Improved in vitro renal tubular drug transport modelling to meet regulatory and development cost challenges

ONE-THIRD OF all drugs and drug candidates are cleared renally.¹ The kidney’s important role in drug clearance makes it prone to damage; drug-induced kidney injury (DIKI) is estimated to be responsible for up to 60 percent of all acute kidney injury (AKI) cases, while 10 percent of post‑market drug attrition is due to DIKI.^2-4 It is estimated that drugs are responsible for approximately 20 percent of acute renal failure episodes and this figure increases dramatically in the elderly where the incidence of drug-induced nephrotoxicity is reported to be as high as 66 percent.^5-8

The renal proximal tubule is the major site of drug transport due to the concerted action of various uptake and efflux mechanisms. Costs of drug development have been increasing since the 1970s to $2.6 billion today.⁹ This underlines the importance of reliable, predictive and cost-efficient in vitro models for use in development and supporting regulatory submission.

Higher uptake of a drug into proximal tubule epithelial cells (PTCs) than efflux results in intracellular accumulation of drugs and may result in toxicity. Furthermore, drug-drug interactions (DDIs) can interfere with drug transport mechanisms and are a huge economic burden on healthcare that is expected to worsen with an ageing population and increasing polypharmacy. DDI most frequently occurs due to interactions with drug metabolising enzymes or drug transporters. Traditional renal in vitro assays often are not sufficient to accurately identify DDIs and nephrotoxicity, while pre-clinical animal models are not predictive due to species differences¹⁰ – these factors present a significant roadblock to the efficient development of safe drugs.

In drug development, existing knowledge of drug distribution routes and possible DDIs is essential for the downstream success of clinic-bound compounds. US Food and Drug Administration (FDA) guidelines, as of 2020, recommend certain in vitro investigational steps concerning a compound’s potential interaction with drug transporters if ADME (absorption, distribution, metabolism and excretion) data suggest that active secretion of the parent drug is greater than or equal to 25 percent of total clearance.¹¹ They define the key transporters as apical-spanning efflux pumps P-glycoprotein (P-gp; ABCB1), breast cancer resistance protein (BCRP; ABCG2) and multidrug and toxin extrusion transporters 1/2 (MATE1/2k; SLC47A1/2), as well as organic anion transporters 1/3 (OAT1/3; SLC22A6/8) and organic cation 2 (OCT2; SLC22A2) expressed on the basolateral membrane of proximal tubular cells. Therefore, to produce relevant and useful data for regulatory submissions, it is crucial to choose your pre-clinical models with care.

The current renal models

Traditionally, animal and human cell lines such as human (HEK293), pig kidney-derived (LLC-PK1) and Madin-Darby canine kidney (MDCK) cell lines^12-16 have been used to model transporter-mediated uptake and to study movement of candidate drug molecules within the kidney. Cells that form polarised monolayers and tight junctions can also be double-transfected, allowing multi‑compartmental evaluation of a compound’s transport.¹³ Though these models are useful for determining uptake kinetics and possible DDIs of new compounds, they only express two transporters at most and therefore do not offer a holistic view of the handling of a compound.

Human-derived cells, such as the immortalised HK-2 cell line, edged the field closer towards a more in vivo model, although they lacked key features and transporters of in vivo tubules.¹⁷ They fall short of true in vivo features to model human clinical relevance, with reported issues that include: tight barrier formation; cells that lack the full complement of transporters in the correct ratios; and the use of animal cells as transporter vectors.^18-21 Using cell models derived from animals can also introduce various discrepancies in transporter expression. For instance, the rodent proximal tubule contains OCT1 at the basolateral membrane, while apical expression of MATE2-K has not been detected.²² Recently, differential protein expression in the proximal tubule between species has been highlighted,^13,23,24 suggesting probable differences in drug handling for a wide range of substrates, which can be problematic when transferring data from pre-clinical species.

Figure 1: A schematic diagram of the major renal proximal tubule epithelial cell transporters. The renal proximal tubule is a specialised polarised cell layer in the kidney that is a major site of drug secretion and absorption mediated by membrane-located transporters. Image used and adapted with permission from SOLVO Biotechnology, a Charles River Company.

To date, the most predictive and representative of in vivo results has been to culture primary cells sourced directly from fresh tissue that retain a higher abundance of functional transporters. In vitro human cell-based kidney models using renal proximal tubule epithelial cells are now becoming popular for early-stage testing of nephrotoxicity.

Functional activity of many of the key transporters has been determined in primary cultures with substrates, including Para-aminohippurate (PAH) and creatinine.^25,26 More recently, a transporter-dependant model of nephrotoxicity has demonstrated functionality across a wide range of key transporters, allowing a high rate of predictivity for nephrotoxins, including test compounds that had been through traditional pre-clinical models.²⁷ Data has also been generated to demonstrate the consistency of cisplatin-induced toxicity over multiple donors and the reproducibility of data generated from primary cells.

The same model has more recently shown species-species differences in the handling of a commercial herbicide, again demonstrating a likely function of OATs in the model²⁸ and exposing the limitations of animal models for transport studies. Criticisms of primary cultures have been the longevity of cultures and the requirement to maintain a consistent supply of fresh human tissue. These aside, freshly isolated proximal tubule cells arguably remain the gold standard of both trans‑epithelial flux and nephrotoxicity prediction assays.

Future modelling options

Generating a model of drug transport within the proximal tubule from stem cells has gained momentum in recent years; theoretically yielding an infinite population of proximal tubule cells with relevant transporters. A challenge is the maturity of the cells after their differentiation, with transcriptomic maturity resembling cells in the first-trimester kidney being reported.²⁹ There have been attempts to increase the maturity of kidney organoids, while also reducing their time in culture; however, transporter expression levels and function have not yet been investigated.³⁰ An interesting recent development has seen the collection of adult stem cells from urine samples, which require less complex medium components and culture time when compared with directing the differentiation of iPSCs.³¹ Currently, however, the stem-cell-derived models lack the extensive functional expression of transporters validation data of primary cell culture models. When this hurdle is overcome, the application of stem-cell-derived models holds great promise for the investigation of renal drug transport.

To mimic the aspects of the PTC microenvironment, in vitro culture platforms can range from two-dimensional (2D) to advanced models that recreate the cells’ microenvironment. Culture of PTCs on inserts creates a bi‑compartmental model and improved formation of a PTC monolayer.³² The increasing complexity of three-dimensional (3D) models could limit throughput in renal drug transport studies; most published models contain one tubule per device and use pumps to induce fluid shear stress (FSS)^19,33-36 although more chips could be combined on one device to allow medium-throughput testing of multiple compounds (Vriend, et al, unpublished).

Focus has shifted towards transferability of advanced renal in vitro models of nephrotoxicity and drug transport in microphysiological systems using primary human PTCs. These systems have demonstrated a more physiological response and function when compared to immortalised PTCs.³⁷ Tubular absorption was successfully predicted by in vitro to in vivo extrapolation (IVIVE)³⁸ and transport kinetics in chronic kidney disease were accurately predicted by IVIVE translation of active secretion of indoxyl sulfate in a hollow fibre membrane.³⁹ The marked increase in the development of and transferability of advanced renal in vitro 3D models in recent years is promising, but no model has yet provided sufficient validation data. Furthermore, advanced renal in vitro models are often not compatible with conventional lab equipment, are low throughput and are not proven to be cost efficient. However, these recent advances are promising and could pave the way for a new wave of improved models to study renal drug transport.

As the requirement for in vivo pre-clinical testing is driven by regulatory and cost consideration we have multiple cell and culture models available for renal drug transport and an improved understanding of renal drug transport. It becomes clear that various types of complexity can be added to mimic PTC drug transport in vitro and we should allow the research question to determine the level of complexity required and establish which in vitro model is fit for purpose. For instance, profiling of renal clearance of a single compound requires a model of higher in vivo resemblance, preferably in primary PTCs. On the other hand, possible DDIs are best studied in more simplified models, such as transfected cell lines. Improving the predictability of renal drug transport and DDIs is not limited by using advanced in vitro models but could be extended by combining IVIVE and physiologically based pharmacokinetic (PBPK) modelling.

References

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