The influence of substrate structure on organic cation transport

Using epifluorescence microscopy I have characterized the transport of the fluorescent organic cation NBD-TMA⁺, [2-(4-nitro-2,1,3-benzoxadiaxol-7-yl)aminoethyl]trimethylammonium. NBD-TMA⁺ has structural characteristics common to other secreted organic cations and it is fluorescent (λₑₓ = 458 nm; λ(em) = 530 nm). Transport of NBD-TMA⁺ by rabbit renal proximal tubules was time dependent, saturable, and inhibited by TEA⁺, cimetidine, and procainamide. These results provide strong evidence that the fluorescent organic cation, NBD-TMA⁺, is transported by the classical organic cation transporter and can be used to monitor organic cation transport in renal proximal tubules or cells expressing organic cation transporters. Additionally, a stable line of HeLa cells expressing hOCT1 was used to investigate the structural and chemical characteristics influencing substrate binding to the transporter. I found that the systematic rotation of a planar hydrophobic mass about a vertical axis aligned with the positively charged nitrogen reduced the binding affinity of substrate for hOCT1 suggesting that hOCT1 possesses a lateral steric limitation not seen in the apical organic cation transporter. Furthermore, the addition of supraplanar hydrophobic mass to a planar chemical structure was found to increase the binding affinity for a substrate substantially more that expected for the increased hydrophobicity suggesting that the binding site for hOCT1 is not simply planar in nature, but may be better described as a pocket

Passive influx and ion trapping are more relevant to the cellular accumulation of highly permeable low molecular weight acidic drugs than is Organic Anion Transporter 2 (OAT2)

Recently published work suggests that highly permeable low molecular weight (LMW) acidic drugs are transported by Organic Anion Transporter 2 (OAT2). However, an asymmetric distribution of ionizable drugs in subcellular organelles where pH gradients are significant may occur in the presence of an inhibitor relative to its absence (e.g. lysosomal trapping). In the present study, OAT2-mediated transport of highly permeable LMW anions could not be demonstrated using OAT2 transfected cells, despite robust transport of the OAT2 substrate penciclovir. Moreover, a rifamycin SV (RifSV) dependent reduction in the accumulation of highly permeable LMW anions previously observed in hepatocytes could be qualitatively reproduced using HepG2 cells and also in MDCK cells which lack expression of OAT2. Neither HepG2 nor MDCK cells demonstrated meaningful penciclovir transport, nor was the cellular accumulation of the highly permeable LMW anions sensitive to competitive inhibition by the neutral OAT2 substrate penciclovir. Both cell lines however demonstrated sensitivity to the mitochondrial uncoupler p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) in a manner similar to RifSV. Furthermore, the transepithelial MDCK permeability of the highly permeable LMW anions was measured in the absence and presence of RifSV and FCCP at concentrations that reduced the cellular accumulation of anions. Neither inhibitor, nor the OAT2 inhibitor ketoprofen, reduced the transepithelial flux of the anions as would be anticipated for transported substrate inhibition. The findings presented here are aligned with cellular accumulation of highly permeable LMW anions being significantly determined by ion trapping sensitive to mitochondrial uncoupling rather than the result of OAT2-mediated transport. Significance Statement The manuscript illustrates that passive influx and ion trapping are more relevant to the cellular accumulation of highly permeable low molecular weight acidic drugs than is the previously proposed mechanism of OAT2-mediated transport. The outcome illustrated here highlights a rare, and perhaps previously not reported, observation of anionic drug trapping in a compartment sensitive to mitochondrial uncoupling (e.g. the mitochondrial matrix) that may be confused for transporter-mediated uptake

The impact of assay recovery on the apparent permeability, a function of lysosomal trapping

1. In vitro permeability assessment tools, like PAMPA, Caco-2, and MDCK, are frequently used to assess permeability and provide input in to various classification systems. Frequently, the measured recovery values in permeability assays are poor. Poor recovery may be a result of lysosomal trapping of compound. It was hypothesized that a relationship existed between diminished assay recovery of compound due to lysosomal trapping and underestimation of the Papp value.2. To examine this hypothesis, a series of experiments were conducted measuring cellular accumulation, percent recovery, and permeability in the absence or presence of an inhibitor of the V-type H+-ATPase, bafilomycin A1, to determine if a quantifiable relationship between lysosomal trapping, recovery, and permeability existed.3. Displacing compounds from lysosomes using bafilomycin A1 resulted in an improved compound recovery in the assay and a corresponding elevated permeability, where for each 10% loss in recovery, a Papp underestimate of ∼2.2 × 10-6 cm/s was observed. The findings highlight the potential for compound misclassification in various classification systems when assay recovery is not considered. Consideration of lysosomal trapping in the context of permeability assays may yield permeability values more reflective of the intrinsic permeability and the appropriate permeability classification

Transport of coproporphyrins I and III by organic anion transporting polypeptides (OATP) 1B1 and 1B3

To better assess drug interaction risk and safety issues, as well as reduce drug development costs and late stage failure within the pharmaceutical industry, the identification of novel biomarkers of transport is desirable. Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are polyspecific transporters that mediate the transport of endogenous compounds and xenobiotics into hepatocytes. Inactivating mutations of both OATP1B1 and OATP1B3 alleles lead to Rotor syndrome, a disease characterized by coproporphyrinuria, an elevated urinary excretion of coproporphyrins I and III. It was hypothesized that the transport of coproporphyrins I and III was mediated by OATP1B1 and OATP1B3. This hypothesis was supported by the current study using CHO cells transfected by OATP1B1 and HEK cells transfected by OATP1B3. The transport of each coproporphyrin by OATP1B1 and OATP1B3 was time-dependent and could be saturated. OATP1B1 mediated high affinity transport of coproporphyrins I and III (Km = 0.49 and 0.64 µM, respectively), as did OATP1B3 (Km = 0.86 and 0.96 µM, respectively). The OATP1B-mediated transport of each coproporphyrin was inhibited by rifamycin SV and atazanavir, with rifamycin SV demonstrating sub-micromolar inhibitor potency towards both transporters using either coproporphyrin as the in vitro probe substrate. The inhibitory potency of atazanavir was 5-10-fold less than that of rifamycin SV. The identification of coproporphyrins I and III as OATP1B substrates may have utility in the in vivo assessment of function and interaction with OATP1B1 and OATP1B3. As biomarkers, the coproporphyrins may enable the early in vivo assessment of OATP1B function and help identify drug interactions

Organic anion transporting polypeptide 2B1 (OATP2B1), an expanded substrate profile, does it align with OATP2B1's hypothesized function?

1. An expanded view of the substrate landscape of organic anion transporting polypeptide (OATP) 2B1 was pursued with the goal of understanding if the identification of novel in vitro substrates could shed additional light on the impact of OATP2B1 on intestinal absorption and brain penetration. 2. To examine this hypothesis, a series of experiments measured the cellular accumulation of a diverse array of compounds. Representative angiotensin II receptor blockers (ARBs) and other compounds of interest were subsequently investigated for inhibition, time dependence, and kinetics. 3. The study identified ARBs as a class of OATP2B1 substrates and found balsalazide, olsalzine, and gavestinel to be novel substrates of OATP2B1 too. Some compounds previously reported to be OATP2B1 substrates in the literature, aliskiren, erlotinib, montelukast, fexofenadine, and taurocholate could not be confirmed as substrates here. 4. Literature describing in vivo outcomes for OATP2B1 substrates, coproporphyrin III, ARBs, balsalazide, olsalzine, and gavestinel highlight the absence of a substantial impact of OATP2B1 on the oral absorption and/or brain penetration of OATP2B1 substrates. Suggestions of including OATP2B1 assessment as part of the drug approval process are likely premature and further mechanistic work with more robust OATP2B1 substrates, which may include some of those described here, is desirable

Using membrane partitioning simulations to predict permeability of forty-nine drug-like molecules

A simple descriptor calculated from molecular dynamics simulations of the membrane partitioning event is found to correlate well with experimental measurements of membrane permeation from the high throughput MDCK-LE assay using a dataset of 49 drug-like molecules. This descriptor approximates the enthalpy cost to membrane flip-flop, which for many molecules limits permeability. Performance is found to be superior in comparison to calculated properties such as clogP, clogD or PSA. Furthermore, the atomistic simulations provide a structural understanding of the partitioned drug membrane complex, facilitating medicinal chemistry optimization of membrane permeability

Simplifying the Extended Clearance Concept Classification System (EC3S) to Guide Clearance Prediction in Drug Discovery.

The Extended Clearance Concept Classification System was established as a development-stage tool to provide a framework for identifying fundamental mechanism(s) governing drug disposition in humans. In the present study, the applicability of the EC3S in drug discovery has been investigated. In its current format, the EC3S relies on low-throughput hepatocyte uptake data, which are not frequently generated in a discovery setting.A relationship between hepatocyte uptake clearance and MDCK permeability was first established along with intrinsic clearance from human liver microsomes. The performance of this approach was examined by categorizing 64 drugs into EC3S classes and comparing the predicted major elimination pathway(s) to that observed in humans. As an extension of the work, the ability of the simplified EC3S to predict human systemic clearance based on intrinsic clearance generated using in-vitro metabolic systems was evaluated.The assessment enabled the use of MDCK permeability and unscaled unbound intrinsic clearance to generate cut-off criteria to categorize compounds into four EC3S classes: Class 12ab, 2cd, 34ab, and 34cd, with major elimination mechanism(s) assigned to each class. The predictivity analysis suggested that systemic clearance could generally be predicted within threefold for EC3S class 12ab and 34ab compounds. For classes 2cd and 34cd, systemic clearance was poorly predicted using in-vitro systems explored in this study.Collectively, our simplified classification approach is expected to facilitate the identification of mechanism(s) involved in drug elimination, faster resolution of in-vitro to in-vivo disconnects, and better design of mechanistic pharmacokinetic studies in drug discovery

Synthesis and fluorescence of N,N,N-trimethyl-2-[methyl (7-nitrobenzo[c][l,2,5]oxadiazol-4-yl) amino]ethanaminium iodide, a pH-insensitive reporter of organic cation transport

A synthesis of the title compound, its characterization, and the pH dependence of its fluorescence properties are described. This compound serves as a pH-insensitive real-time reporter of organic cation transport in biological systems. Copyright © Taylor & Francis Group, LLC

Synthesis and Fluorescence of N,N,N-Trimethyl-2-[methyl(7-nitrobenzo[c] [1,2,5]oxadiazol-4-yl)amino]ethanaminium Iodide, a pH-Insensitive Reporter of Organic Cation Transport.

A synthesis of the title compound, its characterization, and the pH dependence of its fluorescence properties are described. This compound serves as a pH-insensitive real-time reporter of organic cation transport in biological systems. Copyright © Taylor & Francis Group, LLC