Reported predictions of human in vivo hepatic clearance from in vitro data have used a variety of values for the scaling factors human microsomal protein (MPPGL) and hepatocellularity (HPGL) per gram of liver, generally with no consideration of the extent of their inter-individual variability. We have collated and analysed data from a number of sources, to provide weighted meangeo values of human MPPGL and HPGL of 32 mg g-1 (95% Confidence Interval (CI); 29-34 mg.g-1) and 99x10(6) cells.g-1 (95% CI; 74-131 mg.g-1), respectively. Although inter-individual variability in values of MPPGL and HPGL was statistically significant, gender, smoking or alcohol consumption could not be detected as significant covariates by multiple linear regression. However, there was a weak but statistically significant inverse relationship between age and both MPPGL and HPGL. These findings indicate the importance of considering differences between study populations when forecasting in vivo pharmacokinetic behaviour. Typical clinical pharmacology studies, particularly in early drug development, use young, fit, healthy male subjects of around 30 years of age. In contrast, the average age of patients for many diseases is about 60 years of age. The relationship between age and MPPGL observed in this study estimates values of 40 mg.g-1 for a 30 year old individual and 31 mg.g-1 for a 60 year old individual. Investigators may wish to consider the reported covariates in the selection of scaling factors appropriate for the population in which estimates of clearance are being predicted. Further studies are required to clarify the influence of age (especially in paediatric subjects), donor source and ethnicity on values of MPPGL and HPGL. In the meantime, we recommend that the estimates (and their variances) from the current meta-analysis be used when predicting in vivo kinetic parameters from in vitro data.

Aims To assess the utility of human hepatic microsomes for predicting in vivo intrinsic clearance (CL int ) via the use of four cytochrome P450 2C9 substrates: phenytoin, tolbutamide (S)-ibuprofen (two pathways) and diclofenac, and to examine the role of exogenous albumin within the microsomal incubation. Methods V max , K m and CL int (defined as V max /K m ratio) were estimated under initial rate conditions for five pathways of metabolism in a bank of 15 human hepatic microsomal samples and were scaled to in vivo units using the microsomal protein index. Non-metabolic related binding in microsomes was measured for phenytoin and tolbutamide in the presence and absence of albumin. Results Microsomal CL int values differed by over two orders of magnitude, with the means ranging from 0.18 ( phenytoin) to 40.70 (diclofenac) ml minmicrosomal protein. When these data were scaled and compared with published in vivo studies a similar rank order was obtained, however, the actual CL int tended to be underpredicted. While the in vivo unbound K m for phenytoin, 1-5 mm is substantially lower than the value determined in microsomes based on total concentrations (56 mm), correction for the in vitro binding reduces this value to 20 mm and 6 mm in the absence and presence of albumin, respectively. Similar trends were seen with tolbutamide K m . Conclusions An appreciation of the utility of in vitro prediction can be best achieved when the range of CL int values predicted from the individual hepatic microsomal samples are compared with the range of individual in vivo CL int values reported in the literature. The degree of underprediction is less evident using the range than the mean data and no consistent advantage in adding albumin to the incubation media is apparent. Keywords: CYP2C9, human microsomes, intrinsic clearance, in vitro predictionsin vitro and in vivo clearances of 25 drugs and were able Introduction to demonstrate a significant correlation despite the numerous sources of these data and the low sample A general strategy to quantitatively predict in vivo drug clearance from CL int values estimated from in vitro drug numbers upon which the parameters were based (see later). There remains a need for more indepth examinmetabolising systems has recently been explored and evaluated in the rat using a data base of 35 drugs which ations of the utility of human in vitro kinetic data for the prediction of the in vivo situation. show a range of turnovers covering four orders of magnitude [1][2][3]. The ultimate application of such aAs there is a shortage of good quality human liver material from which hepatocytes can be successfully prediction strategy is with human tissue, and although there have been many studies with human in vitro tissue, isolated, the vast majority of researchers employ the hepatic microsomal fraction as their enzyme source due often sufficient methodological detail is lacking from the original report to allow a systematic analysis. Iwatsubo to its ease of preparation from tissue of varying quality and i...

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Recent years have seen increasing usage of large particle size stationary phases and ultra‐high flow rate liquid chromatography/mass spectrometry (LC/MS) for rapid determination of pharmaceuticals in plasma without prior sample preparation. This lack of sample preparation prior to analysis, together with the extremely high throughput of the chromatography, makes the technique extremely attractive to the bioanalyst. Further, the introduction of multiple sprayer interfaces to mass spectrometers provides the potential for even higher throughput. In this paper, we present parallel ultra‐high flow rate liquid chromatography using four columns in parallel and a four‐way multiple sprayer interface to the mass spectrometer. We have applied this on both the narrow‐bore and capillary scale. This technique enables the quantification of drugs from four plasma samples simultaneously, at nanogram per millilitre concentrations, from small aliquots of plasma without sample preparation and with throughputs of up to 120 samples per hour. Copyright © 2000 John Wiley & Sons, Ltd.

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ABSTRACT:The pulmonary and hepatic expression and catalytic activities of phase I and II drug-metabolizing enzymes were compared using human lung and liver tissue, and lung parenchymal cells (LPCs) and cryopreserved hepatocytes. Cytochrome P450 gene expression was generally lower in lung than in liver and CYP3A4 expression in lung was negligible. Esterase gene expression was similar in lung and liver. Expression of all sulfotransferase isoforms in lung was similar to or higher than that in liver. Lung tissue expressed low levels of UGT. However, the expression of UGT2A1 in lung was higher than that in liver. There was a range of catalytic activities in LPCs, including cytochrome P450, esterase, and sulfation pathways. Phase I activities were generally less than 10% of those determined in hepatocytes. Rates of ester hydrolysis and sulfation in LPCs were similar to those in hepatocytes. When measurable, glucuronidation in LPCs was present at very low levels, reflecting the gene expression data. The metabolism of salbutamol, formoterol, and budesonide was also investigated. Production of salbutamol-4-O-sulfate and budesonide oleate was observed in LPCs from at least two of three donor preparations studied. Formoterol sulfate and low levels of formoterol glucuronide were detected in one of three donors. In general, drug-metabolizing capability of LPCs is low compared with liver, although some evidence for substantial sulfation and deesterification capacity was observed. Therefore, these data support the use of this cell-based system for the investigation of key routes of xenobiotic metabolism in human lung parenchyma.

Isolated hepatocytes and liver slices, in short-term suspension or longer-term culture, offer the prospect of providing qualitative metabolic information and quantitative pharmacokinetic parameters from key animal species and man at early stages of the drug discovery-development continuum. The propensity for changes in the fidelity of drug metabolism after removal of hepatocytes from the organ has long been recognized. The many and varied approaches which have been undertaken in an attempt to compensate for physiological shortcomings of in vitro hepatocyte systems are reviewed. In this respect, short-term suspension culture may provide a baseline against which to measure the success of extended culture methods, but it should be remembered that even freshly isolated hepatocyte preparations have deficiencies and liabilities that may affect the nature of information gathered. This article discusses the current advances and shortcomings of hepatocyte suspensions and cultures, along with liver slice technology, at both quantitative and qualitative levels.