Introduction 1 2 3 Sorangium cellulosum 3 4 5 6 7 1 6 2 8 11 8 9 12 13 14 15 Fig. 1 Chemical structures of epothilone B and its lactam analogue ixabepilone  14 16 14 14 14 17 19 14 14 12 14 14 Materials and methods Study design 14 Patients n Study medication 14 14 Sorangium cellulosum 14 14 14 1 2 Sample collection Blood was collected at 0 (pre-dose), 1.5, 3 (prior to end of infusion), 3.25, 3.5, 3.75, 4, 5, 6, 8, 12, 24, 48, 72, 120, and 168 h after start of infusion. An indwelling catheter contra-lateral to the administration site was used for serial blood sampling. Separate, but simultaneous, blood samples were taken for the quantitation of total radioactivity and unchanged ixabepilone, respectively. Plasma was obtained by immediate centrifugation of the blood (10 min, 1000 × g, 4°C). The plasma layer was aspirated and stored below −20°C until analysis. We collected complete urinary and faecal output up to 7 days after administration. Urine was collected over 24-h intervals in refrigerated collection jugs. At the end of each collection interval, the respective urine samples were mixed and total volume was recorded. Aliquots were stored below −20°C until analysis. Faecal samples were collected and stored at −20°C per portion and combined per 24-h interval. Faeces were homogenized after addition of water (2:1, w/w). Aliquots were stored below −20°C until analysis. AMS analysis of total radioactivity (TRA) in plasma, urine and faeces 20 21 22 + 4+ 12 13 14 14 12 14 12 14 14 14 12 14 Analysis of ixabepilone in plasma and urine Concentrations of ixabepilone parent drug in plasma and urine were determined with a validated liquid chromatography assay equipped with tandem mass spectrometric detection (Bristol-Myers Squibb, Princeton, NJ). Briefly, after addition of internal standard (BMS-212188) to 0.2 ml of each sample, calibration standard and quality control sample, the samples were precipitated with acetone. The supernatant was further extracted with 1-chlorobutane. The organic layer was removed and evaporated to dryness. The residue was reconstituted and injected into the LC-MS/MS system. Chromatographic separation was achieved, isocratically, on a YMC ODS-AQ column (4.6 × 50 mm internal diameter) at a flow rate of 0.3 ml/min with detection by electrospray tandem mass spectrometry. The mobile phase contained acetonitrile—0.01 M ammonium acetate (pH 5.0) (65:35, v/v). The standard curve, which ranged from 2 to 500 ng/ml, was constructed with a 1/x weighted quadratic regression model. The within-run precision for ixabepilone in plasma and urine was within 15 and 9%, respectively. The between-run precision for ixabepilone in plasma and urine was within 13 and 9%, respectively. The accuracy was within 11 and 6% of the nominal values in plasma and urine, respectively. Pharmacokinetic analyses max max inf k k t 1/2 k inf inf inf 2 Outliers were statistically assessed using Dixon’s Q test. Results 14 2 Fig. 2 14 filled square 14 open square  1 14 14 1/2 3 max Table 1 14 Nr Ixabepilone Radioactivity (ixabepilone equiv.) last a max last inf t 1/2 Cl (l/h) ss max last inf t 1/2 1 277 3.06 3.35 51.7 20.9 1275 322 1.89 1.96 8.3 b 2 287 2.75 2.97 53.0 23.6 1174 497 11.0 11.8 48.7 0.25 3 433 2.83 2.99 51.7 23.4 929 681 10.9 11.3 36.5 0.26 4 486 3.82 4.32 67.4 16.2 1027 940 20.1 21.6 46.9 0.19 5 266 2.42 2.52 23.5 27.8 756 503 20.0 44.3 185.2 0.12 6 273 2.17 2.29 36.7 30.5 912 573 11.9 16.3 99.8 0.18 7 201 2.25 2.49 71.5 28.1 1520 449 8.53 9.59 61.2 0.26 8 187 1.93 2.22 47.2 31.6 1635 1502 32.5 34.4 34.4 0.06 Mean 301 2.65 2.89 50.3 25.3 1154 683 b b b b SD 105 0.60 0.69 15.4 5.2 308 378 b b b b a last last last b t 1/2 last Fig. 3 14 filled square open square  4 5 14 14 2 14 Q p Q p Fig. 4 open square open circle filled square 14  Fig. 5 14 filled square 14 open square  Table 2 14 a Recovery (% of dose) c renal Ixabepilone Total radioactivity Urine Urine Faeces Total 1 5.07 14.56 59.69 74.25 0.24 1.16 2 4.66 23.28 74.78 98.06 0.31 1.19 3 7.76 14.77 38.29 53.06 0.39 1.92 4 3.32 25.08 12.71 37.79 1.97 0.61 5 5.46 19.66 65.02 84.68 0.30 1.58 6 6.12 30.78 64.59 95.37 0.48 1.98 7 6.03 25.05 86.72 111.77 0.29 1.87 8 4.02 47.65 15.62 63.27 3.05 1.46 Mean 5.30 25.10 52.18 77.28 b 1.47 SD 1.38 10.63 27.17 24.94 b 0.47 a b c On average, more than 77% of the drug was excreted over the 7-day period with the majority of 52% being excreted in faeces and 25% in urine. Excretion in faeces occurred at a relatively constant rate over the entire collection period, with only 10 and 12% of dose recovered in the 0–24 and 24–48 h collection intervals, respectively. In contrast, urinary excretion displayed two distinct phases with 65% of total urinary radioactivity (corresponding to 17% of dose) and 76% of total urinary ixabepilone being excreted in the first 24 h. From 24 to 168 h, urinary excretion occurred at a relatively slow constant rate. This may reflect the biphasic plasma curve, with high concentrations in the distribution phase and lower concentrations in the terminal elimination phase. At first urinary excretion predominates and after 24 h, faecal excretion becomes the dominant route of excretion. Discussion 14 14 16 14 14 14 19 17 23 25 26 2 9 inf max t 1/2 16 16 14 2 16 27 28 wt Given the normal ixabepilone plasma concentrations of patient 8 while experiencing a blocked bile duct, biliary excretion of unchanged ixabepilone into faeces may not be a very important elimination route, and the results suggest that ixabepilone was equally well metabolised but that the biliary excretion of these metabolites was hindered by the blocked bile duct stent, resulting in a shift to urinary excretion of total radioactivity. The importance of metabolism in the elimination of ixabepilone has been confirmed by this study as apparent from the low contribution of unchanged ixabepilone to TRA in plasma and urine. Ixabepilone related radioactivity is predominantly excreted in the faeces. Future investigations must be aimed at elucidating the metabolic fate of ixabepilone, and determining the activity of the metabolites. Subsequent identification of drug metabolizing enzymes involved in ixabepilone metabolism may result in explaining pharmacokinetic variability of ixabepilone in individual patients.