A microfluidic system that replicates pharmacokinetic (PK) profiles in vitro improves prediction of in vivo efficacy in preclinical models

Abstract
Test compounds used on in vitro model systems are conventionally delivered to cell culture wells as fixed concentration bolus doses; however, this poorly replicates the pharmacokinetic (PK) concentration changes seen in vivo and reduces the predictive value of the data. Herein, proof-of-concept experiments were performed using a novel microfluidic device, the Microformulator, which allows in vivo like PK profiles to be applied to cells cultured in microtiter plates and facilitates the investigation of the impact of PK on biological responses. We demonstrate the utility of the device in its ability to reproduce in vivo PK profiles of different oncology compounds over multiweek experiments, both as monotherapy and drug combinations, comparing the effects on tumour cell efficacy in vitro with efficacy seen in in vivo xenograft models. In the first example, an ERK1/2 inhibitor was tested using fixed bolus dosing and Microformulator-replicated PK profiles, in 2 cell lines with different in vivo sensitivities. The Microformulator-replicated PK profiles were able to discriminate between cell line sensitivities, unlike the conventional fixed bolus dosing. In a second study, murine in vivo PK profiles of multiple Poly(ADP-Ribose) Polymerase 1/2 (PARP) and DNA-dependent protein kinase (DNA-PK) inhibitor combinations were replicated in a FaDu cell line resulting in a reduction in cell growth in vitro with similar rank ordering to the in vivo xenograft model. Additional PK/efficacy insight into theoretical changes to drug exposure profiles was gained by using the Microformulator to expose FaDu cells to the DNA-PK inhibitor for different target coverage levels and periods of time. We demonstrate that the Microformulator enables incorporating PK exposures into cellular assays to improve in vitro–in vivo translation understanding for early therapeutic insight.

Introduction
The challenges faced by the pharmaceutical industry in developing novel medicines, including attrition rates from early discovery to clinical validation, have been well described [1]. Kola and Landis [2] highlighted the most common causes of R&D attrition as being a lack of efficacy and/or safety and suggested that more attention be paid to reducing toxicity risks, improving preclinical models and demonstrating adequate proof of mechanism and proof of concept in the clinic. It can be hypothesised that the introduction of novel predictive preclinical testing techniques could improve the speed and reduce attrition in developing therapeutic compounds

Materials and methods
Microformulator—Components and system
The microfluidic device, referred to here as the Microformulator (Vanderbilt Institute for Integrative Biosystems Research and Education (VIIBRE), Vanderbilt University) is a system comprised of media bottles, computer-controlled microfluidic rotary planar peristaltic micropumps and rotary planar valves, tubing, and needles that enable real-time control of the formulation of media in individual wells within a microtiter plate (Fig 2). The device is split into 2 independent sides for simultaneous fluid addition and removal. On the dosing side, Tygon tubing (Cole-Parmer, USA) connects the input bottles to a 5-port valve, pump, a 25-port valve, and then to the individual blunt dosing needles situated above the liquid level within a well. The 5-port valve enables the selection of input media bottles, and the 25-port valve enables the selection of individual wells. The pump draws medium from the selected input media bottle (containing either media or drug stock solutions prepared in media), through the 5-port valve and to the selected port of the 25-port valve, allowing for the addition of media to an individual well. Similarly, on the aspiration side of the device, the 25-port valve selects the blunt needle, situated near the bottom of a well, and the pump draws medium from the well and into a waste bottle. All blunt needles are fixed in position 0.5 mm above the bottom of a well using a needle plate assembly lid, which ensures that wells are never left empty when fully aspirated.

Results
Replicating PK profiles—The Microformulator
The Microformulator is a microfluidic device comprised of valves, pumps, and fluid reservoirs under computer control that enable mixing microliter volumes of cell culture media and drug solutions and delivering these mixtures to individual wells within a microtiter plate (Fig 2). A replicate set of valves and pumps aspirates fluid out of the wells; coordinating the aspiration and dispensing enables changing the drug concentration in each well. A controller is programmed to generate a defined drug exposure profile over a set time interval (24-hour) cycle using predefined intervals of drug/media refresh to achieve the desired time-dependent change in drug concentrations [19]. Each well is individually addressable, and 1 or 2 compounds can be administered simultaneously, each with its own PK profile. The unit operates within a standard cell culture incubator (S3 Fig) and can continue delivering compounds for experiments lasting multiple weeks if desired. The ability of the fluidics system to change drug concentrations was verified using fluorescein as the test compound and performing 3X steps of increasing and decreasing concentrations over a 3,000-fold concentration range (S1 Fig), which is sufficient to characterise pharmacological exposure response profiles. Experiments also were performed with 2 of the pharmacological agents used in this study to confirm the delivery of the targeted concentrations by the Microformulator (S1 Fig).

Discussion
Multiwell microtiter plates are the backbone of in vitro life sciences and drug discovery research. Enormous infrastructure exists to facilitate their use and includes liquid handling and signal detection instruments compatible with a wide range of cell types (primary cells, cell lines, and organoids), formats (monolayer, sandwich culture, spheroids, and 3D scaffold), and readouts (fluorescence, high content imaging, soluble and cell extracted biomarkers, and omics) [28]. However, these in vitro systems have limitations when replicating human biology, a fundamental one being that they allow only fixed concentration bolus drug exposure. This fixed administration profile can be readily achieved in all research labs at present, but what has till now remained absent is the ability to replicate PK like drug concentrations profiles for humans (and animals) in microtiter plates, which are important factors in the successful development of a drug. One approach to testing compounds in vitro with varying concentrations has been the development of organ-on-chip multi-organ systems, which utilise the metabolic capability of cells often hepatocytes, to clear the compound from a system, creating concentration changes with time [29,30]. This approach, while closely mimicking physiology, is relatively complex requiring the simultaneous culture of multiple tissue types and is typically low throughput. We have designed an array of microfluidic pumps and valves that offer a drug exposure solution, which allows (1) individually addressable wells within a standard microtiter plate; (2) time division multiplex dosing of wells to generate PK profiles; (3) the ability to simultaneously deliver 2 drugs, each with their own PK profiles, to a single well (for combination studies), all using (4) a platform that fits within a standard incubator and is capable of running multiweek experiments. With cancer drug attrition rates being higher than other therapeutic areas, we chose to replicate the PK profiles of oncology compounds [20]. Here, we set out to demonstrate that the integration of microfluidics with multiwell plates enables the generation of PK like profiles in vitro for one or more drugs and that this facilitates prediction of mouse xenograft tumour responses as well as exploration of how alterations in PK parameters may influence drug efficacy.

Acknowledgments
We are indebted to John Fellenstein for fabricating Microformulator components and Allison Price for her editorial assistance.

Citation: Singh D, Deosarkar SP, Cadogan E, Flemington V, Bray A, Zhang J, et al. (2022) A microfluidic system that replicates pharmacokinetic (PK) profiles in vitro improves prediction of in vivo efficacy in preclinical models. PLoS Biol 20(5): e3001624. https://doi.org/10.1371/journal.pbio.3001624

Academic Editor: Chaitan Khosla, Stanford University, UNITED STATES

Received: July 12, 2021; Accepted: April 11, 2022; Published: May 26, 2022

Copyright: © 2022 Singh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data is within the paper and its supporting information files.

Funding: Research which has been reported in this publication was supported by the following; the NIH National Center for Advancing Translational Sciences (NCATS), UH3TR000491 (JPW), U01TR002383 (JPW); the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) award UH3TR002097 (JPW); through the Vanderbilt University Medical Center) UL1TR002243 (JPW); NCATS contracts (through CFD Research Corporation) HHSN271201600009C (JPW) and HHSN271201700044C (JPW); the National Cancer Institute (NCI) award U01CA202229 (JPW); the Defense Threat Reduction Agency (DTRA) grants HDTRA1-09-0013 (JPW); the Los Alamos National Laboratory CBMXCEL-XL1-2-001 (JPW); and the National Science Foundation (NSF) grant CBET-1706155 (JPW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: DS, AB, TK, DH are employees of CN Bio and hold stock or options in the company. CN Bio licenses intellectual property from Vanderbilt University related to the microfluidic device described in this study. SPD, EC, VF, JZ, FG, ARM, CEC, EJD, JHLF, KF, MPW, CWS are or were employees of AstraZeneca at the time of conducting these studies. EC, VF, JZ, ARM, CEC, CS hold stock in AstraZeneca.

Abbreviations: DNA-PK, DNA-dependent protein kinase; KO, knockout; NSCLC, non-small cell lung cancer; PARP, Poly(ADP-Ribose) Polymerase; PD, pharmacodynamics; PK, pharmacokinetic; TGI, tumour growth inhibition; VIIBRE, Vanderbilt Institute for Integrative Biosystems Research and Education

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