This article is available online at http://dmd.aspetjournals.org ABSTRACT:Sixty-four compounds with diverse structures were used in evaluation of intrinsic clearance by various hepatocyte preparations from rats, dogs, monkeys, and humans. Intrinsic clearance (CL int ) was calculated from the ratio of the initial amount of the test compound minus the amount remaining after 2 h of incubation and the corresponding area under the concentration versus time curve. The predictive potential of this in vitro model was tested by comparing the intrinsic clearance with in vivo clearance using linear regression analysis. In addition, the intrinsic clearance values obtained from three different types of hepatocytes (cryopreserved, fresh, and sandwich-cultured) from the same species were compared to determine the influence of preservation and culture conditions. It seems that intrinsic clearance determined from human cryopreserved hepatocyte (R 2 ‫؍‬ 0. In the early phase of drug discovery, animal pharmacokinetic data are used for advancing drug candidates to the clinical phase of drug development. According to a study by Prentis et al. (1988), approximately 40% of drug candidates failed because of poor human pharmacokinetic characteristics. For this reason, numerous attempts have been performed in recent years to find a reliable way of predicting in vivo hepatic clearance in humans. For example, Lavé et al. (1997) demonstrated a correlation between intrinsic clearance in human hepatocytes and hepatic extraction ratio in humans, and used the correlation to classify compounds into low, intermediate, or high hepatic extraction ratio in humans. Obach (1997a) used hepatic microsomal intrinsic clearance to predict human in vivo clearance and Houston and Carlile (1997) compared the use of microsomes, hepatocytes, and liver slices for prediction of hepatic clearance in rats. They showed that both microsomes and hepatocytes might be useful for obtaining the rank order of clearances. Schneider et al. (1999) used multivariate statistical models and artificial neural networks to predict the hepatic drug clearance in humans from in vitro hepatocyte data. These in vitro models yielded a wide range of in vivo-in vitro correlation indexes that, in general, suggest the potential for prediction of in vivo human clearance using in vitro clearance data.In addition to developing various in vitro physiological models for the evaluation of in vivo-in vitro correlations, considerable effort has been made toward methods for calculation of intrinsic clearance. In some studies, intrinsic clearance was obtained by determining the enzyme kinetic parameters (V max and K m ). The ratio of these two parameters was then equated with intrinsic clearance. In the second method, referred to as the "in vitro t 1/2 method", intrinsic clearance (CL int ) was calculated by measuring the first order rate constant for consumption of the substrate at low concentration. In a third method, intrinsic clearance was calculated from the ratio of the initial amount of the t...

Human insulin was the first mammalian protein produced in bacteria using recombinant DNA technology. Two technologies were developed; the first based on the separate expression of precursors of chains A and B of insulin, and the second based on the expression of a precursor of proinsulin as a Trp-E fusion protein. Both technologies utilized Escherichia coli as an expression system. Later, a third technology was developed based on a strain of yeast that can secrete a precursor of insulin. The second E. coli process, a variation of which has been commercialized by Eli Lilly and Co., is analyzed in this article from a process design and economic evaluation viewpoint. The objective of this work is to elucidate the technical complexity and high cost associated with the manufacturing of biopharmaceuticals. Human insulin is a good example of a protein-based biopharmaceutical produced in large quantities (a fex tons per year) that requires large scale equipment and presents a multitude of scale-up challenges. Based onthe analysis, a number of conclusions are drawn regarding the cost breakdown and cost dependency on process parameters. Recommendations are made for cost reduction and environmental impact minimization. This analysis was performed using a software tool for computer-aided bioprocess design. (c) 1995 John Wiley & Sons, Inc.

The present data show that in vitro extraction ratios in monkey microsomes are predictive of the in vivo hepatic extraction ratios in monkeys. All compounds with high extraction ratio (>70%) in vivo were successfully classified as high-extraction-ratio compounds based on the in vitro monkey microsomal stability data. From the results of this study, it appears that the parallel tube model provided a slightly better classification than the well-stirred model. CONCULUSIONS: The present method appears to be a valuable tool to rapidly screen and prioritize compounds with respect to liver first-pass metabolism in monkeys at an early phase of drug discovery.

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