Mechanistic Model for the Prediction of Small-Molecule Vitreal Clearance Combining Diffusion-Limited and Permeability-Limited Clearance.

The discovery of new small-molecule drugs for intravitreal administration would benefit from simple models to predict vitreal clearance (CL). The current models available have limitations in their applicability to small-molecule drugs and translatability to humans. We developed a mechanistic model combining the diffusion rate of the molecule in the vitreous and permeability across posterior segment tissues and applied it to 30 small molecules with observed CL available mostly from literature. We used Caco-2 permeability as a surrogate for ocular tissue permeability. The model predicted rabbit vitreal CL well, with 80% of the predictions being within a 2-fold range of the observed CL. For an accurate prediction, it was crucial to consider the anterior diffusion CL from the vitreous to the aqueous and a limiting diffusion CL for the whole eye. We observed no major differences in model accuracy when using literature permeability values from retinal pigment epithelial cell models. Importantly, by adopting the specific dimensions of the human eye, the model was able to accurately predict vitreal CL of four compounds for which human vitreal CL data are available. In summary, this mechanistic model enables a simple, accurate, and translatable estimation of small-molecule vitreal CL.

Quantification of Transporter and Receptor Proteins in Dog Brain Capillaries and Choroid Plexus: Relevance for the Distribution in Brain and CSF of Selected BCRP and P-gp Substrates.

Transporters at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a pivotal role as gatekeepers for efflux or uptake of endogenous and exogenous molecules. The protein expression of a number of them has already been determined in the brains of rodents, nonhuman primates, and humans using quantitative targeted absolute proteomics (QTAP). The dog is an important animal model for drug discovery and development, especially for safety evaluations. The purpose of the present study was to clarify the relevance of the transporter protein expression for drug distribution in the dog brain and CSF. We used QTAP to examine the protein expression of 17 selected transporters and receptors at the dog BBB and BCSFB. For the first time, we directly linked the expression of two efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to regional brain and CSF distribution using specific substrates. Two cocktails, each containing one P-gp substrate (quinidine or apafant) and one BCRP substrate (dantrolene or daidzein) were infused intravenously prior to collection of the brain. Transporter expression varied only slightly between the capillaries of different brain regions and did not result in region-specific distribution of the investigated substrates. There were, however, distinct differences between brain capillaries and choroid plexus. Largest differences were observed for BCRP and P-gp: both were highly expressed in brain capillaries, but no BCRP and only low amounts of P-gp were detected in the choroid plexus. Kp,uu,brain and Kp,uu,CSF of both P-gp substrates were indicative of drug efflux. Also, Kp,uu,brain for the BCRP substrates was low. In contrast, Kp,uu,CSF for both BCRP substrates was close to unity, resulting in Kp,uu,CSF/Kp,uu,brain ratios of 7 and 8, respectively. We conclude that the drug transporter expression profiles differ between the BBB and BCSFB in dogs, that there are species differences in the expression profiles, and that CSF is not a suitable surrogate for unbound brain concentrations of BCRP substrates in dogs.

Addressing the ADME Challenges of Compound Loss in a PDMS-Based Gut-on-Chip Microphysiological System.

Microphysiological systems (MPSs) are promising in vitro technologies for physiologically relevant predictions of the human absorption, distribution, metabolism, and excretion (ADME) properties of drug candidates. However, polydimethylsiloxane (PDMS), a common material used in MPSs, can both adsorb and absorb small molecules, thereby compromising experimental results. This study aimed to evaluate the feasibility of using the PDMS-based Emulate gut-on-chip to determine the first-pass intestinal drug clearance. In cell-free PDMS organ-chips, we assessed the loss of 17 drugs, among which testosterone was selected as a model compound for further study based on its substantial ad- and absorptions to organ chips and its extensive first-pass intestinal metabolism with well-characterized metabolites. A gut-on-chip model consisting of epithelial Caco-2 cells and primary human umbilical vein endothelial cells (HUVECs) was established. The barrier integrity of the model was tested with reference compounds and inhibition of drug efflux. Concentration-time profiles of testosterone were measured in cell-free organ chips and in gut-on-chip models. A method to deduce the metabolic clearance was provided. Our results demonstrate that metabolic clearance can be determined with PDMS-based MPSs despite substantial compound loss to the chip. Overall, this study offers a practical protocol to experimentally assess ADME properties in PDMS-based MPSs.

Discovery of BI-9508, a Brain-Penetrant GPR88-Receptor-Agonist Tool Compound for In Vivo Mouse Studies.

Decreased activity and expression of the G-protein coupled receptor GPR88 is linked to many behavior-linked neurological disorders. Published preclinical GPR88 allosteric agonists all have in vivo pharmacokinetic properties that preclude their progression to the clinic, including high lipophilicity and poor brain penetration. Here, we describe our attempts to improve GPR88 agonists' drug-like properties and our analysis of the trade-offs required to successfully target GPR88's allosteric pocket. We discovered two new GPR88 agonists: One that reduced morphine-induced locomotor activity in a murine proof-of-concept study, and the atropoisomeric BI-9508, which is a brain penetrant and has improved pharmacokinetic properties and dosing that recommend it for future in vivo studies in rodents. BI-9508 still suffers from high lipophilicity, and research on this series was halted. Because of its utility as a tool compound, we now offer researchers access to BI-9508 and a negative control free of charge via Boehringer Ingelheim's open innovation portal opnMe.com.

A selective and orally bioavailable VHL-recruiting PROTAC achieves SMARCA2 degradation in vivo.

Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules.

A Bidirectional Permeability Assay for beyond Rule of 5 Compounds.

Bidirectional permeability measurement with cellular models grown on Transwell inserts is widely used in pharmaceutical research since it not only provides information about the passive permeability of a drug, but also about transport proteins involved in the active transport of drug substances across physiological barriers. With the increasing number of investigative drugs coming from chemical space beyond Lipinski's Rule of 5, it becomes more and more challenging to provide meaningful data with the standard permeability assay. This is exemplified here by the difficulties we encountered with the cyclic depsipeptides emodepside and its close analogs with molecular weight beyond 1000 daltons and cLogP beyond 5. The aim of this study is to identify potential reasons for these challenges and modify the permeability assays accordingly. With the modified assay, intrinsic permeability and in vitro efflux of depsipeptides could be measured reliably. The improved correlation to in vivo bioavailability and tissue distribution data indicated the usefulness of the modified permeability assay for the in vitro screening of compounds beyond the Rule of 5.

Monolayer platform using human biopsy-derived duodenal organoids for pharmaceutical research.

The human small intestine is the key organ for absorption, metabolism, and excretion of orally administered drugs. To preclinically predict these reactions in drug discovery research, a cell model that can precisely recapitulate the in vivo human intestinal monolayer is desired. In this study, we developed a monolayer platform using human biopsy-derived duodenal organoids for application to pharmacokinetic studies. The human duodenal organoid-derived monolayer was prepared by a simple method in 3-8 days. It consisted of polarized absorptive cells and had tight junctions. It showed much higher cytochrome P450 (CYP)3A4 and carboxylesterase (CES)2 activities than did the existing models (Caco-2 cells). It also showed efflux activity of P-glycoprotein (P-gp) and inducibility of CYP3A4. Finally, its gene expression profile was closer to the adult human duodenum, compared to the profile of Caco-2 cells. Based on these findings, this monolayer assay system using biopsy-derived human intestinal organoids is likely to be widely adopted.

In-Depth Characterization of EpiIntestinal Microtissue as a Model for Intestinal Drug Absorption and Metabolism in Human.

The Caco-2 model is a well-accepted in vitro model for the estimation of fraction absorbed in human intestine. Due to the lack of cytochrome P450 3A4 (CYP3A4) activities, Caco-2 model is not suitable for the investigation of intestinal first-pass metabolism. The purpose of this study is to evaluate a new human intestine model, EpiIntestinal microtissues, as a tool for the prediction of oral absorption and metabolism of drugs in human intestine. The activities of relevant drug transporters and drug metabolizing enzymes, including MDR1 P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), CYP3A4, CYP2J2, UDP-glucuronosyltransferases (UGT), carboxylesterases (CES), etc., were detected in functional assays with selective substrates and inhibitors. Compared to Caco-2, EpiIntestinal microtissues proved to be a more holistic model for the investigation of drug absorption and metabolism in human gastrointestinal tract.

Muscle to Brain Partitioning as Measure of Transporter-Mediated Efflux at the Rat Blood-Brain Barrier and Its Implementation into Compound Optimization in Drug Discovery.

Movement of xenobiotic substances across the blood-brain barrier (BBB) is tightly regulated by various transporter proteins, especially the efflux transporters P-glycoprotein (P-gp/MDR1) and breast cancer resistance protein (BCRP). Avoiding drug efflux at the BBB is a unique challenge for the development of new central nervous system (CNS) drugs. Drug efflux at the BBB is described by the partition coefficient of unbound drug between brain and plasma (Kp,uu,brain) which is typically obtained from in vivo and often additionally in vitro measurements. Here, we describe a new method for the rapid estimation of the in vivo drug efflux at the BBB of rats: the measurement of the partition coefficient of a drug between brain and skeletal muscle (Kp,brain/muscle). Assuming a closely similar distribution of drugs into the brain and muscle and that the efflux transporters are only expressed in the brain, Kp,brain/muscle, similar to Kp,uu,brain, reflects the efflux at the BBB. The new method requires a single in vivo experiment. For 64 compounds from different research programs, we show the comparability to other approaches used to obtain Kp,uu,brain. P-gp- and BCRP-overexpressing cell systems are valuable in vitro tools for prescreening. Drug efflux at the BBB can be most accurately predicted based on a simple algorithm incorporating data from both in vitro assays. In conclusion, the combined use of our new in vivo method and the in vitro tools allows an efficient screening method in drug discovery with respect to efflux at the BBB.

Identification and functional characterization of the natural variant MRP3-Arg1297His of human multidrug resistance protein 3 (MRP3/ABCC3).

The human multidrug resistance protein 3 (MRP3, symbol ABCC3) is an ATP-binding cassette transporter that mediates the efflux of organic anions, including lipophilic substances conjugated with glucuronate, sulphate or glutathione, across the basolateral membrane of polarized cells (e.g. hepatocytes) into blood. Genetic variants of MRP3 may affect the transport of these substances out of cells. The aims of this study were: (i) to identify MRP3 polymorphisms; (ii) to functionally characterize one relatively frequent MRP3 polymorphism; and (iii) to establish whether MRP3 transports bilirubin glucuronosides. Exonic nucleotide variants in the ABCC3 gene were identified by single-strand conformation polymorphism analysis. The 3890G>A mutation, resulting in MRP3-ArgHis, was introduced into the ABCC3 cDNA which was stably transfected into MDCKII cells. For the functional characterization of MRP3-ArgHis in comparison with MRP3, ATP-dependent transport was analysed in isolated membrane vesicles. Two non-synonymous MRP3 variants were identified with an allele frequency of 0.003 for 1643T>A (MRP3-LeuGln) and 0.08 for 3890G>A (MRP3-ArgHis). Because of the high frequency of the 3890G>A mutation, and because of the close proximity of Arg to the second nucleotide-binding domain, we pursued the functional characterization of the MRP3-ArgHis polymorphic variant. MRP3-ArgHis was correctly localized to the basolateral membrane of polarized MDCKII cells. We identified monoglucuronosyl bilirubin, bisglucuronosyl bilirubin and leukotriene C4 as substrates for both MRP3 and MRP3-ArgHis. Dehydroepiandrosterone-3-sulphate and 17beta-glucuronosyl oestradiol were transported with similar kinetics by MRP3 and MRP3-ArgHis. This experimental setup provides a useful tool to analyse the functional consequences of polymorphic variants of MRP3.

Influence of albumin binding on the substrate transport mediated by human hepatocyte transporters OATP2 and OATP8.

BACKGROUND: Despite their strong binding to albumin while circulating in blood, many organic anions, such as bilirubin and fatty acids, are removed efficiently by the liver. The uptake transporters of human hepatocytes, OATP2 (symbol, SLC21A6) and OATP8 (SLC21A8), play important roles in the hepatic uptake of endogenous substances and drugs. The two transporters show different affinities for the organic anion sulfobromophthalein (BSP), which binds with high affinity to albumin in blood. METHODS: In this study, we investigated whether a direct interaction of albumin with OATP2 or OATP8 occurs during the uptake of BSP. The uptake of BSP, at varying concentrations of human serum albumin (HSA), into transfected HEK293 cells expressing recombinant human OATP2 or OATP8 was measured. The influence of other organic anions on the uptake of albumin-bound BSP by OATP2 or OATP8 was also studied. RESULTS: OATP8-mediated transport was affected more strongly by HSA than OATP2-mediated transport. Albumin affected both transporters in the manner of a noncompetitive inhibitor. Uptake studies using OATP2-transfected MDCKII cells indicated that a direct interaction between albumin and OATP2 is not necessary for uptake, a finding that was further confirmed by the effects of bilirubin and palmitate on the binding of BSP to albumin and on the uptake of BSP by OATP2 or OATP8. CONCLUSIONS: Our results indicated that uptake of albumin-bound BSP occurs only from the pool of unbound ligand.

Characterization of the transport of the bicyclic peptide phalloidin by human hepatic transport proteins.

Phalloidin, the major phallotoxin of the mushroom Amanita phalloides, enters hepatocytes by a carrier-mediated mechanism. The molecular identity of the transport proteins mediating phalloidin uptake was so far unknown. Earlier studies in rat liver indicated that phalloidin may share a common mechanism of uptake with organic anions like bile salts. In the current study on human transporters, we analyzed the uptake of phalloidin into transfected HEK293 cells stably expressing the recombinant hepatocyte-specific organic anion uptake transporters OATP2 (also termed OATP1B1, OATP-C, LST1, symbol SLC21A6) or OATP8 (OATP1B3 or SLC21A8). Time-dependent uptake of phalloidin was observed with SLC21A6-expressing cells and was inhibited by typical substrates of SLC21A6 such as bromosulfophthalein or cholyltaurine. A K(m) value of 39+/-11 micro M was determined for SLC21A6-mediated phalloidin uptake. Additional inhibitors of phalloidin uptake mediated by SLC21A6 included the immunosuppressive drugs cyclosporin A, FK506, and rapamycin, whereas alpha-amanitin was only a weak inhibitor. Cyclosporin A was a most potent competitive inhibitor for SLC21A6-mediated phalloidin transport with a K(i) value of 51 nM.

The absence of mrp4 has no effect on the recruitment of neutrophils and eosinophils into the lung after LPS, cigarette smoke or allergen challenge.

The multidrug resistance protein 4 (Mrp4) is an ATP-binding cassette transporter that is capable of exporting the second messenger cAMP from cells, a process that might regulate cAMP-mediated anti-inflammatory processes. However, using LPS- or cigarette smoke (CS)-inflammation models, we found that neutrophil numbers in the bronchoalveolar lavage fluid (BALF) were similar in Mrp4(-/-) and Mrp4(+/+) mice treated with LPS or CS. Similarly, neutrophil numbers were not reduced in the BALF of LPS-challenged wt mice after treatment with 10 or 30 mg/kg of the Mrp1/4 inhibitor MK571. The absence of Mrp4 also had no impact on the influx of eosinophils or IL-4 and IL-5 levels in the BALF after OVA airway challenge in mice sensitized with OVA/alum. LPS-induced cytokine release in whole blood ex vivo was also not affected by the absence of Mrp4. These data clearly suggest that Mrp4 deficiency alone is not sufficient to reduce inflammatory processes in vivo. We hypothesized that in combination with PDE4 inhibitors, used at suboptimal concentrations, the anti-inflammatory effect would be more pronounced. However, LPS-induced neutrophil recruitment into the lung was no different between Mrp4(-/-) and Mrp4(+/+) mice treated with 3 mg/kg Roflumilast. Finally, the single and combined administration of 10 and 30 mg/kg MK571 and the specific breast cancer resistance protein (BCRP) inhibitor KO143 showed no reduction of LPS-induced TNFα release into the BALF compared to vehicle treated control animals. Similarly, LPS-induced TNFα release in murine whole blood of Mrp4(+/+) or Mrp4(-/-) mice was not reduced by KO143 (1, 10 µM). Thus, BCRP seems not to be able to compensate for the absence or inhibition of Mrp4 in the used models. Taken together, our data suggest that Mrp4 is not essential for the recruitment of neutrophils into the lung after LPS or CS exposure or of eosinophils after allergen exposure.

Vectorial transport of enalapril by Oatp1a1/Mrp2 and OATP1B1 and OATP1B3/MRP2 in rat and human livers.

Although Oatp1a1 (rat organic anion-transporting polypeptide 1a1) was the transporter found responsible for the hepatocellular entry of enalapril (EN) into the rat liver, the canalicular transporter involved for excretion of EN and the metabolite, enalaprilat (ENA), was unknown. The Eisai hyperbilirubinemic rat (EHBR) that lacks Mrp2 (multidrug resistance-associated protein 2) was used to appraise the role of Mrp2 in the excretion of [3H]EN and its metabolite [3H]ENA in single-pass rat liver preparations. Although the total and metabolic clearances and hepatic extraction ratios at steady-state were virtually unaltered for EN in EHBR compared with published values of Sprague-Dawley rats, the biliary clearances of EN and ENA were significantly reduced almost to zero (P<0.05). Involvement of human OATP1B1, OATP1B3, and MRP2 in EN transport was further assessed in single- or double-transfected mammalian cells. Human embryonic kidney 293 cells that expressed OATP1B1 or OATP1B3 showed that OATP1B3 transport of EN (20-500 microM) was of low affinity, whereas transport of EN by OATP1B1 was associated with the Km of 262+/-35 microM, a value similar to that for Oatp1a1 (214 microM). The transcellular transport of EN via human OATP1B1 and MRP2, investigated with the double-transfected Madin-Darby canine kidney (MDCK) II cells in the Transwell system, showed that the sinusoidal to canalicular flux of EN in the OATP1B1/MRP2/MDCK cells was significantly higher (P<0.05) than that of mock/MDCK and OATP1B1/MDCK cells. EN was transported by Oatp1a1 and Mrp2 in rats and OATP1B1/OATP1B3 and MRP2 in humans.

Structural requirements for the apical sorting of human multidrug resistance protein 2 (ABCC2).

The human multidrug resistance protein 2 (MRP2, symbol ABCC2) is a polytopic membrane glycoprotein of 1545 amino acids which exports anionic conjugates across the apical membrane of polarized cells. A chimeric protein composed of C-proximal MRP2 and N-proximal MRP1 localized to the apical membrane of polarized Madin-Darby canine kidney cells (MDCKII) indicating involvement of the carboxy-proximal part of human MRP2 in apical sorting. When compared to other MRP family members, MRP2 has a seven-amino-acid extension at its C-terminus with the last three amino acids (TKF) comprising a PDZ-interacting motif. In order to analyze whether this extension is required for apical sorting of MRP2, we generated MRP2 constructs mutated and stepwise truncated at their C-termini. These constructs were fused via their N-termini to green fluorescent protein (GFP) and were transiently transfected into polarized, liver-derived human HepG2 cells. Quantitative analysis showed that full-length GFP-MRP2 was localized to the apical membrane in 73% of transfected, polarized cells, whereas it remained on intracellular membranes in 27% of cells. Removal of the C-terminal TKF peptide and stepwise deletion of up to 11 amino acids did not change this predominant apical distribution. However, apical localization was largely impaired when GFP-MRP2 was C-terminally truncated by 15 or more amino acids. Thus, neither the PDZ-interacting TKF motif nor the full seven-amino-acid extension were necessary for apical sorting of MRP2. Instead, our data indicate that a deletion of at least 15 C-terminal amino acids impairs the localization of MRP2 to the apical membrane of polarized cells.

A naturally occurring mutation in the SLC21A6 gene causing impaired membrane localization of the hepatocyte uptake transporter.

The organic anion transporter SLC21A6 (also known as OATP2, OATP-C, or LST-1) is involved in the hepatocellular uptake of a variety of endogenous and xenobiotic substances and drugs. We analyzed 81 human liver samples by immunoblotting and found one with a strongly reduced amount of SLC21A6 protein suggesting mutations in the SLC21A6 gene. The SLC21A6 cDNA from this sample contained five base pair changes in one allele; three of the mutations resulted in amino acid substitutions designated SLC21A6-N130D, SLC21A6-P155T, and SLC21A6-L193R. The former two were polymorphisms (SLC21A6*1b and SLC21A6*4), whereas SLC21A6-L193R represents the first naturally occurring mutation identified in one allele of the SLC21A6 gene, which affects protein maturation and organic anion transport. We introduced each of the mutations into the SLC21A6 cDNA and established stably transfected MDCKII cells expressing the respective mutant SLC21A6 protein. Immunofluorescence microscopy and uptake measurements were used to study localization and transport properties of the mutated proteins. Both proteins carrying the polymorphisms were sorted to the lateral membrane like wild-type SLC21A6, but their transport properties for the substrates cholyltaurine and 17beta-glucuronosyl estradiol were altered. Importantly, most of the mutant protein SLC21A6-L193R was retained intracellularly, and this single amino acid exchange abolished transport function.

Detection of the human organic anion transporters SLC21A6 (OATP2) and SLC21A8 (OATP8) in liver and hepatocellular carcinoma.

Transport proteins mediating the selective uptake of organic anions into human hepatocytes include the organic anion transporters SLC21A6 (also termed OATP2, OATP-C, or LST-1) and SLC21A8 (OATP8). Both transporters are localized to the basolateral membrane of human hepatocytes. Because of the importance of these transporters for hepatobiliary elimination, including the removal of bilirubin and its conjugates from the blood circulation, we have generated monoclonal antibodies for studies on the expression and localization of these transport proteins. We describe two antibodies, designated monoclonal antibody MDQ (mMDQ) and monoclonal antibody ESL (mESL), directed against the amino terminus and the carboxyl terminus of human SLC21A6, respectively. Both antibodies have been characterized by immunoblot analysis, immunoprecipitation, and immunofluorescence microscopy. While mESL reacted specifically with SLC21A6, mMDQ detects both SLC21A6 and SLC21A8. Neither of the two antibodies reacted with other human, or with dog, rat, or mouse liver SLC21A family members. Antibody mMDQ may be used for the simultaneous detection of SLC21A6 and SLC21A8 in immunoblotting because of its immunoreactivity with both molecules and because of the different molecular masses of both glycosylated proteins in human hepatocytes. This is exemplified in hepatocellular carcinomas where SLC21A6 and SLC21A8 were differentially synthesized and showed an irregular staining pattern. Both transport proteins have not been detected in human hepatoma HepG2 cells. In routine paraffin sections, 10 of 12 hepatocellular carcinomas were focally positive with antibody mMDQ. In contrast, cholangiocarcinomas and liver metastases of colorectal and pancreatic adenocarcinoma were negative without exception. This suggests the usefulness of SLC21A6/SLC21A8 within a panel of tumor markers for hepatocellular carcinomas. Moreover, both antibodies should be useful in studies on the expression and localization of two important uptake transporters of human hepatocytes under physiologic and pathophysiologic conditions.