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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.

Accurate profiling of pharmacokinetic (PK) and pharmacodynamic (PD) parameters is critical in the discovery and development of new oncology drugs, schedules and combinations. Predicting these parameters pre-clinically is essential to determine efficacious doses required in clinical treatment. The PK/PD/efficacy relationship has traditionally been characterized in xenograft models, owing to an absence of viable in vitro alternatives. However, murine/human PK parameters can vary for many compounds. To explore PK/PD/efficiency relationships on 3D tumor models and organoids, we have developed a microphysiological system (MPS) able to mimic or reverse translate clinical/in vivo drug exposure (PK) profiles on an in vitro system. The MPS can deliver up to two different drugs to tumor models in monotherapy or combinations, using customizable PK profiles and treatment schedules, by stepwise addition and removal of cell culture medium in a regular 24-well plate format. We first recapitulated the in vivo PK/PD relationship for a PI3K inhibitor (BYL719- t1/2 6 hours, equivalent to 50 mg/kg murine oral dose), used to treat an A549 non-small cell lung carcinoma model. Following BYL719 exposure in the MPS device cellular p-AKT levels (biomarker of PI3K pathway activity) fell by up to 90%, but then recovered to pre-dosed levels over a 24-hour period as drug concentrations in the tumor reduce, as observed in vivo. This effect lacks in standard in vitro bolus experiments. Next, we explored whether the MPS could recapitulate in vivo effects of combining topoisomerase inhibitors with DNA-damage-response inhibitors (DDRi) in the treatment of colorectal tumors. Being almost non-toxic to normal cells, DDRi’s enhance DNA damage induced by topoisomerase inhibitors. 3D colorectal tumor models were treated by the MPS device for six days with monotherapy or combination therapy of the two compounds (SN38 Cmax = 20 nM, DDRi Cmax = 162 nM), recapitulating their in vivo PK profiles. Viability assays showed the benefits of combination therapy (viability 48.5% vs controls) over topoisomerase inhibitor monotherapy (viability 58.3% compared vs controls), with efficacy rates consistent with in vivo observations, rather than static 2D in vitro cultures which were over predictive. Overall, our MPS is able to accurately capture PK/PD/efficacy relationships using in vitro cultures. This approach will enable a greater understanding of drug properties and optimal treatment regimens to ultimately enable better design of clinical studies. Citation Format: Tudor Petreus, Tomasz Kostrzewski, Dharaminder Singh, David Hughes. Bridging gaps in translational biology: Exploring pharmacokinetic/pharmacodynamic/efficacy relationships and combination treatments in 3D tumor models using a microphysiological system [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 182.

Characterizing the relationship between pharmacokinetics (PK), pharmacodynamics (PD) and efficacy is critical in the discovery and development of new drugs, schedules and combinations. The PK/PD/efficacy relationship has historically been characterized in xenograft models, owing to an absence of viable alternatives. The study of this relationship in vitro, has to date been problematic as the generation of time varying concentrations in multi-well plates has not been possible. We have explored an in vitro methodology utilizing a device (Microformulator, Viibre, Vanderbilt University) capable of precision addition and removal of medium from the wells of a microtiter cell culture plate. Through stepwise addition and removal of medium the device was able to recapitulate PK-like, time varying concentration profiles of one, or more drugs in individual wells. We used this approach to explore the effects of PI3K pathway inhibitors, on p-AKT levels, and viability in a number of cancer lines. The PI3K/mTOR pathway is a central oncogenic pathway deregulated in cancer and p-AKT is a marker of PI3K pathway activity. BYL719 and PI-103, are PI3K inhibitors with varying PK profiles and varying target coverage against PI3K isoforms. The compounds were tested on a pair of cell lines, T47D (breast, sensitive) and Colo205 (gut, insensitive). Both compounds were potent in a bolus dose 72hr assay with BYL719 having an IC50 of 0.63 µM for T47D and 15.8 µM for Colo205. Whilst PI-103 gave IC50 of 0.72 µM for T47D and 4.16 µM for Colo205. Both compounds also reduced, p-AKT in a dose-dependent manner after 2 hours. Using the microfluidic device, we generated PK-like dosing profiles for the two compounds, (BYL719 - t1/2 6 hours) (PI-103 - t1/2 3 hours) which mimicked their in vivo clearance profiles over a 24-hour period. p-AKT levels were initially reduced by both compounds but recovered at 24 hours as the concentration of the compound in the well declined. This replicates findings in xenograph models. The shorter t1/2 of PI-103, resulted in a more rapid recovery of p-AKT than observed for BYL719. Efficacy tests were conducted in which a 24 hour PK-like profile was applied for 3 consecutive days, mimicking daily dosing. For both compounds in T47D cells, the inhibition of cell growth was reduced as compared to a bolus addition at the Cmax concentration. These data demonstrate how PI-103 initially appeared to be an efficacious molecule but its rapid in vivo metabolism meant it could not be progressed in clinical studies. In conclusion, this study demonstrates the microfluidic addition and removal device can be used to recapitulate PK-like profiles in vitro and allow exploration of the PK/PD/efficacy relationship. The availability of an in vitro method will enable these important parameters to be determined at an earlier stage of the drug discovery process. Citation Format: Tomasz M. Kostrzewski, Dharaminder Singh, Paul Golby, Alysha Bray, David Hughes. Investigating the PK/PD/efficacy relationship of PI3K inhibitors in vitro, enabled by a microfluidic addition and removal device [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1931.

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