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December 15, 2022

Lysosomal trapping assays using a HCI platform

While the plasma concentration of a drug is the most readily accessible measure of its bioavailability, its disposition – and often its effect, too – may ultimately depend on free intracellular drug concentrations in the respective tissues. Therefore, understanding the principles and mechanisms that govern cellular unbound drug concentration are key to drug discovery. For this purpose, SOLVO has recently set up new service for a  lysosomal trapping assessment, and an assay for Kp,uu, (unbound liver-to-blood partition coefficient) determination is also in development.

Lysosomal trapping is caused by pH-driven accumulation of a drug in lysosomes and mostly affects lipophilic or amphiphilic compounds with a basic moiety. In the acidic environment of the lysosome (pH 4-5), a basic compound’s protonated form becomes predominant, which has reduced membrane permeability, leading to its the entrapment in the lysosome. As a result, the overall intracellular concentration of the drug increases, while the unbound drug concentration in the cytosol decreases, pontentially affecting target engagement, the rate of clearance and metabolic processes. In our new assay, lysosomal trapping is determined using an indirect method where the effect of the test compound on the lysosomal accumulation of LysoTracker Red, a fluorescent dye is assessed. Compounds that accumulate in the lysosome, competitively extrude the dye from the lysosomes, leading to a reduction in LysoTracker Red fluorescence signal intensity. Fluorescence signals are analyzed using High Content Imaging (HCI) that allows specific detection of individual lysosomes making this method more sensitive than the more conventional microplate reader-based readouts. A scientific poster on the assay performance is available on our website.

Kp,uu (unbound liver-to-blood partition coefficient) and cellular unbound fraction (fu) of a drug should be considered for a reliable prediction of in vivo hepatic clearance using in vitro data, especially where active and passive uptake and efflux are the main clearance mechanisms. A Kp,uu determination assay, using an estimation of intra- and extracellular concentrations of the compound of interest in plated human hepatocytes is planned for a launch in Q1 2023.

New Metabolism Services

UGT inhibition assays

If direct glucuronidation is a major elimination pathway of an investigational drug or its expected co-medications, it is recommended to study in vitro whether the drug can inhibit uridine diphosphate (UDP) glucuronosyltransferase enzymes (UGTs). This in vitro assay is now available at SOLVO-CRL for the 7 most relevant UGT isoforms including UGT1A1 and UGT2B7. The assay utilizes recombinant UGTs or human liver microsomes (HLM) and relatively isoform-selective substrates. Our protocol was established following recommendations from the M12 draft Guidance on Drug Interaction Studies of the International Council for Harmonisation (ICH).

CYP induction screening.

The potential of drug candidates to induce metabolizing enzymes via activation of nuclear receptors is often assessed at an early phase of drug development to identify risks and rank-order compounds. A CYP induction assay based on cryopreserved human hepatocytes with a simplified assay setup was introduced recently by SOLVO. This provides a more reliable and definitive high-throughput alternative to the nuclear receptor assays that are commonly applied at this stage.. This assay offers the reliability of primary human hepatocytes at a reduced cost, and data delivery within 10 working days for up to 20 test compounds in one round.

Recent new cell line launches

New members of our uptake transporter portfolio include the rat sodium-iodide symporter (Nis) and the rat equilibrative nucleoside transporter 1 (Ent1). Nis supplies iodide for thyroid hormone synthesis, and interference with its function may disrupt thyroid homeostasis. Like its human counterpart, the HEK293-based rat Nis assay measures cellular accumulation of iodide by means of a colorimetric reaction. Ent1, the ubiquitous sodium-independent nucleoside transporter is not only a major regulator of physiological adenosine levels but also important to pharmacology as it is engaged by some nucleoside analog drugs while inhibited by others. Our MDCKII-based uptake assay for rat Ent1 operates with adenosine as probe substrate and the highly specific and potent S-(4-Nitrobenzyl)-6-thioinosine (a.k.a. NBMPR) as the reference inhibitor. Cell lines overexpressing rat or other pre-clinical species orthologs of human transporters can be used to assess species-differences in compound handling.

Bioanalytics updates

Our bioanalytics team had an exciting 18th of October at the Solvo Budapest site. A rather large package arrived containing a Sciex 6500+ triple quadrupole mass spectrometer, the first piece of a high throughput (HT), high sensitivity system, called LS-1. The LS-1 is set with eight UHPLC capable valves and is built around a high-speed sampling robot to support the increasing volumes of fast turnaround screen-type automated transporter assays’ bioanalyses. The remaining elements for the complete configuration, two Agilent Infinity II HPLC pumps and the LS-1 robot, are arriving before the end of the year. We can’t wait to fire up our second HT system beside the ADDA to support our partners with Screen-type needs from January 2023!

Events

In addition to the many new developments by the R&D team, SOLVO closes a successful year on the PR field as well. We had the opportunity to support two very esteemed conferences, the North American ISSX Meeting and the DMDG-GMP-SMP Joint Meeting, as Gold sponsor. Two posters were presented at these events, you can access and download them from the website.

  • Adaptation of in vitro Methods of Determine Unbound Cell to Medium Partition Coefficient, Kp,uu
  • Lysosomal Trapping Measurement in Multiple Cell Types Using High Content Screening System

Four online events were organized as part of our Meet the Experts webinar series with two excellent invited speakers and two in-house experts. These webinars and previous ones are available at the website as well.

  • ICH M12 Draft Guidance on Drug Interaction Studies, Noémi Szili, PhD, SOLVO Biotechnology
  • Gaining Mechanistic Insight in Drug Induced Cholestasis, Pieter Annaert, PhD, KU Leuven
  • Assessing Drug Interactions Using Solute Carrier (SLC) Biomarkers, David Rodrigues, PhD, Pfizer
  • Efflux Transporters – Challenges, Failures, and Success Stories, Zsuzsanna Gáborik, PhD, SOLVO Biotechnology

Previous entry: ISSX Webinar Series - New ICH M12 Guidelines on Drug Interactions Studies presented by Dr. Noémi Szili


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