Development and evaluation of a multiple-plate fraction collector for sample processing: application to radioprofiling in drug metabolism studies.

Microplate scintillation counters are utilized routinely in drug metabolism laboratories for the off-line radioanalysis of fractions collected during HPLC radioprofiling. In this process, the current fraction collection technology is limited by the number of plates that can be used per injection as well as the potential for sample loss due to dripping or spraying as the fraction collector head moves from well to well or between plates. More importantly, sample throughput is limited in the conventional process, since the collection plates must be manually exchanged after each injection. The Collect PAL, an innovative multiple-plate fraction collector, was developed to address these deficiencies and improve overall sample throughput. It employs a zero-loss design and has sub-ambient temperature control. Operation of the system is completely controlled with software and up to 24 (96- or 384-well) fraction collection plates can be loaded in a completely automated run. The system may also be configured for collection into various-sized tubes or vials. At flow rates of 0.5 or 1.0 mL/min and at collection times of 10 or 15s, the system precisely delivered 83-µL fractions (within 4.1% CV) and 250-µL fractions (within 1.4% CV), respectively, of three different mobile phases into 12 mm × 32 mm vials. Similarly, at a flow rate of 1 mL/min and 10s collection times, the system precisely dispensed mobile phase containing a [(14)C]-radiolabeled compound across an entire 96-well plate (% CV was within 5.3%). Triplicate analyses of metabolism test samples containing [(14)C]buspirone and its metabolites, derived from three different matrices (plasma, urine and bile), indicated that the Collect PAL produced radioprofiles that were reproducible and comparable to the current technology; the % CV for 9 selected peaks in the radioprofiles generated with the Collect PAL were within 9.3%. Radioprofiles generated by collecting into 96- and 384-well plates were qualitatively comparable; however, the peak resolution was greater in the profiles that were collected in 384-well plates due to the collection of a larger number of fractions per minute. In conclusion, this new and innovative fraction collector generated radioprofile results that were comparable to current technology and should provide a major improvement in capacity and throughput for radioprofiling studies.

In vitro inhibition of UDP glucuronosyltransferases by atazanavir and other HIV protease inhibitors and the relationship of this property to in vivo bilirubin glucuronidation.

Several human immunodeficiency virus (HIV) protease inhibitors, including atazanavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir, were tested for their potential to inhibit uridine 5'-diphospho-glucuronosyltransferase (UGT) activity. Experiments were performed with human cDNA-expressed enzymes (UGT1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) as well as human liver microsomes. All of the protease inhibitors tested were inhibitors of UGT1A1, UGT1A3, and UGT1A4 with IC(50) values that ranged from 2 to 87 microM. The IC50 values found for all compounds for UGT1A6, 1A9, and 2B7 were >100 microM. The inhibition (IC50) of UGT1A1 was similar when tested against the human cDNA-expressed enzyme or human liver microsomes for atazanavir, indinavir, and saquinavir (2.4, 87, and 7.3 microM versus 2.5, 68, and 5.0 microM, respectively). By analysis of the double-reciprocal plots of bilirubin glucuronidation activities at different bilirubin concentrations in the presence of fixed concentrations of inhibitors, the UGT1A1 inhibition by atazanavir and indinavir was demonstrated to follow a linear mixed-type inhibition mechanism (Ki = 1.9 and 47.9 microM, respectively). These results suggest that a direct inhibition of UGT1A1-mediated bilirubin glucuronidation may provide a mechanism for the reversible hyperbilirubinemia associated with administration of atazanavir as well as indinavir. In vitro-in vivo scaling with [I]/Ki predicts that atazanavir and indinavir are more likely to induce hyperbilirubinemia than other HIV protease inhibitors studied when a free Cmax drug concentration was used. Our current study provides a unique example of in vitro-in vivo correlation for an endogenous UGT-mediated metabolic pathway.