBS from samples. 2.3. Synthesis of PEI-Functionalized Graphene Nanosheets (PEI-RGO). GO was prepared according to previous reports.17 First, graphite oxide was exfoliated under ultrasonication in water for 2 h. Then, the obtained brown dispersion was centrifuged at 3000 rpm for 30 min to remove the unexfoliated graphite oxide. Finally, dialysis was carried out for 1 week in order to make residual salts and acids completely removed.
CYP2D6 (Scheme 1). CYP2D6 plays a vital role in the drugs metabolism, which catalyzes a variety of common used drugs, Scheme 1. Schematic of the Fabrication of the Electrochemical-Driven Drug Metabolism Platform
including antiarrhythmic drugs, neuroleptics, antidepressants, β-blockers, and opiates.22−24 Meanwhile, CYP2D6 is selectively inhibited by quinidine, cimetidine, paroxetine. In this work, the direct electron transfer between CYP2D6 and the electrode, drug metabolism via CYP2D6 in vitro and the inhibitive impact of quinidine on the drug metabolism process were investigated based on the as-constructed electrochemical platform.
2. EXPERIMENTAL SECTION 2.1. Materials. Cytochrome P450 2D6*1 isozyme protein (CYP2D6 1 nmol, 0.5 mL) contained cytochrome P450reductase (CPR) in 0.1 M, pH 7.4 potassium phosphate buffer was obtained from Bioscience Discovery Gene Co., Ltd. (Nanjing, China). Graphite oxide was purchased from Nanjing XFNANO Materials Tech Co., Ltd. (Nanjing, China). Polyethylenimine (PEI, Mw = 10000, 99%), glutaraldehyde (GA, 25% in water), phenformin hydrochloride, yohimbine hydrochloride and phenacetin were obtained from Aladdin Reagent Co., Ltd. (Shanghai, China). Mexiletine hydrochloride and metoclopramide hydrochloride were received from J&K Chemical Co., Ltd. (Shanghai, China). Metoprolol tartrate salt, tramadol hydrochloride, Nafion (10%, in water) and clomipramine hydrochloride were received from Sigma-Aldrich Co., Ltd. (Shanghai, China). Phosphate buffer solution was prepared by mixing 0.1 M NaH2PO4 and Na2HPO4. Other reagents were of analytical reagent grade and were used as received. Milli-Q ultrapure water (Millipore, resistance >18 MΩ cm) was used throughout the experiment.
Figure 1. (A) UV−vis spectra of (a) GO and (b) PEI-RGO. Inset: the corresponding optical photographs of GO (left side) and PEI-RGO (right side). (B) Raman spectra of (a) GO and (b) PEI-RGO. (C) TEM images of (a) GO and (b) PEI-RGO. B
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Figure 2. (A) CV curves of (a) CYP2D6/PEI-RGO/GCE prepared with GA as linkage, (b) CYP2D6/PEI-RGO/GCE prepared by electrostatic interaction, (c) PEI-RGO/GCE in anaerobic 0.1 M, pH 7.4 PBS at a scan rate of 100 mV s−1. (B) CV curves of CYP2D6/PEI-RGO/GCE in 0.1 M, pH 7.4 PBS (a) under nitrogen; (b) air saturated; (c) addition 200 μM tramadol to the air saturated at 100 mV s−1.
which corresponded to the D band and G band of graphene, respectively. The intensity ratio of the D band to the G band (ID/IG) increased from 0.95 to 1.12 after PEI reduction. Since the ID/IG is inversely proportional to the average size of the sp2 domains, the increase of ID/IG suggested the smaller sp2 domains in PEI-RGO, which confirmed the successful reduction of graphene oxide to graphene.18 Moreover, the nucleophilic addition of −NH2 functionalities of PEI to the epoxy functionalities of GO also made the increase of ID/IG.19 All the results indicated that GO was successfully reduced by PEI. Further, EDS analysis of GO and PEI-RGO (Supporting Information Figure S1.) confirmed that the element of N was detected in graphene after the reduction by PEI. This revealed that PEI had attached on graphene as hoped. TEM images (Figure 1C) showed that both GO and PEI-RGO were the forms of nanosheets. Differently, the size of RGO was smaller than GO, which may be due to the sonication during the reduction process. 3.2. Direct Electrochemistry of CYP2D6/PEI-RGO/GCE. The RGO hybrided with PEI had a number of amino groups, which could be activated by GA and conjugated with CYP2D6 through amide bond. The immobilization of CYP2D6 on PEIRGO film could be monitored by the impedance change of electrode in the electrochemical impedance spectroscopy (Figure S2). Once PEI-RGO casted on the bare GCE, charge-transfer resistance decreased obviously, which confirmed the excellent conductivity of PEI-RGO. Upon immobilization of CYP2D6, the charge-transfer resistance increased dramatically because of the poor conductivity of protein, demonstrating the successful linkage of CYP2D6 to the GCE surface. Cyclic voltammogram showed that the CYP2D6 coupling to PEI-RGO via GA as linkage displayed a pair of stable and welldefined redox peaks located at around −0.454 and −0.512 V with the peak currents of −0.674 and 0.726 μA, respectively (Figure 2A, curve a). When CYP2D6 was coated on PEI-RGO/ GCE through electrostatic interaction (in the absence of GA), a couple of weak redox peaks were also observed (Figure 2A, curve b). However, the peak currents (−0.288 and 0.298 μA) were only 40% of the currents for CYP2D6/PEI-RGO/GCE prepared by covalent binding with GA as linkage. This suggested that the coating of CYP2D6 on PEI-RGO presented covalent binding with GA linkage and electrostatic adsorption. No redox peaks were observed for PEI-RGO/GCE in the absence of CYP2D6 (Figure 2A, curve c). Therefore, the response was attributed to the redox of the electroactive centers in the immobilized proteins, which were the heme cofactor in
PEI-functionalized graphene was obtained by reducing the as-prepared graphene oxide with PEI.25 Briefly, 25.5 mL of the stable graphene oxide solution (0.12 mg mL−1) was added into 4.5 mL of the PEI solution (0.2 wt %) under strong stirring. The mixture was then stirred and sonicated for 10 min, respectively. After that, the mixture was heated under reflux at 135 oC for 3.5 h. During the reduction process, the brown dispersion was observed to turn black quickly (inset in Figure 1A). Positively charged PEI molecules were used to serve as reducing agent and surface modifier. The obtained black solution was centrifuged and washed for several times. The asprepared PEI-RGO was full of amino groups (−NH2) on the surface and could be used for further functionalization. 2.4. Construction of CYP2D6/PEI-RGO/GCE. The glassy carbon electrode (GCE) with a diameter of 3 mm was successively polished using 1.0 and 0.3 μm alumina powder, followed by rinsing thoroughly with water. After successive sonication in 1:1 nitric acid, acetone and water, the electrode was rinsed with water and dried at room temperature. Then, 10 μL of PEI-RGO was dropped onto the cleaned GCE surface and dried at room temperature. After activating of the PEIRGO by GA for 1 h, 5 μL of CYP2D6 was coated on PEIRGO/GCE and allowed to dry at 4 oC overnight. The electrode was rinsed thoroughly to remove any unbounded CYP2D6 and stored at 4 oC. In addition, Nafion solution (0.05% in ethanol, 5 μL) was coated atop the CYP2D6/PEIRGO/GCE while RDE was used as the working electrode. For comparison, the CYP2D6/PEI-RGO/GCE was prepared through electrostatic absorption by dropping 5 μL of CYP2D6 on PEI-RGO/GCE without activating of PEI-RGO by GA.
3. RESULTS AND DISCUSSION 3.1. Characterization of PEI-RGO Nanocomposites. The formation of PEI-RGO was characterized by UV−vis and Raman spectroscopy. As shown in Figure 1A, GO displays a characteristic absorption peak at 230 nm corresponding to the typical π−π* transition of aromatic sp2 domains, and a little bump around 300 nm attributed to electron transition of n → π*. After reduced by PEI, the characteristic absorption peak shifted from 230 to 270 nm, and the bump around 300 nm disappeared. Compared with GO, the baseline of PEI-RGO raised obviously, which implies that the π electronic conjugation within the graphene nanosheets was restored.18,19 Raman spectroscopy showed two prominent peaks at 1345 cm−1 and 1588 cm−1 for both GO and PEI-RGO (Figure 1B), C
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Figure 3. (A) Rotating disk voltammograms (1000 rpm) of CYP2D6/PEI-RGO modified rotating disk electrode in aerobic 0.1 M pH 7.4 PBS containing 25, 50, 100, 150, 400, and 640 μM tramadol (from bottom to top). Inset: CV curves of CYP2D6/PEI-RGO modified rotating disk electrode obtained by subtracting the background before any addition of tramadol. (B) Plots of ΔI at −0.483 V versus tramodol concentration. (C) Linear sweep voltammograms of CYP2D6/PEI-RGO/GCE in aerobic 0.1 M pH 7.4 PBS with various rotation rates (400 to 3000 rpm) at the scan rate of 100 mV s−1. (D) Koutecky−Levich plot of i−1 versus ω‑1/2 at different electrode potentials.
CYP2D6 and the flavin cofactor in CPR.8,16 On the other hand, only very weak redox response was observed for directly immobilization of CYP2D6 on bare GCE or RGO modified GCE (data not shown), indicating that PEI-RGO played a key role in promoting the direct electrochemistry of the immobilized enzyme. An estimate of the formal potential of the CYP2D6/PEI-RGO/GCE was −0.483 V with peak-to-peak separation of 58 mV, while reduction current to oxidation current ratio was approximately equal to 1. With the increasing of scan rates, redox peak current showed good linear relationship with the scan rates while peak-to-peak separation gradually increased (Figure S3). These indicated that the redox process of CYP2D6 on PEI-RGO film was a quasi-reversible surface controlled process. The apparent rate constant of direct electron transfer Ks was calculated26 to be 0.47 s−1 at the scan rate of 100 mV s−1. On the basis of a previous report,27 low electron transfer rate is essential for the system of electrochemically driven catalysis via cyt P450s, because it allows for the fine-tuning of the electron delivery from the reductase to the haem catalytic site. 3.3. Metabolism of Tramadol by CYP2D6/PEI-RGO/ GCE. Previous studies demonstrated that the cyt P450s mediated drug metabolism is oxygen-dependent.9,28 As shown in Figure 2B, the shape of the cyclic voltammogram curve of CYP2D6/PEI-RGO/GCE changed greatly in aerobic PBS (curve b), with an increased reduction current and a decreased oxidation current, compared with its cyclic voltammogram curve in anaerobic PBS (curve a). This was attributed to the typical electrocatalytic oxygen reduction process.15 The electrochemically-driven drug metabolism by CYP2D6 on the PEI-RGO modified GCE was investigated by using tramadol as substrate. Tramadol is one of the opioid drugs and
used as analgesic. It is mainly metabolized by CYP2D6, CYP2B6 and CYP3A4 in human liver microsomes. CYP2D6 is responsible for the formation of O-demethyl tramadol.29 Upon addition of tramadol into the air saturated 0.1 M, pH 7.4 PBS, the cyclic voltammogram curve of the CYP2D6/PEI-RGO/ GCE showed increase of the reduction current and decrease of the oxidation current (Figure 2B, curve c), confirming the effective electrocatalysis of the immobilized CYP2D6 to tramadol. According to previous studies,17,30 RDE (rotating disk electrode) possesses many advantages over the stationary electrode in study of electrochemically driven drugs metabolism. The most important one is that parameters during the measurements of the RDE are in a steady state. Because of the invariable rotation rate of RDE, the diffusion condition on its surface is stable, and the impact of the mass transfer process on the electrode kinetics is reduced, even can be ignored. Based on these superiorities, RDE was used throughout the studies of drugs metabolism. Upon successive addition of tramadol into aerobic 0.1 M, pH 7.4 PBS, the reduction current of the CYP2D6/PEI-RGO/ GCE was increased gradually at around −0.48 V (Figure 3A). After subtracting the background recorded before any addition of tramadol, the reduction current increased obviously and only one peak located at around −0.483 V observed corresponding to the enzymatic reaction of tramadol (inset in Figure 3A). For control experiments, no obvious responses were observed from the cyclic voltammogram curves when an equivalent aliquot (10 μL) of other reagents (water, ethanol, PBS) was injected into the same solution (Figure S4). Besides, only slightly response was detected on PEI-RGO/GCE upon the addition of tramadol into PBS (data not shown). Thus, the increase of reduction D
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Figure 4. (A) The ion chromatogram of pure tramadol. (B) The total ion chromatogram of the reaction mixture after 2 h of electrolysis (−0.520 V vs SCE) using CYP2D6/PEI-RGO/GCE in 20 mL of 0.1 M PBS pH 7.4 containing 300 μM tramadol. (C) Mass spectra of pure tramadol. (D) The corresponding mass spectra of the peak at 22.28 min in B.
The effective electrochemically-driven drug metabolism was confirmed by high performance liquid chromatography-mass spectrometry (HPLC-MS) analysis. At an applied potential of −0.520 V (vs SCE), electrolysis was carried out by the CYP2D6/PEI-RGO/GCE in the presence of 300 μM tramadol for 2 h. The total ion chromatograms of the tramadol solution before and after electrolysis were illustrated in Figure 4A and Figure 4B, respectively. There were two peaks at 21.47, 22.28 min after electrolysis, whereas only one peak at 21.44 min was observed in pure tramadol solution. The peak at 22.28 min should be attributed to the electric catalytic product of Odemethyl tramadol. Furthermore, the formation of the metabolized product, o-demethyl-tramadol, was confirmed by MS (Figure 4D). Corresponding to the peak at 22.28 min in total ion chromatogram, the MS peak at 250.18 (m/z) was just the molecular weight of o-demethyl-tramadol plus hydrogen ion. Results were in accordance with previous reports on the metabolic reactions of tramadol catalyzed by CYP2D6 in vivo.34 According to the peak area ratio in Figure 4B, the o-demethyltramadol yield approximated to 14.5%. Factors affected the yield may include two aspects. Through the linkage of GA, the electrochemical response of CYP2D6 on the electrode improved. However, covalent coupling may change the conformation of enzyme which affects product formation directly.35 On the other hand, oxygen is the necessary material when drug metabolism reaction happens via cyt P450s, and its amount that available to react is the indirect reason. 3.4. Inhibition of CYP2D6 Catalyzed Metabolism with Quinidine. Quinidine is identified as a strong inhibitor of CYP2D6. Its inhibitory influence on tramadol metabolism was investigated by gradually adding varying concentrations of quinidine (from 0 to 5.5 μM) into the aerobic PBS with the constant tramadol concentration of 200 μM. The inhibition curve (current inhibition rate at −0.483 V of the reduction
current in the presence of tramadol mainly resulted from the metabolism driven by the CYP2D6/PEI-RGO/GCE. In order to eliminate the influence of the dissolved oxygen on the reduction current, the current difference (ΔI) between the reduction currents in air saturated solution (I0) and after addition of substrate (I) was defined. Plots of ΔI at −0.483 V (vs SCE) versus the concentrations of the successive added tramadol showed a hyperbolic behavior which could be fitted well with the Michaelis−Menten model (Figure 3B). According to the meaning of the Michaelis−Menten equation,31 the apparent Michaelis−Menten constant Kmapp was calculated to be 23.85 μM. It was smaller than 210 μM, the value in previous report.29 This variability is, on one hand, related to the source of the cyt P450s (purified cyt P450s, microsomes, or hepatocytes), and on the other hand, associated with the conditions of the assay, for example the ratio between the cyt P450s and its reductase.32 The small Kmapp value revealed that CYP2D6 immobilized on the PEI-RGO/GCE had strong affinity toward tramadol. It is further suggested that CYP2D6 kept high enzymatic activity in this system. To investigate the effect of RDE rotation speeds on the mass transfer of enzymatic reaction, electrode rotation speed was changed during the electrocatalytic process by CYP2D6. The linear sweep voltammetry curves showed that the current was increased along with increasing of the electrode rotation speed (Figure 3C). The corresponding Koutecky−Levich plots (i−1 vs ω‑1/2) at different potentials showed good linearity (Figure 3D), which was consistent with the pseudo first-order reaction kinetics form. The heterogeneous reaction rate constant for the electrocatalytic oxygen reduction process was calculated33 to be 1.96 cm s−1 at −0.5 V, revealing that the equilibrium of this electrocatalytic oxygen reduction process can be achieved in a short time. E
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The direct electron transfer between electrode and the immobilized enzyme, electrochemical-driven tramadol conversion to o-demethyl-tramadol and inhibitory research of quinidine on tramadol metabolism were realized in this platform. Furthermore, this system was applied to other clinical drugs. The immobilized CYP2D6 on the PEI-RGO/GCE displayed high enzymatic activity and strong affinity toward various substrates. This electrochemical platform may have potentials for the assessment of interactions between drugs and food. Also, it may offer value in building vitro multienzyme system, which may be much closer to the model in vivo.
segment versus the concentration of quinidine) is plotted in Figure 5. The IC50 value (concentration of inhibitor producing
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ASSOCIATED CONTENT
S Supporting Information *
Figure S1−S4, the electrochemical responses and HPLC-MS analysis of seven clinical drugs. This material is available free of charge via the Internet at http://pubs.acs.org.
Figure 5. Inhibition effect of quinidine on the conversion from tramadol to o-demethyl-tramadol by CYP2D6/PEI-RGO/GCE. Changing in bioelectrocatalytic current at −0.483 V (vs SCE) upon addition of increasing concentration of quinidine.
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50% inhibition) of quinidine for the inhibition on O-demethyl of tramadol was calculated to be 0.693 μM. This was in good agreement with the IC50 value reported in the literature.36 3.5. The Application of CYP2D6/PEI-RGO/GCE to Other Drugs Metabolism. To test the potential application of the present method to other drugs metabolism, seven clinical drugs or chemicals were chosen and the electrochemical responses of CYP2D6/PEI-RGO/GCE were recorded after addition of these drugs into the electrolyte. The data were fitted to the Michaelis−Menten model. The Kmapp values for each drug were summarized in Table 1. The detailed electrochemical
*E-mail
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the National Basic Research Program of China (Grant 2010CB732400), the Key Program (Grant 21035002) from the National Natural Science Foundation of China, and the National Natural Science Foundation of China (Grant 21375014 and 21175021).
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Table 1. Comparison of Kmapp Values Measured in This Work with the Literature Reports drug tramadol metoprolol mexiletine metoclopramide phenacetin clomipramine phenformin yohimbine a
major metabolite O-demethyl tramadol29,30 hydroxy metoprolol37,38 hydroxy mexiletine39,40 deethylated metoclopramide41,42 deethylation phenacetin43 hydroxy clomipramine44 hydroxy phenformin45,46 hydroxy yohimbine47
Kmapp (μM)
Kmapp (μM) in literature
23.85
21029
9.85 14.67 8.65
26 ± 937 19.53 ± 4.9539 6.41±0.4241
43.46
785 ± 12543
7.86 26.22 42.95
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AUTHOR INFORMATION
Corresponding Author
ABBREVIATIONS GCE, glassy carbon electrode; CYP2D6, cytochrome P450 2D6; cyt P450s, cytochrome P450 enzymes; CPR, cyt P450 reductase; NADPH, nicotinamide adenine dinucleotide phosphate; GO, oxide graphene; RGO, reduced oxide graphene; PEI, polyethylenimine; PEI-RGO, PEI-functionalized graphene; PBS, Phosphate buffer solution; UV−vis, ultraviolet visible; TEM, transmission electron microscope; EDS, energy dispersive spectroscopy; SCE, saturated calomel electrode; RDE, rotating ring-disk electrode; HPLC, high performance liquid chromatography; MS, mass spectrometry
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REFERENCES
(1) Krishnan, S.; Schenkman, J. B.; Rusling, J. F. Bioelectronic delivery of electrons to cytochrome P450 enzymes. J. Phys. Chem. B 2011, 115, 8371−8380. (2) Gilardi, G.; Fantuzzi, A.; Sadeghi, S. J. Engineering and design in the bioelectrochemistry of metalloproteins. Curr. Opin. Struc. Biol. 2001, 11, 491−499. (3) Evans, W. E.; Relling, M. V. Pharmacogenomics: Translating functional genomics into rational therapeutics. Science 1999, 286, 487−491. (4) Willner, B.; Katz, E.; Willner, I. Electrical contacting of redox proteins by nanotechnological means. Curr. Opin. Biotechnol. 2006, 17, 589−596. (5) Mie, Y.; Suzuki, M.; Komatsu, Y. Electrochemically driven drug metabolism by membranes containing human cytochrome P450. J. Am. Chem. Soc. 2009, 131, 6646−6647. (6) Bistolas, N.; Wollenbergera, U.; Jung, C.; Scheller, F. W. Cytochrome P450 biosensorsA review. Biosens. Bioelectron 2005, 20, 2408−2423.
No reports had been found for comparison.
responses and HPLC-MS analysis were shown in the Supporting Information. From Table 1, the Kmapp values were different from that of the previous reports. The possible reasons were due to the use of different sources of the cyt P450s such as purified cyt P450s, microsomes, or hepatocytes, and the conditions of the assay.32 Anyhow, this strategy on the immobilization of CYP2D6 could keep high enzymatic activity and display strong affinity toward various substrates.
4. CONCLUSIONS In this work, a novel electrochemical platform for monitoring drug metabolism in vitro has been constructed by using PEIRGO nanocomposite as matrix for immobilization of CYP2D6. F
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