Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects.

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.75 ± 1.22 µM. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

A Human Stem Cell-Derived Brain-Liver Chip for Assessing Blood-Brain-Barrier Permeation of Pharmaceutical Drugs.

Significant advancements in the field of preclinical in vitro blood-brain barrier (BBB) models have been achieved in recent years, by developing monolayer-based culture systems towards complex multi-cellular assays. The coupling of those models with other relevant organoid systems to integrate the investigation of blood-brain barrier permeation in the larger picture of drug distribution and metabolization is still missing. Here, we report for the first time the combination of a human induced pluripotent stem cell (hiPSC)-derived blood-brain barrier model with a cortical brain and a liver spheroid model from the same donor in a closed microfluidic system (MPS). The two model compounds atenolol and propranolol were used to measure permeation at the blood-brain barrier and to assess metabolization. Both substances showed an in vivo-like permeation behavior and were metabolized in vitro. Therefore, the novel multi-organ system enabled not only the measurement of parent compound concentrations but also of metabolite distribution at the blood-brain barrier.

Metabolism and plasma protein binding of 16 straight- and branched-chain parabens in in vitro liver and skin models.

Parabens are alkyl esters of 4-hydroxybenzoic acid (4-HBA), with short-chain parabens used as antimicrobials in cosmetics. We investigated the impact of chain structure on skin and liver metabolism. Incubations with primary human hepatocytes and human liver S9 indicated that methyl-, ethyl-, propyl- and butylparaben were rapidly metabolized to similar metabolites, including 4-HBA plus the corresponding alcohols. Liver and EpiSkin™ S9 were used to investigate the metabolism of 16 short and long straight- and branched-chain parabens. The rate of hydrolysis generally decreased with increasing chain length in liver S9, whereas the reverse was true for EpiSkin™ S9. Chain length also correlated with the number of metabolites, with more oxidized metabolites detected from longer chain parabens. The identity of the alcohol group impacted metabolism the most, in terms of the rate of metabolism and the contribution of cofactors. The majority of parabens (13/16) exhibited high plasma protein binding (PPB) (>90%); whereas, 4-HBA PPB was 38%. PPB was related to the LogP of the parabens. In conclusion, the major and common paraben metabolite in PHH, liver S9 and EpiSkin™ S9 was 4-HBA. The rate of metabolism, type of metabolite and contribution of hydrolysis was tissue-specific (liver, skin) and was influenced by the chain length (and hence LogP), structural isomeric form (straight vs branched), and/or the identity of the alkyl group. SHORT ABSTRACT: We investigated how the chain structure of parabens affects their metabolism by liver and EpiSkin™ S9. The major and common metabolite in primary human hepatocytes, liver S9 and EpiSkin™ S9 was 4-HBA plus the corresponding alcohols. The rate of metabolism, type of metabolite and contribution of hydrolysis was tissue-specific and influenced by the chain length, structural isomeric form (straight vs branched), and/or the identity of the alkyl group. Most parabens exhibited high PPB (>90%), whereas the PPB of 4-HBA was 38%.

HepaRG human hepatic cell line utility as a surrogate for primary human hepatocytes in drug metabolism assessment in vitro.

INTRODUCTION: Primary human hepatocytes are considered as a highly predictive in vitro model for preclinical drug metabolism studies. Due to the limited availability of human liver tissue for cell isolation, there is a need of alternative cell sources for pharmaceutical research. METHODS: In this study, the metabolic activity and long-term stability of the human hepatoma cell line HepaRG were investigated in comparison to primary human hepatocytes (pHH). Hepatocyte-specific parameters (albumin and urea synthesis, galactose and sorbitol elimination) and the activity of human-relevant cytochrome P450 (CYP) enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) were assayed in both groups over a period of 14 days subsequently to a two week culture period in differentiated state in case of the HepaRG cells, and compared with those of cryopreserved hepatocytes in suspension. In addition, the inducibility of CYP enzymes and the intrinsic clearances of eleven reference drugs were determined. RESULTS: The results show overall stable metabolic activity of HepaRG cells over the monitored time period. Higher albumin production and galactose/sorbitol elimination rates were observed compared with pHH, while urea production was not detected. CYP enzyme-dependent drug metabolic capacities were shown to be stable over the cultivation time in HepaRG cells and were comparable or even higher (CYP2C9, CYP2D6, CYP3A4) than in pHH, whereas commercially available hepatocytes showed a different pattern The intrinsic clearance rates of reference drugs and enzyme induction of most CYP enzymes were similar in HepaRG cells and pHH. CYP1A2 activity was highly inducible in HepaRG by ß-naphthoflavone. DISCUSSION: In conclusion, the results from this study indicate that HepaRG cells could provide a suitable alternative to pHH in pharmaceutical research and development for metabolism studies such as CYP induction or sub-chronic to chronic hepatotoxicity studies.

High throughput, non-invasive and dynamic toxicity screening on adherent cells using respiratory measurements.

A dynamic respiration assay based on luminescence decay time detection of oxygen for high throughput toxicological assessment is presented. The method uses 24-well plates (OxoDishes) read with the help of a sensor dish reader placed in a humidified CO(2)-incubator. Adherent primary rat hepatocytes and the human hepatic cell line Hep G2 were exposed to known toxic compounds. Dissolved oxygen concentration, a measure of respiration, was measured with an oxygen sensor optode immobilized in the centre of each well. The cells were maintained in the dishes during the assay period and can afterwards be processed for further analyses. This dynamic, non-invasive measurement allowed calculation of 50% lethal concentrations (LC(50)) for any incubation time point giving concentration-time-dependent responses without further manipulation or removal of the cells from the incubator. Toxicokinetic profiles are compared with Sulforhodamine B assay, a common cytotoxicity assay. The novel assay is robust and flexible, very easy to carry out and provides continuous online respiration data reflecting dynamic toxicity responses. It can be adapted to any cell-based system and the calculated kinetics contributes to understanding of cell death mechanisms.