Wenfang B. Fang

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.

Gender Differences in Pharmacokinetics of Maintenance Dosed Buprenorphine

AIMS Gender differences are known to occur in the pharmacokinetics of many drugs. Mechanisms may include differences in body composition, body weight, cardiac output, hormonal status, and use of different co-medications. Recently subtle gender-dependent differences in cytochrome P450 (CYP) 3A-dependent metabolism have been demonstrated. Buprenorphine N-dealkylation to norbuprenorphine is primarily performed by CYP3A. We therefore asked whether gender-dependent differences occur in the pharmacokinetics of buprenorphine. METHODS A retrospective examination was made of control (buprenorphine/naloxone-only) sessions from a number of drug interaction studies between buprenorphine and antiretroviral drugs. Twenty males and eleven females were identified who had a negative cocaine urine test prior to participation in the control session and were all on the same maintenance dose (16/4 mg) of sublingual buprenorphine/naloxone. Pharmacokinetic data from their control sessions (buprenorphine/naloxone only) were sorted by gender and compared using the two-sample t-test. RESULTS Females had significantly higher area under the plasma concentration curve (AUC) and maximum plasma concentrations for buprenorphine, norbuprenorphine and norbuprenorphine-3-glucuronide. AUCs relative to dose per body weight and surface area were significantly higher for only norbuprenorphine. AUCs relative to lean body mass were, however, not significantly different. CONCLUSIONS Gender-related differences exist in the pharmacokinetics of buprenorphine; differences in body composition appear to have a major impact; differences in CYPA-dependent metabolism may also contribute.

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.

Pharmacokinetics of Intranasal Crushed OxyContin and Intravenous Oxycodone in Nondependent Prescription Opioid Abusers

Prescription opioid misuse and unintentional overdose deaths involving these analgesics are increasing public health concerns among all ages in the United States.1-4 According to the US Centers for Disease Control and Prevention, prescription opioid-related overdose deaths have become the leading cause of injury deaths, outpacing motor-vehicle crashes in 6 US states and Washington, DC, in 2006.5 The increases in illicit prescription opioid use and related deaths are associated with a marked increase in number of opioid prescriptions written.6,7 OxyContin (Purdue Pharma) is an extended-release oxycodone formulation approved by the US Food and Drug Administration (FDA) for treatment of moderate to severe pain. It is a Schedule II controlled substance in the United States with a significant history of aggressive commercial marketing as well as abuse and diversion.8,9 Pharmacokinetic studies of orally administered OxyContin report a mean terminal half-life (t1/2) of approximately 4.5 to 6.5 hours, mean time to maximum concentration (Tmax) of 2.5 to 5 hours, and oral bioavailability equivalent to immediate-release oxycodone (mean 60%, standard deviation [SD] 20%).10-12 Oxycodone is metabolized by N-demethylation via cytochrome P450 3A4 to noroxycodone, its primary metabolite. It is unlikely that noroxycodone contributes to the pharmacodynamic response in humans, because it has 4-fold lower affinity for the receptor than oxycodone and low μ-opioid receptor activation in GTPγS binding studies.13 However, oxycodone also is metabolized by O-demethylation via cytochrome P450 2D6 to oxymorphone,13 a μ-opioid agonist with analgesic potency approximately 10 times that of parenteral morphine.14 Immediate-release oxycodone products are prescribed approximately 5 times more frequently than extended-release oxycodone products; however, emergency department visits report the nonmedical use of extended-release oxycodone products approximately 4 times more often than immediate-release oxycodone products.15 OxyContin tablets can be easily crushed and snorted or injected to bypass the sustained-release feature, which likely contributes to its abuse liability. In 2008, 15% of publically funded substance abuse treatment admissions with prescription opioid abuse (eg, abuse defined as substance causing problems leading to the admission) reported snorting prescription opioids.16 However, there are no intranasal (IN) pharmacokinetic data demonstrating that OxyContin tablets have immediate-release characteristics when crushed, and the IN bioavailability of OxyContin is unknown despite its common misuse by this route. Thus, this study evaluated the pharmacokinetics of oxycodone and its key metabolites when administered as IN crushed OxyContin tablets compared with intravenous (IV) immediate-release oxycodone solution.

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.

Determination of Oxycodone, Noroxycodone and Oxymorphone by High-Performance Liquid Chromatography–Electrospray Ionization-Tandem Mass Spectrometry in Human Matrices: In vivo and In vitro Applications

The opioid analgesic oxycodone is widely abused and increasingly associated with overdose deaths. A sensitive analytical method was developed for oxycodone and its metabolites, noroxycodone and oxymorphone, in human plasma, urine (±enzymatic hydrolysis at 50°C for 16 h) and liver microsomes (HLMs). Liquid-liquid extraction was followed by high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. The calibration range was 0.2-250 ng/mL for plasma and HLM and 10-5000 ng/mL for urine. Intra- and interrun accuracies were within 13.3% of target; precisions were within 12.8% for all matrices. Recoveries from plasma were: oxycodone, 75.6%; noroxycodone, 37.4% and oxymorphone, 18.2%. Analytes exhibited room temperature stability in plasma and urine up to 24 h, and freeze-thaw stability in plasma up to three cycles. In 24-h hydrolyzed urine from subjects administered intranasal oxycodone (30 mg/70 kg, n = 5), mean concentrations (ng/mL) and % daily doses excreted were: oxycodone, 1150, 6.53%; noroxycodone, 1330, 7.81% and oxymorphone, 3000, 17.1%. Oxycodone incubated with HLM produced more noroxycodone than oxymorphone. With a panel of recombinant human cytochrome P450s (CYPs), CYP2C18 and CYP3A4 produced the most noroxycodone, whereas CYP2D6 produced the most oxymorphone. These results demonstrate a new method suitable for both in vivo and in vitro metabolism and pharmacokinetic studies of oxycodone.

Azole antifungal inhibition of buprenorphine, methadone and oxycodone in vitro metabolism.

Opioid-related mortality rates have escalated. Drug interactions may increase blood concentrations of the opioid. We therefore used human liver microsomes (HLMs) and cDNA-expressed human cytochrome P450s (rCYPs) to study in vitro inhibition of buprenorphine metabolism to norbuprenorphine (CYP3A4 and 2C8), oxycodone metabolism to noroxycodone (CYP3A4 and 2C18) and oxymorphone (CYP2D6), and methadone metabolism to R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP; CYP3A4 and 2B6). In this study, we have examined the inhibitory effect of 12 (mostly antifungal) azoles. These compounds have a wide range of solubility; to keep organic solvent ≤1%, there was an equally wide range of highest concentration tested (e.g., itraconazole 5 µM to fluconazole 1000 µM). Inhibitors were first incubated with HLMs at three concentrations with or without preincubation of inhibitor with reducing equivalents to also screen for time-dependent inhibition (TDI). Posaconazole displayed evidence of TDI; metronidazole and albendazole had no significant effect. Azoles were next screened at the highest achievable concentration for non-CYP3A4 pathways. IC50 values (µM) were determined for most CYP3A4 pathways (ranges) and other pathways as dictated by screen results: clotrimazole (0.30 - 0.35; others >30 µM); econazole (2.2 - 4.9; 2B6 R-EDDP - 9.5, S-EDDP - 6.8; 2C8 - 6.0; 2C18 - 1.0; 2D6 - 1.2); fluconazole (7.7 - 66; 2B6 - 313, 361; 2C8 - 1240; 2C18 - 17; 2D6 - 1000); itraconazole (2.5 to >5; others >5); ketoconazole (0.032 - 0.094; 2B6 - 12, 31; 2C8 - 78; 2C18 - 0.98; 2D6 - 182); miconazole (2.3 - 7.6; 2B6 - 2.8, 2.8; 2C8 - 5.3; 2C18 - 3.1; 2D6 - 5.9); posaconazole (3.4 - 20; 2C18 - 3.8; others >30); terconazole (0.48 to >10; 2C18 - 8.1; others >10) and voriconazole (0.40 - 15; 2B6 - 2.4, 2.5; 2C8 - 170; 2C18 - 13; 2D6 >300). Modeling based on estimated Ki values and plasma concentrations from the literature suggest that the orally administered azoles, particularly ketoconazole and voriconazole, have the greatest potential for inhibiting CYP3A4 pathways, as does voriconazole for the CYP2B6 pathways. Azoles used for mucosal and topical applications did not exceed the modeling threshold.

Tipranavir/ritonavir induction of buprenorphine glucuronide metabolism in HIV-negative subjects chronically receiving buprenorphine/naloxone.

Background: Previous reports on the pharmacokinetic of tipranavir (TPV) and buprenorphine (BUP)/ naloxone found that coadministration resulted in an 80% reduction in the area under the curve AUC of the primary BUP metabolite, norBUP, without any pharmacodynamic consequences. This study was conducted to characterize how tipranivir/ritonavir effects the glucuronide metabolites of BUP and may explain the reduction in the norBUP. Methods: HIV-seronegative subjects stabilized on at least 3 weeks of BUP/naloxone sequentially underwent baseline and steady-state pharmacokinetic evaluation of twice daily TPV 500 mg coadministered with ritonavir 200 mg (TPV/r). Results: Twelve subjects were enrolled and ten completed the study. The steady-state pharmacokinetics for BUP-3-glucuronide (BUP-3G) and norBUP-3-glucuronide (norBUP-3G) in the presence and absence of steady-state TPV/r were analyzed. The Cmax of BUP-3G was 8.78 ± 5.23 ng/mL without TPV/r and increased to 12.7 ± 11.7 after steady state of TPV/r was achieved. The AUC of BUP-3G was 31.1 ± 19.4 (ng/mL) (h) without TPV/r and increased to 58. 6 ± 49.5 after steady state of TPV/r was achieved (p = .0966). In contrast, steady-state norBUP-3G AUC0–24 h (p = .0216) and Cmax (p = .0088) were significantly decreased in the presence of steady-state TPV/r. Conclusions and Scientific Significance: This study further elucidates the effects of TPV/r on glucuronidation. The current evaluation of glucuronide metabolites of BUP and norBUP are suggestive of combined inhibition of Uridine diphosphate (UDP)-glucuronosyltransferase of the 1A family and cytochrome P450 3A4 that spares UGT2B7 leading to a shunting of BUP away from production of norBUP and toward BUP-3G as seen by a statistically significant increase in the AUC of BUP-3G.

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.

In vitro Inhibition of Methadone and Oxycodone Cytochrome P450-Dependent Metabolism: Reversible Inhibition by H2-Receptor Agonists and Proton-Pump Inhibitors

In vitro inhibition of oxycodone metabolism to noroxycodone and oxymorphone and R- and S-methadone metabolism to R- and S-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) was measured for four H2-receptor antagonists and five proton-pump inhibitors (PPIs) using human liver microsomes (HLM) and cDNA-expressed human cytochrome P450s (rCYPs). Inhibitors were first incubated with HLM at three concentrations with and without preincubation of inhibitor, enzyme source and reducing equivalents to also screen for time-dependent inhibition (TDI). Cimetidine and famotidine (10-1,000 µM) inhibited all the four pathways >50%. Nizatidine and ranitidine did not. All the five PPIs (1-200 µM) inhibited one or more pathways >50%. Half maximal inhibitory concentrations (IC50s) were then determined using rCYPs. Cimetidine and famotidine both inhibited CYP3A4-mediated formation of noroxycodone and CYP2D6-mediated formation of oxymorphone, and famotidine inhibited CYP3A4-mediated formation of R- and S-EDDP, but IC50s were so high that only >10× therapeutic concentrations may have potential for reversible in vivo inhibition. The PPIs were more potent inhibitors; many have the potential for reversible in vivo inhibition at therapeutic concentrations. Omeprazole, esomeprazole and pantoprazole had greater effects on CYP3A4-mediated reactions, whereas lansoprazole was selective for CYP2D6-mediated formation of oxymorphone. Preincubation enhanced cimetidine inhibition of noroxycodone formation and rabeprazole inhibition of all pathways. Future studies will explore irreversible TDI.