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Enzyme-Based Amperometric Platform to Determine the Polymorphic Response in Drug Metabolism by Cytochromes P450 Paola Panicco,† Vikash R Dodhia,† Andrea Fantuzzi,† and Gianfranco Gilardi*,‡ † ‡
Division of Molecular Biosciences, Imperial College London, South Kensington, London SW7 2AZ, U.K. Department of Human and Animal Biology, University of Torino, Torino, Italy ABSTRACT: “Personalized medicine” is a new concept in health care, one aspect of which defines the specificity and dosage of drugs according to effectiveness and safety for each patient. Dosage strongly depends from the rate of metabolism which is primarily regulated by the activity of cytochrome P450. In addition to the need for a genetic characterization of the patients, there is also the necessity to determine the drug-clearance properties of the polymorphic P450 enzyme. To address this issue, human P450 2D6 and 2C9 were engineered and covalently linked to an electrode surface allowing fast, accurate, and reliable measurements of the kinetic parameters of these phase-1 drug metabolizing polymorphic enzymes. In particular, the catalytic activity of P450 2C9 on the electrode surface was found to be improved when expressed from a gene-fusion with flavodoxin from Desulfovibrio vulgaris (2C9/FLD). The results are validated using marker drugs for these enzymes, bufuralol for 2D6, and warfarin for 2C9/FLD. The platform is able to measure the same small differences in KM, and it allows a fast and reproducible mean to generated the product identified by HPLC from which the kcat is calculated.
I
t is well-recognized that patients administered a particular drug will exhibit significant interindividual variability in their response to treatment. Unfortunately some patients will fail to respond to the therapy entirely, while some others will suffer dose-related side effects, resulting in significant costs and fatalities1-4 (Figure 1). For these reasons, polymorphism in genes encoding the drug metabolizing cytochromes P450 is a very important factor that can no longer be neglected in the development of new drugs.5-8 Progress in the human genome analysis has recently made it possible to identify a patient’s cytochromes P450 make up by genotype analysis using the AmpliChip CYP450 Test available from Roche Diagnostics.9 However, genotyping needs a parallel enzyme-based platform capable of rapidly measuring a drug’s pharmacokinetics and clearance by the polymorphic P450 enzymes typical for a given genotype, and to this date, such platform is not available; this is the subject of this paper. Such a platform could be used during the drug discovery process to determine the performance of the polymorphic P450 with respect to new potential drugs. This information would then be translated in to a specific dosage for each polymorphic variant leading to tailored dosages for each patient with known genotypic makeup. Two P450 isoforms are key in polymorphism in drug metabolism: CYP2C9 and CYP2D6 (Figure 2a and b). Interindividual variability in the enzymatic activity of CYP2D6 due to genetic polymorphisms was first recognized as early as the 1970s,10,11 and this often results in concentrations of drugs in the plasma at levels that are either subtherapeutic or toxic.12-14 Two allelic r 2011 American Chemical Society
variants, 2D6.2 (R296C, S486T) and 2D6.17 (T107I, R296C and S486T), have been linked to a decrease in enzymatic activity in vitro and in vivo.15-22 These alleles differ significantly in their interethnic prevalence, with CYP2D6*17 being particularly high in Black Africans (up to 34%), and , in Caucasians (22-34%).23 Previous in vitro studies on the clearance of the beta-blocker bufuralol by the 2D6.2 and 2D6.17 polymorphic variants have shown that, when compared to the wild-type (2D6.1), the KM of bufuralol is increased up to two-fold in the case of the 2D6.2 and up to four-fold in the case of the 2D6.17.15,18,19 Furthermore, these studies showed that the kcat is unchanged with 2D6.2 and is reduced by 50% for the 2D6.17. CYP2C9 is involved in the metabolism of ∼16% of therapeutically important drugs such as the anticoagulant warfarin, hypoglycemic tolbutamide and glipzide, anticonvulsant phenytoin, and a number of nonsteroidal anti-inflammatory drugs.24 Several in vitro studies identified CYP2C9.2 (R144C) and CYP2C9.3 (I359L) as the two main allelic variants of this isoform and showed a reduced catalytic activity with increased KM values and/or decreased maximum rate of metabolism (kcat) with a resulting decreased intrinsic clearance.25-29 Their interethnic distribution appears to be similar, with the two allelic variants being mainly identified in Caucasians (35%). Few cases have been observed in African-American and Asiatic population (below 3%) for the CYP2C9.3, while the CYP2C9.2 has been found absent in the East-Asian population.30 Received: November 20, 2010 Accepted: February 4, 2011 Published: February 24, 2011 2179
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Figure 1. Impact of cytochromes P450 pharmacogenomics on drug dosage. The P450 genotype identification (top) allows separating patients (left) in different groups (right). For a given drug dose, they will have different concentrations of drug in the blood. The proposed amperometric platform allows the determination of the performance of the polymorphic P450 (bottom) to ensure effective and safe action of the drug.
Figure 2. Construction of P450 2C9 and 2D6 polymorphic variants. (a) Structure of CYP2C9 highlighting the mutations for the CYP2C9.2 (R144C) and CYP2C9.3 (I359L) allelic variants. The heme is shown in red, the I359, in yellow, the R144, in blue (PDB: 1R90).52 (b) Structure of CYP2D6 highlighting the mutations for the 2D6.2 (R296C, S486T) and 2D6.17 (T107I, R296C, and S486T) allelic variants. The heme is shown in red, the R296, in blue, the T107, in green, the S486, in yellow (PDB: 2F9Q).53 (c) Typical absorption spectra of CYP2C9.1FLD and its allelic variants (CYP2C9.2FLD and CYP2C9.3FLD) in their oxidized (black line), reduced (dotted line), and reduced after CO bubbling (gray line) forms. Here CYP2C9.3FLD is shown.
Several reports can be found in the most recent literature on the electrochemical characterization of cytochrome P450s, and in some cases, their potential exploitation for the characterization of P450 specific substrates.31-34 Our laboratory has previously reported the successful covalent immobilization of these enzymes on gold electrodes by using self-assembled monolayers to achieve controlled orientation.35 The immobilized enzymes are catalytically active by using the electrode as a source of electrons in place of NADPH. The proportionality between the measured current and the substrate turnover allows the determination of the kinetic parameters characteristic of the P450 for the particular substrate.36,37 This paper presents a new amperometric platform that allows metabolic profiling for polymorphic P450 enzymes allowing the direct measurement of KM and the indirect determination of kcat leading to link the genotype profile of an individual with the personalized dosage of a drug (Figure 3).
The platform presented consists of a disposable strip of eight wells each with three electrodes. The central working electrode has a covalently linked polymorphic P450 enzyme. The catalytic current generated by the P450 allelic variant when interacting with a drug allows the calculation of its kinetic parameters. We show that the small differences in the metabolism of bufuralol and warfarin by 2D6.2 and 2D6.17 or 2C9.2 and 2C9.3 allelic variants, respectively, are detected by the platform. This opens new avenues in the drug discovery process for the benefit of personalized medicine.
’ EXPERIMENTAL SECTION Materials. Chemicals were purchased from Sigma-Aldrich (UK) unless otherwise stated. Electrochemical measurements were carried out at 25 °C in 50 mM potassium phosphate pH 7.4 2180
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Figure 3. Schematic representation of the polymorphic P450 amperometric platform. The P450 enzyme is immobilized on the polycrystalline gold working electrode (WE, yellow) at the bottom of each of the eight parallel 400 μL wells. Platinum counter-electrode (CE, black) and carbon referenceelectrodes (RE, gray) are also present in each well. The catalytic current is measured upon substrate titration by a potentiostat simultaleously for the eight wells. Michaelis-Menten curves are elaborated by a PC, and from their fitting, the KMapp is calculated. Further HPLC analysis of product formation leads to the calculation of kcat from which the intrinsic clearance Clint can be derived as kcat/KMapp.
containing 500 mM potassium chloride and a 500 μM stock solution of (()-bufuralol hydrochloride and (S)-warfarin in 50 mM potassium phosphate pH 7.4 containing 500 mM potassium chloride was used throughout. Electrochemical measurements were performed using an Autolab PGSTAT12 potentiostat and the GPES software (Eco Chemie, The Netherlands). All potentials are reported versus the standard hydrogen electrode (NHE). Data fittings were carried out using Sigmaplot (Sytat Inc., USA) Expression and Purification of the Variants. The CYP2D6*1 gene in the pCW2D6 (4xHis) plasmid was modified using PCR site-directed mutagenesis to introduce the R296C and S486T mutations resulting in the CYP2D6*2 gene and the T107I, R296C, and S486T mutations resulting in the CYP2D6*17 gene. All the genes were cloned in the pCW vector. The 2D6.1, 2D6.2, and 2D6.17 enzymes were expressed and purified as previously described38,39 except that 1 mM tris(2carboxyethyl)phosphine (Thermo Fisher, UK) was used as the reducing agent. We previously established that CYP2C9 gives a better electrochemical response when genetically fused to the electron transfer protein flavodoxin (FLD) from Desulfovibrio vulgaris.36 Therefore the CYP2C9*1 gene in the pCW2C9FLD (4xHis) plasmid carrying the 2C9FLD chimera gene was modified using site-directed mutagenesis to introduce the R144C mutation resulting in the CYP2C9*2 gene and the I359L mutation resulting in the CYP2C9*3 gene. The 2C9.1FLD, 2C9.2FLD, and 2C9.3FLD were expressed and purified as previously described.38,39 Protein purity and integrity was verified by SDS-PAGE and UV-visible spectroscopy, respectively.
Electrode Preparation, Protein Immobilization, and Voltammetric Studies. The purified proteins were immobilized on
an amperometric platform designed and developed by Conductive Technologies Inc. The prototype consisted of a polypropylene strip with eight wells containing a sputtered polycrystalline gold working electrodes, a screen printed silver chloride reference electrodes, and a screen printed carbon paste counter electrode. The gold surface was electrochemically cleaned as previously described.40 Different methods of immobilization were performed for each isoform. In the case of 2D6, 2D6.2, and 2D6.17, the cleaned surface was modified sequentially to render the surface reactive toward cysteine groups on the protein, with an ethanolic solution of 1 mM 6-aminohexanethiol (NBS Biologicals, UK) for 48 h and 3 mM N-succinimidyl-3-maleimido propionic acid (in acetonitrile) for 2 h. Following modification, protein immobilization on the electrode proceeded by immersion in a 40 μM solution of protein overnight at 4 °C. In the case of 2C9.1FLD, 2C9.2FLD, and 2C9.3FLD, the cleaned surface was modified by immersion in 1 mM 6-hexanethiol and 7-mercapto-heptanoic acid in ethanol for 48 h. The electrode was then sequentially rinsed with ethanol, Milli-Q water, and buffer MES. The electrode was then immersed into a solution of 2 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and 5 mM NHS (N-hydroxysuccinimide) for 15 min. Immediately after, the electrode surface was rinsed with Milli-Q water and 30 μL of the protein solution (5 μM) were deposited. The electrode was then left at 4 °C for 1 h. 2181
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Figure 4. (a) Three step immobilization of CYP2D6 (P450) to gold electrodes (Au) after sequential deposition of 6-aminohexanethiol (6AHT) and Nsuccinimidyl-3-maleimido propionic acid (MALM) as spacers. (b) Two step immobilization of CYP2C9FLD (P450) to gold electrode (Au) after deposition of a mixture of 6-hexanethiol (6HT) and 7-mercaptoheptanoic acid (7MHA). The carboxy group of the 7MHA was activated by 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide) (EDC) and N-hydroxysuccinimide (NHS) prior binding the cytochrome P450 enzyme (P450).
The protein-modified electrodes were washed and stored in 50 mM potassium phosphate pH 7.4 containing 500 mM potassium chloride at 4 °C. The surface chemistry was verified and confirmed at all steps of the immbolisation procedure by FT-IR spectroscopy using a Bruker Tensor 27 FT-IR spectrometer with a p-polarized light incident at grazing angle (75° from the surface normal) with grazing angle accessory (Harrick, USA) as previously describe in Mak et al.35 Anaerobic studies were carried out at 2C9.2 > 2C9.3] (Figure 5g-i). Interestingly, this ranking is in agreement with previous literature data obtained from conventional enzyme incubation studies in solution.26 This finding is very encouraging because it indicates that the bioelectrochemical method described in this paper is able to easily extract the same information obtained from lengthy conventional procedures. Similarly CYP2D6 and its allelic variants showed a typical Michaelis-Menten behavior upon addition of increasing concentrations of the substrate (()-bufuralol (Figure 5d-f). Values of KMapp of 8.89, 16.62, and 26.24 μM were calculated for the ( ()-bufuralol 10 -hydroxylation by the immobilized 2D6.1, 2D6.2, and 2D6.17, respectively. It is striking to observe how the amperometry allows discerning the small differences in affinity between the allelic variants, with values that closely approach those previously reported in the literature by using traditional methodologies19 (Table 1). Moreover it is also important to note that also for the CYP2D6 the ranking of the KMapp values [2D6.17 > 2D6.2 > 2D6.1] is in agreement with previous literature reports on the proteins studies in solution.15,19,46 The kcat of the immobilized enzymes was determined from the measurement of the product formed in the well during the amperometric measurement and not from the maximal bioelectrocatalytic 2184
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Analytical Chemistry current of the Michaelis-Menten plot as the latter is affected by the uncoupling intrinsic to the P450 turnover.36,37 Therefore the formation of 7-hydroxy warfarin from S-warfarin by immobilized CYP2C9 was measured by HPLC. Control experiments carried out with the SAM derivatized electrodes in the absence of immobilized CYP2C9 did not lead to product formation. Quantification of the 7-hydroxy warfarin formed by immobilized CYP2C9FLD allowed the calculation of the kcat values for 2C9.1FLD, 2C9.2FLD, and 2C9.3FLD of 15.75, 6.85, and 2.17 min-1, respectively (Table 1). It must be noted that the kcat values available in the literature are rather dispersed. Nevertheless the constant pattern observed when comparing these values is their ranking,26 that in the specific case studied here is in the order [2C9.1 > 2C9.2 > 2C9.3]. This same ranking is measured in the present study by amperometry. Moreover our data indicate that the turnover of the 2C9.2FLD and 2C9.3FLD is decreased by 56% and 86% respectively in line with what observed in the literature.26,28,47 The intrinsic clearance (CLint) of S-warfarin by the immobilized 2C9.1, 2C9.2, and 2C9.3 allelic variants calculated as kcat/KMapp resulted to be 18.75, 3.89, and 0.35 μL 3 min-1 3 pmol-1 for 2C9.1FLD, 2C9.2FLD, and 2C9.3FLD, respectively. Therefore, the CLint for the 2C9.2 and 2C9.3 variants are decreased by 80% and 98%, respectively, when compared to the wild-type 2C9.1. These changes in the intrinsic clearance of S-warfarin by 2C9.2 and 2C9.3 are comparable to those reported in previous studies.26,28 HPLC was also used to quantify the formation of 1-hydroxy bufuralol by the immobilized CYP2D6. Also in this case no product formation was observed in the absence of immobilized protein. The kcat values determined amperometrically for this reaction were found to be 24.3, 20.9, and 14.7 min-1 for 2D6.1, 2D6.2, and 2D6.17, respectively. The kcat values measured for the bufuralol 10 -hydroxylation by the immobilized 2D6.1 were found to be comparable to those observed previously in the literature where kcat values of 21, 22.4, and 26.2 min-1 have been reported.48-50 The bioelectrocatalytic kcat for the immobilized 2D6.2 allelic variant was comparable to that of the wild-type enzyme (2D6.1) indicating that the R296C and S486T mutations in the 2D6.2 allelic variant result only in an increased KMapp and have no effect on kcat: this has also been shown to be the case in previous in vitro literature.15,18,19 As for 2D6.17, the kcat measured from the amperometric catalysis was 60% of that measured for the wild-type enzyme (2D6.1). Therefore, the T107I, R296C, and S486T mutations in the 2D6.17 allelic variant affected both the turnover and the KMapp: also, these results are in line with previous in vitro literature studies that have demonstrated that in the 2D6.17 allelic variant the single nucleotide mutations result in an increased KM and decreased kcat.18,19 The intrinsic clearances (CLint) of (()-bufuralol by the immobilized 2D6.1, 2D6.2, and 2D6.17 allelic variants (i.e., kcat/ KMapp) were calculated to be 2.79, 1.26, and 0.57 μL 3 min-1 3 pmol-1 for the 2D6.1, 2D6.2, and 2D6.17, respectively. Therefore, with respect to the wild-type 2D6.1 variant, the CLint of the 2D6.2 and 2D6.17 variants are reduced by 55% and 80%, respectively. Again, these changes in the intrinsic clearance of (()-bufuralol by 2D6.2 and 2D6.17 are comparable to those reported in previous studies and represent variants with moderately and significantly reduced activity.18,19,21
’ CONCLUSIONS The amperometric platform presented in this paper can rapidly and accurately determine the changes in the KM, and it
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generates product for the kcat and intrinsic clearance (CLint) determination of bufuralol 10 -hydroxylation and warfarin 7-hydroxylation linked to the effect of genetic polymorphism in the human cytochromes P450 2D6 and 2C9 enzymes, affecting the pharmacokinetics and clearance of drugs that are substrates of these enzymes. At present, pharmacogenomics is used to remove new drugs whose metabolism depends primarily on polymorphic drug metabolizing enzymes in order to eliminate a potential source of adverse drug reactions. However, this deprives some patients of potentially life saving treatments. The use of the amperometric platform, used in parallel with the genotype characterization, offers a new approach where the one-dose-fits-all “dogma” is substituted with a personalized dosage.5 For decades, clinicians have used a trial and error approach to determine drug dosage. Today there is a strong drive to move away from the trial and error approach to an individualized therapy with well-controlled and monitored clinical trials, but in reality the evaluation of the genetic risk factors for adverse drug reaction in marketed drugs is based on voluntary reporting at clinician level.51 The use of the amperometric platform of Figure 3 is not envisaged to be at the level of routine clinical practice, where gene-typing is necessary, but it would fit at the level of the pharmaceutical research lab. Here it will allow the routine evaluation of the effect of cytochromes P450 polymorphisms on a drug’s pharmacokinetics and clearance during the drug development process in a standardized manner. The information collected with the proposed approach would be crucial in defining the dosage setting for subsets of the population based on cytochromes P450 genotype. This provides data for inclusion in the safety sheets of all drugs. This “personalized” dose setting would result in significant decrease in either poor clinical responses or adverse drug reactions due to subtherapeutic or toxic plasma drug concentrations respectively.
’ AUTHOR INFORMATION Corresponding Author
*Phone: (þ) 39 011 6704593. Fax: (þ) 39 011 6704643. E-mail:
[email protected].
’ ACKNOWLEDGMENT This work was supported by NanoBioDesign Ltd. (UK) and the CIPE programme of the Piedmont Region (Italy). ’ REFERENCES (1) Lazarou, J.; Pomeranz, B. H.; Corey, P. N. J.A.M.A 1998, 279, 1200–1205. (2) Pirmohamed, M.; James, S.; Meakin, S.; Green, C.; Scott, A. K.; Walley, T. J.; Farrar, K.; Park, B. K.; Breckenridge, A. M. B.M.J. 2004, 329, 15–19. (3) Kirchheiner, J.; Brockmoller J. Clin. Pharmacol. Ther. 2005, 77, 1–16. (4) Kirchheiner, J.; Seeringer, A. Biochim. Biophys. Acta 2007, 1770, 489–494. (5) Evans, W. E.; Relling, M. V. Nature 2004, 429, 464–468. (6) Eichelbaum, M.; Ingelman-Sundberg, M.; Evans, W. E. Annu. Rev. Med. 2006, 57, 119–137. (7) Zanger, U. M.; Turpeinen, M.; Klein, K.; Schwab, M. Anal. Bioanal. Chem. 2008, 392, 1618–2642. (8) Ingelman-Sundberg, M.; Sim, S. C.; Gomez, A.; RodriguezAntona, C. Pharmacol. Ther. 2007, 116, 496–526. 2185
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