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ANESTHETIC PHARMACOLOGY

The Effect of a Long–Term Epidural Infusion of Ropivacaine on CYP2D6 Activity

Jeroen Wink, MD^*, Bernadette Th. Veering, MD, PhD^*, Michel Kruit, MD, Anton G. L. Burm, PhD^*, Gunilla A. I. Huledal, MSc Pharm, Gunilla Y. Ekström, MD, PhD, Rudolf Stienstra, MD, PhD^*, and Jack W. van Kleef, MD, PhD^*

From the *Department of Anesthesiology, Leiden University Medical Centre, Leiden, The Netherlands; Department of Anesthesiology, Rijnland Hospital, Leiderdorp, The Netherlands; and AstraZeneca R&D, Södertälje, Sweden.

Address correspondence to Jeroen Wink, MD, Department of Anesthesiology, Leiden University Medical Centre, P.O. Box 9600, 2300 RC, Leiden, The Netherlands. Address e-mail to j.wink{at}lumc.nl.

	Abstract

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BACKGROUND: Ropivacaine and one of its metabolites, pipecoloxylidide, inhibit CYP2D6 in. human liver microsomes in vitro with K_i values of 5 µM (1.4 mg/L) and 13 µM (3.6 mg/L), respectively.

We investigated the effect of a 50 h continuous epidural infusion of ropivacaine 2 mg/mL at a rate of 14 mL/h on CYP2D6 activity.

METHODS: Nineteen patients (41–85 yr) undergoing hip or knee replacement, all extensive metabolizers with respect to CYP2D6 activity, were included. Medications known to inhibit or be metabolized by CYP2D6, or known to be strong inhibitors/inducers of CYP1A2 or CYP3A4 were not allowed. Patients received 10 mg debrisoquine (a marker for CYP2D6 activity) before surgery and after 40 h epidural infusion. The metabolic ratio (MR) for debrisoquine hydroxylation was calculated as the amount of debrisoquine/amount of 4-OH-debrisoquine excreted in 0–10 h urine.

RESULTS: The median (range) of MR before and after ropivacaine were 0.54 (0.1–3.4) and 1.79 (0.3–6.7), respectively. The Hodges Lehman estimate of the ratio MR after/MR before ropivacaine was 2.2 with a 95% confidence interval 1.9–2.7 (P < 0.001).

CONCLUSION: A continuous epidural infusion of ropivacaine inhibits CYP2D6 activity in patients who are extensive metabolizers resulting in a twofold increase in the MR for debrisoquine hydroxylation. However, since none of the patients was converted into a functional poor metabolizer (MR >12.6), the effect on the metabolism of other drugs metabolized by CYP2D6 is unlikely to be of major clinical importance.

	Introduction

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CYP2D6 is an isoenzyme of cytochrome P450 that exhibits genetic polymorphism. Different alleles for CYP2D6 have been identified, explaining the existence of poor (PM) and extensive metabolizers.1 Drugs metabolized by CYP2D6 often encountered in daily anesthesia practice include 5-HT₃ receptor antagonists, antiarrhythmics, µ-opioid receptor agonists, and β-receptor antagonists.2 Debrisoquine, a selective substrate for CYP2D6, is commonly used as a marker for CYP2D6 activity.3

Ropivacaine (S(-)-propyl-2',6'-pipecoloxylidide), a long-acting amide local anesthetic, mainly undergoes oxidative metabolism in humans with two metabolites N-depropylated ropivacaine (pipecoloxylidide, PPX) and 3-hydroxy ropivacaine (3-OH-ropivacaine). An in vitro study in human liver microsomes has shown that ropivacaine and PPX inhibit CYP2D6 competitively with K_i values of 5 µM (1.4 mg/L) and 13 µM (3.6 mg/L), respectively (Ekström G., AstraZeneca, Sweden, personal communication).

The objective of this study was to evaluate the inhibitory effect of ropivacaine given as a continuous epidural infusion for at least 50 h on CYP2D6 activity in surgical patients who were extensive metabolizers for CYP2D6 activity.

	METHODS

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Patients
Nineteen patients, aged 69 ± 12 yr, scheduled for hip or knee replacement, were included in this open, no treatment controlled, two-center study. The study protocol was approved by the Ethics Committees at both centers and written informed consent was obtained from all patients after full explanation of the study.

Inclusion criteria were ASA risk category I or II, age 18 yr, weight 50 and 110 kg and genotyped as extensive metabolizers for CYP2D6 activity.

Exclusion criteria were contraindication to epidural anesthesia/analgesia or to patient-controlled analgesia with morphine; contraindication to debrisoquine; intake of drugs known to cause inhibition of CYP2D6, intake of drugs known to be metabolized by CYP2D6 or intake of drugs known to be inhibitors or inducers of CYP1A2 or CYP3A4 within 1 wk before the first administration of debrisoquine; and parenteral administration of ropivacaine or bupivacaine in the week before surgery.

Restrictions were intake of grapefruit juice, grapefruit and alcohol was not allowed during the study; patients had to fast from 2 h before up to 2 h after the intake of debrisoquine, but intake of water was permitted; and tea, coffee, and caffeine-containing beverages were not allowed from 2 h before up to 10 h after administration of debrisoquine.

Drugs
On the day before surgery and 40–50 h after the start of the postoperative continuous infusion of ropivacaine, oral debrisoquine 10 mg was administered. After each administration, urine was collected at 10 h for determination of debrisoquine and 4-OH-debrisoquine concentrations in urine in order to calculate the metabolic ratio (MR) of debrisoquine.

On the day of surgery, an epidural catheter was inserted at the L2–3 or L3–4 interspace. Before surgery, a 15-mL bolus of ropivacaine 10 mg/mL was given. After surgery, a continuous epidural infusion of ropivacaine 2 mg/mL was started at a rate of 14 mL/h for at least 50 h. IV patient-controlled analgesia morphine was the only rescue analgesic.

Procedures
At a screening visit 6–2 wk before surgery, patients were screened for eligibility according to the inclusion and exclusion criteria. A routine physical examination was performed, including an electrocardiogram and a blood sample for genotyping CYP2D6 polymorphisms using polymerase chain reaction.

Sensory block was determined by assessing temperature perception changes using an ice cube. Motor block was determined using the modified Bromage scale. Adverse events were recorded from the administration of debrisoquine until a follow-up by telephone 2–3 wk after surgery.

Blood and Urine Sampling
The peripheral venous blood sample for genotyping was collected into EDTA-containing tubes. The sample was stored at room temperature until assayed within 3 days by Professional Genetic Laboratory AB, Uppsala, Sweden.

Peripheral venous blood samples were taken before the start of administration of ropivacaine, and immediately before and 5 and 10 h after the second dose of debrisoquine, for determination of plasma concentrations of ₁-acidglycoprotein (AAG), ropivacaine (total and unbound), and PPX (total and unbound). Plasmaconcentrations of total and unbound ropivacaine and PPX were measured to confirm the exposure of ropivacaine and PPX in each individual. The blood samples were transferred into Venoject® tubes and centrifuged at room temperature at 3000 rpm for 10 min within 60 min of collection. The plasma was separated and stored in polypropylene Cryotube® tubes at –20°C until analysis.

Before each dose of debrisoquine, the bladder was emptied which constituted a blank urine sample. Urine was then collected in the interval 0–10 h after the administration of debrisoquine. The urine was weighed and the weight was converted to volume (1:1.02). After thorough mixing, 2 5 mL samples were taken from each urine fraction and transferred to Cryotubes® and stored at –20°C until assay.

Bioanalytical Methods
Genotyping for CYP2D6 polymorphism was done using polymerase chain reaction and multiple solid phase minisequencing.4 This method allows for determination of deficient alleles (CYP2D6*1, *3, *4, *5, *6). Based on the results each patient was characterized as being an extensive metabolizer or a PM.

The total concentration of ropivacaine was determined by a method based on liquid-liquid extraction, followed by gas chromatography with nitrogen-sensitive detection.5 Using 0.1 mL plasma, the inter-assay coefficient of variation (CV) was 6.8% and the limit of quantification was 0.0027 mg/L. The total concentration of PPX in plasma was determined by a method based on ultrafiltration of acidified plasma, followed by coupled-column liquid chromatography and mass spectrometry detection with electro spray ionization.6 Using 0.5 mL plasma, the inter-assay CV was 4.3% and limit of quantification was 0.0023 mg/L. The unbound concentration of ropivacaine and PPX in plasma was determined by a method based on coupled-column liquid chromatography and mass spectrometry detection using electro spray ionization after ultrafiltration of plasma pH-adjusted to 7.4 at 37°C.6 Inter-assay CVs were unbound ropivacaine 4.8% and unbound PPX 4.3%. The limits of quantification were unbound ropivacaine 0.0027 mg/L and unbound PPX 0.0023 mg/L. The concentration of AAG was measured by an immunoturbidometric method. The concentration of debrisoquine and 4-OH-D in urine were determined using a method based on liquid chromatography and fluorescence detection.7

Data Analysis
The sample size was not based on considerations of statistical power, since no variability information was available for the primary variable. Sample size was considered adequate based on previous, similar studies. The MR for debrisoquine hydroxylation was calculated as

This ratio was used to describe CYP2D6 activity before and during the epidural infusion of ropivacaine. Subsequently, the Hodges Lehman estimate of the ratio of the MRs after and before the start of the epidural ropivacaine infusion (MRafter/MRbefore) was calculated. The Hodges Lehman estimate is defined as the median of all possible pairwise ratios MR_after/MR_before and provides a suitable point estimate of the ratio. The key statistical entity used to describe the within-patient treatment difference ropivacaine-debrisoquine versus debrisoquine with respect to the MR of debrisoquine was a 95% two-sided nonparametric confidence interval. The exact distribution was used in the calculation of the confidence interval, which was based on the Wilcoxon Signed Rank Test. Linear regression analysis was performed to examine the relationships between MR_after/MR_before and the sum of the unbound ropivacaine and PPX concentrations (at 10 h after intake of the debrisoquine tablet) divided by the corresponding K_i values for the in vitro inhibition of CYP2D6 (C_ropivacaine/K_{i,ropivacaine} + C_PPX/K_i,PPX). Values are expressed as mean ± sd (standard deviation) or median (range), as appropriate, and considered statistically significant when P < 0.05.

	RESULTS

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Data from six patients were excluded from analysis because of incorrect placement of the epidural catheter (n = 2), inadequate epidural analgesia (n = 2), and displacement of the epidural catheter (n = 2). Consequently, 19 patients were included in the safety analysis and 13 patients in the pharmacokinetic analysis.

Steady-state plasma concentrations were reached for ropivacaine and PPX at the time of intake of the second tablet of debrisoquine (Table 1). There was a postoperative increase in AAG plasma concentrations in all patients. AAG ranged between 15.5 and 28.5 µmol/L before surgery and between 25.3 and 43.8 µmol/L 10 h after intake of the second tablet of debrisoquine.

The median (range) MRs for debrisoquine hydroxylation before and after 40–50 h epidural infusion of ropivacaine were 0.54 (0.1–3.4) and 1.79 (0.3–6.7), respectively (Fig. 1). The Hodges Lehman estimate of the ratio MR_after/MR_before administration of ropivacaine was 2.2 with a 95% confidence interval 1.9–2.7 (P = 0.00024). The slope of the regression line relating MR_after/MR_before to the sum of the total ropivacaine and PPX concentrations divided by the corresponding K_i values did not differ from 0. However, the slope of the regression line relating MR_after/MR_before to the sum of the unbound ropivacaine and PPX concentrations divided by the corresponding K_i values (Fig. 2) differed significantly from 0 (P < 0.05). The coefficient of determination, characterizing the relationship (R²) equaled 0.40, indicating that 40% of the variability in the MR_after/MR_before ratios can be explained by variations in the unbound ropivacaine and PPX concentrations.

View larger version (13K): [in this window] [in a new window]   Figure 1. Individual metabolic ratios of debrisoquine and 4-OH-debrisoquine before (Day 1) and after at least 40 h of ropivacaine infusion (Day 4).

View larger version (10K): [in this window] [in a new window]   Figure 2. Ratio of the metabolic ratio of debrisoquine and 4-OH-debrisoquine before and 40–50 h after the start of ropivacaine infusion plotted as a function of the sum of the unbound ropivacaine and pipecoloxylidide (PPX) concentrations divided by their respective Ki values. Dots represent individual values. The line was obtained by linear regression analysis. The slope of the line was significantly different from zero (P < 0.05). The coefficient of determination (R2) was 0.40.

Postoperative pain visual analog pain scale scores were low both at rest and on movement of the operated leg.

Overall there were no major safety concerns. The most common adverse event was hypotension which was treated with ephedrine.

	DISCUSSION

Top Abstract Introduction METHODS RESULTS DISCUSSION REFERENCES

 
The aim of this study was to investigate the effects of a continuous epidural infusion of ropivacaine on the CYP2D6 activity in vivo. This study showed that a 50-h continuous epidural infusion of ropivacaine in surgical patients inhibits CYP2D6 activity, resulting in a twofold increase in the MR of debrisoquine. These observations are in accordance with a previous in vitro study.

All medications known to inhibit CYP2D6 or metabolized by CYP2D6 were avoided in addition to strong inhibitors of CYP1A2, CYP3A4, and inducers of CYP1A2. However, given the pre-and postoperative situation, a number of medications were given to these patients and, although most of these drugs have been on the market for decades, the potential for drug interactions is not always known.

The results of this study are consistent among patients, which indicates that the observed increase in the MR is indeed related to the administration of ropivacaine. This is further substantiated by the coefficient of determination (R²), which demonstrates that 40% of the variability in the increase in the MR can be explained by unbound plasma concentrations of ropivacaine and PPX.

Administration of drugs primarily metabolized by polymorphic enzymes may be associated with marked variability in plasma drug concentrations and, consequently, in the clinical effect, leading to the risk of side effects and drug toxicity, prolonged therapeutic effect or lack of effect.8–12 Besides genetic factors, nongenetic factors, including enzyme inhibition, may affect CYP2D6 activity.1 Turning a patient into a PM by administration of an inhibiting drug could lead to clinically important changes in drug response. The clinical importance of polymorphism or inhibition of enzymes depends, however, on a number of factors, including therapeutic index of the drug, activity of the compound or metabolite, and dependence of metabolic pathway on enzyme activity.13 As such, pharmacogenetics and knowledge of drug interactions may help us understand individual drug response and drug interactions and possibly predict toxicity, all of which will improve clinical outcome.

The changes in the MR of debrisoquine induced by ropivacaine are relatively small compared with the very large (30-fold) interindividual variability in the MR.14 In this study, none of the patients was converted into a functional PM (MR >12.6)15 and the effect on the metabolism of other drugs with CYP2D6-dependent metabolism is not expected to be of any major clinical importance.

	ACKNOWLEDGMENTS

 
We thank Professor L Bertilsson, Karolinska Institute, for valuable discussions. We also thank the anesthetists, orthopedic surgeons and nurses from the Rijnland Hospital for their invaluable assistance and Y. Askemark, AstraZeneca R&D, for the bioanalytical work.

	Footnotes

 
Accepted for publication September 13, 2007.

This study was financially supported by AstraZeneca, Södertälje, Sweden.

Deceased.

Reprints will not be available from the author.

	REFERENCES

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a colleague Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Wink, J. Articles by van Kleef, J. W. Search for Related Content PubMed PubMed Citation Articles by Wink, J. Articles by van Kleef, J. W. Related Collections Clinical Pharmacology Regional Anesthesia Pharmacology