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Dry age-related macular degeneration (AMD) is a currently untreatable vision threatening disease. Impaired proteasomal clearance and autophagy in the retinal pigment epithelium (RPE) and subsequent photoreceptor damage are connected with dry AMD, but detailed pathophysiology is still unclear. In this paper, we discover inhibition of cytosolic protease, prolyl oligopeptidase (PREP), as a potential pathway to treat dry AMD. We showed that PREP inhibitor exposure induced autophagy in the RPE cells, shown by increased LC3-II levels and decreased p62 levels. PREP inhibitor treatment increased total levels of autophagic vacuoles in the RPE cells. Global proteomics was used to examine the phenotype of a commonly used cell model displaying AMD characteristics, oxidative stress and altered protein metabolism, in vitro. These RPE cells displayed induced protein aggregation and clear alterations in macromolecule metabolism, confirming the relevance of the cell model. Differences in intracellular target engagement of PREP inhibitors were observed with cellular thermal shift assay (CETSA). These differences were explained by intracellular drug exposure (the unbound cellular partition coefficient, Kpuu). Importantly, our data is in line with previous observations regarding the discrepancy between PREP's cleaving activity and outcomes in autophagy. This highlights the need to further explore PREP's role in autophagy so that more effective compounds can be designed to battle diseases in which autophagy induction is needed. The present work is the first report investigating the PREP pathway in the RPE and we predict that the PREP inhibitors can be further optimized for treatment of dry AMD.

Human hepatocytes show marked differences in cell size, gene expression, and function throughout the liver lobules, an arrangement termed liver zonation. However, it is not clear if these zonal size differences, and the associated phenotypic differences, are retained in isolated human hepatocytes, the “gold standard” for in vitro studies of human liver function. Here, we therefore explored size differences among isolated human hepatocytes and investigated whether separation by size can be used to study liver zonation in vitro. We used counterflow centrifugal elutriation to separate cells into different size fractions and analyzed them with label-free quantitative proteomics, which revealed an enrichment of 151 and 758 proteins (out of 5163) in small and large hepatocytes, respectively. Further analysis showed that protein abundances in different hepatocyte size fractions recapitulated the in vivo expression patterns of previously described zonal markers and biological processes. We also found that the expression of zone-specific cytochrome P450 enzymes correlated with their metabolic activity in the different fractions. In summary, our results show that differences in hepatocyte size matches zonal expression patterns, and that our size fractionation approach can be used to study zone-specific liver functions in vitro.

Purpose

The intracellular fraction of unbound compound (f(u,cell)) is an important parameter for accurate prediction of drug binding to intracellular targets. f(u,cell) is the result of a passive distribution process of drug molecules partitioning into cellular structures. Initial observations in our laboratory showed an up to 10-fold difference in the f(u,cell) of a given drug for different cell types. We hypothesized that these differences could be explained by the phospholipid (PL) composition of the cells, since the PL cell membrane is the major sink of unspecific drug binding. Therefore, we determined the f(u,cell) of 19 drugs in cell types of different origin.

Method

The cells were characterized for their total PL content and we used mass spectrometric PL profiling to delineate the impact of each of the four major cellular PL subspecies: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). The cell-based experiments were compared to cell-free experiments that used beads covered by PL bilayers consisting of the most abundant PL subspecies.

Results

PC was found to give the largest contribution to the drug binding. Improved correlations between the cell-based and cell-free assays were obtained when affinities to all four major PL subspecies were considered. Together, our data indicate that f(u,cell) is influenced by PL composition of cells.

Conclusion

We conclude that cellular PL composition varies between cell types and that cell-specific mixtures of PLs can replace cellular assays for determination of f(u,cell) as a rapid, small-scale assay covering a broad dynamic range.

This thesis work investigates factors influencing intracellular drug disposition. An experimental method for measurement of intracellular bioavailability (F_ic), was used throughout. F_ic is defined as the ratio between the unbound drug concentration inside the cell and the compound concentration in the cell exterior.

First, the impact of transporter proteins—such as the uptake transporter OATP-1B1 and the efflux transporter P-gp—on F_ic was assessed in isolation in singly transfected, well-characterized cell models. The net impact of ADME proteins on F_ic, including drug transporter proteins and metabolic enzymes, was assessed in primary human hepatocytes. The results indicated that the F_ic measurement accurately reflected system-dependent functionality of these proteins.

Second, the impact of cellular lipids on F_ic was studied, in particular phospholipids (a major constituent of cellular membranes) and neutral lipids (in the form of neutral lipid droplets in adipocytes). Drug partitioning to phospholipids was found to be the major determinant of intracellular fraction of unbound drug (f_u,cell), while neutral lipid droplets and cellular proteins played a relatively smaller role. Therefore, the importance of phospholipids, and their major four subspecies—phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI)—was investigated in a cell-free approach with purified phospholipids.

Finally, F_ic was applied in two ways to drug discovery settings. First, F_ic successfully harmonized system-dependent CYP450 enzyme inhibition values (IC₅₀) obtained in human hepatocytes and human liver microsomes. F_ic measured in suspended human hepatocytes also reflected hepatic enrichment factors of CYP450 inhibitors used in physiologically-based pharmacokinetic modelling. Second, F_ic was used as a complementary tool to study the effect of cell-penetrating peptides on intracellular disposition of targeted antisense oligonucleotide conjugates.

Overall, the thesis contributes to the mechanistic understanding of F_ic and demonstrates its use for drug compound profiling at an early stage in drug discovery settings.

Accurate prediction of drug-drug interactions (DDI) is a challenging task in drug discovery and development. It requires determination of enzyme inhibition in vitro which is highly system-dependent for many compounds. The aim of this study was to investigate whether the determination of intracellular unbound concentrations in primary human hepatocytes can be used to bridge discrepancies between results obtained using human liver microsomes and hepatocytes. Specifically, we investigated if Kp(uu) could reconcile differences in CYP enzyme inhibition values (K-i or IC50). Firstly, our methodology for determination of Kp(uu) was optimized for human hepatocytes, using four well-studied reference compounds. Secondly, the methodology was applied to a series of structurally related CYP2C9 inhibitors from a Roche discovery project. Lastly, the Kp(uu) values of three commonly used CYP3A4 inhibitorsketoconazole, itraconazole, and posaconazolewere determined and compared to compound-specific hepatic enrichment factors obtained from physiologically based modeling of clinical DDI studies with these three compounds. Kp(uu) obtained in suspended human hepatocytes gave good predictions of system-dependent differences in vitro. The Kp(uu) was also in fair agreement with the compound-specific hepatic enrichment factors in DDI models and can therefore be used to improve estimations of enrichment factors in physiologically based pharmacokinetic modeling.

Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability (F-ic) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower F-ic. The induction of NL did not further increase drug binding but led to altered F-ic due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly.