Enhancing Throughput of In Vitro DDI Assays with Echo^®MS+ Platform - PBSS 2024

High throughput screening (HTS) of in vitro drug-drug interactions (DDIs) can greatly reduce assay turnaround time, thereby accelerating development. At the recent PBSS DMPK/Clinical Pharmacology for IND/NDA Filings workshop in San Diego – David Johnson, PhD, Senior Director, Drug Metabolism and Pharmacokinetics at BioAgilytix, presented data demonstrating that the Echo^®MS+ system has powerful advantages to support HTS for transporter and other DDI assays.

DDI studies, which evaluate the effects of one drug on the pharmacokinetics or pharmacodynamics of another, are critical to the development of new therapeutics. The US Food and Drug Administration (FDA) typically requires developers to perform DDI studies to support investigational new drug (IND) applications, particularly for drugs that are substrates or inhibitors of key transporters and metabolic enzymes. Timely delivery of DDI reports is required to meet filing deadlines, and BioAgilytix is always investigating how new technologies and processes can reduce turnaround time.

Acoustic ejection mass spectrometry (AEMS) is a cutting-edge technique that harnesses acoustic waves to propel sample droplets into a flow stream for mass spectrometric analysis, achieving very high sampling rates. In this poster summary, we describe a case study utilizing the Sciex Echo^®MS+, which couples AEMS and open port interface (OPI) sampling with the SCIEX ZenoTOF 7600 mass spectrometry system, to conduct efflux transporter assays.

Purpose

The purpose of this case study was to evaluate the feasibility of using the Echo^®MS+ system coupled to a ZenoTOF 7600 to enhance throughput and reduce the turnaround time of in vitro efflux transporter assays. Efflux transporters such as breast cancer resistance protein (BCRP) and P-glycoprotein 1 (P-gp), also known as multidrug resistance protein 1 (MDR1), are membrane-bound proteins that use adenosine triphosphate (ATP) to transport xenobiotics across cell membranes. FDA and International Council for Harmonisation (ICH) guidelines outline recommended substrates and inhibitors for use in in vitro transporter studies.^1,2

	Prototypical Substrate	Prototypical Inhibitor
BCRP	Estrone-3-sulfate (E3S)	Ko143
P-gp	N-methyl quinidine (NMQ)	Elacridar

Method

HEK293-derived P-gp and BCRP vesicles, containing substrate with and without inhibitor, were preincubated in assay buffer at 37°C for 10 minutes prior to the addition of ATP or AMP. After the addition of ATP or AMP, the reactions were incubated for 2 minutes at 37°C and then terminated by the addition of ice-cold wash buffer followed by filtering through a glass fiber filter plate. The filter plate was washed 5 times with ice-cold buffer and then dried. The samples were eluted from the filter plate and then processed by solid-phase extraction. After extraction, the samples were transferred to a 384-well plate and analyzed using the Echo^®MS+ system coupled to a ZenoTOF 7600.

Results

The Echo^®MS+ system demonstrated 384 ejections in approximately 10 minutes (see Figure 1).

Calibration curves were established with E3S (range: 2.29 to 5,000 nM) for BCRP and NMQ (range: 2.29 to 5000 nM) for P-gp, achieving high linearity (see Figure 2), with analytical performance comparable to LC-MS/MS results. The lower limit of quantitation was suitable for the assay requirements.

Prototypical inhibitors demonstrated concentration-dependent inhibition of substate transport for both BCRP and P-gp (see Figure 3). Ko143 inhibited transport of E3S by BCRP with an IC₅₀ of 0.12 µM. Elacridar inhibited transport of NMQ with an IC₅₀ of 0.58 µM. These values were comparable to values found in the literature.

Conclusion

To date, liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has been the gold standard for conducting DDI studies. While this method is highly accurate, it is time-intensive and can prolong the drug development process.

Use of the Echo^®MS+ system coupled to the ZenoTOF 7600 demonstrated a robust linear range for various transporter substrates for BCRP and P-gp. In addition, the system reproduced IC₅₀ data that agrees with reported values, confirming the validity of this high throughput approach. Based on these findings, the Echo^®MS+ system offers suitable performance and greater speed compared to traditional LC-MS/MS, positioning it as a powerful advancement for transporter and other DDI assays with the potential to save time while maintaining scientific rigor. Ongoing work is investigating the utility of AEMS in other transporter and enzyme assays, including cytochrome P450 (CYP) inhibition and metabolic stability.

BioAgilytix offers a range of services to evaluate the potential for drug-drug interactions, including cytochrome P450 (CYP450) induction studies, UDP-glycosyltransferase (UGT) induction studies, CYP/UGT inhibition studies, CYP/UGT reaction phenotyping, and transporter substrate/inhibition studies. In vitro drug-drug interaction studies are performed in accordance with appropriate global industry and regulatory expectations. Learn more about our DDI services. At BioAgilytix, we offer industry-leading bioanalytical solutions for all therapeutic modalities, from small molecules to advanced therapy medicinal products (ATMPs), specializing in several different instrument platforms, staying abreast of the latest technologies to accelerate drug development from discovery through IND and approval. To learn more about BioAgilytix’s extensive capabilities, speak to our scientists.

¹ FDA Guidance: In Vitro Drug Interaction Studies — P450 Enzyme- and Transporter-Mediated Drug Interactions. 2020.

² ICH M12: Drug Interaction Studies. 2024.