Empirical validation of a rat in vitro organ slice model as a tool for in vivo clearance prediction

Tissue slices have been shown to be a valuable tool to predict metabolism of novel drugs. However, besides the numerous advantages of their use for this purpose, some potential drawbacks also exist, including reported poor penetration of drugs into the inner cell layers of slices and loss of metabolic capacity during prolonged incubation, leading to underprediction of metabolic clearance. In the present study, we empirically identified ( and quantified) sources of underprediction using rat tissue slices of lung, intestine, kidney, and liver and found that thin liver slices ( +/- 100 mu m) metabolized model substrates ( 7- hydroxycoumarin, testosterone, warfarin, 7- ethoxycoumarin, midazolam, haloperidol, and quinidine) as rapidly as isolated hepatocytes. Furthermore, it was found that organ slices remain metabolically active for sufficient periods of incubation, enabling study of the kinetics of low clearance compounds. In addition, we determined the influence of albumin on the clearance prediction of six model substrates. For three of these substrates, the intrinsic clearance in the presence of albumin was approximately 3 times higher than that obtained from incubations without albumin, but corrected for unbound fraction. This resulted in a much more accurate prediction of in vivo whole body metabolic clearance for these compounds. Collectively, these results show that drawbacks of the use of slices for clearance prediction are largely surmountable. Provided that thin liver slices and physiological albumin concentration are used, whole body metabolic clearance is predicted with acceptable ( 2- fold) accuracy with organ slices. These results emphasize the applicability of organ slices in this field of research

Bovine liver slices combined with an androgen transcriptional activation assay: an in-vitro model to study the metabolism and bioactivity of steroids

Previously we described the properties of a rapid and robust yeast androgen bioassay for detection of androgenic anabolic compounds, validated it, and showed its added value for several practical applications. However, biotransformation of potent steroids into inactive metabolites, or vice versa, is not included in this screening assay. Within this context, animal-friendly in-vitro cellular systems resembling species-specific metabolism can be of value. We therefore investigated the metabolic capacity of precision-cut slices of bovine liver using 17β-testosterone (T) as a model compound, because this is an established standard compound for assessing the metabolic capacity of such cellular systems. However, this is the first time that slice metabolism has been combined with bioactivity measurements. Moreover, this study also involves bioactivation of inactive prohormones, for example dehydroepiandrosterone (DHEA) and esters of T, and although medium extracts are normally analyzed by HPLC, here the metabolites formed were identified with more certainty by ultra-performance liquid chromatography time-of-flight mass spectrometry (UPLC–TOFMS) with accurate mass measurement. Metabolism of T resulted mainly in the formation of the less potent phase I metabolites 4-androstene-3,17-dione (4-AD), the hydroxy-T metabolites 6α, 6β, 15β, and 16α-OH-T, and the phase II metabolite T-glucuronide. As a consequence the overall androgenic activity, as determined by the yeast androgen bioassay, decreased. In order to address the usefulness of bovine liver slices for activation of inactive steroids, liver slices were exposed to DHEA and two esters of T. This resulted in an increase of androgenic activity, because of the formation of 4-AD and T

Iodine-125 brachytherapy for brain tumours - a review

Iodine-125 brachytherapy has been applied to brain tumours since 1979. Even though the physical and biological characteristics make these implants particularly attractive for minimal invasive treatment, the place for stereotactic brachytherapy is still poorly defined

DRUG TRANSPORT, VIABILITY AND MORPHOLOGY OF ISOLATED RAT HEPATOCYTES PRESERVED FOR 24 HOURS IN UNIVERSITY-OF-WISCONSIN SOLUTION

Isolated hepatocytes are a valuable tool to study liver functions. Suitable methods to preserve the isolated cells with good maintenance of viability and functions are crucial to extend experiments with hepatocytes from a single isolation over 2 or more consecutive days. We investigated whether University of Wisconsin (UW) solution, which was designed to preserve organs for transplantation, is also suitable for preservation of isolated rat hepatocytes. Viability, as determined by Trypan blue exclusion and reduction of the tetrazolium 3(4,5-dimethyl-thiazoyl-2-yl)2,5 diphenyltetrazolium bromide to a purple formazan, morphological appearance using electron microscopy, ATP levels and uptake and storage of three model drugs ([H-3]vecuronium, [H-3]taurocholic acid and [H-3]ouabain) were determined directly after isolation and after 22 hr of storage in UW solution at 0-4-degrees. The present study shows that cold storage of rat hepatocytes for 22 hr in UW can be performed without significant loss of viability and with maintenance of proper morphology, cellular ATP and transport functions. In contrast, after storage in Krebs-Henseleit buffer the normal morphology, ATP content and transport functions were strongly affected. These results imply that hepatocytes from a single isolation and stored in UW solution can be used for experiments on 2 consecutive days