Materials & Methods
Study Design & Subjects
This study was approved by the Human Research Protection Office and Institutional Review Board of the Medical College of Wisconsin and Froedtert Hospital (WI, USA). Written informed consent was obtained from all subjects during their routine visits at one of the three area pain management clinics (Midwest Comprehensive Pain Care, Zablocki VA Hospital [WI, USA], and Froedtert Hospital). Male and female patients who were being treated for chronic pain and were prescribed an opioid, or opioid-like medication (hydrocodone, methadone, oxycodone or tramadol) metabolized by CYP2D6, were eligible to participate in the study. Therefore, patients on long-term opioid therapy for chronic painful conditions, such as osteoarthritis of the spine, were included. Other inclusion criteria included that the patient was at least 18 years of age and willing to comply with the study protocol. Exclusion criteria included patients receiving methadone maintenance therapy.
Patients who were newly prescribed (<1 month) an opioid were required to have two blood samples drawn. The initial blood sample was used to determine the CYP2D6 genotype of the patient, while the second blood sample (at least 1 month after reaching a stable titration for pain relief) was used to determine the Css of the opioid in the patient's plasma. Established patients who were already on opioids for at least 1 month and had reached a stable titration for pain relief had only one blood sample drawn, which was used to determine both the plasma drug concentration and CYP2D6 genotype of the patient.
Clinical assessment of each patient included a detailed history and physical examination with emphasis on neurological, cognitive and functional assessment. Standard scales such as the verbal/visual analog scale (VAS) were employed for the assessment of pain at every patient's clinic visit. A VAS scale is used with the categorization of relief from beginning of titration to stable dose. Pain relief was defined as follows: no pain relief was no change in the VAS score from baseline, partial pain relief was a decrease in the VAS score of 2 or more from the initial visit, while complete pain relief was a VAS score of 0. The categories of partial, complete and no relief were chosen and defined (as is common in other pain studies). However, pain assessment is subjective so a 9 to 7 may be another person's 3 to 1. In addition, change from each patient was evaluated for possible side effects using an investigator-developed questionnaire at the initiation and subsequent visits. Common opioid side effects included: drowsiness, trouble concentrating, constipation and nausea/vomiting. Currently, self report is the primary and best indicator of side effects that physicians have. Furthermore, the consumption of prn (as needed) analgesic medication for breakthrough pain and the information recorded in the patient's daily pain log (or self-reported pain) was used to determine the effectiveness of the pain management therapy. Functional improvement, the ability to perform daily living tasks and range of motion were also used to determine the effectiveness of therapy.
DNA was extracted from ethylenediaminetetraacetic acid (EDTA) whole blood using the Puregene® DNA isolation kit (Gentra Systems, MN, USA). The DNA was then quantified and checked for purity by spectrophotometric methods.
All samples were analyzed for variants in CYP2D6 (*3, *4, *6, *7 and *8) using Pyrosequencing® (Qiagen, Hilden, Germany) technology as previously described. Briefly, nested PCR was performed with a single fragment in the primary PCR and two separate reactions in the second PCR. The Pyrosequencing analysis was then run as a duplex (*3 and *7) and triplex (*4, *6 and *8) assay using a PSQ™ HS96A system and software (Biotage, Uppsala, Sweden). In addition, the CYP2D6 gene deletion (*5) and duplication (*2xn) were detected using Pyrosequencing technology as previously described. Briefly, an equivalent region of CYP2D6 and the pseudogene CYP2D8 were amplified by PCR and analyzed in two common Pyrosequencing reactions in which peaks specific for each respective gene were generated and peak heights compared. The heights of specific peaks from CYP2D8, which occur as a single copy in all alleles, are used as a reference for CYP2D6-specific peak heights. This relative quantification approach allows the distinction of zero to four CYP2D6 genome copies.
Hydrocodone, hydrocodone-D3, oxycodone, oxymorphone, methadone and tramadol were purchased from Cerilliant (TX, USA). HPLC-grade methanol was purchased from Fischer Scientific (NJ, USA). Ammonium acetate was purchased from ICN Biomedicals Inc. (OH, USA). Formic acid was purchased from Mallinckrodt Baker Inc. (KY, USA). A Barnstead™ Fi-streem glass still (Thermo Scientific, MA, USA) water purification system was used to obtain purified water for the HPLC solvent.
Hydrocodone, oxycodone, oxymorphone, methadone and tramadol were extracted in a single step from alkalized plasma into N-hexane/isoamylalcohol (99/1, v/v) following a previously published protocol. Briefly, sample aliquots (500 µl plasma) were mixed with 100 µl of 1 M potassium carbonate and 800 µl of N-hexane/isoamylalcohol containing the internal standard (50 µg/l hydrocodone-D3) in a 1.5 ml microcentrifuge tube. After vortex mixing, the samples were centrifuged for 5 min at 13,000 rpm. The supernatant was transferred to another microcentrifuge tube and loaded onto a 24-well sample plate for HPLC-mass spectrometry (MS)-MS analysis. A Waters Alliance 2795 HPLC system was used. Chromatography was achieved using a Discovery® Cyano HPLC column (Sigma Aldrich, MO, USA; 15 cm × 4.6 mm, 5 µm), maintained at 30°C. The injection volume was 20 µl with a flow rate of 500 µl/min. The opioids were eluted isocratically using 2 mM ammonium acetate containing 0.1% formic acid in water:methanol with 0.1% formic acid (60:40). The total analysis time was 5 min. All aspects of the system operation and data acquisition were controlled using MassLynx NT 3.5 software.
A Micromass Quattro micro™ triple quadrupole tandem mass spectrometer (Waters Corp., MA, USA) was used for all analyses. Ionization was achieved using electrospray in the positive ion mode. The following conditions were found to be optimal for the analysis of the opioids: capillary voltage, 1.0 kV; source block temperature, 100°C; and desolvation gas (nitrogen) heated to 300°C. The mass spectrometers parameters were: hydrocodone m/z 300.1 → m/z 199.0, cone 35 V, collision energy 30 eV; hydrocodone-D3 m/z 303.1 → m/z 199.0, cone 35 V, collision energy 30 eV; oxycodone m/z 316.1 → m/z 298.0, cone 35 V, collision energy 20 eV; oxymorphone m/z 302.1 → m/z 284.1, cone 30 V, collision energy 20 eV; methadone m/z 310.1 → m/z 265.0, cone 20 V, collision energy 15 eV; and tramadol m/z 264.2 → m/z 58.1, cone 20 V, collision energy 15 eV. Calibration was linear from 1 to 1000 ng/ml for all the opioids, with linear regression analysis showing an R2 of at least 0.95.
A Fisher's exact test was used to compare the prevalence of variant CYP2D6 alleles in the chronic pain patients in the pain management clinics to those previously published in for the general population. In addition, the average Css of each drug (oxycodone, hydrocodone, tramadol and methadone) was compared between each of the CYP2D6 genotypes (EM, IM and PM) using the one-way analysis of variance (ANOVA). Statistically significant results had a p-value of less than 0.05.
Pharmacogenomics. 2009;10(7):1157-1167. © 2009 Future Medicine Ltd.
Cite this: Utilization of Pharmacogenomics and Therapeutic Drug Monitoring for Opioid Pain Management - Medscape - Jul 01, 2009.