Drug-drug interactions and cooperative effects detected in electrochemically driven human cytochrome P450 3A4

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Drug-drug interactions and cooperative effects detected in electrochemically driven human cytochrome P450 3A4

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Drug-drug interactions and cooperative effects detected in

electrochemically driven human cytochrome P450 3A4

CORRESPONDING AUTHOR

*G. Gilardi, Department of Life Sciences and Systems Biology, Via Accademia Albertina 13, 10123 Turin, Italy. Phone: +39-011-6704593, Fax: +39-011-6704643, E-mail: gianfranco.gilardi@unito.it

ABSTRACT

Inhibition of cytochrome P450-mediated drug metabolism by a concomitantly administered second drug is one of the major causes of drug–drug interactions in humans. The present study reports on the first electrochemically-driven drug-drug interactions of human cytochrome P450 3A4 probed with erythromycin, ketoconazole, cimetidine, diclofenac and quinidine. Cytochrome P450 3A4 was immobilized on glassy carbon electrodes in the presence of a cationic polyelectrolyte, PDDA (poly(diallyldimethylammonium chloride). Inhibition of the turnover of its substrate, erythromycin, was subsequently measured using chronoamperometry at increasing concentrations of different known inhibitors of this enzyme namely ketoconazole, cimetidine and diclofenac for which IC50 values of 135

nM, 80 µM and 311 µM were measured, respectively. Furthermore, heterotrophic cooperativity where the turnover of a first substrate is enhanced in the presence of a second, was tested for the immobilized P450 3A4 enzyme. In this case, diclofenac 5-hydroxylation was stimulated by the presence of quinidine resulting in doubling of the potency of this inhibitor i.e. lowering the measured IC50 of

diclofenac from 311 µM down to 157 µM. The results obtained in this work confirm that bioelectrochemistry can be imployed for in vitro studies of not only drug-drug interactions but also prediction of adverse drug reactions in this important P450 isozyme.

KEYWORDS

Protein immobilization, glassy carbon, cytochrome P450, inhibition, chronoamperometry.

1. Introduction

In humans, majority of drugs and xenobiotics are cleared by the hepatic cytochromes P450 (P450) that are the most important Phase I drug metablising enzymes. Inhibition of P450-mediated drug metabolism by a concomitantly administered second drug is one of the major causes of drug–drug interactions (DDI) in humans and can cause serious adverse drug reactions (ADR) or an increase in toxic side effects, in some cases leading to the removal of the drug from the market[1-3]. In pharmacokinetic terms, the interacting drug (“perpetrator”) binds to the P450 causing a change in the metabolism of the second drug (“victim”) leading to its toxicity [4]. The availability of an in vitro test able to predict the inhibitory effect of a drug is therefore very attractive, especially if the experimental procedures involved are rapid and simple.

In general, inhibition of P450-dependent metabolism can be divided into three categories: competitive, non-competitive and mechanism-based. In the latter case, turnover of the initial compound leads to a reactive metabolite(s) that may covalently bind to the enzyme causing its inactivation. Furthermore, a significant number of P450 reactions exhibit “atypical” non-Michaelis-Menten kinetics [5] with multiple ligands simultaneously binding the active site. Several models have been proposed to explain this cooperativity but the mechanism is far from being fully understood[5]. These “atypical” kinetics further complicate the prediction of DDI in P450 enzymes.

Activity of P450 enzymes is currently determined from the rates of formation of metabolites using human liver microsomes (HLM) or reconstituted systems in vitro. Another in vitro methodology that has received considerable attention in recent years is electrochemistry [6,7]. The electrochemically driven catalysis of P450 enzymes bypasses the need for NADPH regenerating system, is rapid and can be standardized. However, a key question remains open: can the immobilized P450 respond to substrate-substrate or substrate-inhibitor interactions in the same way as the native protein? This paper addresses the question by investigating DDI of the immobilized human P450 3A4, the isoform thought to be the most important in terms of drug metabolism [8].

2. Materials and methods

All chemicals and reagents including erythromycin, cimetidine, ketoconazole and diclofenac were purchased from Sigma-Aldrich (Italy) and used as received.

2.2. P450 3A4 expression and activity

The P450 3A4 enzyme was expressed in E. coli over a period of 48 hours at 28 ºC post-induction. The harvested cells were sonicated and the membrane fractions solubilized by the addition of surfactant (octylphenoxypolyethoxyethanol) after clarification by ultracentrifugation. The protein was then purified by a two-step chromatography procedure including ion exchange (DEAE-Sepharose) followed by affinity chromatography (Nickel chelating Sepharose) [9].

2.3. Preparation of enzyme electrodes

Direct electrochemistry of P450 3A4 was achieved by immobilization on glassy carbon (GC) electrodes (0.07cm2 –BASi, UK) polished with aqueous slurries of alumina and then by sonication. Subsequently the GC electrodes were modified with the polymer poly (diallyldimethylammonium) (PDDA) in a 1:1 mixture of PDDA:protein (15 µM) and allowed to set overnight at 4°C. The solution used for the latter preparation was 100 mM potassium phosphate buffer pH 7.4. The protein film was not dry after the overnight incubation and prior to use it was rinsed with 100 mM potassium phosphate

buffer pH 7.4 containing 100 mM potassium chloride

2.4. Electrochemical Measurements

The electrochemical experiments were performed using an AUTOLAB PGSTAT 12 potentiostat (Eco Chemie -Netherlands). Measurements were carried out with a three-electrode system with a platinum wire as the counter electrode, an Ag/AgCl (3M KCl) as the reference and the GC electrode as the working. For cyclic voltammetry (CV) experiments the potential range 0f -0.6 to +0.4 V (vs Ag/AgCl) was chosen. The measurements were performed in 100mM potassium phosphate buffer pH 7.4 containing 100mM potassium chloride.

Integration of the reduction peak observed for electron transfer by CV under anaerobic conditions

(achieved using a glove box (Belle Technology Ltd, UK) under a nitrogen atmosphere where the oxygen levels were kept below 2ppm) gave the amount of charge (Q) transferred on reduction of P450 3A4. The surface concentration of electroactive enzyme on the electrode was then estimated by assuming the one electron transfer by using Faraday’s Law:

Q = nF (1)

where Q, charge (C); n, amount of electroactive species (mol); F, Faraday’s constant (96485 C/mol). Chronoamperometric measurements were carried out in fully oxygenated, constantly stirred electrochemical cell containing 100 mM potassium phosphate buffer pH 7.4 and 100 mM potassium chloride at 37°C for 40 min. For catalysis experiments an excess of marker substrate, erythromycin (100 µM) was used and the reactions were conducted for 40 min at the defined potential of -650mV (vs Ag/AgCl). Control experiments were carried out in the absence of the substrate.

2.5. Kinetic Measurements

In the process of N- or O-demethylation of drugs by P450 enzymes, formaldehyde is liberated and it can be quantified using a method based on the Hantzsch reaction where it is reacted with the NASH reagent to form the coloured product diacetyldihydrolutidine [10]. Therefore the N-demethylated product of erythromycin formed during chronoamperometric experiments with immobilized P450 3A4 was measured spectrophotometrically (HP 8452A diode array, Agilent Technologies, Italy) at 412 nm after incubation with NASH reagent [10].

For the inhibition experiments, the amount of erythromycin substrate was kept constant (100 µM) whilst varying the concentration of the inhibitory drug: ketoconazole (0-1250 nM), cimetidine (0-5 mM), diclofenac (0-500 µM). Stock solutions of all inhibitors were made in acetonitrile. In particular in the case of the mechanism-based inhibitor diclofenac, a 15 min pre-incubation was applied in the absence of substrate. The stimulation effect of quinidine on the inhibition of P450 3A4 by 5-hydroxy diclofenac was investigated by pre-incubating the different amounts of diclofenac in presence of a fixed concentration of quinidine (2.5 µM).

The IC50 values were calculated by plotting the residual activity (%) as function of the logarithm of the

substrate concentration added. Data were fitted to non-linear regression with SigmaPlot software (SYSTAT software, USA) using the sigmoidal eqaution to determine IC50 values:

y = y0 + a/[1 + (x/IC50)-slope] (2)

where “y0”is the minimum of residual activity and “a” is the maximum minus the minimum activity

observed. All inhibition experiments were done in triplicates and the mean values were plotted with error bars indicative of the standard error of the mean of the three separate measurements. The final

organic solvent concentration was less than 2% (v/v) in all incubations.

3. Results and Discussion

Direct electrochemistry of P450 3A4 was achieved by immobilization on glassy carbon (GC) electrodes (0.07cm2) modified with the polymer poly (diallyldimethylammonium) (PDDA) in a 1:1 mixture of PDDA:protein (15 µM), allowed to set overnight at 4°C. The P450 3A4 was N-terminally modified, heterologously expressed in Escherichia coli and purified as previously reported [9]. The immobilized protein showed two waves in cyclic voltammetry (CV) with a midpoint potential of -319± 7 mV vs Ag/AgCl (Figure 1-inset) at 25°C under anaerobic conditions [11].

Cyclic voltammetry was carried out in the electrochemical cell containing 100mM potassium phosphate buffer pH 7.4 with 100mM potassium chloride in a final volume of 500µL, at room temperature. The working electrode was cycled between initial and switch potentials of +400 and -500mV (vs Ag/AgCl), respectively. Voltammograms were taken in the absence and in presence of oxygen and/or substrate to observe the enzyme-oxygen-substrate interaction. A typical cyclic voltammogram of the immobilized P450 3A4 is shown in Figure 1A with the visible peaks attributed to the FeIII_/FeII _{redox couple of the haem iron of this enzyme. In the presence of substrate (and oxygen)} an increase in the cathodic peak current was observed (Figure 1B) due to the coupling of the reduction of the haem group to the monooxygenation (hydroxylation) of the substrate.

“Here Figure 1”

This phenomenon of protein film voltammetry [12] has been a successful method for not only electron transfer measurements of enzymes but also their catalytic processes. The latter has been employed to human P450 isoenzymes [7]. The oxidation and reduction peak currents observed for the immobilized P340 3A4 in cyclic voltammetry experiments were linear with scan rates up to 100 mV s-1

, consistent with a surface-confined reaction. Integration of the reduction peak observed in CV gave the amount of charge (Q) transferred upon reduction of P450 3A4. The surface concentration of electroactive enzyme on the electrode was then estimated by assuming one electron transfer by using Faraday’s Law and was found to be 4.6 x 1013

molecules per cm2

, indicating a multilayer formation. The molecular diameter of P450 3A4 is taken as 4 nm [13] with the geometric electrode area of 0.07 cm2 resulting in a theoretical coverage of 5.6x1011 _{molecules per electrode. From the electrochemical data we have} calculated 3.2 x 1012 molecules hence, a multilayer of around 5.7. However, the real surface area of glassy carbon electrodes is always higher than the geometric area therefore the number of layers is lower than the calculated one.

Addition of the marker substrate, erythromycin [14-15], in the presence of oxygen, gave a typical catalytic current (Figure 1B). An estimate of the enzyme turnover gave a value of around 6 min-1 [16]. An apparent Km value of 86±3 µM was calculated for the immobilized P450 3A4 by addition of increasing amounts of erythromycin, in excellent agreement with literature report of 88 µM for the microsomal enzyme [17]. All these results are consistent with previous work from our group showing that the immobilized P450 enzyme is active [11,18-20].

Once it was confirmed that the immobilized P450 3A4 was electrochemically active capable of transferring electrons to and from the GC electrode, chronoamperometry experiments were carried out in the presence of its known substrate erythromycin. A bias of -0.65 V (vs Ag/AgCl) was applied for a total of 40 min after which time the presence of the N-demethylated product was measured spectroscopically at 412 nm using the NASH reagent [10]. In the presence of erythromycin, an increasing current was observed in the chronoamperometric measurements whereas the same current decayed in the absence of the substrate (Figure 2).

“Here Figure 2”

In order to test the possibility of measuring the inhibition of the immobilized P450 3A4, three known inhibitors of this enzyme were selected with their chemical structures shown in Figure 3. The first inhibitor, ketoconazole, is an antifungal drug used to prevent and treat fungal skin infections and is widely used in inhibition studies of P450 3A4 due to its potency and selectivity [16]. The second inhibitor, cimetidine, is a histamine H2-receptor antagonist which is used in the treatment of stomach

ulcers and has been shown to be a weak inhibitor of this enzyme. The third inhibitor, diclofenac, is a nonsteroidal anti-inflammatory drug used for the treatment of pain.

“Here Figure 3”

For the inhibition experiments, the amount of erythromycin (substrate) was kept constant (100 µM) whilst varying the concentration of the inhibitory drug. In particular, in the case of the mechanism-based inhibitor diclofenac, a 15min pre-incubation was applied in the absence of substrate, due to the known fact that P450 3A4 converts this drug into its product 5’-hydroxydiclofenac, that acts as the inhibitor [21]. Conversely, pre-incubation was not required for ketoconazole or cimetidine since they are not mechanism-based inhibitors of P450 3A4 [22-24]. The inhibition data obtained are shown in Figure 4. The results reported are in good agreement with the previously published data where ketoconazole exhibits a high inhibitory capacity (IC50= 135 ± 10 nM) [25,26] with respect to the less

potent inhibitor cimetidine (IC50= 80 ± 5 µM) [24]. The highest measured IC50 was that of diclofenac

(311 ± 20 µM) inline with this drug being a weak mechanism-based inhibitor [21]. “Here Figure 4”

In addition to inhibition, P450 enzymes have also been shown to be prone to heterotrophic cooperativity where the turnover of a first substrate is enhanced in the presence of a second one. In the specific case of P450 3A4, there are reports that diclofenac 5-hydroxylation is stimulated, through a mechanism of positive heterotrophic cooperativity, by quinidine, an important antimalarial drug [21,27]. The stimulation effect of quinidine on the inhibition of immobilized P450 3A4 by 5-hydroxy diclofenac was therefore investigated with our electrochemical set up. The results of pre-incubation of different amounts of diclofenac in the presence of a fixed concentration of quinidine (2.5 µM) resulted in doubling of the potency of the inhibitor, i.e. lowering the IC50 of diclofenac from 311 ± 20 µM to

157 ± 20 µM (Figure 4C). These results show that diclofenac is able to inactivate the immobilized P450 3A4 and that the presence of quinidine stimulates this inhibition with respect to the catalysis of erythromycin. This is comparable to a previous literature report [21] where the same stimulation effect by quinidine was observed but using testosterone as the substrate for microsomal P450 3A4. Using a similar analogy, it can be hypothesized that diclofenac binds to the same binding site of erythromycin, it is metabolized to reactive intermediates, which then covalently bind to the same site therefore making it inaccessible to the substrate. Quinidine stimulates this inhibition by binding to a different second site [21]. The above-mentioned hypothesis is shown schematically in Figure 5. The presence of multiple binding sites within the active site of P450 3A4 has been demonstrated by several research groups based on inhibition studies [28,29], site-directed mutagenesis [30] and X-ray crystallography [31]and is one of the prevailing models for explaining the cooperativity seen in this isozyme.

“Here Figure 5”

In conclusion, the electrochemical inhibition data presented in this work show that the substrate-binding pocket of the immobilized P450 3A4 has similar characteristics to those of the native microsomal enzyme, which confirms that the immobilization has had no effect on the enzyme’s substrate and inhibitor binding sites and its functionality in terms of enzymatic activity. In a recent work on immobilized P450 2C9 [32] the authors also concluded that there had been no significant alteration of the active site of this cytochrome due to immobilization based on kinetic measurements and the regioselectivity of P450 2C9-mediated metabolism of warfarin. The heterotrophic cooperativity data are also consistent with the presence of multiple binding sites within the active site of P450 3A4. Therefore, bioelectrochemistry can be a methodology of choice for in vitro studies of not only drug-drug interactions but also prediction of adverse drug reactions in this important P450 isozyme.

Acknowledgements

The authors wish to thank Dr. V. Dodhia (Imperial College, London) for useful discussions. This work was supported by the Region Piedmont CIPE 2006 (CYP-TECH project, Italy) and Nanobiodesign Italy.

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Figure Captions

Figure 1. Cyclic voltammograms of P450 3A4 on GC electrode in the absence (A) and the catalytic current in the presence of erythromycin and oxygenated buffer (B). Inset: anaerobic cyclic voltammogram of P450 3A4 immobilized on PDDA modified GC electrodes. The dashed-line is the background cyclic voltammogram of buffer in the absence of any protein. Scan rate 50 mVs-1 _{in 100}

mM potassium phosphate buffer with 100 mM KCl, pH 7.4 at 25ºC. In the case of cyclic voltammograms in the presence of substrate, the temperature of the electrochemical cell was increased to 37ºC.

Figure 2. Chronoamperometry experiments of the P450 3A4 immobilized on PDDA modified GC electrodes in the absence (grey line) and presence (black line) of the substrate erythromycin, and presence of inhibitor ketoconazole (purple line). The potential of the working electrode was kept at -650 mV (vs Ag/AgCl) whilst the current was recorded for 40 min. Experiments were carried out at 37ºC in 100 mM potassium phosphate buffer with 100 mM KCl, pH 7.4.

Figure 3. Chemical structures of the inhibitors used in this work.

Figure 4. Concentration-dependent inhibition of erythromycin N-demethylation by (A) ketoconazole, (B) cimetidine and (C) diclofenac. The latter shows the effect of quinidine on diclofenac inhibition of erythromycin N-demethylation (No quinidine = filled circles, 2.5 µM quinidine = open squares). Error bars indicate the standard error of the mean of three separate measurements.

Figure 5. Schematic representation of heterotropic cooperativity in immobilized cytochrome P450 3A4. Erythromycin as substrate binds to site A (left), pre-incubation with diclofenac where this mechanism-based inhibitor also binds site A (center) and pre-incubation with both diclofenac and quinidine (right).

Figure 1

13 Figure 4. Erythromycin Fe Diclofenac Quinidine Erythromycin Fe Fe Diclofenac Quinidine Erythromycin Diclofenac Fe Erythromycin Diclofenac Fe Fe Erythromycin Fe Erythromycin Fe Fe Binding site A Binding site B