Oxidative Drug Metabolism Research Articles

Altered protein S-glutathionylation identifies a potential mechanism of resistance to acetaminophen-induced hepatotoxicity.

Acetaminophen (APAP) is the most commonly used over-the-counter analgesic. However, hepatotoxicity induced by APAP is a major clinical issue, and the factors that define sensitivity to APAP remain unclear. We have previously demonstrated that mice nulled for glutathione S-transferase Pi (GSTP) are resistant to APAP-induced hepatotoxicity. This study aims to exploit this difference to delineate pathways of importance in APAP toxicity. We used mice nulled for GSTP and heme oxygenase-1 oxidative stress reporter mice, together with a novel nanoflow liquid chromatography-tandem mass spectrometry methodology to investigate the role of oxidative stress, cell signaling, and protein S-glutathionylation in APAP hepatotoxicity. We provide evidence that the sensitivity difference between wild-type and Gstp1/2(-/-) mice is unrelated to the ability of APAP to induce oxidative stress, despite observing significant increases in c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation in wild-type mice. The major difference in response to APAP was in the levels of protein S-glutathionylation: Gstp1/2(-/-) mice exhibited a significant increase in the number of S-glutathionylated proteins compared with wild-type animals. Remarkably, these S-glutathionylated proteins are involved in oxidative phosphorylation, respiratory complexes, drug metabolism, and mitochondrial apoptosis. Furthermore, we found that S-glutathionylation of the rate-limiting glutathione-synthesizing enzyme, glutamate cysteine ligase, was markedly increased in Gstp1/2(-/-) mice in response to APAP. The data demonstrate that S-glutathionylation provides an adaptive response to APAP and, as a consequence, suggest that this is an important determinant in APAP hepatotoxicity. This work identifies potential novel avenues associated with cell survival for the treatment of chemical-induced hepatotoxicity.

Optimization of reaction parameters for the electrochemical oxidation of lidocaine with a Design of Experiments approach

Identification of potentially toxic oxidative drug metabolites is a crucial step in the development of new drugs. Electrochemical methods are useful to study oxidative drug metabolism, but are not widely used to synthesize metabolites for follow-up studies. Careful optimization of reaction parameters is important for scaling up the electrochemical synthesis of metabolites. In the present study, lidocaine was used as a drug compound in order to optimize electrochemical reaction parameters employing a design of experiments approach to improve the yield of N-dealkylated lidocaine, a major in vivo metabolite. pH and electrode material were found to have a major effect on the final yield.

Advance in studies on natural antidepressant drugs and cytochrome P450

In the fast pace of modern life and under the heavy work pressure, the prevalence of depression has increased year by year. Meanwhile, the demands for antidepressant drugs have also grown, especially the high-efficiency and low-toxicity natural antidepressant drugs, mainly including polyphenols, flavonoids, organic acids, alkaloids and terpenoids. Cytochrome P450 (CYP450) is a type of enzymes involving oxidative metabolism of drugs in vivo, and can change the pharmacokinetics and efficacy of drugs. Therefore, it is of important significant to define the effect of natural antidepressant drugs on CYP450 in human bodies, in order to improve the rational clinical medication, avoid drug interactions and reduce adverse reactions.

Prospects on the Hyphenated Electrochemistry and Mass Spectrometry as a Practical Analytical Technique in the Assessment of Oxidative Drug Metabolism

For several years, electrochemistry in combination with mass spectrometry has been the subject of attention and development for studying the oxidative drug metabolism at early stages of new drug development. Though the technique could successfully imitate the in vivo oxidative drug metabolism initiated by electron transfer, it lags in the imitation of reactions initiated by either hydrogen atom transfer or oxygen atom transfer. The prospect of using electrochemistry as a practical analytical technique in the imitation of oxidative drug metabolism, therefore, relies on the extension of its utility toward covering those reactions initiated by hydrogen atom transfer and oxygen atom transfer. In this brief critical review, I discuss potential electrochemical techniques that can benefit the application of electrochemistry beyond electron transfer reactions.

Professor Folke Sjöqvist is the 2013 BCPT Nordic Prize Winner in Basic & Clinical Pharmacology & Toxicology

Professor Folke Sjöqvist is the 2013 <scp>BCPT</scp> Nordic Prize Winner in Basic &amp; Clinical Pharmacology &amp; Toxicology

Frequency of CYP450 enzyme gene polymorphisms in the Greek population: review of the literature, original findings and clinical significance.

The cytochrome P450 (CYP450) enzyme family is involved in the oxidative metabolism of many therapeutic drugs and various endogenous substrates. These enzymes are highly polymorphic. Prevalence of CYP450 enzyme gene polymorphisms vary among different populations and substantial inter- and intra-ethnic variability in frequency of CYP450 enzyme gene polymorphisms has been reported. This paper provides an overview and investigation of CYP450 genotypic and phenotypic reports published in the Greek population.

Effects of artemisinin antimalarials on Cytochrome P450 enzymes in vitro using recombinant enzymes and human liver microsomes: potential implications for combination therapies

1. Cytochrome P450 enzyme system is the most important contributor to oxidative metabolism of drugs. Modification, and more specifically inhibition, of this system is an important determinant of several drug–drug interactions (DDIs).2. Effects of the antimalarial agent artemisinin and its structural analogues, artemether, artesunate and dihydroartemisinin, on seven of the major human liver CYP isoforms (CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6 and 3A4) were evaluated using recombinant enzymes (fluorometric assay) and human liver microsomes (LC–MS/MS analysis). Inhibitory potency (IC50) and mechanisms of inhibition were evaluated using nonlinear regression analysis. In vitro–in vivo extrapolation using the [I]/Ki ratio was applied to predict the risk of DDI in vivo.3. All compounds tested inhibited the enzymatic activity of CYPs, mostly through a mixed type of inhibition, with CYP1A2, 2B6, 2C19 and 3A4 being affected. A high risk of interaction in vivo was predicted if artemisinin is coadministrated with CYP1A2 or 2C19 substrates.4. With respect to CYP1A2 inhibition in vivo by artemisinin compounds, our findings are in line with previously published data. However, reported risks of interaction may be overpredicted and should be interpreted with caution.

Reduction of Hepatotoxicity Induced by Acetaminophen Overdoses in a Mouse Model of Inflammation Induced by Freund´s Adjuvants

Acetaminophen (APAP) is often used as an analgesic and antipyretic during the inflammatory process. Its toxicity in overdoses depends on the integrity of the hepatic cytochrome P450 (CYP). The oxidative drug metabolism mediated by CYP can be inhibited during inflammatory diseases or after use of immuno-stimulants drugs and vaccines. The objective of this work was to evaluate if inflammation is able to modulate the toxicity of APAP. Five female Balb/c mice were injected subcutaneously with Freund Complete Adjuvant (FCA), and boosted with Freund’s Incomplete Adjuvant (FIA) at 14th day. Then, they were treated with 360 mg/kg of acetaminophen orally during the 14th, 15th and 16th days. Convenient control groups were included with APAP administration without immuno-stimulation. Serum levels of IL-1β, TNFα, IFNγ, α-1-acid glycoprotein (α-1-AGP), alanine transaminase (ALT), aspartic acid aminotransferase (AST), lactate dehydrogenase (LDH) and hepatic CYP2E1 expression were measured. Inoculation site of adjuvants and liver histopathological responses were also evaluated. FCA/FIA injection produced acute inflammatory response in the inoculation site and increased serum levels of the pro-inflammatory cytokines, α-1-AGP and LDH with reduction of hepatic CYP2E1 expression. A reduction of liver damage induced by APAP overdoses was also observed, suggesting that inflammatory processes can be protective against APAP hepatotoxicity.

Oxidative Stress and Drugs of Abuse: An Update

Drug addiction is a public health and social burden. Presently, the most abused illicit substance is cannabis, fol- lowed by amphetamines, cocaine and opioids, with different prevalence in different countries. Several evidences support a role for oxidative stress in the toxicity induced by many drugs of abuse in different organs, such as the brain, heart, liver or kidneys. This leads to oxidation of important cellular macromolecules, and may culminate in cell dysfunction and death. In this review we describe the evidences for oxidative damage and depletion of antioxidants upon exposure to drugs of abuse, especially amphetamines, cocaine and opiates. We also discuss the sources of oxidative stress induced by drugs of abuse, including oxidative metabolism of drugs, oxidative metabolism of monoamines by monoamine oxidases or by auto-oxidation, mitochondrial dysfunction, excitotoxicity, microglial activation, inflammation, hyperthermia and the ef- fects of drug interactions. These consolidate oxidative stress as a relevant mechanism contributing for the cytotoxicity of drugs of abuse and for behavioral changes associated with drug addiction.

Protein Restoration in Low-Birth-Weight Rat Offspring Derived from Maternal Low-Protein Diet Leads to Elevated Hepatic CYP3A and CYP2C11 Activity in Adulthood

The World Health Organization has identified hypercholesterolemia to be one of the major symptoms encompassing the metabolic syndrome. Moreover, epidemiologic evidence indicates that low-birth-weight offspring are at greater risk of developing the metabolic syndrome. Previous work in our laboratory demonstrated that maternal protein restriction (MPR) results in impaired fetal growth and hypercholesterolemia in adulthood. This was attributed to repression of hepatic CYP7A1, a rate-limiting enzyme that catabolizes cholesterol to bile acids. Another important function of hepatic cytochrome P450 enzymes is the phase I oxidative metabolism of drugs (i.e., statins for hypercholesterolemia), which can significantly impact pharmacokinetics. We hypothesized that MPR offspring may have altered ability to metabolize drugs in adulthood. To address this hypothesis, we maintained Wistar rats on a 20% protein diet (control) or a low 8% protein diet throughout prenatal and postnatal life (LP1) or exclusively during prenatal life and weaning (LP2). Intriguingly CYP3A and CYP2C11 intrinsic clearance (Vmax/Km) was significantly increased exclusively in LP2 offspring at postnatal day 130 compared with control or LP1 offspring, as evaluated by testosterone enzyme kinetics in liver microsomes. The increase in activity was secondary to an increase in CYP3A23 and CYP2C11 mRNA. Collectively, these findings suggest that a low-birth-weight offspring with postnatal catch-up growth may have a diminished response to xenobiotics metabolized by CYP3A and CYP2C11 enzymes.

Computational analysis of CYP3A4-mediated metabolism to investigate drug interactions between anti-TB and anti-HIV drugs in HIV/TB co-infection

The treatment of human immunodeficiency virus (HIV)-related tuberculosis (TB) includes combination therapy, in which antiretroviral drugs and anti-TB drugs are co-administered. The complexities associated with the treatment of dual infection by Mycobacterium tuberculosis and HIV include occurrence of drug–drug interactions, in addition to pill burden, overlapping drug toxicity, and immune reconstitution inflammatory syndrome. Drug–drug interactions can occur between these drugs toward cytochrome P450 3A4 (CYP3A4), a drug-metabolizing enzyme. A thorough understanding of these interactions can prevent occurrence of treatment failures and drug toxicity. Molecular docking studies were carried out for FDA-approved drugs, to predict binding mechanism of anti-TB drugs with CYP3A4 and to compare them with our previous studies on antiretroviral drugs, in order to infer possible occurrence of drug–drug interactions. The core regimen of anti-TB treatment viz., rifampin (RIF), isoniazid (INH), and pyrazinamide (PZA) showed similar binding mode (i.e., competitive binding) via utilizing the same binding residue, Arg212 in CYP3A4. This regimen also shared similar binding mode with antiretroviral protease inhibitors except tipranavir, nelfinavir, lopinavir, and atazanavir. Contraindicated drug interactions were not observed between non-nucleoside reverse transcriptase inhibitors, delavirdine, efavirenz, and etravirine; and anti-TB drugs, RIF, INH, and PZA. The contraindications occurring within anti-TB drugs can be negated with inhibitory effect of INH to induction effect of RIF toward CYP3A4. We evaluated the importance of Arg212 along with Ser119, Ala370, Arg372, and heme moiety for mediating oxidative metabolism of drugs. These drug interaction details were incorporated into our web server by creating a database called “CYP3A4 DDIDB” and it is open accessible from http://cyp.bicpu.edu.in .

A proposal for a problem‐oriented pharmacobiochemistry course in dental education

Problem-oriented learning is an effective method of learning that increases students' learning motivation, improves the relationship amongst students and results in open-minded discussions. In this study, a new problem-oriented pharmacobiochemistry course related to 'oxidative metabolism of drugs by cytochrome P450 (CYP450) systems' was designed. Students were divided into seven groups. Three keywords related to drug interaction through CYP450 were provided to each group in order for them to conduct research on the information given. After 1month, the groups attended a session under the supervision of a tutor to solve a simulated problem case that was designed using the keywords. At the end of the integrated course, a multiple-choice examination was given. The success rate of 76 students who attended the course was found to be significantly higher than the success rate of the students who received the lecture-based course only (P<0.0001). A questionnaire containing 20 items (Cronbach's alpha: 0.92) was administered to the students to learn about their perception regarding this educational model. The questionnaire was evaluated using the Likert scale. Student feedback was very positive, with fourteen answers rated as 'agree' and the remaining six rated as 'strongly agree'. Students thought that the problem-oriented model was very enjoyable and useful in regard to dental education. Based on these results, we conclude that this course model may help achieve an integrated curriculum for dental school programmes.

Electrocatalytic oxidation of hydrogen peroxide on a platinum electrode in the imitation of oxidative drug metabolism of lidocaine

Electrochemistry in combination with mass spectrometry has shown promise as a versatile technique not only in the analytical assessment of oxidative drug metabolism, but also for small-scale synthesis of drug metabolites. However, electrochemistry is generally limited to reactions initiated by direct electron transfer. In the case of substituted-aromatic compounds, oxidation proceeds through a Wheland-type intermediate where resonance stabilization of the positive charge determines the regioselectivity of the anodic substitution reaction, and hence limits the extent of generating drug metabolites in comparison with in vivo oxygen insertion reactions. In this study, we show that the electrocatalytic oxidation of hydrogen peroxide on a platinum electrode generates reactive oxygen species, presumably surface-bound platinum-oxo species, which are capable of oxygen insertion reactions in analogy to oxo-ferryl radical cations in the active site of Cytochrome P450. Electrochemical oxidation of lidocaine at constant potential in the presence of hydrogen peroxide produces both 3- and 4-hydroxylidocaine, suggesting reaction via an arene oxide rather than a Wheland-type intermediate. No benzylic hydroxylation was observed, thus freely diffusing radicals do not appear to be present. The results of the present study extend the possibilities of electrochemical imitation of oxidative drug metabolism to oxygen insertion reactions.

Novel CYP3A4 intron 6 single nucleotide polymorphism is associated with simvastatin-mediated cholesterol reduction in The Rotterdam Study

CYP3A4 is involved in the oxidative metabolism of many drugs and xenobiotics including the HMG-CoA reductase inhibitor simvastatin. The objective of this study was to investigate whether a new CYP3A4 functional single nucleotide polymorphism (SNP) in intron 6 (CYP3A4*22) modifies the effect of simvastatin on total cholesterol (TOTc) or LDL cholesterol (LDLc) reduction in a population-based cohort study. In a total of 80 incident simvastatin users, the association between the CYP3A4 intron 6 C>T SNP (rs35599367) and reduction in cholesterol levels was analyzed using linear regression analysis and adjusting for potential confounding factors. The CYP3A4*22 allele was associated with a trend towards a stronger simvastatin lipid-lowering response, as reflected by the greater reduction in both TOTc and LDLc levels when compared with homozygous wild type. We observed that the CYP3A4*22 allele carriers had an increased reduction in TOTc and LDLc: -0.25 mmol/l (95% confidence interval [CI(95%)]=[-0.52; 0.01], P=0.058) and -0.29 mmol/l (CI(95%)=[-0.58; 0.01], P=0.054) when compared with homozygous CC. When we adjusted the model for potential confounding factors, the corresponding reduction in TOTc was -0.31 mmol/l (CI(95%)=[-0.59;-0.04], P=0.028) and for LDLc -0.34 mmol/l (CI(95%)=[-0.66; -0.02], P=0.034) greater for CYP3A4*22 allele carriers when compared with homozygotes wild type. The CYP3A4*22 intron 6 SNP T-variant allele was associated with reduced CYP3A4 activity, resulting in a better lipid lowering response to simvastatin, when data were adjusted for confounding factors. This observation is a step towards the clarification of the reasons of interindividual variability in statins response and may potentially lead to improved tailoring of simvastatin therapy.

Oxidative Metabolism of a Quinoxaline Derivative by Xanthine Oxidase in Rodent Plasma

As part of efforts directed at the G protein-coupled receptor 119 agonist program for type 2 diabetes, a series of cyanopyridine derivatives exemplified by isopropyl-4-(3-cyano-5-(quinoxalin-6-yl)pyridine-2-yl)piperazine-1-carboxylate (1) were identified as novel chemotypes worthy of further hit-to-lead optimization. Compound 1, however, was found to be unstable in plasma (37 °C, pH 7.4) from rat (T(1/2) = 16 min), mouse (T(1/2) = 61 min), and guinea pig (T(1/2) = 4 min). Lowering the temperature of plasma incubations (4-25 °C) attenuated the degradation of 1, implicating the involvement of an enzyme-mediated process. Failure to detect any appreciable amount of 1 in plasma samples from protein binding and pharmacokinetic studies in rats was consistent with its labile nature in plasma. Instability noted in rodent plasma was not observed in plasma from dogs, monkeys, and humans (T(1/2) > 370 min at 37 °C, pH 7.4). Metabolite identification studies in rodent plasma revealed the formation of a single metabolite (M1), which was 16 Da higher than the molecular weight of 1 (compound 1, MH(+) = 403; M1, MH(+) = 419). Pretreatment of rat plasma with allopurinol, but not raloxifene, abolished the conversion of 1 to M1, suggesting that xanthine oxidase (XO) was responsible for the oxidative instability. Consistent with the known catalytic mechanism of XO, the source of oxygen incorporated in M1 was derived from water rather than molecular oxygen. The formation of M1 was also demonstrated in incubations of 1 with purified bovine XO. The structure of M1 was determined by NMR analysis to be isopropyl-4-(3-cyano-5-(3-oxo-3,4-dihydroquinoxalin-6-yl)pyridine-2-yl)piperazine-1-carboxylate. The regiochemistry of quinoxaline ring oxidation in 1 was consistent with ab initio calculations and molecular docking studies using a published crystal structure of bovine XO. A close-in analogue of 1, which lacked the quinoxaline motif (e.g., 5-(4-cyano-3-methylphenyl)-2-(4-(3-isopropyl-1,2,4-oxadiazol-5-yl)piperidin-1-yl)nicotinitrile (2)) was stable in rat plasma and possessed substantially improved GPR119 agonist properties. To the best of our knowledge, our studies constitute the first report on the involvement of rodent XO in oxidative drug metabolism in plasma.

Effect of a New Functional CYP3A4 Polymorphism on Calcineurin Inhibitors‘ Dose Requirements and Trough Blood Levels in Stable Renal Transplant Patients

CYP3A4 is involved in the oxidative metabolism of many drugs and xenobiotics including the immunosuppressants tacrolimus (Tac) and cyclosporine (CsA). The objective of the study was to assess the potential influence of a new functional SNP in CYP3A4 on the pharmacokinetic parameters assessed by dose requirements and trough blood levels of both calcineurin inhibitors (CNI) in stable renal transplant patients. A total of 99 stable renal transplant patients receiving either Tac (n = 49) or CsA (n = 50) were genotyped for the CYP3A4 intron 6 C>T (rs35599367) and CYP3A5*3 SNPs. Trough blood levels ([Tac](0) or [CsA](0) in ng/ml), dose-adjusted [Tac](0) or [CsA](0) (ng/ml per mg/kg bodyweight) as well as doses (mg/kg bodyweight) required to achieve target concentrations were compared among patients according to allelic status for CYP3A4 and CYP3A5. Dose-adjusted concentrations were 2.0- and 1.6-fold higher in T-variant allele carriers for the CYP3A4 intron 6 C>T SNP compared with homozygous CC for Tac and CsA, respectively. When CYP3A4/CYP3A5 genotypes were combined, the difference was even more striking as the so-defined CYP3A poor metabolizer group presented dose-adjusted concentration 1.6- and 4.1-fold higher for Tac, and 1.5- and 2.2-fold higher for CsA than the intermediate metabolizer and extensive metabolizer groups, respectively. Multiple linear regression analysis revealed that, taken together, both CYP3A4 intron 6 and CYP3A5*3 SNPs explained more than 60 and 20% of the variability observed in dose-adjusted [Tac](0) and [CsA](0), respectively. The CYP3A4 intron 6 C>T polymorphism is associated with altered Tac and CsA metabolism. CYP3A4 intron 6 C>T along with CYP3A5*3 (especially for Tac) pharmacogenetic testing performed just before transplantation may help identifying patients at risk of CNI overexposure and contribute to limit CNI-related nephrotoxicity by refining the starting dose according to their genotype. Original submitted 5 May 2011; Revision submitted 29 June 2011.

Oxidative stress and hematological profiles of advanced breast cancer patients subjected to paclitaxel or doxorubicin chemotherapy

Several adverse effects of chemotherapy treatments have been described, and most of these effects are associated with direct interactions between blood cells and indirect effects generated during the oxidative metabolism of antineoplastic drugs. In this study we evaluated the oxidative systemic status and hematological profiles of breast cancer patients with advanced ductal infiltrative carcinoma treated with doxorubicin (DOX) or paclitaxel (PTX) within 1 h after chemotherapy. Blood analyses included evaluation of hemogram, pro-oxidative markers, and antioxidant status. The results showed that advanced breast cancer diseased (AD) patients without previous chemotherapy presented anemia and high oxidative stress status characterized by elevated levels of lipid peroxidation and nitric oxide, and reduced catalase activity when compared with controls. DOX-treated patients exhibited increased anemia and reduced antioxidant status, which was revealed by decreases in reduced glutathione levels and the total antioxidant capacity of plasma; however, these changes did not lead to further increases in lipid peroxidation or carbonyl proteins when compared with the AD group. PTX-treated patients also showed increased anemia, lactate dehydrogenase leakage, and enhanced lipid peroxidation. These data reveal for the first time that patients subjected to chemotherapy with DOX or PTX present immediate systemic oxidative stress and red blood cell oxidative injury with anemia development. These findings provide a new perspective on the systemic redox state of AD and patients subjected to chemotherapy regarding oxidative stress enhancement and its possible involvement in the aggravation of chronic anemia.

Electrochemical Oxidation by Square-Wave Potential Pulses in the Imitation of Oxidative Drug Metabolism

Electrochemistry combined with mass spectrometry (EC-MS) is an emerging analytical technique in the imitation of oxidative drug metabolism at the early stages of new drug development. Here, we present the benefits of electrochemical oxidation by square-wave potential pulses for the oxidation of lidocaine, a test drug compound, on a platinum electrode. Lidocaine was oxidized at constant potential and by square-wave potential pulses with different cycle times, and the reaction products were analyzed by liquid chromatography-mass spectrometry [LC-MS(/MS)]. Application of constant potentials of up to +5.0 V resulted in relatively low yields of N-dealkylation and 4-hydroxylation products, while oxidation by square-wave potential pulses generated up to 50 times more of the 4-hydroxylation product at cycle times between 0.2 and 12 s (estimated yield of 10%). The highest yield of the N-dealkylation product was obtained at cycle times shorter than 0.2 s. Tuning of the cycle time is thus an important parameter to modulate the selectivity of electrochemical oxidation reactions. The N-oxidation product was only obtained by electrochemical oxidation under air atmosphere due to reaction with electrogenerated hydrogen peroxide. Square-wave potential pulses may also be applicable to modulate the selectivity of electrochemical reactions with other drug compounds in order to generate oxidation products with greater selectivity and higher yield based on the optimization of cycle times and potentials. This considerably widens the scope of direct electrochemistry-based oxidation reactions for the imitation of in vivo oxidative drug metabolism.

Bioelectronic Delivery of Electrons to Cytochrome P450 Enzymes

Cytochrome P450s (cyt P450s) are the major oxidative enzymes in human oxidative metabolism of drugs and xenobiotic chemicals. In nature, the iron heme cyt P450s utilize oxygen and electrons delivered from NADPH by a reductase enzyme to oxidize substrates stereo- and regioselectively. Significant research has been directed toward achieving these events electrochemically. This Feature Article discusses the direct electrochemistry of cyt P450s in thin films and the utilization of such films for electrochemically driven biocatalysis. Maintaining and confirming structural integrity and catalytic activity of cyt P450s in films is an essential feature of these efforts. We highlight here our efforts to elucidate the influence of iron heme spin state and secondary structure of human cyt P450s on voltammetric and biocatalytic properties, using methodologies to quantitatively describe the dynamics of these processes in thin films. We also describe the first cyt P450/reductase films that accurately mimic the natural biocatalytic pathway and show how they can be used with voltammetry to elucidate key mechanistic features. Such bioelectronic cyt P450 systems have high value for future drug development, toxicity screening, fundamental investigations, and chemical synthesis systems.

Electrochemistry in the Mimicry of Oxidative Drug Metabolism by Cytochrome P450s

Prediction of oxidative drug metabolism at the early stages of drug discovery and development requires fast and accurate analytical techniques to mimic the in vivo oxidation reactions by cytochrome P450s (CYP). Direct electrochemical oxidation combined with mass spectrometry, although limited to the oxidation reactions initiated by charge transfer, has shown promise in the mimicry of certain CYP-mediated metabolic reactions. The electrochemical approach may further be utilized in an automated manner in microfluidics devices facilitating fast screening of oxidative drug metabolism. A wide range of in vivo oxidation reactions, particularly those initiated by hydrogen atom transfer, can be imitated through the electrochemically-assisted Fenton reaction. This reaction is based on O-O bond activation in hydrogen peroxide and oxidation by hydroxyl radicals, wherein electrochemistry is used for the reduction of molecular oxygen to hydrogen peroxide, as well as the reduction of Fe(3+) to Fe(2+). Metalloporphyrins, as surrogates for the prosthetic group in CYP, utilizing metallo-oxo reactive species, can also be used in combination with electrochemistry. Electrochemical reduction of metalloporphyrins in solution or immobilized on the electrode surface activates molecular oxygen in a manner analogous to the catalytical cycle of CYP and different metalloporphyrins can mimic selective oxidation reactions. Chemoselective, stereoselective, and regioselective oxidation reactions may be mimicked using electrodes that have been modified with immobilized enzymes, especially CYP itself. This review summarizes the recent attempts in utilizing electrochemistry as a versatile analytical and preparative technique in the mimicry of oxidative drug metabolism by CYP.