Cryopreserved human hepatocytes: characterization of drug-metabolizing activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug–drug interaction potential

Li, Albert P.; Lu, Chuang; Brent, Julie A; Pham, Chuong; Fackett, Andrew; Ruegg, Charles E.; Silber, Paul M.

doi:10.1016/s0009-2797(99)00088-5

Cited by 276 publications

(176 citation statements)

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“…This limitation has been overcome in the past decade due to the advancements in the procurement of human livers for research and the commercial availability of isolated human hepatocytes. The application of human hepatocytes in drug metabolism studies is also greatly aided by the successful cryopreservation of human hepatocytes to retain drug metabolism activities (Li et al, 1999(Li et al, , 1999a(Li et al, , 1999b. Recently, the usefulness of cryopreserved human hepatocytes is further extended through the development of technologies to cryopreserve human hepatocytes to retain their ability to be cultured as attached cultures (plateable cryopreserved hepatocytes) that can be used for longer term studies such as enzyme induction studies (Li, 2007).…”

Section: Hepatocytesmentioning

confidence: 99%

In Vitro Evaluation of Metabolic Drug–Drug Interactions: Concepts and Practice

2007

Drug–Drug Interactions in Pharmaceutical Development

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Section: Hepatocytesmentioning

confidence: 99%

In Vitro Evaluation of Metabolic Drug–Drug Interactions: Concepts and Practice

2007

Drug–Drug Interactions in Pharmaceutical Development

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“…[24][25][26] As an alternative to HepG2 cells, primary human hepatocytes represent the best predictive in vitro model to determine liver function for metabolism, 27,28 drug-drug interactions, 29,30 and potential hepatotoxicity of compounds. [30][31][32] Hepatocytes can be utilized in suspension for assays lasting a few hours or may be maintained in collagen-coated tissue culture plates for extended culturing. Traditionally, the use of hepatocytes has been limited to low density well formats such as 24-well for enzyme induction studies 29 or culture tubes for drug metabolism assays.…”

Section: Introductionmentioning

confidence: 99%

“…The low availability of freshly isolated human hepatocytes and plateable cryopreserved human hepatocytes had made them impractical for screening protocols and had limited assays to short-term incubations of six hours or less. 32 However, recent improvements in availability and quality of plateable cryopreserved human hepatocytes have increased the opportunity for their use in HTS. Inclusion of primary hepatocytes in an HTS format would provide relevant data from the screening of large chemical libraries for the assessment of hepatotoxicity.…”

Section: Introductionmentioning

confidence: 99%

Assessment of Compound Hepatotoxicity Using Human Plateable Cryopreserved Hepatocytes in a 1536-Well-Plate Format

Moeller¹,

Shukla

Xia

2012

ASSAY and Drug Development Technologies

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Hepatotoxicity is a major concern for both drug development and toxicological evaluation of environmental chemicals. The assessment of compound-induced hepatotoxicity has traditionally relied on in vivo testing; however, it is being replaced by human in vitro models due to an emphasis on the reduction of animal testing and species-specific differences. Since most cell lines and hybridomas lack the full complement of enzymes at physiological levels found in the liver, primary hepatocytes are the gold standard to study liver toxicities in vitro due to the retention of most of their in vivo activities. Here, we optimized a cell viability assay using plateable cryopreserved human hepatocytes in a 1536-well-plate format. The assay was validated by deriving inhibitory concentration at 50% values for 12 known compounds, including tamoxifen, staurosporine, and phenylmercuric acetate, with regard to hepatotoxicity and general cytotoxicity using multiple hepatocyte donors. The assay performed well, and the cytotoxicity of these compounds was confirmed in comparison to HepG2 cells. This is the first study to report the reliability of using plateable cryopreserved human hepatocytes for cytotoxicity studies in a 1536-well-plate format. These results suggest that plateable cryopreserved human hepatocytes can be scaled up for screening a large compound library and may be amenable to other hepatocytic assays such as metabolic or drug safety studies.

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“…33 The metabolic stability was defined as the percentage of parent compound lost over time in the active test system. Vorinostat was used as a reference compound, together with 7-ethoxycoumarin and 7-hydroxycoumarin as the positive controls of hepatocyte competence for phase I and II metabolism, respectively (0.42% left after 90 min at 1 μM concentration and 2.08% left under the same conditions).…”

mentioning

confidence: 99%

“…Vorinostat was used as a reference compound, together with 7-ethoxycoumarin and 7-hydroxycoumarin as the positive controls of hepatocyte competence for phase I and II metabolism, respectively (0.42% left after 90 min at 1 μM concentration and 2.08% left under the same conditions). 33 Although a lower stability was observed for (R/S)-2 as compared to Vorinostat (23% left after 90 min at 1 μM concentration vs 61% left under the same conditions for Vorinostat), measures for improving its pharmacokinetic profile can be taken based on the chemical modification at specific sites. 34 In conclusion, we have studied the structural and stereochemical effects of an optimized ω-alkoxy analogue of Vorinostat as a probe to assess its in vitro activity profile against 11 isoforms of HDAC.…”

mentioning

confidence: 99%

Vorinostat-Like Molecules as Structural, Stereochemical, and Pharmacological Tools

Hanessian

Auzzas

Larsson

et al. 2010

ACS Med. Chem. Lett.

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The inhibitory activity of an ω-alkoxy analogue of the HDAC inhibitor, Vorinostat (SAHA), against the 11 isoforms of HDAC is described and evaluated with regard to structural biology information retrieved through computational methods. Preliminary absorption and metabolism studies were performed, which positioned this compound as a potential candidate for further preclinical studies and delineated measures for improving its pharmacokinetic profile.KEYWORDS HDAC promiscuous inhibitors, SAHA analogues, HDAC1-11 profile, class IIa-specific profile, absorption and metabolism I n spite of the significant progress made in HDAC research, 1-4 the quest for isoform-selective inhibitors continues to present a major challenge. [5][6][7] To date, detailed biostructural information is available only regarding a few among the known 11 metallo-enzymes. 1,[8][9][10][11] Consequently, the design of isoform-and class-selective inhibitors is somewhat arbitrary and derivative. 1,7,[12][13][14] In addition, complete data regarding the factual HDAC profile for most of the early discovered agents are not available, since the development of effective isozyme-based assays is relatively recent. [5][6][7][15][16][17][18] As a consequence of the complex, pleiotropic nature of cancer, promiscuous HDAC inhibitors such as Vorinostat (1) (SAHA, suberoylanilide hydroxamic acid) 19 (Figure 1) seem superior and as safe in the clinic as compared to the few class-specific agents available. [5][6][7]15 Ligand-based approaches are a first-choice solution to delineate the structural requirements for HDAC selectivity, especially with the emergence of ω-aryl alkanoyl hydroxamates such as Vorinostat as clinical candidates. 1,7,[12][13][14] In this regard, simple and readily accessible surrogates are worthy of study, provided that they bear pharmacophoric determinants as close as possible to a maximum common substructural consensus required to target the whole HDAC panel. The judicious inclusion of specific appendages capable of interacting with specific residues in the cap region of HDACs can provide valuable structural, functional, and stereochemical insight into the design of new inhibitors.In a previous study, we determined the optimal alkyl chain length for activity against a single isoform, HDAC2. 20 Designed for understanding the role of the chirality in simple ω-alkoxy analogues of Vorinostat, compound (R/S)-2 emerged as a promising lead for its preliminary cytotoxic activity against leukemia, colon, and lung tumor cell lines. Here, we describe the details for the improved and shortened synthesis of 2, together with the biostructural insights through molecular modeling and a preliminary study of its in vitro activity against an extended panel of HDACs.Although relatively simple in conception, the previous nine-step synthesis of racemic 2 20 was shortened and adapted to produce gram-scale material for pharmacological studies (Supporting Information) (Scheme 1). The required 8-carbon chain of (R/S)-2 was assembled by a cross metathesis reac...

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Cryopreserved human hepatocytes: characterization of drug-metabolizing activities and applications in higher throughput screening assays for hepatotoxicity, metabolic stability, and drug–drug interaction potential

Cited by 276 publications

References 20 publications

In Vitro Evaluation of Metabolic Drug–Drug Interactions: Concepts and Practice

In Vitro Evaluation of Metabolic Drug–Drug Interactions: Concepts and Practice

Assessment of Compound Hepatotoxicity Using Human Plateable Cryopreserved Hepatocytes in a 1536-Well-Plate Format

Vorinostat-Like Molecules as Structural, Stereochemical, and Pharmacological Tools

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