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Introduction The organic cation transporter 3 (OCT3, SLC22A3) is ubiquitously expressed and interacts with a wide array of compounds including endogenous molecules, environmental toxins and prescription drugs. Understudied as a determinant of pharmacokinetics and pharmacodynamics, OCT3 has the potential to be a major determinant of drug absorption and disposition and to be a target for drug-drug interactions (DDIs). Goal The goal of the current study was to identify prescription drug inhibitors of OCT3. Methods We screened a compound library consisting of 2556 prescription drugs, bioactive molecules, and natural products using a high throughput assay in HEK-293 cells stably expressing OCT3. Results We identified 210 compounds that at 20 mu M inhibit 50% or more of OCT3-mediated uptake of 4-Di-1-ASP (2 mu M). Of these, nine were predicted to inhibit the transporter at clinically relevant unbound plasma concentrations. A Structure-Activity Relationship (SAR) model included molecular descriptors that could discriminate between inhibitors and non-inhibitors of OCT3 and was used to identify additional OCT3 inhibitors. Proteomics of human brain microvessels (BMVs) indicated that OCT3 is the highest expressed OCT in the human blood-brain barrier (BBB). Conclusions This study represents the largest screen to identify prescription drug inhibitors of OCT3. Several are sufficiently potent to inhibit the transporter at therapeutic unbound plasma levels, potentially leading to DDIs or off-target pharmacologic effects.

Introduction The organic cation transporter 3 (OCT3, SLC22A3) is ubiquitously expressed and interacts with a wide array of compounds including endogenous molecules, environmental toxins and prescription drugs. Understudied as a determinant of pharmacokinetics and pharmacodynamics, OCT3 has the potential to be a major determinant of drug absorption and disposition and to be a target for drug-drug interactions (DDIs). Goal The goal of the current study was to identify prescription drug inhibitors of OCT3. Methods We screened a compound library consisting of 2556 prescription drugs, bioactive molecules, and natural products using a high throughput assay in HEK-293 cells stably expressing OCT3. Results We identified 210 compounds that at 20 mu M inhibit 50% or more of OCT3-mediated uptake of 4-Di-1-ASP (2 mu M). Of these, nine were predicted to inhibit the transporter at clinically relevant unbound plasma concentrations. A Structure-Activity Relationship (SAR) model included molecular descriptors that could discriminate between inhibitors and non-inhibitors of OCT3 and was used to identify additional OCT3 inhibitors. Proteomics of human brain microvessels (BMVs) indicated that OCT3 is the highest expressed OCT in the human blood-brain barrier (BBB). Conclusions This study represents the largest screen to identify prescription drug inhibitors of OCT3. Several are sufficiently potent to inhibit the transporter at therapeutic unbound plasma levels, potentially leading to DDIs or off-target pharmacologic effects.

Introduction The organic cation transporter 3 (OCT3, SLC22A3) is ubiquitously expressed and interacts with a wide array of compounds including endogenous molecules, environmental toxins and prescription drugs. Understudied as a determinant of pharmacokinetics and pharmacodynamics, OCT3 has the potential to be a major determinant of drug absorption and disposition and to be a target for drug-drug interactions (DDIs). Goal The goal of the current study was to identify prescription drug inhibitors of OCT3. Methods We screened a compound library consisting of 2556 prescription drugs, bioactive molecules, and natural products using a high throughput assay in HEK-293 cells stably expressing OCT3. Results We identified 210 compounds that at 20 mu M inhibit 50% or more of OCT3-mediated uptake of 4-Di-1-ASP (2 mu M). Of these, nine were predicted to inhibit the transporter at clinically relevant unbound plasma concentrations. A Structure-Activity Relationship (SAR) model included molecular descriptors that could discriminate between inhibitors and non-inhibitors of OCT3 and was used to identify additional OCT3 inhibitors. Proteomics of human brain microvessels (BMVs) indicated that OCT3 is the highest expressed OCT in the human blood-brain barrier (BBB). Conclusions This study represents the largest screen to identify prescription drug inhibitors of OCT3. Several are sufficiently potent to inhibit the transporter at therapeutic unbound plasma levels, potentially leading to DDIs or off-target pharmacologic effects.

Introduction The organic cation transporter 3 (OCT3, SLC22A3) is ubiquitously expressed and interacts with a wide array of compounds including endogenous molecules, environmental toxins and prescription drugs. Understudied as a determinant of pharmacokinetics and pharmacodynamics, OCT3 has the potential to be a major determinant of drug absorption and disposition and to be a target for drug-drug interactions (DDIs). Goal The goal of the current study was to identify prescription drug inhibitors of OCT3. Methods We screened a compound library consisting of 2556 prescription drugs, bioactive molecules, and natural products using a high throughput assay in HEK-293 cells stably expressing OCT3. Results We identified 210 compounds that at 20 mu M inhibit 50% or more of OCT3-mediated uptake of 4-Di-1-ASP (2 mu M). Of these, nine were predicted to inhibit the transporter at clinically relevant unbound plasma concentrations. A Structure-Activity Relationship (SAR) model included molecular descriptors that could discriminate between inhibitors and non-inhibitors of OCT3 and was used to identify additional OCT3 inhibitors. Proteomics of human brain microvessels (BMVs) indicated that OCT3 is the highest expressed OCT in the human blood-brain barrier (BBB). Conclusions This study represents the largest screen to identify prescription drug inhibitors of OCT3. Several are sufficiently potent to inhibit the transporter at therapeutic unbound plasma levels, potentially leading to DDIs or off-target pharmacologic effects.

Introduction The organic cation transporter 3 (OCT3, SLC22A3) is ubiquitously expressed and interacts with a wide array of compounds including endogenous molecules, environmental toxins and prescription drugs. Understudied as a determinant of pharmacokinetics and pharmacodynamics, OCT3 has the potential to be a major determinant of drug absorption and disposition and to be a target for drug-drug interactions (DDIs). Goal The goal of the current study was to identify prescription drug inhibitors of OCT3. Methods We screened a compound library consisting of 2556 prescription drugs, bioactive molecules, and natural products using a high throughput assay in HEK-293 cells stably expressing OCT3. Results We identified 210 compounds that at 20 mu M inhibit 50% or more of OCT3-mediated uptake of 4-Di-1-ASP (2 mu M). Of these, nine were predicted to inhibit the transporter at clinically relevant unbound plasma concentrations. A Structure-Activity Relationship (SAR) model included molecular descriptors that could discriminate between inhibitors and non-inhibitors of OCT3 and was used to identify additional OCT3 inhibitors. Proteomics of human brain microvessels (BMVs) indicated that OCT3 is the highest expressed OCT in the human blood-brain barrier (BBB). Conclusions This study represents the largest screen to identify prescription drug inhibitors of OCT3. Several are sufficiently potent to inhibit the transporter at therapeutic unbound plasma levels, potentially leading to DDIs or off-target pharmacologic effects.

Human liver microsomes (HLM) and human hepatocytes (HH) are important in vitro systems for studies of intrinsic drug clearance (CLint) in the liver. However, the CLint values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kpuu) effects on the CLint were investigated for five prototypical probe substrates (bupropion–CYP2B6, diclofenac–CYP2C9, omeprazole–CYP2C19, bufuralol–CYP2D6, and midazolam–CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CLint. Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CLint with CYP amounts and Kpuu could only partly explain the discordance in absolute values of CLint for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.

Human liver microsomes (HLM) and human hepatocytes (HH) are important in vitro systems for studies of intrinsic drug clearance (CLint) in the liver. However, the CLint values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kpuu) effects on the CLint were investigated for five prototypical probe substrates (bupropion–CYP2B6, diclofenac–CYP2C9, omeprazole–CYP2C19, bufuralol–CYP2D6, and midazolam–CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CLint. Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CLint with CYP amounts and Kpuu could only partly explain the discordance in absolute values of CLint for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.

Human liver microsomes (HLM) and human hepatocytes (HH) are important in vitro systems for studies of intrinsic drug clearance (CLint) in the liver. However, the CLint values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kpuu) effects on the CLint were investigated for five prototypical probe substrates (bupropion–CYP2B6, diclofenac–CYP2C9, omeprazole–CYP2C19, bufuralol–CYP2D6, and midazolam–CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CLint. Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CLint with CYP amounts and Kpuu could only partly explain the discordance in absolute values of CLint for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.

Human liver microsomes (HLM) and human hepatocytes (HH) are important in vitro systems for studies of intrinsic drug clearance (CLint) in the liver. However, the CLint values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kpuu) effects on the CLint were investigated for five prototypical probe substrates (bupropion–CYP2B6, diclofenac–CYP2C9, omeprazole–CYP2C19, bufuralol–CYP2D6, and midazolam–CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CLint. Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CLint with CYP amounts and Kpuu could only partly explain the discordance in absolute values of CLint for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.

Human liver microsomes (HLM) and human hepatocytes (HH) are important in vitro systems for studies of intrinsic drug clearance (CLint) in the liver. However, the CLint values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kpuu) effects on the CLint were investigated for five prototypical probe substrates (bupropion–CYP2B6, diclofenac–CYP2C9, omeprazole–CYP2C19, bufuralol–CYP2D6, and midazolam–CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CLint. Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CLint with CYP amounts and Kpuu could only partly explain the discordance in absolute values of CLint for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.

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.

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.

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.

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.

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.

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.

Conformational flexibility has been proposed to significantly affect drug properties outside rule-of-5 (Ro5) chemical space. Here, we investigated the influence of dynamically exposed polarity on cell permeability and aqueous solubility for a structurally diverse set of drugs and clinical candidates far beyond the Ro5, all of which populated multiple distinct conformations as revealed by X-ray crystallography. Efflux-inhibited (passive) Caco-2 cell permeability correlated strongly with the compounds’ minimum solvent-accessible 3D polar surface areas (PSA), whereas aqueous solubility depended less on the specific 3D conformation. Inspection of the crystal structures highlighted flexibly linked aromatic side chains and dynamically forming intramolecular hydrogen bonds as particularly effective in providing “chameleonic” properties that allow compounds to display both high cell permeability and aqueous solubility. These structural features, in combination with permeability predictions based on the correlation to solvent-accessible 3D PSA, should inspire drug design in the challenging chemical space far beyond the Ro5.

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.

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.

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.

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.

Intracellular drug exposure is influenced by cell-and tissue-dependent expression of drug-transporting proteins and metabolizing enzymes. Here, we introduce the concept of intracellular bioavailability (F-ic) as the fraction of extracellular drug available to bind intracellular targets, and we assess how Fic is affected by cellular drug disposition processes. We first investigated the impact of two essential drug transporters separately, one influx transporter (OATP1B1; SLCO1B1) and one efflux transporter (P-gp; ABCB1), in cells overexpressing these proteins. We showed that OATP1B1 increased Fic of its substrates, while P-gp decreased Fic. We then investigated the impact of the concerted action of multiple transporters and metabolizing enzymes in freshly-isolated human hepatocytes in culture configurations with different levels of expression and activity of these proteins. We observed that Fic was up to 35-fold lower in the configuration with high expression of drug-eliminating transporters and enzymes. We conclude that Fic provides a measurement of the net impact of all cellular drug disposition processes on intracellular bioavailable drug levels. Importantly, no prior knowledge of the involved drug distribution pathways is required, allowing for high-throughput determination of drug access to intracellular targets in highly defined cell systems (e.g., single-transporter transfectants) or in complex ones (including primary human cells).

Intracellular drug exposure is influenced by cell-and tissue-dependent expression of drug-transporting proteins and metabolizing enzymes. Here, we introduce the concept of intracellular bioavailability (F-ic) as the fraction of extracellular drug available to bind intracellular targets, and we assess how Fic is affected by cellular drug disposition processes. We first investigated the impact of two essential drug transporters separately, one influx transporter (OATP1B1; SLCO1B1) and one efflux transporter (P-gp; ABCB1), in cells overexpressing these proteins. We showed that OATP1B1 increased Fic of its substrates, while P-gp decreased Fic. We then investigated the impact of the concerted action of multiple transporters and metabolizing enzymes in freshly-isolated human hepatocytes in culture configurations with different levels of expression and activity of these proteins. We observed that Fic was up to 35-fold lower in the configuration with high expression of drug-eliminating transporters and enzymes. We conclude that Fic provides a measurement of the net impact of all cellular drug disposition processes on intracellular bioavailable drug levels. Importantly, no prior knowledge of the involved drug distribution pathways is required, allowing for high-throughput determination of drug access to intracellular targets in highly defined cell systems (e.g., single-transporter transfectants) or in complex ones (including primary human cells).

Intracellular drug exposure is influenced by cell-and tissue-dependent expression of drug-transporting proteins and metabolizing enzymes. Here, we introduce the concept of intracellular bioavailability (F-ic) as the fraction of extracellular drug available to bind intracellular targets, and we assess how Fic is affected by cellular drug disposition processes. We first investigated the impact of two essential drug transporters separately, one influx transporter (OATP1B1; SLCO1B1) and one efflux transporter (P-gp; ABCB1), in cells overexpressing these proteins. We showed that OATP1B1 increased Fic of its substrates, while P-gp decreased Fic. We then investigated the impact of the concerted action of multiple transporters and metabolizing enzymes in freshly-isolated human hepatocytes in culture configurations with different levels of expression and activity of these proteins. We observed that Fic was up to 35-fold lower in the configuration with high expression of drug-eliminating transporters and enzymes. We conclude that Fic provides a measurement of the net impact of all cellular drug disposition processes on intracellular bioavailable drug levels. Importantly, no prior knowledge of the involved drug distribution pathways is required, allowing for high-throughput determination of drug access to intracellular targets in highly defined cell systems (e.g., single-transporter transfectants) or in complex ones (including primary human cells).

Intracellular drug exposure is influenced by cell-and tissue-dependent expression of drug-transporting proteins and metabolizing enzymes. Here, we introduce the concept of intracellular bioavailability (F-ic) as the fraction of extracellular drug available to bind intracellular targets, and we assess how Fic is affected by cellular drug disposition processes. We first investigated the impact of two essential drug transporters separately, one influx transporter (OATP1B1; SLCO1B1) and one efflux transporter (P-gp; ABCB1), in cells overexpressing these proteins. We showed that OATP1B1 increased Fic of its substrates, while P-gp decreased Fic. We then investigated the impact of the concerted action of multiple transporters and metabolizing enzymes in freshly-isolated human hepatocytes in culture configurations with different levels of expression and activity of these proteins. We observed that Fic was up to 35-fold lower in the configuration with high expression of drug-eliminating transporters and enzymes. We conclude that Fic provides a measurement of the net impact of all cellular drug disposition processes on intracellular bioavailable drug levels. Importantly, no prior knowledge of the involved drug distribution pathways is required, allowing for high-throughput determination of drug access to intracellular targets in highly defined cell systems (e.g., single-transporter transfectants) or in complex ones (including primary human cells).

Intracellular drug exposure is influenced by cell-and tissue-dependent expression of drug-transporting proteins and metabolizing enzymes. Here, we introduce the concept of intracellular bioavailability (F-ic) as the fraction of extracellular drug available to bind intracellular targets, and we assess how Fic is affected by cellular drug disposition processes. We first investigated the impact of two essential drug transporters separately, one influx transporter (OATP1B1; SLCO1B1) and one efflux transporter (P-gp; ABCB1), in cells overexpressing these proteins. We showed that OATP1B1 increased Fic of its substrates, while P-gp decreased Fic. We then investigated the impact of the concerted action of multiple transporters and metabolizing enzymes in freshly-isolated human hepatocytes in culture configurations with different levels of expression and activity of these proteins. We observed that Fic was up to 35-fold lower in the configuration with high expression of drug-eliminating transporters and enzymes. We conclude that Fic provides a measurement of the net impact of all cellular drug disposition processes on intracellular bioavailable drug levels. Importantly, no prior knowledge of the involved drug distribution pathways is required, allowing for high-throughput determination of drug access to intracellular targets in highly defined cell systems (e.g., single-transporter transfectants) or in complex ones (including primary human cells).

Caco-2 cells are widely used in studies of intestinal cell physiology and drug transport. Here, the global proteome of filter-grown Caco-2 cells was quantified using the total protein approach and compared with the human colon and jejunum proteomes. In total, 8096 proteins were identified. In-depth analysis of proteins defining enterocyte differentiation—including brush-border hydrolases, integrins, and adherens and tight junctions—gave near-complete coverage of the expected proteins. Three hundred twenty-seven absorption, distribution, metabolism and excretion proteins were identified, including 112 solute carriers and 20 ATP-binding cassette transporters. OATP2B1 levels were 16-fold higher in Caco-2 cells than in jejunum. To investigate the impact of this difference on in vitro-in vivo extrapolations, we studied the uptake kinetics of the OATP2B1 substrate pitavastatin in Caco-2 monolayers, and found that the contribution of OATP2B1 was 60%-70% at clinically relevant intestinal concentrations. Pitavastatin kinetics was combined with transporter concentrations to model the contribution of active transport and membrane permeation in the jejunum. The lower OATP2B1 expression in jejunum led to a considerably lower transporter contribution (<5%), suggesting that transmembrane diffusion dominates pitavastatin absorption in vivo. In conclusion, we present the first in-depth quantification of the filter-grown Caco-2 proteome. We also demonstrate the crucial importance of considering transporter expression levels for correct interpretation of drug transport routes across the human intestine.

Macrocycles are of increasing interest as chemical probes and drugs for intractable targets like protein-protein interactions, but the determinants of their cell permeability and oral absorption are poorly understood. To enable rational design of cell-permeable macrocycles, we generated an extensive data set under consistent experimental conditions for more than 200 nonpeptidic, de novo-designed macrocycles from the Broad Institute's diversity-oriented screening collection. This revealed how specific functional groups, substituents and molecular properties impact cell permeability. Analysis of energy-minimized structures for stereo- and regioisomeric sets provided fundamental insight into how dynamic, intramolecular interactions in the 3D conformations of macrocycles may be linked to physicochemical properties and permeability. Combined use of quantitative structure-permeability modeling and the procedure for conformational analysis now, for the first time, provides chemists with a rational approach to design cell-permeable non-peptidic macrocycles with potential for oral absorption., Delat förstaförfattarskap: Bjorn Over, Pär Matsson(Shared first authorship: Bjorn Over, Pär Matsson)

Madin-Darby canine kidney II cells transfected with one or several transport proteins are commonly used models to study drug transport. In these cells, however, endogenous transporters such as canine Mdr1/P-glycoprotein (Abcb1) complicate the interpretation of transport studies. The aim of this investigation was to establish a Madin-Darby canine kidney II cell line using CRISPR-Cas9 gene-editing technology to knock out endogenous canine Mdr1 (cMdr1) expression. CRISPR-Cas9-mediated Abcb1 homozygous disruption occurred at frequencies of around 20% and resulted in several genotypes. We selected 1 clonal cell line, cMdr1 KO Cl2, for further examination. Consistent with an on-target effect of CRISPR-Cas9 in specific regions of the endogenous canine Abcb1 gene, we obtained a cell clone with Abcb1 gene alterations and without any cMdr1 expression, as confirmed by genome sequencing and quantitative protein analysis. Functional studies of these cells, using digoxin and other prototypic MDR1 substrates, showed close to identical transport in the apical-to-basolateral and basolateral-to-apical directions, resulting in efflux ratios indistinguishable from unity.

The conventional model of drug permeability has recently been challenged. An alternative model proposes that transporter-mediated flux is the sole mechanism of cellular drug permeation, instead of existing in parallel with passive transmembrane diffusion. We examined a central assumption of this alternative hypothesis; namely, that transporters can give rise to experimental observations that would typically be explained with passive transmembrane diffusion. Using systems-biology simulations based on available transporter kinetics and proteomic expression data, we found that such observations are possible in the absence of transmembrane diffusion, but only under very specific conditions that rarely or never occur for known human drug transporters.

The conventional model of drug permeability has recently been challenged. An alternative model proposes that transporter-mediated flux is the sole mechanism of cellular drug permeation, instead of existing in parallel with passive transmembrane diffusion. We examined a central assumption of this alternative hypothesis; namely, that transporters can give rise to experimental observations that would typically be explained with passive transmembrane diffusion. Using systems-biology simulations based on available transporter kinetics and proteomic expression data, we found that such observations are possible in the absence of transmembrane diffusion, but only under very specific conditions that rarely or never occur for known human drug transporters.

The conventional model of drug permeability has recently been challenged. An alternative model proposes that transporter-mediated flux is the sole mechanism of cellular drug permeation, instead of existing in parallel with passive transmembrane diffusion. We examined a central assumption of this alternative hypothesis; namely, that transporters can give rise to experimental observations that would typically be explained with passive transmembrane diffusion. Using systems-biology simulations based on available transporter kinetics and proteomic expression data, we found that such observations are possible in the absence of transmembrane diffusion, but only under very specific conditions that rarely or never occur for known human drug transporters.

The conventional model of drug permeability has recently been challenged. An alternative model proposes that transporter-mediated flux is the sole mechanism of cellular drug permeation, instead of existing in parallel with passive transmembrane diffusion. We examined a central assumption of this alternative hypothesis; namely, that transporters can give rise to experimental observations that would typically be explained with passive transmembrane diffusion. Using systems-biology simulations based on available transporter kinetics and proteomic expression data, we found that such observations are possible in the absence of transmembrane diffusion, but only under very specific conditions that rarely or never occur for known human drug transporters.

The conventional model of drug permeability has recently been challenged. An alternative model proposes that transporter-mediated flux is the sole mechanism of cellular drug permeation, instead of existing in parallel with passive transmembrane diffusion. We examined a central assumption of this alternative hypothesis; namely, that transporters can give rise to experimental observations that would typically be explained with passive transmembrane diffusion. Using systems-biology simulations based on available transporter kinetics and proteomic expression data, we found that such observations are possible in the absence of transmembrane diffusion, but only under very specific conditions that rarely or never occur for known human drug transporters.

Profiling of eight stereoisomeric T. cruzi growth inhibitors revealed vastly different in vitro properties such as solubility, lipophilicity, pK(a), and cell permeability for two sets of four stereoisomers. Using computational chemistry and NMR spectroscopy, we identified the formation of an intramolecular NH -> NR3 hydrogen bond in the set of stereoisomers displaying lower solubility, higher lipophilicity, and higher cell permeability. The intramolecular hydrogen bond resulted in a significant pKa difference that accounts for the other structure property relationships. Application of this knowledge could be of particular value to maintain the delicate balance of size, solubility, and lipophilicity required for cell penetration and oral administration for chemical probes or therapeutics with properties at, or beyond, Lipinski's rule of 5.

Optimization of drug efficacy in the brain requires understanding of the local exposure to unbound drug at the site of action. This relies on measurements of the unbound drug fraction (f(u,brain)), which currently requires access to brain tissue. Here, we present a novel methodology using homogenates of cultured cells for rapid estimation of f(u,brain). In our setup, drug binding to human embryonic kidney cell (HEK293) homogenate was measured in a small-scale dialysis apparatus. To increase throughput, we combined drugs into cassettes for simultaneous measurement of multiple compounds. Our method estimated f(u,brain) with an average error of 1.9-fold. We propose that our simple method can be used as an inexpensive, easily available and high-throughput alternative to brain tissues excised from laboratory animals. Thereby, estimates of unbound drug exposure can now implemented at a much earlier stage of the drug discovery process, when molecular property changes are easier to make.