Cyclosporine-inhibitable Cerebral Drug Transport Does Not Influence Clinical Methadone Pharmacodynamics

Background: Interindividual variability and drug interaction studies suggest that blood–brain barrier drug transporters mediate human methadone brain biodistribution. In vitro and animal studies suggest that methadone is a substrate for the efflux transporter P-glycoprotein, and that P-glycoprotein–mediated transport influences brain access and pharmacologic effect. This investigation tested whether methadone is a transporter in humans sample contents. Methods: Healthy volunteers received oral (N = 16) or IV (N = 12) methadone in different crossover protocols after nothing (control) or the validated P-glycoprotein inhibitor cyclosporine (4.5 mg/kg orally twice daily for 4 days, or 5 mg/kg IV over 2 h). Plasma and urine methadone and metabolite concentrations were measured by mass spectrometry. Methadone effects were measured by miosis and thermal analgesia (maximally tolerated temperature and verbal analog scale rating of discreet temperatures). Results: Cyclosporine marginally but significantly decreased methadone plasma concentrations and apparent oral clearance, but had no effect on methadone renal clearance or on hepatic N-demethylation. Cyclosporine had no effect on miosis or on R-methadone concentration–miosis relationships after either oral or IV methadone. Peak miosis was similar in controls and cyclosporine-treated subjects after oral methadone (1.4 ± 0.4 and 1.3 ± 0.5 mm/mg, respectively) and IV methadone (3.1 ± 1.0 and 3.2 ± 0.8 mm, respectively). Methadone increased maximally tolerated temperature, but analgesia testing was confounded by cyclosporine-related pain. Conclusions: Cyclosporine did not affect methadone pharmacodynamics. This result does not support a role for cyclosporineinhibitable transporters mediating methadone brain access and biodistribution. (Anesthesiology 2014; 121:1281-91) Submitted for publication January 8, 2014. Accepted for publication June 23, 2014. From the Department of Anesthesiology, Universitätsmedizin Greifswald, Greifswald, Germany (K.M.); and Departments of Anesthesiology (K.M., J.B., A.M.F., V.Y., E.D.K.) and of Biochemistry and Molecular Biophysics (E.D.K.), Washington University in St. Louis, St. Louis, Missouri. Cyclosporine-inhibitable Cerebral Drug Transport Does Not Influence Clinical Methadone Pharmacodynamics Konrad Meissner, M.D., Jane Blood, R.N., B.S.N., Amber M. Francis, R.N., B.S.N., Viktar Yermolenka, Ph.D., Evan D. Kharasch, M.D., Ph.D. PAIN MEDICINE Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/931155/ on 10/15/2017 Anesthesiology 2014; 121:1281-91 1282 Meissner et al. Methadone Brain Transport and Pharmacodynamics an ABC transporter substrate.10,11 Methadone did not accumulate in ABCB1-transfected pig kidney cells compared with controls, suggesting methadone was a P-gp substrate.12 In human P-gp-overexpressing cells, the P-gp inhibitors verapamil and GF120918 (elacridar) significantly decreased basal-to-apical methadone transport.13 In vivo, and consistent with these data, methadone brain uptake clearance or concentrations were approximately three-fold higher in multidrug-resistant (mdr)-deficient mdr1a/b(−/−) mice relative to wild-type mdr1a/b(+/+) mice, and methadone produced greater analgesia.13–15 Cerebral methadone concentrations were substantially greater in mdr1a (−/−) compared with wild-type mice.16 Upregulation of BBB P-gp activity in wild-type mice reduced methadone antinociception.17 In rats, methadone coadministration with the ABC transport inhibitor PSC833 (valspodar) increased methadone brain concentrations and antinociception, and reduced the dose for half-maximal effect (ED50). 18 Together, these studies suggest that methadone is a substrate for P-gp, and brain P-gp-mediated transport influences brain access and pharmacologic effect. In contrast to cellular and animal studies, little information exists on the role of P-gp in determining methadone brain access in humans. Indirect evidence from a pharmacogenetic study of P-gp genetic variants and dose requirements in methadone-maintained patients suggested P-gp substrate potential for methadone.19 In contrast, the P-gp inhibitor quinidine did not alter IV methadone-dependent changes in pupil diameter (miosis) or methadone concentration–effect relationships.20 Although quinidine did increase miosis after oral methadone, this was attributed to intestinal P-gp inhibition, increased methadone absorption, and increased plasma concentrations rather than enhanced brain penetration and altered BBB P-gp activity.20 It was recognized that quinidine is a nonpotent P-gp inhibitor, and plasma quinidine concentrations possibly insufficient to inhibit brain P-gp and P-gpmediated methadone transport (if present).20 Therefore, the potential role of BBB P-gp in influencing human methadone brain penetration is unknown. A recent study in human volunteers, conducted because in vitro and animal studies implicated P-gp in morphine transport, suggested a role for P-gp or other efflux transporters in morphine brain access and pharmacodynamics.21 Specifically, morphine miosis was more pronounced and prolonged in subjects pretreated with cyclosporine, reported to be an effective inhibitor of human BBB P-gp activity.21–23 The current study, therefore, tested the hypothesis that methadone is a substrate for human BBB drug transporters, such as P-gp, and that transport activity influences methadone plasma concentration–effect relationships (pharmacodynamics). The secondary aim was to evaluate the role of intestinal and renal transporters in the oral absorption and renal excretion of methadone. Cyclosporine was used as a drug transport inhibitor. Methadone concentration–effect relationships were studied using pupil diameter and analgesia as primary and secondary effect measures, in a single-center, open-label, crossover study in healthy volunteers. Materials and Methods Clinical Protocol The clinical investigation comprised two separate protocols, for oral and IV drug administration, in healthy volunteers (fig. 1). Both were approved by the Institutional Review Board of Washington University in St. Louis. The protocols were two-period sequential crossovers in healthy volunteers (control session first, for logistical considerations) with each subject as their own control. All subjects provided written informed consent. Healthy males and females, aged 18 to 40 yr and body mass index 20 to 33 kg/m2, were eligible. Exclusion criteria were a history of major medical problems, including a history of liver or kidney disease, use of prescription or nonprescription medications, herbals, or foods known to be substrates of P-gp or to affect its activity, pregnant or nursing females, and a known history of addiction to drugs or alcohol. For both protocols, IV catheters were inserted for drug administration and blood sampling, and subjects received IV ondansetron (4 mg) for antiemetic prophylaxis. Subjects were monitored with a pulse oximeter and automated blood pressure cuff, and received supplemental oxygen for saturations less than 94%. Subjects were fed a standard breakfast 2 h after drug dosing and had free access to food and water thereafter. Methadone doses were chosen to target a small change (2 to 3 mm) in pupil diameter based on previous studies. Protocol 1 (oral methadone) consisted of two sessions at least 10 days apart, the second of which was preceded by oral cyclosporine 4.5 mg/kg twice per day (maximally used therapeutic dose) (Gengraf; Abbott, Abbott Park, IL) for 4 days before and on the morning of the study day. The first four subjects were given 10 or 8 mg of racemic methadone hydrochloride orally for the control (session 1) or cyclosporine (session 2) sessions, respectively, in anticipation of Fig. 1. Protocol scheme. IV = intravenous. Downloaded From: http://anesthesiology.pubs.asahq.org/pdfaccess.ashx?url=/data/journals/jasa/931155/ on 10/15/2017 Anesthesiology 2014; 121:1281-91 1283 Meissner et al. PAIN MEDICINE a potentially increased methadone effect when coadministered with cyclosporine. The 10 mg dose was chosen to target a small change (2 to 3 mm) in pupil diameter. Methadone was administered 2 h after the final oral cyclosporine dose. Because of greater than anticipated intersubject variability in weight, the remaining 12 subjects received weight-based dosing (0.175 and 0.14 mg/kg methadone hydrochloride, respectively, in control and cyclosporine sessions) to diminish potential interindividual variability in plasma concentrations. For protocol 2 (IV methadone), also on two occasions at least a week apart, 12 subjects received 0.1 mg/kg methadone as a 1 h IV infusion for both control (session 1) and cyclosporine (session 2) sessions. In session 2, subjects received an IV infusion of 2.5 mg kg−1 h−1 cyclosporine (Bedford Laboratories, Bedford, OH) for 2 h. This cyclosporine dose produced a 79% increase in intracerebral concentrations of the P-gp substrate verapamil,23 and was used in the previous investigation of morphine pharmacodynamics.21 Methadone was administered starting at the beginning of the second hour of the cyclosporine infusion. Dark-adapted pupil diameter was measured in triplicate coincident with blood sampling using a handheld infrared pupillometer (Neuroptics, Irvine, CA).24 Pupil diameter change from predrug baseline (miosis) was determined at each time. Analgesia was assessed by response to thermal stimulus (Pathway; Medoc Advanced Medical Systems, Ramat Yishai, Israel) using both the maximum-tolerated temperature (method of limits) and the verbal analog pain rating of several predetermined temperatures (ramp-andhold method). Thermode temperature started at 36°C and increased 0.5°C/s, and subjects pressed a button when the maximum tolerable temperature was reached. The average result of three stimuli was recorded in degree Celsius. Subjects then rated pain intensity on a verbal analog scale (VAS, 0 to 100) in response to discrete stimuli (41.0°, 43.0°, 44.8°, 46.5°, 48.2°, and 50.0°C in random order). The probe was moved to a different region for each thermal stimulus. Sixteen subjects (eight males, eight females, 79 ± 14 kg, body mass index 27 ± 4) completed both arms of protocol 1. The average oral racemic methadone hydrochloride doses were 13 ± 3 and 10 ± 2 mg (control and cyclosporine sessions), corresponding to 5.8 ± 1.2 and 4.7 ± 1.0 mg of each methadone enantiomer base, and the cyclosporine dose was 361 ± 64 mg two times per day for 4 days. Twelve subjects (eight males, four females, 75 ± 14 kg, body mass index 25 ± 4) completed both arms of protocol 2. The average IV methadone hydrochloride dose was 7.5 ± 1.5 mg for control and cyclosporine sessions, corresponding to 3.4 ± 0.7 mg of each methadone enantiomer base. The cyclosporine dose was 424 ± 84 mg IV over 2 h. Analytical Methods Venous blood samples were obtained before and periodically for 96 (protocol 1) and 24 h (protocol 2) after methadone administration, and all urine was collected during these times. Plasma was separated and stored at −20°C for later analysis. Cyclosporine blood concentrations were determined by the clinical laboratory of Barnes-Jewish Hospital as trough (predose) and peak (2 h after oral cyclosporine) concentrations on the methadone study day (protocol 1), and 1 (mid-infusion), 2 (end-infusion), and 4 h after starting the IV cyclosporine infusion (protocol 2). Serum creatinine was determined before and after the study. Methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in plasma and urine were quantified by solid-phase extraction and stereoselective high-pressure liquid chromatography–mass spectrometry using a previous method.25 Interday coefficients of variation in plasma averaged 6 and 6% for EDDP enantiomers (0.2, 2 ng/ml) and 7, 5, and 4% for methadone enantiomers (1, 10, and 80 ng/ ml). Data and Statistical Analysis The intended primary outcome measure was the EC50 (plasma concentration causing 50% attenuation of response to thermal stimulation) and secondarily, EC50 for miosis, determined using a standard sigmoid Emax model, where Emax is the maximum possible effect (e.g., pupil diameter change, miosis) and C is plasma methadone concentration: