December 16, 2021
OAT7 is expressed on the sinusoidal membrane domain of hepatocytes, where it exchanges sulfate-conjugated steroids, including estrone-3-sulfate, and sulfate conjugates of xenobiotics for intracellular short-chain fatty acids such as butyrate. Due to its role in steroid transport, it has been associated with conditions like osteoporosis. Among its substrates, we also find pravastatin, so while OAT7 is currently less characterized than other members of the OAT family, it may play a role in the disposition of certain drugs. Remarkably, OAT7 does not prefer prototypical OAT substrates like p-aminohippuric acid and is not affected by the otherwise generic OAT inhibitor probenecid. Our recently launched OAT7 assay, based on HEK293 cells overexpressing OAT7, utilizes estrone 3-sulfate as a probe substrate and bromosulfophthalein as the reference inhibitor.
Our other upcoming cell line, due for release in Q1 2022, features a relatively underappreciated transporter, ASCT1. Whereas its close homologue, the glutamine-preferring transporter ASCT2, has been researched extensively for its overexpression in many cancer types, the importance of ASCT1 in brain amino acid and neurotransmitter homeostasis is just being uncovered. The key role of ASCT1 in the central nervous system is underscored by the devastating effect of its mutations on neural development and function. Unlike ASCT2, ASCT1 does not accept glutamine as a substrate, while it transports both L- and D-serine, the latter being a co-agonist of NMDA-type glutamate receptors. For now, the role of ASCT1 has mostly been investigated in pre-clinical species where it has been associated with fatigue and stroke in rodents, and certain intestinal inflammatory/stress conditions in other mammals. This, together with a recently shown ASCT1 upregulation in human micropapillary carcinoma cells, make it a potential candidate for drug targeting as well as disease models. The HEK293-ASCT1 assay has been set up with L-serine as a probe substrate and a competing natural amino acid, L-cysteine, as the reference inhibitor.
• In collaboration with the University of Szeged, Sáfár et al. have compared several ABCG2/BCRP vesicular transport inhibition assay platforms to investigate how the choice of expression system and probe substrate impacts on transport kinetics and the obtained IC50 values
• SOLVO Senior Scientist Csilla Temesszentandrási-Ambrus has co-authored a paper with colleagues from the Research Centre for Natural Sciences, Budapest, on the interactions of potential anti-COVID-19 compounds with ABC and OATP transporters.
• SOLVO has contributed to the collection of transporter data in a study led by the Washington State University that aims at elucidating the disposition routes of the anticancer drug irinotecan.
• Senior Scientist Péter Tátrai and former Chief Scientific Officer Péter Krajcsi, with researchers from universities in Budapest, have reviewed how common drugs modulate the transport of urate through inhibition or stimulation of key transporters involved in renal urate disposition.
For obtaining a waiver of clinical bioequivalence studies, especially for the development of generic drugs, compounds need to be assessed according to the criteria of the Biopharmaceutics Classification System (BCS). The BCS system can also be used to justify inclusion or omission of certain assays (e.g., efflux transporter substrate assessment assays) as part of drug approval processes. The BCS classifies compounds according to high/low permeability and high/low solubility. SOLVO now offers Permeability services for BCS class determination using a fully validated Caco-2 assay in full agreement with the ICH M9 criteria. As part of assay validation, in vitro permeability of 28 internal permeability standard drugs – chosen for low-, moderate- and high permeability ranges – has been characterized in our Caco-2 system and plotted against their in vivo fraction absorbed resulting in a reliable correlation of in vitro permeability to predict in vivo absorption. The test compound is classified as either high- or low permeable based on direct comparison to the high-low permeability boundary cut-off compound, Minoxidil, as identified in our system. To be suitable for BCS classification, the test compound needs to be mainly passively permeable and not substrate to active (efflux) transport (Efflux ratio (ER) < 2). As part of our BCS permeability study, active transport is assessed and efflux transporter (BCRP, MDR1 and MRP2) functionality is demonstrated in the assay system. SOLVO’s BCS Permeability classification package applies a stepwise approach, including – in addition to compound permeability assessment – a set of supporting assays, such as solubility testing and compound tolerability assessment in the Caco-2 system, to assure conditions selected for the permeability determination are the best adapted to your compound.
In addition to our portfolio expansion, 2021 has been a year of growth and development for our bioanalytical platform as well. Our capabilities now extend to Metabolite Identification – using QE OrbiTrap high resolution Mass Spectrometry and Compound Discoverer software. To support our ADMET services, the BA team has also further fortified our capabilities in quantifying therapeutic oligonucleotides, polypeptides, and oligosaccharides, dedicating their efforts to method development and refinement for these molecules in addition to the more “traditional” small molecule capabilities. We have also expanded our instrument park, including multiple UHPLC instruments and Triple Quadrupole Mass Spectrometers to support high sensitivity as well as high throughput measurements. To match the machine capacities, the team has also welcomed 5 new BA scientists this year, together we make sure to support any request and rise to any challenge brough to SOLVO by our Sponsors!
SOLVO’s last webinar for 2021 was presented by guest speaker Dr. Yurong Lai from Gilead titled, “Endogenous Biomarkers to Detect Transporter Drug-drug Interactions: the Recent Updates".
An important milestone in the history of transporter-function biomarkers was the 2018 publication of an ITC white paper, co- authored by Dr. Lai, boosting the quest for developing endogenous biomarker-based DDI assessment strategies. The talk highlights the most recent advances on the use of clinical biomarkers to track modulation of OATP1B1/1B3 and OAT1/3 transporter functionality. Increased evidence is provided for Coproporphyrin-I (CP-I) as the most promising biomarker for OATP1B1, while for OATs, encouraging results have been obtained with kynurenic acid.
For most transporters, markers that are specific and selective enough still need to be identified, while where good candidates have been selected, as in the case of OATP1B1, the focus is now on obtaining sufficient data for generating a validated model. In addition to human clinical results, the need for characterization of these biomarkers in in vitro systems as well as in multiple preclinical species, has been highlighted to assess IVIVE potential and cross-species applicability.
The webinar series resumes early next year, stay tuned for the newest updates!
Next entry: Join our next webinar on 6 April