Britta Laube

Procédé et appareil pour la détection des interactions des métabolismes de cellules aérobiques et anaérobiques

New Hepatocyte In Vitro Systems for Drug Metabolism: Metabolic Capacity and Recommendations for Application in Basic Research and Drug Development, Standard Operation Procedures

Drug Metabolism Reviews, 2003

Primary hepatocytes represent a well-accepted in vitro cell culture system for studies of drug me... more Primary hepatocytes represent a well-accepted in vitro cell culture system for studies of drug metabolism, enzyme induction, transplantation, viral hepatitis, and hepatocyte regeneration. Recently, a multicentric research program has been initiated to optimize and standardize new in vitro systems with hepatocytes. In this article, we discuss five of these in vitro systems: hepatocytes in suspension, perifusion culture systems, liver slices, co-culture systems of hepatocytes with intestinal bacteria, and 96-well plate bioreactors. From a technical point of view, freshly isolated or cryopreserved hepatocytes in suspension represent a readily available and easy-to-handle in vitro system that can be used to characterize the metabolism of test substances. Hepatocytes in suspension correctly predict interspecies differences in drug metabolism, which is demonstrated with pantoprazole and propafenone. A limitation of the hepatocyte suspensions is the length of the incubation period, which should not exceed 4 hr. This incubation period is sufficiently long to determine the metabolic stability and to allow identification of the main metabolites of a test substance, but may be too short to allow generation of some minor, particularly phase II metabolites, that contribute less than 3% to total metabolism. To achieve longer incubation periods, hepatocyte culture systems or bioreactors are used. In this research program, two bioreactor systems have been optimized: the perifusion culture system and 96-well plate bioreactors. The perifusion culture system consists of collagen-coated slides allowing the continuous superfusion of a hepatocyte monolayer with culture medium as well as establishment of a constant atmosphere of 13% oxygen, 82% nitrogen, and 5% CO 2 . This system is stable for at least 2 weeks and guarantees a remarkable sensitivity to enzyme induction, even if weak inducers are tested. A particular advantage of this system is that the same bioreactor can be perfused with different concentrations of a test substance in a sequential manner. The 96-well plate bioreactor runs 96 modules in parallel for pharmacokinetic testing under aerobic culture conditions. This system combines the advantages of a three-dimensional culture system in collagen gel, controlled oxygen supply, and constant culture medium conditions, with the possibility of high throughput and automatization. A newly developed co-culture system of hepatocytes with intestinal bacteria offers the possibility to study the metabolic interaction between liver and intestinal microflora. It consists of two chambers separated by a permeable polycarbonate membrane, where hepatocytes are cultured under aerobic and intestinal bacteria in anaerobic conditions. Test substances are added to the aerobic side to allow their initial metabolism by the hepatocytes, followed by the metabolism by intestinal bacteria at the anaerobic side. Precision-cut slices represent an alternative to isolated hepatocytes and have been used for the investigation of hepatic metabolism, hepatotoxicity, and enzyme induction. A specific advantage of liver slices is the possibility to study toxic effects on hepatocytes that are mediated or modified by nonparenchymal cells (e.g., by cytokine release from Kupffer cells) because the physiological liver microarchitecture is maintained in cultured slices. For all these in vitro systems, a prevalidation has been performed using standard assays for phase I and II enzymes. Representative results with test substances and recommendations for application of these in vitro systems, as well as standard operation procedures are given.

Download

Cryopreservation of Hepatocytes

Determination of Testosterone Metabolites in Rat Hepatocytes with and without Cryopreservation by On-Line SPE Column-Switching LC and MS Detection

Chromatographia, Dec 12, 2007

In a recently published paper development of a sensitive automated “on-line” solid-phase extracti... more In a recently published paper development of a sensitive automated “on-line” solid-phase extraction (SPE)/RP-HPLC assay for 6β-hydroxytestosterone (6β-OHT) with corticosterone as the internal standard (IS) was reported and its potential for quantification of various testosterone metabolites in culture media reflecting metabolic activity of cultured human and animal hepatocytes demonstrated [1]. In this following contribution the technique has been extended to determination of another five testosterone metabolites in cultured rat hepatocytes using an identical “on-line” SPE/RP-HPLC procedure and detection by tandem MS-MS with an atmospheric pressure chemical ionization (APCI) source in the selected reaction monitoring (SRM) mode as that described in [1]. All six testosterone metabolites, namely 2α-OHT, 2β-OHT, 6α-OHT, 6β-OHT, 7α-OHT and 16α-OHT, could be sufficiently separated from each other and thus an unequivocal assignment to the individual structures was achieved. Validation data are presented specifying the limits of quantitation as well as the mean values of the coefficients of variation (CV) for the target analytes and the accuracy obtained at five different days. Regio- and stereoselective testosterone hydroxylation by rat hepatocytes was measured in a long-term culture system with and without exposure to rifampicin as an inducer of liver CYP 3A4 activity. In addition, testosterone hydroxylation was analyzed in cultures of cryopreserved hepatocytes that had been stored at −196 °C. The rat hepatocytes were cultured after thawing for up to 11 days and induction of testosterone hydroxylase activity could be demonstrated in cultures which underwent a new cryopreservation protocol.

Human relevance of follicular thyroid tumors in rodents caused by non-genotoxic substances

Regulatory Toxicology and Pharmacology, 2018

Download

Kryokonservierung von Hepatocyten

3-Iodo-2-propynyl butylcarbamate (IPBC) [MAK Value Documentations, 2011]

The MAK-Collection for Occupational Health and Safety, 2016

The German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work A... more The German Commission for the Investigation of Health Hazards of Chemical Compounds in the Work Area has re‐evaluated 3‐iodo‐2‐propynyl butylcarbamate (IPBC) to establish a maximum concentration at the workplace (MAK value), considering all toxicity endpoints. Available unpublished study reports and publications are described in detail. IPBC is irritating to the eyes and respiratory tract and has a skin sensitizing potential. New data have substantiated the previous designation with “Sh”. In a 5‐day aerosol inhalation study in rats, hyperplasia, squamous metaplasia and necrosis of the underlying cartilage of the larynx were observed at 1 mg/m3 and above. These effects were observed to an increased extent in the 13‐week study, in which the NOAEC was 0.23 mg/m3 and the LOAEC 0.3 mg/m3. Since the laryngeal irritation is the most sensitive endpoint, a MAK value of 0.1 mg/m3 (0.01 ml/m3) is established. IPBC has been attributed Peak Limitation Category I for local effects with an excursion factor of 2, since there was no direct sensory irritation but rather a tissue alteration and the NOAEC of the 5‐day study was 3 times the MAK value. In rats IPBC caused no tumours in an oral 2‐year carcinogenicity study. Increased number of liver adenomas in male mice might have been caused by liver enzyme induction. Available studies in vitro and in vivo provided no evidence of a specific genotoxic effect. Therefore IPBC is not classified as carcinogen or germ cell mutagen. In rats and mice, there was a sufficiently high difference between the oral NOAEL for developmental toxicity scaled to a concentration at the work place and the MAK value. Therefore IPBC is classified in Pregnancy Risk Group C. Skin contact will not significantly contribute to the systemic toxicity of 3‐iodo‐2‐propynyl butylcarbamate

Cryopreservation of Hepatocytes

Enzyme induction in cryopreserved human hepatocyte cultures

Toxicology, 2006

Download

Determination of Testosterone Metabolites in Rat Hepatocytes with and without Cryopreservation by On-Line SPE Column-Switching LC and MS Detection

Chromatographia, 2007

In a recently published paper development of a sensitive automated “on-line” solid-phase extracti... more In a recently published paper development of a sensitive automated “on-line” solid-phase extraction (SPE)/RP-HPLC assay for 6β-hydroxytestosterone (6β-OHT) with corticosterone as the internal standard (IS) was reported and its potential for quantification of various testosterone metabolites in culture media reflecting metabolic activity of cultured human and animal hepatocytes demonstrated [1]. In this following contribution the technique has been extended to determination of another five testosterone metabolites in cultured rat hepatocytes using an identical “on-line” SPE/RP-HPLC procedure and detection by tandem MS-MS with an atmospheric pressure chemical ionization (APCI) source in the selected reaction monitoring (SRM) mode as that described in [1]. All six testosterone metabolites, namely 2α-OHT, 2β-OHT, 6α-OHT, 6β-OHT, 7α-OHT and 16α-OHT, could be sufficiently separated from each other and thus an unequivocal assignment to the individual structures was achieved. Validation data are presented specifying the limits of quantitation as well as the mean values of the coefficients of variation (CV) for the target analytes and the accuracy obtained at five different days. Regio- and stereoselective testosterone hydroxylation by rat hepatocytes was measured in a long-term culture system with and without exposure to rifampicin as an inducer of liver CYP 3A4 activity. In addition, testosterone hydroxylation was analyzed in cultures of cryopreserved hepatocytes that had been stored at −196 °C. The rat hepatocytes were cultured after thawing for up to 11 days and induction of testosterone hydroxylase activity could be demonstrated in cultures which underwent a new cryopreservation protocol.

Establishment of a novel in vitro system for studying the interaction of xenobiotic metabolism of liver and intestinal microflora

Archives of Toxicology, 2000

We developed a new two-chamber system for the coculture of hepatocytes and fecal microflora under... more We developed a new two-chamber system for the coculture of hepatocytes and fecal microflora under aerobic and anaerobic conditions, respectively, to investigate the sequential metabolism of chemicals by the liver and microflora in vitro. The culture device consisted of two chambers separated by a permeable polycarbonate membrane. In the aerobic compartment, hepatocytes were cultivated as a monolayer on the membrane and in the anaerobic compartment fecal microflora as a suspension. To characterize the metabolic capacity of the microflora and hepatocytes, various marker enzymes were studied. Azoreductase, nitroductase, beta-glucuronidase, beta-glucosidase and sulphatase were tested in the microflora of the feces from three volunteers who had had significantly different eating habits for years (daily meat, mixed diet, vegetarian). The microflora exhibited significant activities and the various enzymes differed only moderately in the samples from the three volunteers. For rat hepatocytes the activities of various cytochrome P450 forms and conjugating enzymes served as markers. The enzyme activities were tested in the coculture system during a 4-h culture period intended for the test protocol. Deethylation of ethoxycoumarin and 2alpha-, 6beta- and 16alpha-hydroxylation of testosterone decreased by about 30%, 25%, 40% and 20%, respectively, while there was no loss of glucuronidation and sulphonation of 3-OH-benzo(a)pyrene nor of glutathione conjugation of 1-chloro-2,4-dinitrobenzene during the 4-h culture period. The activities of the tested hepatic phase I and II enzymes were not changed after coculture of the hepatocytes with the microflora for 4 h. The applicability of the in vitro system for studying the metabolic interaction of liver and microflora was demonstrated using 7-ethoxycoumarin and the developmental drug EMD 57033, a thiadiazinon derivative from Merck KGaA, as model compounds. Both compounds were oxidized and conjugated by liver cells. In the coculture of hepatocytes and fecal microflora the resulting glucuronides and sulphoconjugates were split by hydrolytic enzymes of the intestinal microflora.

Uploads

Papers by Britta Laube

Log In