A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling

Wernevik, Johan; Bergström, Fredrik; Novén, Anna; Hulthe, Johan; Fredlund, Linda; Addison, Dan; Holmgren, Jan; Strömstedt, Per-Erik; Rehnström, Erika; Lundböck, Thomas

doi:10.1089/adt.2020.970

Cited by 26 publications

(32 citation statements)

References 56 publications

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“…CL int is a key parameter for drug optimisation, and both liver microsomes and hepatocytes in different assay formats are widely applied. 28,32,33 The relatively long time to form stable spheroid cultures may appear unattractive for general CL int assessments, however additional benefits from PHH spheroids are worth considering. Our data with a small representation of three slowly cleared compounds confirm the longevity and performance of spheroids to determine low CL int values.…”

Section: Discussionmentioning

confidence: 99%

Primary Human Hepatocyte Spheroid Model as a 3D In Vitro Platform for Metabolism Studies

Kanebratt

Janefeldt

Vilén

et al. 2021

Journal of Pharmaceutical Sciences

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3D cultures of primary human hepatocytes (PHH) are emerging as a more in vivo-like culture system than previously available hepatic models. This work describes the characterisation of drug metabolism in 3D PHH spheroids. Spheroids were formed from three different donors of PHH and the expression and activities of important cytochrome P450 enzymes (CYP1A2, 2B6, 2C9, 2D6, and 3A4) were maintained for up to 21 days after seeding. The activity of CYP2B6 and 3A4 decreased, while the activity of CYP2C9 and 2D6 increased over time (P < 0.05). For six test compounds, that are metabolised by multiple enzymes, intrinsic clearance (CL int) values were comparable to standard in vitro hepatic models and successfully predicted in vivo CL int within 3-fold from observed values for low clearance compounds. Remarkably, the metabolic turnover of these low clearance compounds was reproducibly measured using only 1e3 spheroids, each composed of 2000 cells. Importantly, metabolites identified in the spheroid cultures reproduced the major metabolites observed in vivo, both primary and secondary metabolites were captured. In summary, the 3D PHH spheroid model shows promise to be used in drug discovery projects to study drug metabolism, including unknown mechanisms, over an extended period of time.

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Section: Discussionmentioning

confidence: 99%

Primary Human Hepatocyte Spheroid Model as a 3D In Vitro Platform for Metabolism Studies

Kanebratt

Janefeldt

Vilén

et al. 2021

Journal of Pharmaceutical Sciences

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“…The clearance of compound 14 by human liver microsomes (CL int ) was determined at AstraZeneca as reported previously. 62 …”

Section: Methodsmentioning

confidence: 99%

Mining Natural Products for Macrocycles to Drug Difficult Targets

et al. 2020

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Lead generation for difficult-to-drug targets that have large, featureless, and highly lipophilic or highly polar and/or flexible binding sites is highly challenging. Here, we describe how cores of macrocyclic natural products can serve as a high-quality in silico screening library that provides leads for difficult-to-drug targets. Two iterative rounds of docking of a carefully selected set of natural-product-derived cores led to the discovery of an uncharged macrocyclic inhibitor of the Keap1-Nrf2 protein–protein interaction, a particularly challenging target due to its highly polar binding site. The inhibitor displays cellular efficacy and is well-positioned for further optimization based on the structure of its complex with Keap1 and synthetic access. We believe that our work will spur interest in using macrocyclic cores for in silico -based lead generation and also inspire the design of future macrocycle screening collections.

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“…The panel consists of two metabolic stability assays based on rat hepatocytes and human liver microsomes, respectively, a logD assay investigating partitioning between an aqueous solution and octanol, a solubility assay in PBS at pH 7.4 and a plasma protein binding assay performed in human serum and reporting both the fraction unbound and stability of the compounds. All assays are run in an automated 96‐well format with a UPLC‐MS/MS read‐out …”

Section: Methodsmentioning

confidence: 99%

Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro‐Oxindole Dihydroquinazolinones as IRAP Inhibitors

et al. 2020

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Insulin‐regulated aminopeptidase (IRAP) is a new potential macromolecular target for drugs aimed for treatment of cognitive disorders. Inhibition of IRAP by angiotensin IV (Ang IV) improves the memory and learning in rats. The majority of the known IRAP inhibitors are peptidic in character and suffer from poor pharmacokinetic properties. Herein, we present a series of small non‐peptide IRAP inhibitors derived from a spiro‐oxindole dihydroquinazolinone screening hit (pIC50 5.8). The compounds were synthesized either by a simple microwave (MW)‐promoted three‐component reaction, or by a two‐step one‐pot procedure. For decoration of the oxindole ring system, rapid MW‐assisted Suzuki‐Miyaura cross‐couplings (1 min) were performed. A small improvement of potency (pIC50 6.6 for the most potent compound) and an increased solubility could be achieved. As deduced from computational modelling and MD simulations it is proposed that the S‐configuration of the spiro‐oxindole dihydroquinazolinones accounts for the inhibition of IRAP.

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A Fully Integrated Assay Panel for Early Drug Metabolism and Pharmacokinetics Profiling

Cited by 26 publications

References 56 publications

Primary Human Hepatocyte Spheroid Model as a 3D In Vitro Platform for Metabolism Studies

Primary Human Hepatocyte Spheroid Model as a 3D In Vitro Platform for Metabolism Studies

Mining Natural Products for Macrocycles to Drug Difficult Targets

Synthesis, Evaluation and Proposed Binding Pose of Substituted Spiro‐Oxindole Dihydroquinazolinones as IRAP Inhibitors

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