Shen Nan Lin

Stereo-Selective Metabolism of Methadone by Human Liver Microsomes and cDNA-Expressed Cytochrome P450s: A Reconciliation

In vitro metabolism of methadone was investigated in cytochrome P450 (CYP) supersomes and phenotyped human liver microsomes (HLMs) to reconcile past findings on CYP involvement in stereo-selective metabolism of methadone. Racaemic methadone was used for incubations; (R)- and (S)-methadone turnover and (R)- and (S)-EDDP formation were determined using chiral liquid chromatography-tandem mass spectrometry. CYP supersome activity for methadone use and EDDP formation ranked CYP2B6 > 3A4 > 2C19 > 2D6 > 2C18, 3A7 > 2C8, 2C9, 3A5. After abundance scaling, CYP3A4, 2B6 and 2C19 accounted for 63-74, 12-32 and 1. 4-14% of respective activity. CYP2B6, 2D6 and 2C18 demonstrated a preference for (S)-EDDP formation; CYP2C19, 3A7 and 2C8 for (R)-EDDP; 3A4 none. Correlation analysis with 15 HLMs supported the involvement of CYP2B6 and 3A. The significant correlation of S/R ratio with CYP2B6 activity confirmed its stereo-selectivity. CYP2C19 and 2D6 inhibitors and monoclonal antibody (mAb) did not inhibit EDDP formation in HLM. Chemical and mAb inhibition of CYP3A in high 3A activity HLM reduced EDDP formation by 60-85%; inhibition of CYP2B6 in 2B6 high-activity HLM reduced (S)-EDDP formation by 80% and (R)-EDDP formation by 55%. Inhibition changed methadone metabolism in a stereo-selective manner. When CYP3A was inhibited, 2B6 mediated (S)-EDDP formation predominated; S/R stereo-selectivity increased. When 2B6 was inhibited (S)-EDDP formation fell and stereo-selectivity decreased. The results confirmed the primary roles of CYPs 3A4 and 2B6 in methadone metabolism; CYP2C8 and 2C9 did not appear involved; 2C19 and 2D6 have minimal roles. CYP2B6 is the primary determinant of stereo-selective metabolism; stereo-selective inhibition might play a role in varied plasma concentrations of the two enantiomers.

Repeated Dosing with Oral Cocaine in Humans: Assessment of Direct Effects, Withdrawal and Pharmacokinetics

Cocaine withdrawal symptoms are thought to play a role in relapse; studies characterizing the symptomatology have yielded mixed findings. This study sought to examine the pharmacodynamic/pharmacokinetic profile of repeated high dose exposure to oral cocaine and characterize acute and protracted withdrawal in cocaine abusers. This study employed a repeated-dosing, single-blind design in which subjects (n = 9), resided for 40 days on a closed ward. They were maintained for two 4-day cocaine exposure periods (Days 1-4 & Days 9-12, cocaine 175 mg, p.o.; 5 hourly doses; 875 mg/day) separated by a 4-day matched placebo exposure period (Days 5-8). After these 12 days, an additional period of 28 days of placebo maintenance followed (Days 13-40). Test sessions were conducted during each phase; measures of mood, drug effects, sleep, pharmacokinetics, and prolactin were collected throughout the study. The dosing regimen produced cocaine plasma concentrations (Cmax of 680 ng/mL) two to threefold higher than typically seen in acute dose studies. Prototypic psychostimulant effects, including subjective ratings of euphoric effects (liking, high, good effects) and significant cardiopressor effects, were sustained during the active dosing periods, corresponding to the rise and fall of plasma cocaine. Withdrawal-like symptoms (i.e., disruptions of sleep, increased ratings of anxiety, irritability, crashing) were observed within 24-hr after cessation of dosing. Cocaine reduced prolactin acutely, but no sustained alterations were observed for this measure or for other signs or symptoms during the 28-day abstinence period. These findings indicate that exposure to controlled high doses of cocaine produces modest symptoms consistent with cocaine withdrawal within hours of cessation of dosing but provide no evidence of symptoms persisting beyond 24 hours.

Pharmacokinetic Interactions Between Buprenorphine/Naloxone and Tipranavir/Ritonavir in HIV-Negative Subjects Chronically Receiving Buprenorphine/Naloxone

HIV-infected patients with opioid dependence often require opioid replacement therapy. Pharmacokinetic interactions between HIV therapy and opioid dependence treatment medications can occur. HIV-seronegative subjects stabilized on at least 3 weeks of buprenorphine/naloxone (BUP/NLX) therapy sequentially underwent baseline and steady-state pharmacokinetic evaluation of open-label, twice daily tipranavir 500 mg co-administered with ritonavir 200 mg (TPV/r). Twelve subjects were enrolled and 10 completed the study. Prior to starting TPV/r, the geometric mean BUP AUC(0-24h) and C(max) were 43.9 ng h/mL and 5.61 ng/mL, respectively. After achieving steady-state with TPV/r (> or = 7 days), these values were similar at 43.7 ng h/mL and 4.84 ng/mL, respectively. Similar analyses for norBUP, the primary metabolite of BUP, demonstrated a reduction in geometric mean for AUC(0-24h) [68.7-14.7 ng h/mL; ratio=0.21 (90% CI 0.19-0.25)] and C(max) [4.75-0.94 ng/mL; ratio=0.20 (90% CI 0.17-0.23)]. The last measurable NLX concentration (C(last)) in the concentration-time profile, never measured in previous BUP/NLX interaction studies with antiretroviral medications, was decreased by 20%. Despite these pharmacokinetic effects on BUP metabolites and NLX, no clinical opioid withdrawal symptoms were noted. TPV steady-state AUC(0-12h) and C(max) decreased 19% and 25%, respectively, and C(min) was relatively unchanged when compared to historical control subjects receiving TPV/r alone. No dosage modification of BUP/NLX is required when co-administered with TPV/r. Though mechanistically unclear, it is likely that decreased plasma RTV levels while on BUP/NLX contributed substantially to the decrease in TPV levels. BUP/NLX and TPV/r should therefore be used cautiously to avoid decreased efficacy of TPV in patients taking these agents concomitantly.

Pharmacokinetic interactions between buprenorphine/naloxone and once-daily lopinavir/ritonavir

Background:This study was conducted to examine the pharmacokinetic interactions between buprenorphine/naloxone (BUP/NLX) and lopinavir/ritonavir (LPV/r) in HIV-seronegative subjects chronically maintained on BUP/NLX. Methods:This study was an open labeled pharmacokinetic study in twelve HIV-seronegative subjects stabilized on at least 3 weeks of BUP/NLX therapy. Subjects sequentially underwent baseline and steady-state pharmacokinetic evaluation of once-daily LPV/r (800/200 mg). Results:Compared to baseline values, BUP AUC0-24h (46.8 vs. 46.2 ng*hr/mL) and Cmax (6.54 vs. 5.88 ng/mL) did not differ significantly after achieving steady-state LPV/r. Similar analyses of norBUP, the primary metabolite of BUP, demonstrated no significant difference in norBUP AUC0-24 hours (73.7 vs. 52.7 ng·h/mL); however, Cmax (5.29 vs. 3.11 ng/mL) levels were statistically different (P < 0.05) after LPV/r administration. Naloxone concentrations were similarly unchanged for AUC0-24 hours (0.421 vs. 0.374 ng·hr/mL) and Cmax (0.186 vs. 0.186 ng/mL). Using standardized measures, no objective opioid withdrawal was observed. The AUC0-24 hours and Cmin of LPV in this study did not significantly differ from historical controls (159.6 vs. 171.3 μg·hr/mL) and (2.3 vs. 1.3 μg/mL). Conclusions:The addition of LPV/r to stabilized patients receiving BUP/NLX did not affect buprenorphine pharmacokinetics but did increase the clearance of norbuprenorphine. Pharmacodynamic responses indicate that the altered norbuprenorphine clearance did not lead to opioid withdrawal. Buprenorphine/naloxone and LPV/r can be safely coadministered without need for dosage modification.

Effect of rifampin and nelfinavir on the metabolism of methadone and buprenorphine in primary cultures of human hepatocytes.

We tested the hypothesis that primary cultures of human hepatocytes could predict potential drug interactions with methadone and buprenorphine. Hepatocytes (five donors) were preincubated with dimethyl sulfoxide (DMSO) (vehicle), rifampin, or nelfinavir before incubation with methadone or buprenorphine. Culture media (0–60 min) was analyzed by liquid chromatography-tandem mass spectrometry for R- and S-methadone and R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) or for buprenorphine, norbuprenorphine, and their glucuronides [buprenorphine-3-glucuronide (B-3-G) and norbuprenorphine-3-glucuronide (N-3-G)]. R- and S-EDDP were detected in three of five, four of five, and five of five media from cells pretreated with DMSO, nelfinavir, and rifampin. R-EDDP increased 3.1- and 26.5-fold, and S-EDDP increased 2.5- and 21.3-fold after nelfinavir and rifampin. The rifampin effect was significant. B-3-G production was detected in media of all cells incubated with buprenorphine and accounted for most of the buprenorphine loss from culture media; it was not significantly affected by either pretreatment. Norbuprenorphine and N-3-G together were detected in three of five, four of five, and five of five donors pretreated with DMSO, nelfinavir and rifampin, and norbuprenorphine in one of five, one of five, and two of five donors. Although there was a trend for norbuprenorphine (2.8- and 4.9-fold) and N-3-G (1.7- and 1.9-fold) to increase after nelfinavir and rifampin, none of the changes were significant. To investigate low norbuprenorphine production, buprenorphine was incubated with human liver and small intestine microsomes fortified to support both N-dealkylation and glucuronidation; N-dealkylation predominated in small intestine and glucuronidation in liver microsomes. These studies support the hypothesis that methadone metabolism and its potential for drug interactions can be predicted with cultured human hepatocytes, but for buprenorphine the combined effects of hepatic and small intestinal metabolism are probably involved.