Performance of Population Pharmacokinetic Models in Predicting Polymyxin B Exposures.

Polymyxin B is the last line of defense in treating multidrug-resistant gram-negative bacterial infections. Dosing of polymyxin B is currently based on total body weight, and a substantial intersubject variability has been reported. We evaluated the performance of different population pharmacokinetic models to predict polymyxin B exposures observed in individual patients. In a prospective observational study, standard dosing (mean 2.5 mg/kg daily) was administered in 13 adult patients. Serial blood samples were obtained at steady state, and plasma polymyxin B concentrations were determined by a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. The best-fit estimates of clearance and daily doses were used to derive the observed area under the curve (AUC) in concentration-time profiles. For comparison, 5 different population pharmacokinetic models of polymyxin B were conditioned using patient-specific dosing and demographic (if applicable) variables to predict polymyxin B AUC of the same patient. The predictive performance of the models was assessed by the coefficient of correlation, bias, and precision. The correlations between observed and predicted AUC in all 5 models examined were poor (r2 < 0.2). Nonetheless, the models were reasonable in capturing AUC variability in the patient population. Therapeutic drug monitoring currently remains the only viable approach to individualized dosing.

Development and validation of liquid chromatography tandem mass spectrometry method quantitative determination of polymyxin B1, polymyxin B2, polymyxin B3 and isoleucine-polymyxin B1 in human plasma and its application in clinical studies.

Polymyxin B (PB) is an antibiotic consisting of a cyclic heptapeptide and a tripeptide side chain used in treatment of infections caused by Gram-negative bacteria. Commercial formulations of PB contain multiple structurally related components with major constituents of PB1, PB2, PB3 and ile-PB1. To understand the pharmacokinetics of these major components, we have developed and validated a LC-MS/MS method to quantify PB1, PB2, PB3 and ile-PB1 in human plasma. PB was extracted from plasma by protein precipitation using trichloroacetic acid followed by chromatographic separation on Zorbax Bonus-RP column (100mm×2.1mm, 1.8µm) using stepwise gradient elution of water containing 0.1% of formic acid and 0.1% of trichloroacetic acid (mobile phase A) and 90% acetonitrile with 0.1% formic acid (mobile phase B). Despite of structural similarities, these PBs were completely resolved in the analytical run time of 6.5min. Detection and quantification of PBs were performed by selected reaction monitoring (SRM) under positive ionization mode in the mass spectrometer. Separation of PB1 and ile-PB1, as well as PB2 and PB3, before quantification is crucial because they are structural isomers detected based the same SRM. Excellent linearity was achieved (r2>0.99) in the calibration curves of PB. The developed method was accurate (95.3-111.7%) and precise (CV<5.1%). Recovery of PB from the plasma extraction was between 53 and 76% and reproducible (CV<4.5%). Matrix effect was not observed by post-column infusion of PB in the mass spectrometer. This methodology has been successfully applied to clinical study of patients dosed with intravenous infusions of PB.

Development and validation of liquid chromatography tandem mass spectrometry method for simultaneous quantification of first line tuberculosis drugs and metabolites in human plasma and its application in clinical study.

Rifampicin (RIF) and isoniazid (INH), first line drugs for the treatment of tuberculosis, are known to cause hepatotoxicity as a serious adverse side effect. To further understand the pharmacokinetic parameters of these two drugs, we have developed and validated a rapid, sensitive and selective LC-MS/MS method for simultaneous quantification of RIF, INH and their metabolites 25-desacetylrifampicin (DRIF), acetylisoniazid (AcINH) and isonicotinic acid (INA). Analytes were extracted from 20 µl of plasma using solid-phase extraction (SPE) followed by chromatographic separation on Zorbax SB-Aq column (50 mm × 4.6mm, particle size 5 µm) using stepwise gradient elution of 5mM ammonium acetate and 90% acetonitrile with 0.1% formic acid. Separation of all analytes was achieved in the total run time of 6 min. The analytes were detected under positive ionization mode by multiple reaction monitoring (MRM) and quantification of analytes was performed by using deuterium-labelled internal standard. Excellent linearity (r(2) ≥ 0.995) was achieved for the analytes at different concentration ranges. The method was accurate (90-115%), precise (CV %<14) and specific. Matrix effect was in the range of 93-111% except for INA (40-42%) while recovery from SPE was reproducible (CV %<7.4) in the range of 60-86%. Post-preparative stability (48 h, 6°C autosampler) and freeze-thaw stability (3 cycles) were assessed with mean recovered concentration of >85%. The method was successfully applied to a clinical study of 33 healthy subjects to evaluate the effect of concomitant of INH on the pharmacokinetic parameters of RIF as well as the segregation of the subjects into slow or fast acetylators of INH.

Simultaneous determination of probe drugs, metabolites, inhibitors and inducer in human plasma by liquid chromatography/tandem mass spectrometry and its application to pharmacokinetic study.

Cytochrome P450 3A4 (CYP3A4) and UDP-glucuronosyltransferase 1A1 (UGT1A1) are important enzymes responsible for the metabolism of many xenobiotics. To investigate their induction and inhibition properties, administering probe drugs and monitoring their concentration in plasma under the effects of inducers/inhibitors is the gold standard method. A rapid and sensitive liquid chromatography-tandem mass spectrometry method was developed for simultaneous quantification of midazolam, raltegravir (probe drugs for CYP3A4 and UGT1A1), their major metabolites, 1'-hydroxymidazolam, 1'-hydroxymidazolam glucuronide and raltegravir glucuronide, rifampicin (inducer), ritonavir and ketoconazole (inhibitors). Analytes were extracted from 100µl of plasma using solid-phase extraction followed by chromatographic separation on a reversed-phase C18 column (50mm×2.1mm, particle size 1.8µm). The mass spectrometer was operated under positive ionization mode. Excellent linearity (r(2)≥0.995) was achieved for all. The method was validated and found to be accurate (88-111%), precise (CV%<13) and selective. Matrix effect was acceptable (88-118%) and analytes recovery was reproducible (60-95%). Analytes in plasma were also found to be stable in the autosampler (6°C for 48h) and after two freeze-thaw cycles. We have developed a robust analytical method to simultaneously quantify probes, inducer and inhibitor of important drug metabolism enzymes. The method was successfully applied in a clinical study to investigate the degree of induction and inhibition of CYP3A4 and UGT1A1 among ethnic groups in Singapore.