To support the development of a dynamic in vitro human pharmacokinetic/pharmacodynamic simulation model for biofilm-mediated infections and study stability of meropenem, an LC-MS/MS method for the determination of meropenem in Luria Bertani (LB) media was developed and validated in an API2000 LC-MS/MS system. A partial validation was also performed in M9 media. Sample aliquots of 100μL (or 25μL for M9 media) were mixed with the internal standard (IS) ceftazidime and filtered. The filtrate was directly injected onto a C8 column eluted with ammonium formate (10mM, pH 4) and acetonitrile (0.1% formic acid) in a gradient mode. ESI(+) and MRM with ion pair m/z 384→68 for meropenem and m/z 547→468 for the IS were used for quantification. The calibration curve concentration range was 50 to 25,000ng/mL. The recovery was over 98%. In LB media, significant signal suppression was observed throughout the time period of detection when compared with mobile phase solvents, but the matrix effect was compensated well with the IS. In M9 media, much less signal suppression was observed. The method is simple, fast, and reliable. Using the method, stability of meropenem in LB and M9 media were tested. No significant degradation was observed for at least 8h in both LB media (37°C) and M9 media (30°C), but more than 15% degradation was observed overnight (∼20h). The method was transferred to an API5000 LC-MS/MS system using meropenem-d6 as the IS.

A method for quantification of fludarabine (FDB) and clofarabine (CFB) in human plasma was developed with an API5000 LC-MS/MS system. FDB and CFB were extracted from EDTA plasma samples by protein precipitation with trichloroacetic acid. Briefly, 50 μL plasma sample was mixed with 25 μL internal standard (50 ng/mL aqueous 2-Cl-adensosine) and 25 μL 20% trichloroacetic acid, centrifuged at 25,000 × g (20,000 rpm) for 3 min, and then transfered to an autosampler vial. The extracted sample was injected onto an Eclipse extend C18 column (2.1 mm×150 mm, 5 μm) and eluted with 1mM NH4OH (pH 9.6) - acetonitrile in a gradient mode. Electrospray ionization in positive mode (ESI(+)) and multiple reaction monitoring (MRM) were used, and ion pairs 286/134 for FDB, 304/170 for CFB and 302/134 for the internal standard were selected for quantification. The retention times were typically 3.72 min for FDB, 4.34 min for the internal standard, 4.79 min for CFB. Total run time was 10 min per sample. Calibration range was 0.5-80 ng/mL for CFB and 2-800 ng/mL for FDB. The method was applied to a clinical pharmacokinetic study in pediatric patients.

Objective

African potato (Hypoxis obtusa) is commonly used in Sub-Saharan Africa as a complementary herbal remedy for HIV-infected patients. It is unknown whether or not co-administration of African potato alters the pharmacokinetics of protease inhibitor antiretrovirals. The objective of this study was to investigate the impact of the African potato on the steady-state pharmacokinetics of ritonavir-boosted lopinavir (LPV/r).

Methods

Sixteen adult volunteers were administered LPV/r 400/100 mg twice a day for 14 days, followed by concomitant administration with African potato given once daily for 7 days. Lopinavir plasma exposure as estimated by the area under the concentration-time curve over the 12-h dosing interval (AUC(0-12h), AUCτ) was determined on day 14 and again on day 21. Lopinavir in plasma was analyzed using a validated liquid chromatography with tandem mass spectrometry (LC-MS/MS) method. Steady-state AUCτ and the maximum concentration following dose administration (C(max)) were determined using non-compartmental methods using WinNonlin Professional version 5.2.1. Statistical analyses were performed using Stata version 12.1.

Results

Co-administration of African potato was not associated with any change in lopinavir AUCτ, C(max), or C(trough).

Conclusions

African potato when taken concomitantly with LPV/r is well-tolerated and not associated with clinically significant changes in lopinavir pharmacokinetics.

Piperaquine (PQ) is part of a first-line treatment regimen for Plasmodium falciparum malaria recommended by the World Health Organization (WHO). We aimed to determine the major metabolic pathway(s) of PQ in vitro. A reliable, validated tandem mass spectrometry method was developed. Concentrations of PQ were measured after incubation with both human liver microsomes (HLMs) and expressed cytochrome P450 enzymes (P450s). In pooled HLMs, incubations with an initial PQ concentration of 0.3 µM resulted in a 34.8 ± 4.9% loss of substrate over 60 min, corresponding to a turnover rate of 0.009 min(-1) (r(2) = 0.9223). Miconazole, at nonspecific P450 inhibitory concentrations, resulted in almost complete inhibition of PQ metabolism. The greatest inhibition was demonstrated with selective CYP3A4 (100%) and CYP2C8 (66%) inhibitors. Using a mixture of recombinant P450 enzymes, turnover for PQ metabolism was estimated as 0.0099 min(-1); recombinant CYP3A4 had a higher metabolic rate (0.017 min(-1)) than recombinant CYP2C8 (p < .0001). Inhibition of CYP3A4-mediated PQ loss was greatest using the selective inhibitor ketoconazole (9.1 ± 3.5% loss with ketoconazole vs 60.7 ± 5.9% with no inhibitor, p < .0001). In summary, the extent of inhibition of in vitro metabolism with ketoconazole (83%) denotes that PQ appears to be primarily catalyzed by CYP3A4. Further studies to support these findings through the identification and characterization of PQ metabolites are planned.

Piperaquine (PQ) is part of a first-line treatment regimen for Plasmodium falciparum malaria recommended by the World Health Organization (WHO). We aimed to determine the major metabolic pathway(s) of PQ in vitro. A reliable, validated tandem mass spectrometry method was developed. Concentrations of PQ were measured after incubation with both human liver microsomes (HLMs) and expressed cytochrome P450 enzymes (P450s). In pooled HLMs, incubations with an initial PQ concentration of 0.3 µM resulted in a 34.8 ± 4.9% loss of substrate over 60 min, corresponding to a turnover rate of 0.009 min(-1) (r(2) = 0.9223). Miconazole, at nonspecific P450 inhibitory concentrations, resulted in almost complete inhibition of PQ metabolism. The greatest inhibition was demonstrated with selective CYP3A4 (100%) and CYP2C8 (66%) inhibitors. Using a mixture of recombinant P450 enzymes, turnover for PQ metabolism was estimated as 0.0099 min(-1); recombinant CYP3A4 had a higher metabolic rate (0.017 min(-1)) than recombinant CYP2C8 (p < .0001). Inhibition of CYP3A4-mediated PQ loss was greatest using the selective inhibitor ketoconazole (9.1 ± 3.5% loss with ketoconazole vs 60.7 ± 5.9% with no inhibitor, p < .0001). In summary, the extent of inhibition of in vitro metabolism with ketoconazole (83%) denotes that PQ appears to be primarily catalyzed by CYP3A4. Further studies to support these findings through the identification and characterization of PQ metabolites are planned.

Background

The antiretroviral drug efavirenz (EFV) and the antimalarial artemisinin-based combination therapy artemether-lumefantrine (AL) are commonly co-administered to treat HIV and malaria. EFV is a known inducer of cytochrome P450 3A4, which converts artemether to dihydroartemisinin (DHA) that is also active and metabolizes longer acting lumefantrine (LR). A study in healthy volunteers was completed to address the concern that EFV impacts AL pharmacokinetics (PKs).

Methods

Adults received AL (80/480 mg twice daily) for 3-days before and during EFV co-administration (600 mg daily for 26 days) with intensive PK for artemether, DHA, and LR conducted after the last AL dose for each period. EFV PK was evaluated with and without AL. PK parameters were estimated using noncompartmental methods.

Results

Twelve subjects completed the 2-period study. PK exposure for artemether, DHA, and LR [as estimated by the area under the concentration time curve (AUClast)] decreased or trended toward decrease with EFV, compared with when administered alone [-51% (P = 0.084), -46% (P = 0.005), and -21% (P = 0.102), respectively]. Day-7 LR levels, previously deemed predictive of treatment success, were 46% lower (P = 0.002) with EFV, but the LR half-life was unchanged. EFV PK exposure was minimally altered after AL co-administration [AUC0-24 hrs decreased by 17% (P = 0.034)].

Conclusions

Exposure to DHA, but not LR, was significantly lower during EFV-AL co-administration compared with that during administration of AL alone. These findings may have implications for the treatment efficacy of AL, particularly in children. However, the observed modest changes probably do not warrant dosage adjustment during co-administration of AL with EFV.