Albert P. Li

Screening for human ADME/Tox drug properties in drug discovery

There is no doubt that ADME/Tox drug properties, absorption, distribution, metabolism, elimination and toxicity, are properties crucial to the final clinical success of a drug candidate. It has been estimated that nearly 50% of drugs fail because of unacceptable efficacy, which includes poor bioavailability as a result of ineffective intestinal absorption and undesirable metabolic stability(1). It has also been estimated that up to 40% of drug candidates have failed in the past because of safety issues(2). In this review, the methodologies that are available for use in drug development as in vitro human-based screens for ADME/Tox drug properties are discussed.

Species comparison in P450 induction: effects of dexamethasone, omeprazole, and rifampin on P450 isoforms 1A and 3A in primary cultured hepatocytes from man, Sprague-Dawley rat, minipig, and beagle dog.

Induction of P450 isoforms 1A (CYP1A) and 3A (CYP3A) by model inducers dexamethasone, omeprazole and rifampin was evaluated in primary cultured hepatocytes from man and laboratory animals. Inducer-specific species-differences were observed. Results with human hepatocytes from six human donors consistently show that both rifampin and dexamethasone were inducers of CYP3A activity (measured as testosterone 6beta-hydroxylase activity), with rifampin being more potent. Conversely, in rat hepatocytes, dexamethasone was a potent CYP3A inducer while rifampin was not an inducer. Rifampin but not dexamethasone induced CYP3A in minipig and beagle dog hepatocytes. Omeprazole was a potent inducer of CYP1A activity (measured as ethoxyresorufin-O-deethylase activity) in human, beagle dog and minipig hepatocytes, and not an inducer in rat hepatocytes. The species-differences observed suggest that human hepatocytes represent the most appropriate preclinical experimental system for the evaluation of P450 induction in human.

Optimization of Cryopreservation Procedures for Rat and Human Hepatocytes

1. Rat hepatocytes were cryopreserved using a number of procedures and the viability, attachment, and metabolic activity of the cryopreserved cells were compared to freshly isolated hepatocytes. Several cryopreservation agents (dimethylsulphoxide [DMSO], glycerol, polyvinylpyrrolidone [PVP], dextrans), and combinations of these agents, were examined. Other variables tested included the freezing rate, thawing rate, and the concentration of serum in the freezing medium. 2. Recovery of viable attached cells was optimal using DMSO at concentrations of 10% or higher, a slow stepwise cooling procedure, and a quick thaw. The concentration of serum in the freezing medium (0% to 90%) did not affect cryopreservation results. Using this procedure the recovery of viable hepatocytes was 70%. 3. Levels of hepatocyte ethoxycoumarin-O-deethylase (ECOD) activity did not change following cryopreservation. The rate of decline of ECOD activity with time in culture was similar in freshly isolated and cryopreserved hepatocytes. 4. Hepatocytes isolated from three human livers were cryopreserved and recovered with viabilities similar to those obtained with the rat. A preliminary experiment also showed no loss of metabolic activity in human hepatocytes following cryopreservation.

A review of the common properties of drugs with idiosyncratic hepatotoxicity and the multiple determinant hypothesis for the manifestation of idiosyncratic drug toxicity

Idiosyncratic drug toxicity is generally believed to be a phenomenon that cannot be readily evaluated experimentally. Reasons for this difficulty include the following: 1. It is a rare event (<1/5,000) and therefore impossible to be studied in clinical trials; 2. It is a human-specific event not detectable in experimental animals. To aid the understanding of idiosyncratic toxicity and to develop an experimental strategy for this phenomenon, a hypothesis is proposed. The hypothesis states that the low frequency of idiosyncratic drug toxicity is due to the requirements for the occurrence of multiple critical and discrete events, with the probability for the occurrence of idiosyncratic drug toxicity as a product of the probabilities of each event. The key determinants of these critical events are proposed to be: 1. Chemical properties; 2. exposure; 3. environmental factors; and 4. genetic factors. Based on this hypothesis, idiosyncratic drug toxicity can be evaluated experimentally via studying these key determinants. The chemical properties critical to idiosyncratic drug toxicity are identified via a review of the common properties of drugs that cause idiosyncratic liver toxicity. These properties include: 1. Formation of reactive metabolites. 2. Metabolism by P450 isoforms. 3. Preponderance of P450 inducers, and 4. Occurrence of clinically significant pharmacokinetic interactions with co-administered drugs. Based on this review, it is proposed that these common properties may be useful experimental endpoints for the prediction and therefore avoidance of the selection of drug candidates with idiosyncratic drug toxicity for further development.

Present status of the application of cryopreserved hepatocytes in the evaluation of xenobiotics: consensus of an international expert panel.

Successful cryopreservation of freshly isolated hepatocytes would significantly decrease the need for freshly-procured livers for the preparation of hepatocytes for experimentation. Hepatocytes can be prepared, cryopreserved, and used for experimentation as needed at different times after isolation. Cryopreservation is especially important for research with human hepatocytes because of the limited availability of fresh human livers. Based on the cumulative experience of this international expert panel, a consensus was reached on the various aspects of hepatocyte cryopreservation, including cryopreservation and thawingprocedures and applications of the cryopreserved hepatocytes. Key to successful cryopreservation includes slow addition of cryopreservants, controlled-rate freezing with adjustment for the heat of crystallization, storage at -150 degrees C, and rapid thawing. There is a general consensus that cryopreserved hepatocytes are useful for short-term xenobiotic metabolism and cytotoxicity evaluation.

Isolation and culturing of hepatocytes from human livers

Using a modified collagenase digestion procedure, we were successful in the isolation of viable hepatocytes from human liver surgical wastes, unused transplantable livers, and diseased livers from transplant recipients. The modified procedures for isolation involved collagenase perfusion of a sample of limited size (<50 g) with only one cut surface, perfusion via only one blood vessel, the use of an appropriate amount of collagenase, as well as manual “massaging” of the liver sample during critical stages of the collagenase perfusion. Using this modified procedures, we had close to 100% success rate in the isolation of viable hepatocytes from the human liver samples.

A simplified method for the culturing of primary adult rat and human hepatocytes as multicellular spheroids

SummaryA simple and highly reproducible method was established for the culturing of adult rat and human hepatocytes as multicellular aggregates (spheroids). Purified rat and human liver parenchymal cells were cultured on nontissue culture (bacteriological) polystyrene petri dishes on a rotating platform. After an overnight incubation, the cells were found to form multicellular aggregates. The aggregates became spheroidal in shape after several days in culture. Histological sections of the spheroids showed an organized structure consisting of squamated cells on the outermost layer and cuboidal cells in the interior. Cellular structures characteristic of hepatocytes in the liver in vivo including bile canaliculi, peroxisomes, Golgi boides, abundant mitochondria, and rough and smooth endoplasmic reticulum were observed with electron microscopy. The spheroids were found to be viable up to the longest time studied of approx. 1 month in culture as demonstrated by their adherence and growth on collagen-coated substratum. The morphological resemblance between hepatocytes cultured as spheroids and the liver in vivo suggests that the spheroids may be a useful in vitro experimental model of the liver. Our simple method should allow hepatocytes to be cultured as spheroids easily in any laboratory equipped for cell culture. Our study here also is the first to report the culturing of human hepatocytes as spheroids.

Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: A potential bioartificial liver

SummaryConventional culture systems for hepatocytes generally involve cells cultured as flat, monolayer cells, with limited cell-cell contact, in a static pool of medium, unlike the liver in vivo where the parenchymal cells are cuboidal, with extensive cell-cell contact, and are continuously perfused with blood. We report here a novel bioreactor system for the culturing of primary hepatocytes with cuboidal cell shape, extensive cell-cell contact, and perfusing medium. The hepatocytes were inoculated into the bioreactor and allowed to recirculate at a rate optimal for them to collide and form aggregates. These newly-formed aggregates were subsequently entrapped in a packed bed of glass beads. The bioreactor was perfused with oxygenated nutrient medium, with controlled oxygen tension, pH, and medium perfusion rate. The hepatocytes were viable for up to the longest time point studied of 15 days in culture based on urea synthesis, albumin synthesis and cell morphology. Light microscopy studies of hepatocytes cultured for 15 days in the bioreactor showed interconnecting three-dimensional structures resembling the hepatic cell plate in the liver organ. Electron microscopy studies on the same cells revealed ultrastructure similar to the hepatocytes in vivo, including the presence of plentiful mitochondria, rough and smooth endoplasmic reticulum, glycogen granules, peroxisomes, and desmosomes. We believe that our hepatocyte bioreactor is a major improvement over conventional culture systems, with important industrial applications including toxicology, drug metabolism, and protein/peptide synthesis. The hepatocyte bioreactor concept may also be used as the basis for the development of a bioartificial liver to provide extracorporeal hepatic support to patients with hepatic failure.

Identification of glutathione conjugates of troglitazone in human hepatocytes.

Troglitazone (TGZ) is an orally active antihyperglycemic agent used in the treatment of noninsulin-dependent diabetes mellitus. Several cases of liver failure following TGZ administration led to its withdrawal from the market. The mechanism of toxicity is still not understood. The formation of toxic metabolites is believed to play an important role. Herein, we report the biotransformation of TGZ in human hepatocytes. TGZ at 50 microM concentration was incubated with cryopreserved human hepatocytes. Four metabolites were found-glucuronide, sulfate, and two glutathione (GSH) conjugates of TGZ. The two GSH metabolites could be conjugation at the 6-hydroxychromane nucleus and the thiazolidinedione ring. Alternatively, the conjugation could be one of the two rings, with the two GSH metabolites are diastereomers. The sulfate conjugate was the major metabolite found. The cytochrome P450 (CYP) inhibitors furafylline (CYP1A1/2), omeprazole (CYP2C19), ketoconazole (CYP3A4), and sulfaphenazole (CYP2C9) had no inhibitory effect on the TGZ metabolism suggesting that several P450s may play a role in the TGZ metabolic pathway. Previous studies in our laboratory have shown a large interindividual variation between different donors in cytotoxicity after dosing with TGZ. Based on EC(50) values, donors were classified as sensitive or resistant. The sensitive human donors were found to form significantly less troglitazone GSH conjugates and glucuronides than the resistant donors.

An evaluation of the genotoxic potential of glyphosate

The potential genotoxicity of glyphosate, the active ingredient in Roundup herbicide, was tested in a variety of well-established in vitro and in vivo assays including the Salmonella typhimurium and Escherichia coli WP-2 reversion assays, recombination (rec-assay) with Bacillus subtilis. Chinese hamster ovary cell gene mutation assay at the hypoxanthine/guanine phosphoribosyl transferase gene locus, hepatocyte primary culture/DNA repair assay, and in vivo cytogenetics assay in rat bone marrow. No genotoxic activity was observed in the assays performed. The data suggest that glyphosate should not pose a genetic risk to man.