The current drug discovery process for anti-parasitics initially focuses on identifying compounds which are active against parasites cultured in vitro. To do this parasites are cultured at a set drug concentration. This is repeated over a range of drug concentrations allowing the EC50, the concentration required to kill 50% of the parasites, to be measured. From this the EC99 (concentration required to kill 99% of the parasites) is calculated. Potent (greatest effect at lowest concentration) anti-parasitic compounds which also meet other required criteria for likely successful development are then progressed into an in vivo pharmacokinetic study to assess blood exposure over time. This is followed by a maximum tolerated dose (MTD) study to determine the maximum concentration of compound which does not cause unacceptable side effects. Ideally, the tolerated circulating blood free concentration will reach at least the EC99. The efficacy at the dose level delivering this maximum tolerated blood concentration is then determined, to allow proof of concept (PoC) for the drug. Following successful PoC, the minimal effective dose and the optimal dosing regimen are then determined in the mouse model of disease and understanding of the pharmacokinetic/pharmacodynamic (PKPD) is developed. This can involve multiple groups of animals.
In recent years, there has been a surge of interest in the use of hollow fibre cell culture (HFCC) as a tool to better understand PKPD relationship in vitro. The benefit of this system is that as fresh media containing drug is perfused through the cartridge, the concentration of drug can be varied over the course of the experiment, better mimicking the in vivo situation of different dose levels and dose regimens. This allows multiple rounds of dose optimisation to be performed in vitro, avoiding the associated animal usage required. Current HFCC has been successful with a number of both intra- and extracellular pathogens. The major limitation of this model is that the host cells are ideally grown in suspension to allow convenient sampling and imaging of the infected cells. If the host cells adhere to the hollow fibres, the cartridge must be disassembled and the cells freed prior to imaging. A process which is time consuming and dangerous when dealing with infectious agents. As both human peripheral blood mononuclear cells, hPBMCs, the gold-standard in vitro host cell of L. donovani, and Vero cells, the in vitro T. cruzi host cell, are adherent they would not be suitable for HFCC. It would therefore be invaluable to further develop this HFCC approach with the use of a flow through cell system (FTCS) which would have all the benefits of the HFCC but in a more convenient platform; suitable for adherent mammalian cells infected with intracellular pathogens and more amenable to a higher throughput workflow. In this WCAIR project we plan to combine the FTCS with microscopic imaging using an IncuCyte live cell imager (Essen Bioscience).
Mammalian cells infected with L. donovani or T. cruzi will be incubated in a commercially available flow-through chamber slide (e.g. IBIDI), allowing fresh media, with or without drug, to be perfused over the cells. The chamber slides will be continuously monitored microscopically on an IncuCyte live cell imager, allowing real time visualisation of the effect the drug is having on both the mammalian cell and the intracellular parasites. Delivery of media will be precisely controlled by an elveflow microfluidic device. By co-infusing drug-spiked media with control media, the exact concentration of drug reaching the cells can be dynamically controlled and the in vitro drug concentration/time (CT) profile can be easily programmed to mimic the unbound in vivo CT profile measured from a preliminary mouse pharmacokinetic study. The effects of increasing dose and changes in dose regimen can then be explored in this device to build PKPD in vitro. This allows determination of the optimal drug concentration time course regimen that can be translated back to an in vivo efficacy dose and dose regimen study design using the preliminary PK profile. This removes the current need for multiple dose groups of animals to identify this same optimal dosing regimen. If systemic drug levels are not a good surrogate for efficacy, the CT profile in vitro can be further modified to factor in tissue accumulation (measured from a simple tissue distribution study and tissue free fraction) for tissues relevant to where the parasites reside. The levels of compounds in all FTCS experiments will be monitored from spot sampling from the device and UPLC-MSMS analysis.
This research is based on modernising an existing system which has a track history of success with a number of other pathogens (notwithstanding the host cells were in suspension as opposed to adherent). So the risk of unsuccessful progress is considered low. Current examples of HFCC used successfully for in vitro PKPD and translation to in vivo include anti-malarial (Bakshi et al., 2013) and anti-tuberculosis (Gumbo et al., 2015) programmes. Indeed, the European Medicines Agency has approved the use of HFCC, or “glass mouse” as they colloquially refer to it, as a tool to replace the existing in vivo dose-ranging studies.