James P. Byers

Prediction of in vivo hepatic clearance from in vitro data using cryopreserved human hepatocytes

1. Cryopreserved human hepatocytes were used to predict in vivo hepatic clearance (CL hepatic) from estimates of in vitro intrinsic clearance (). 2. was estimated for phenytoin, valproic acid, carbamazepine, theophylline, quinidine and procainamide after their addition to hepatocytes suspended either in human serum or in serum-free media. was estimated from in vitro concentration versus time data fitted to a monoexponential decay model. was estimated from concentrations measured at four time points and from just two-point measures, namely the initial concentration (C 0) and the final concentration measurement (C last). 3. Predicted CL hepatic was within twofold of reported in vivo values of CL hepatic for all substrates. Moreover, predictions were not significantly different whether derived from hepatocytes suspended in serum or in serum-free medium. 4. Two-point estimates of were just as accurate in predicting CL hepatic as were multipoint estimates of. 5. Although the data set was limited, the findings suggest that the measurement of the disappearance of xenobiotics from serum or serum-free media in which primary human hepatocytes have been suspended provides a physiologically relevant estimate of hepatic clearance that can be employed early in the drug development process to eliminate xenobiotics with unacceptable clearances.

Microplate Screening of the Differential Effects of Test Agents on Hoechst 33342, Rhodamine 123, and Rhodamine 6G Accumulation in Breast Cancer Cells that Overexpress P-Glycoprotein:

A microplate screening method has been developed to evaluate the effects of test agents on the accumulation of the fluorescent P-glycoprotein (Pgp) substrates Hoechst 33342, rhodamine 123, and rhodamine 6G in multidrug-resistant (MDR) breast cancer cells that overexpress Pgp. All three substrates exhibit substantially higher accumulation in MCF7 non-MDR cells versus NCI/ADR-RES MDR cells, while incubation with 50 μM reserpine significantly reduces or eliminates these differences. Rhodamine 123 shows the lowest substrate accumulation efficiency in non-MDR cells relative to the substrate incubation level. The effects of several chemosensitizing agents and a series of paclitaxel analogs on the accumulation of each fluorescent substrate suggest that there are distinct differences in the substrate interaction profiles exhibited by these different agents. The described methods may be useful in Pgp-related research in the areas of cancer MDR, oral drug absorption, the blood-brain barrier, renal/hepatic transport processes, and drug-drug interactions.

Application of a convective-dispersion model to predict in vivo hepatic clearance from in vitro measurements utilizing cryopreserved human hepatocytes.

Growing interest in the prediction of in vivo pharmacokinetic data from purely in vitro data has grown into a process known as the in vitro-in vivo correlation (IVIC). IVIC can be used to determine the viability of new chemical entities in the early drug development phases, leading to a reduction of resource spending by many large pharmaceutical companies. Here, a convective-dispersion model was developed to predict the total hepatic clearance of six drugs using pharmacokinetic data obtained from in vitro metabolism studies in which the drug disappearance from suspensions of human cryopreserved hepatocytes was measured. Predicted in vivo hepatic clearances estimated by the convective-dispersion model were ultimately compared to the actual clearance values and to in vivo hepatic clearances that were scaled based on the well-stirred model. Finally, sensitivity studies were performed to determine the dependence of hepatic clearance on a number of physiological model parameters. Results reaffirmed that low clearance drugs exhibit rate-limited metabolism, and their hepatic clearances are thus independent of blood flow characteristics, whereas drugs with relatively higher clearance values show a more pronounced dependence on the flood flow properties of dispersion and convection. Absent a priori knowledge about the flow-dependent properties of a drugs clearance, the convective dispersion model applied to disappearance data acquired from cryopreserved human hepatocytes is likely to provide satisfactory estimates of hepatic drug clearance.

Demonstration of pH control in a commercial immobilized glucose isomerase

The synthesis of a variety of important biochemicals involves multistep enzyme-catalyzed reactions. In many cases, the optimal operating pH is much different for the individual enzymatic steps of such synthesis reactions. Yet, it may be beneficial if such reaction steps are combined or paired, allowing them to occur simultaneously, in proximity to one another, and at their respective optimal pH. This can be achieved by separating the micro-environments of the two steps of a reaction pathway using a thin urease layer that catalyzes an ammonia-forming reaction. In this article, the pH control system in a commercial immobilized glucose (xylose) isomerase pellet, which has an optimal pH of 7.5, is demonstrated. This system allows the glucose isomerase to have near its optimal pH activity when immersed in a bulk solution of pH 4.6. A theoretical analysis is also given for the effective fraction of the immobilized glucose isomerase, which remains active when the bulk pH is at 4.6 in the presence of 20 mM urea versus when the bulk pH is at its optimal pH of 7.5. Both theoretical and experimental results show that this pH control system works well in this case. (c) 1996 John Wiley & Sons, Inc.

A FEASIBILITY ANALYSIS OF A NOVEL APPROACH FOR THE CONVERSION OF XYLOSE TO ETHANOL

Economic production of ethanol from plant biomass could be significantly increased if the feedstock for the fermentation is more completely utilized. Currently, simple sugars (mostly D-glucose and D-xylose) can be recovered from lignocellulose by enzymatic or acid hydrolysis. However, while glucose can be readily converted to ethanol by yeasts, the xylose is not fermentable by many of the same species of yeasts that are able to convert glucose into ethanol. Nevertheless, xylose can be converted to its ketose isomer, xylulose, by the enzyme xylose isomerase and this isomer can be converted to ethanol. A major obstacle, however, in converting the xylose to xylulose and then simultaneously converting the xylulose to ethanol is that the pH at which xylose isomerase displays its optimal activity (pH of 7.0-8.0) is much different from the pH at which the fermentation of the xylulose and glucose is best carried out (pH of 4.0-5.0). Herein we propose a novel scheme to provide a means by which the isomerization an...

Chapter 10 – Pharmacokinetic Modeling

Publisher Summary This chapter describes pharmacokinetic (PK) models that focus on how the concentration of drug in the body changes with time. Blood flow and membrane permeation are both important in PK processes. During absorption, drug molecules must permeate into the vasculature of the administration site after which blood flow carries the drug away. There are two types of parameters that can be employed to represent the extent of distribution in PK models. The first is a tissue partition coefficient (KT), and the second is a volume of distribution (V). It should be noted that different types of volumes of distribution can be defined for different types of PK models and in some cases for different time points during the distribution process. All PK models involve several assumptions that allow the amazingly complex interactions between drug molecules and body systems to be simplified into a series of solvable equations. The plasma concentration versus time equation for the two-compartment first-order absorption model contains three exponential decay terms and three corresponding phases.

Optimal pH Control in Sequential Biochemical Reaction Systems via Generating a pH Gradient Across an Immobilized Enzyme Film

Multistep synthesis reactions form the basis for the manufacture of a variety of important biochemicals. However, in many cases, the optimal operating pH is much different for each individual step of these reactions. Even so, conducting all or some of these reaction steps in the same reactor often may have technical (and/or economic) benefits, as when one of the intermediate steps has a very low product to reactant ratio at equilibrium. When it is required to perform these sequential reaction steps in the same vessel, the usual method is to use a compromise pH. However, this approach is neither satisfactory nor feasible always because the rates of most enzyme-catalyzed reactions decay exponentially as one drifts away from the enzyme’s optimal pH. Thus, a technique which permits one to conduct the individual steps at their respective optimal pH and, yet, in close proximity to one another is extremely desirable. We recently proposed such a technique: a method for conducting, in a single reactor, the sequential reaction A ⇔ B → C. The first step is an enzyme-catalyzed reaction that takes place in a basic environment and the second step is either an enzyme-catalyzed reaction or a fermentation that occurs in an acidic environment. The approach suggested was to coat the pellets, within which the enzyme catalyzing the A ⇔ B step was immobilized, with a very thin (< lµm) layer of urease, before suspending the pellets in an acidic bulk solution in which the step B → C takes place. Now, adding a small concentration of urea to the bulk solution allows one to sustain the necessary pH difference between the bulk solution and the interior of the pellets, thereby enabling the two steps to take place in close proximity and also at their respective optimal pH.

An estimate of the number of hepatocyte donors required to provide reasonable estimates of human hepatic clearance from in vitro experiments.

Cryopreserved human hepatocytes in suspension were used to estimate in vivo hepatic clearances for six different drugs. In vitro intrinsic clearances were measured on the basis of substrate depletion. The number of different hepatocyte donors required for a reasonable estimate of in vivo hepatic clearance--within twice or (1/2) of the actual value--was determined. Depending upon the desired level of confidence, anywhere from 9-20 donors are required by this method.