Use of a Double-Transfected System to Predict hOCT2/hMATE1-Mediated Renal Drug-Drug Interactions.

Accurate predictions of renal drug-drug interactions (DDIs) mediated by the human organic cation transporter 2 (hOCT2) and multidrug and toxin extrusion proteins (hMATEs) remain challenging. Current DDI evaluation using plasma maximal unbound inhibitor concentrations (Imax,u) and IC50 values determined in single transporter-transfected cells frequently leads to false or overprediction especially for hMATE1. Emerging evidence suggests intracellular unbound inhibitor concentration may be more relevant for hMATE1 inhibition in vivo. However, determination of intrarenal inhibitor concentrations is impractical. Here, we explored the use of hOCT2/hMATE1 double-transfected Madin-Darby canine kidney (MDCK) cells as a new in vitro tool for DDI risk assessment. Our results showed that potent in vitro hMATE1 inhibitors (hydroxychloroquine, brigatinib, and famotidine) failed to inhibit metformin B-to-A flux in the double-transfected system. On the other side, the classic hOCT2/hMATE1 inhibitors, pyrimethamine and cimetidine, dose-dependently inhibited metformin apparent B-to-A permeability (Papp). The different behaviors of these hMATE1 inhibitors in the double-transfected system can be explained by their different ability to gain intracellular access either via passive diffusion or transporter-mediated uptake. A new parameter (IC50,flux) was proposed reflecting the inhibitor's potency on overall hOCT2/hMATE1-mediated tubular secretion. The IC50,flux values significantly differ from the IC50 values determined in single transporter-transfected cells. Importantly, the IC50,flux accurately predicted in vivo DDIs (within 2-fold) when used in a static model. Our data demonstrated that the IC50,flux approach circumvents the need to measure intracellular inhibitor concentrations and more accurately predicted hOCT2/hMATE1-mediated renal DDIs. This system represents a new approach that could be used for improved DDI assessment during drug development. SIGNIFICANCE STATEMENT: This study demonstrated that flux studies in double-transfected MDCK cells and the IC50,flux represents a better approach to assess in vivo DDI potential for the renal organic cation secretion system. This study highlights the importance of inhibitor intracellular accessibility for accurate prediction of hMATE1-mediated renal DDIs. This approach has the potential to identify in vitro hMATE1 inhibitors that are unlikely to result in in vivo DDIs, thus reducing the burden of unnecessary and costly clinical DDI investigations.

Tamoxifen Upregulates Collagenase Gene Expression in Human Dermal Fibroblasts.

Background: Tamoxifen is a known inhibitor of fibroblast transforming growth factor beta biosynthesis and wound scar formation. Tamoxifen is also known to be an estrogen antagonist and protein kinase C (PKC) inhibitor. Cells treated with tamoxifen and other PKC/calmodulin inhibitors depolymerize their membrane focal adhesion complexes and cytoskeletal protein structures. These effects result in substrate detachment, cell shape rounding, and upregulation of collagenase synthesis and extracellular matrix degradation. The purpose of our study was to test the hypothesis that tamoxifen treatment of human foreskin fibroblasts results in alteration of cytoskeletal protein organization, cell detachment and rounding, and increased collagenase synthesis similar to known PKC/calmodulin inhibitors such as H-7. Methods: We characterized the effects of PKC/calmodulin inhibitors tamoxifen and H-7 on human dermal fibroblast morphology, cytoskeletal protein organization, and collagenase gene expression in monolayer culture and within collagen gels. Results: We found that fibroblasts responded to tamoxifen by initiation of actin filament depolymerization followed by alteration from spindle to spheroidal shapes. This change in cell shape led to increased collagenase synthesis in cells treated with either tamoxifen or H-7 compared with controls. There was also a 23% increase of hydroxyproline release from tamoxifen-treated fibroblast-populated collagen matrices. Conclusions: Tamoxifen may reduce scarring by inhibiting fibroblast PKC/calmodulin activity, which down-regulates pro-fibrotic transforming growth factor beta signaling and upregulates collagenase production. These effects mimic those of the known PKC/calmodulin inhibitor H-7. Overall, these findings suggest that tamoxifen and its analogues are promising agents for clinical investigation as small molecule regulators of fibrosis and scarring disorders.

Interaction and Transport of Benzalkonium Chlorides by the Organic Cation and Multidrug and Toxin Extrusion Transporters.

Humans are chronically exposed to benzalkonium chlorides (BACs) from environmental sources. The U.S. Food and Drug Administration (FDA) has recently called for additional BAC safety data, as these compounds are cytotoxic and have great potential for biochemical interactions. Biodistribution studies revealed that BACs extensively distribute to many tissues and accumulate at high levels, especially in the kidneys, but the underlying mechanisms are unclear. In this study, we characterized the interactions of BACs of varying alkyl chain length (C8 to C14) with the human organic cation transporters (hOCT1-3) and multidrug and toxin extrusion proteins (hMATE1/2K) with the goal to identify transporters that could be involved in BAC disposition. Using transporter-expressing cell lines, we showed that all BACs are inhibitors of hOCT1-3 and hMATE1/2K (IC50 ranging 0.83-25.8 µM). Further, the short-chain BACs (C8 and C10) were identified as substrates of these transporters. Interestingly, although BAC C8 displayed typical Michaelis-Menten kinetics, C10 demonstrated a more complex substrate-inhibition profile. Transwell studies with transfected Madin-Darby canine kidney cells revealed that intracellular accumulation of basally applied BAC C8 and C10 was substantially higher (8.2- and 3.7-fold, respectively) in hOCT2/hMATE1 double-transfected cells in comparison with vector-transfected cells, supporting a role of these transporters in mediating renal accumulation of these compounds in vivo. Together, our results suggest that BACs interact with hOCT1-3 and hMATE1/2K as both inhibitors and substrates and that these transporters may play important roles in tissue-specific accumulation and potential toxicity of short-chain BACs. Our findings have important implications for understanding human exposure and susceptibility to BACs due to environmental exposure. SIGNIFICANCE STATEMENT: Humans are systemically exposed to benzalkonium chlorides (BACs). These compounds broadly distribute through tissues, and their safety has been questioned by the FDA. Our results demonstrate that hOCT2 and hMATE1 contribute to the renal accumulation of BAC C8 and C10 and that hOCT1 and hOCT3 may be involved in the tissue distribution of these compounds. These findings can improve our understanding of BAC disposition and toxicology in humans, as their accumulation could lead to biochemical interactions and deleterious effects.

Inborn errors of immunity in adulthood.

Inborn errors of immunity (IEIs) are a group of conditions whereby parts of the immune system are missing or dysfunctional. Once thought to primarily be a pediatric disorder, it is now estimated that more than 50% of worldwide incident IEI cases are accounted for by adults. Delayed diagnosis, late symptom onset, and IEI phenocopies can all lead to adult-onset recognition of IEIs. Lack of awareness regarding the diversity of IEI manifestations in adults contributes to diagnostic and treatment delays. Prompt referral to immunology is critical so that patients can receive a precise molecular diagnosis and targeted therapy when available. This article serves as a primer on IEIs in adulthood, highlighting the pathophysiology, epidemiology and clinical features. We present clinical vignettes of three key IEIs to assist clinicians in building illness scripts on their presentations. We provide a framework for the laboratory evaluation of IEIs and their initial treatment, with the aim of improving recognition and management of these conditions.

Interaction of ALK Inhibitors with Polyspecific Organic Cation Transporters and the Impact of Substrate-Dependent Inhibition on the Prediction of Drug-Drug Interactions.

Small molecules targeting aberrant anaplastic lymphoma kinase (ALK) are active against ALK-positive non-small-cell lung cancers and neuroblastoma. Several targeted tyrosine kinase inhibitors (TKIs) have been shown to interact with polyspecific organic cation transporters (pOCTs), raising concerns about potential drug-drug interactions (DDIs). The purpose of this study was to assess the interaction of ALK inhibitors with pOCTs and the impact of substrate-dependent inhibition on the prediction of DDIs. Inhibition assays were conducted in transporter-overexpressing cells using meta-iodobenzylguanidine (mIBG), metformin, or 1-methyl-4-phenylpyridinium (MPP+) as the substrate. The half-maximal inhibitory concentrations (IC50) of brigatinib and crizotinib for the substrates tested were used to predict their potential for in vivo transporter mediated DDIs. Here, we show that the inhibition potencies of brigatinib and crizotinib on pOCTs are isoform- and substrate-dependent. Human OCT3 (hOCT3) and multidrug and toxin extrusion protein 1 (hMATE1) were highly sensitive to inhibition by brigatinib and crizotinib for all three tested substrates. Apart from hMATE1, substrate-dependent inhibition was observed for all other transporters with varying degrees of dependency; hOCT1 inhibition showed the greatest substrate dependency, with differences in IC50 values of up to 22-fold across the tested substrates, followed by hOCT2 and hMATE2-K, with differences in IC50 values of up to 16- and 12-fold, respectively. Conversely, hOCT3 inhibition only showed a moderate substrate dependency (IC50 variance < 4.8). Among the substrates used, metformin was consistently shown to be the most sensitive substrate, followed by mIBG and MPP+. Pre-incubation of ALK inhibitors had little impact on their potencies toward hOCT2 and hMATE1. Our results underscore the complexity of the interactions between substrates and the inhibitors of pOCTs and have important implications for the clinical use of ALK inhibitors and their DDI predictions.

Functional Evaluation of P-gp and Bcrp at the Murine Blood-Cerebrospinal Fluid Barrier.

PURPOSE: The brain is protected from circulating metabolites and xenobiotics by the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier. Previous studies report that P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) are expressed apically or subapically at the blood-CSF barrier (BCSFB), implying a paradoxical function to mediate blood-to-CSF transport of xenobiotics. As evidence of P-gp and Bcrp activity at the BCSFB is limited, the goal of this study is to investigate functional activity of P-gp and Bcrp at the murine BCSFB using a live tissue imaging approach. METHODS: The choroid plexuses (CP) forming the BCSFB were freshly isolated from mouse brain ventricles and incubated with fluorescent probes calcein-AM and BODIPY FL-Prazosin. Using quantitative fluorescence microscopy, the functional contributions of Bcrp and P-gp were examined using inhibitors and mice with targeted deletion of the Abcb1a/b or Abcg2 gene. RESULTS: Apical transport of calcein-AM in choroid plexus epithelial (CPE) cells is sensitive to inhibition by elacridar and Ko143 but is unaffected by P-gp deletion. In wild-type mice, elacridar increased CPE accumulation of BODIPY FL-Prazosin by 220% whereas deletion of Bcrp increased BODIPY FL-Prazosin accumulation by 43%. There was no change in Mdr1a/1b mRNA expression in CP tissues from the Bcrp-/- mice. CONCLUSIONS: This study demonstrated functional activity of Bcrp at the BCSFB apical membrane and provided evidence supporting an additional contribution by P-gp. These findings contribute to the understanding of transport mechanisms that regulate CSF drug concentrations, which may benefit future predictions of CNS drug disposition, efficacy, and toxicity.

Incorporating Uremic Solute-mediated Inhibition of OAT1/3 Improves PBPK Prediction of Tenofovir Renal and Systemic Disposition in Patients with Severe Kidney Disease.

BACKGROUND: Dose modification of renally secreted drugs in patients with chronic kidney disease (CKD) has relied on serum creatinine concentration as a biomarker to estimate glomerular filtration (GFR) under the assumption that filtration and secretion decline in parallel. A discrepancy between actual renal clearance and predicted renal clearance based on GFR alone is observed in severe CKD patients with tenofovir, a compound secreted by renal OAT1/3. Uremic solutes that inhibit OAT1/3 may play a role in this divergence. METHODS: To examine the impact of transporter inhibition by uremic solutes on tenofovir renal clearance, we determined the inhibitory potential of uremic solutes hippuric acid, indoxyl sulfate, and p-cresol sulfate. The inhibition parameters (IC50) were incorporated into a previously validated mechanistic kidney model; simulated renal clearance and plasma PK profile were compared to data from clinical studies. RESULTS: Without the incorporation of uremic solute inhibition, the PBPK model failed to capture the observed data with an absolute average fold error (AAFE) > 2. However, when the inhibition of renal uptake transporters and uptake transporters in the slow distribution tissues were included, the AAFE value was within the pre-defined twofold model acceptance criterion, demonstrating successful model extrapolation to CKD patients. CONCLUSION: A PBPK model that incorporates inhibition by uremic solutes has potential to better predict renal clearance and systemic disposition of secreted drugs in patients with CKD. Ongoing research is warranted to determine if the model can be expanded to include other OAT1/3 substrate drugs and to evaluate how these findings can be translated to clinical guidance for drug selection and dose optimization in patients with CKD.

The Plasma Membrane Monoamine Transporter is Highly Expressed in Neuroblastoma and Functions as an mIBG Transporter.

Neuroblastoma (NB) is a pediatric cancer with low survival rates in high-risk patients. 131I-mIBG has emerged as a promising therapy for high-risk NB and kills tumor cells by radiation. Consequently, 131I-mIBG tumor uptake and retention are major determinants for its therapeutic efficacy. mIBG enters NB cells through the norepinephrine transporter (NET), and accumulates in mitochondria through unknown mechanisms. Here we evaluated the expression of monoamine and organic cation transporters in high-risk NB tumors and explored their relationship with MYCN amplification and patient survival. We found that NB mainly expresses NET, the plasma membrane monoamine transporter (PMAT), and the vesicular membrane monoamine transporter 1/2 (VMAT1/2), and that the expression of these transporters is significantly reduced in MYCN-amplified tumor samples. PMAT expression is the highest and correlates with overall survival in high-risk NB patients without MYCN amplification. Immunostaining showed that PMAT resides intracellularly in NB cells and co-localizes with mitochondria. Using cells expressing PMAT, mIBG was identified as a PMAT substrate. In mitochondria isolated from NB cell lines, mIBG uptake was reduced by ∼50% by a PMAT inhibitor. Together, our data suggest that PMAT is a previously unrecognized transporter highly expressed in NB and could impact intracellular transport and therapeutic response to 131I-mIBG. SIGNIFICANCE STATEMENT: This study identified that plasma membrane monoamine transporter (PMAT) is a novel transporter highly expressed in neuroblastoma and its expression level is associated with overall survival rate in high-risk patients without MYCN amplification. PMAT is expressed intracellularly in neuroblastoma cells, transports meta-iodobenzylguanidine (mIBG) and thus could impact tumor retention and response to 131I-mIBG therapy. These findings have important clinical implications as PMAT could represent a novel molecular marker to help inform disease prognosis and predict response to 131I-mIBG therapy.

Molecular Mechanisms of Organic Anion Transporting Polypeptide-Mediated Organic Anion Clearance at the Blood-Cerebrospinal Fluid Barrier.

The blood-cerebrospinal fluid barrier (BCSFB), formed by the choroid plexus epithelial (CPE) cells, plays an active role in removing drugs and metabolic wastes from the brain. Recent functional studies in isolated mouse choroid plexus (CP) tissues suggested the presence of organic anion transporting polypeptides (OATPs, encoded by SLCOs) at the apical membrane of BCSFB, which may clear large organic anions from the cerebrospinal fluid (CSF). However, the specific OATP isoform involved is unclear. Using quantitative fluorescence imaging, we showed that the fluorescent anions sulforhodamine 101 (SR101), fluorescein methotrexate (FL-MTX), and 8-fluorescein-cAMP (fluo-cAMP) are actively transported from the CSF to the subepithelial space in CP tissues isolated from wild-type mice. In contrast, transepithelial transport of these compounds across the CPE cells was abolished in Oatp1a/1b-/- mice due to impaired apical uptake. Using transporter-expressing cell lines, SR101, FL-MTX, and fluo-cAMP were additionally shown to be transported by mouse OATP1A5 and its human counterpart OATP1A2. Kinetic analysis showed that estrone-3-sulfate and SR101 are transported by OATP1A2 and OATP1A5 with similar Michaelis-Menten constants (Km). Immunofluorescence staining further revealed the presence of OATP1A2 protein in human CP tissues. Together, our results suggest that large organic anions in the CSF are actively transported into CPE cells by apical OATP1A2 (OATP1A5 in mice), then subsequently effluxed into the blood by basolateral multidrug resistance-associated proteins (MRPs). As OATP1A2 transports a wide array of endogenous compounds and xenobiotics, the presence of this transporter at the BCSFB may imply a novel clearance route for drugs and neurohormones from the CSF. SIGNIFICANCE STATEMENT: Drug transporters at the blood-cerebrospinal fluid (CSF) barrier play an important but understudied role in brain drug disposition. This study revealed a functional contribution of rodent organic anion transporting polypeptide (OATP) 1A5 towards the CSF clearance of organic anions and suggested a similar role for OATP1A2 in humans. Delineating the molecular mechanisms governing CSF organic anion clearance may help to improve the prediction of central nervous system (CNS) pharmacokinetics and identify drug candidates with favorable CNS pharmacokinetic properties.

Association between wait time and behavioral health appointment attendance across patient characteristics.

Adequate access to behavioral health (BH) services is a critical issue. Many patients who are referred to BH care miss their appointments. One barrier to BH care is that longer wait times decrease the likelihood of appointment attendance. The present study examines the relationship between the wait time for BH services and appointment attendance, overall and by multiple patient characteristics. Logistic regression was performed to analyze the association between wait time and patient attendance for BH referrals made from March 1, 2016, to February 28, 2019, at an urban academic medical center. In total, 1,587 referrals were included. Most patients were female (72%) and of non-Hispanic/Latinx Black race (55%). Each additional week of waiting between the referral and scheduled appointment was associated with a 5% decrease in odds of attendance. In adjusted race/ethnicity-stratified analyses, Hispanic/Latinx patients had a 9% lower odds of attendance per week of waiting. Non-Hispanic/Latinx White and Black patients had a 5% lower odds of attendance per week of waiting. Patients with private insurance had a 7% lower odds of attendance per week of waiting, and patients with Medicare had a 6% lower odds of attendance per week of waiting. Limiting scheduling may improve BH care utilization by decreasing the rate of "no shows." (PsycInfo Database Record (c) 2023 APA, all rights reserved).

Solute Carrier Nucleoside Transporters in Hematopoiesis and Hematological Drug Toxicities: A Perspective.

Anticancer nucleoside analogs produce adverse, and at times, dose-limiting hematological toxicities that can compromise treatment efficacy, yet the mechanisms of such toxicities are poorly understood. Recently, cellular nucleoside transport has been implicated in normal blood cell formation with studies from nucleoside transporter-deficient mice providing additional insights into the regulation of mammalian hematopoiesis. Furthermore, several idiopathic human genetic disorders have revealed nucleoside transport as an important component of mammalian hematopoiesis because mutations in individual nucleoside transporter genes are linked to various hematological abnormalities, including anemia. Here, we review recent developments in nucleoside transporters, including their transport characteristics, their role in the regulation of hematopoiesis, and their potential involvement in the occurrence of adverse hematological side effects due to nucleoside drug treatment. Furthermore, we discuss the putative mechanisms by which aberrant nucleoside transport may contribute to hematological abnormalities and identify the knowledge gaps where future research may positively impact treatment outcomes for patients undergoing various nucleoside analog therapies.

Challenges and Opportunities for Improved Drug-Drug Interaction Predictions for Renal OCT2 and MATE1/2-K Transporters.

Transporters contribute to renal elimination of drugs; therefore drug disposition can be impacted if transporters are inhibited by comedicant drugs. Regulatory agencies have provided guidelines to assess potential drug-drug interaction (DDI) risk for renal organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 and 2-K (MATE1/2-K) transporters. Despite this, there are challenges with translating in vitro data using currently available tools to obtain a quantitative assessment of DDI risk in the clinic. Given the high number of drugs and new molecular entities showing in vitro inhibition toward OCT2 and/or MATE1/2-K and the lack of translation to clinically significant effects, it is reasonable to question whether the current in vitro assay design and modeling practice has led to unnecessary clinical evaluation. The aim of this review is to assess and discuss available in vitro and clinical data along with prediction models intended to provide clinical context of risk, including static models proposed by regulatory agencies and physiologically-based pharmacokinetic models, in order to identify best practices and areas of future opportunity. This analysis highlights that different in vitro assay designs, including substrate and cell systems used, strongly influence the derived concentration of drug producing 50% inhibition values and contribute to high variability observed across laboratories. Furthermore, the lack of sensitive index substrates coupled with specific inhibitors for individual transporters necessitates the use of complex models to evaluate clinical DDI risk.

Evaluation of Blood-CSF Barrier Transport by Quantitative Real Time Fluorescence Microscopy.

PURPOSE: Transporters at the blood-cerebrospinal fluid (CSF) barrier (BCSFB) play active roles in removing drugs and toxins from the CSF. The goal of this study is to develop a fluorescence microscopy approach to quantitatively study the transepithelial transport processes at the murine BCSFB in real time. METHODS: Choroid plexus (CP) tissues were isolated from mouse lateral ventricles and incubated with anionic (fluorescein-methotrexate, 8-fluorescein-cAMP) or cationic (IDT307) fluorescent probes. The CSF-to-blood transport was imaged and quantified using compartmental segmentation and digital image analysis. Real time images were captured and analyzed to obtain kinetic information and identify the rate-limiting step. The effect of transporter inhibitors was also evaluated. RESULTS: The transport processes of fluorescent probes can be captured and analyzed digitally. The intra- and inter- animal variability were 20.4% and 25.7%, respectively. Real time analysis showed distinct transport kinetics and rate-limiting step for anionic and cationic probes. A CP efflux index was proposed to distinguish between transepithelial flux and intracellular accumulation. Rifampin and MK571 decreased the overall transepithelial transport of anionic probes by more than 90%, indicating a possible involvement of organic anion transporting polypeptides (Oatps) and multidrug resistance-associated proteins (Mrps). CONCLUSIONS: A CP isolation method was described, and a quantitative fluorescence imaging approach was developed to evaluate CSF-to-blood transport in mouse CP. The method is consistent, reproducible, and capable of tracking real time transepithelial transport with temporal and spatial resolution. The approach can be used to evaluate transport mechanisms, assess tissue drug accumulation, and assay potential drug-drug interactions at the BCSFB.

3D-printed porous condylar prosthesis for temporomandibular joint replacement: Design and biomechanical analysis.

BACKGROUND: Customized prosthetic joint replacements have crucial applications in severe temporomandibular joint problems, and the combined use of porous titanium scaffold is a potential method to rehabilitate the patients. OBJECTIVE: The objective of the study was to develop a design method to obtain a titanium alloy porous condylar prosthesis with good function and esthetic outcomes for mandibular reconstruction. METHODS: A 3D virtual mandibular model was created from CBCT data. A condylar defect model was subsequently created by virtual condylectomy on the initial mandibular model. The segmented condylar defect model was reconstructed by either solid or porous condyle with a fixation plate. The porous condyle was created by a density-driven modeling scheme with an inhomogeneous tetrahedral lattice structure. The porous condyle, supporting fixation plate, and screw locations were topologically optimized. Biomechanical behaviors of porous and solid condylar prostheses made of Ti-6Al-4V alloy were compared. Finite element analysis (FEA) was used to evaluate maximum stress distribution on both prostheses and the remaining mandibular ramus. RESULTS: The FEA results showed levels of maximum stresses were 6.6%, 36.4% and 47.8% less for the porous model compared to the solid model for LCI, LRM, and LBM loading conditions. Compared to the solid prosthesis, the porous prosthesis had a weight reduction of 57.7% and the volume of porosity of the porous condyle was 65% after the topological optimization process. CONCLUSIONS: A custom-made porous condylar prosthesis with fixation plate was designed in this study. The 3D printed Ti-6Al-4V porous condylar prosthesis had reduced weight and effective modulus of elasticity close to that of cortical bone. The.

Inactivated and live-attenuated seasonal influenza vaccines boost broadly neutralizing antibodies in children.

The induction of broadly neutralizing antibodies (bNAbs) that target the hemagglutinin stalk domain is a promising strategy for the development of "universal" influenza virus vaccines. bNAbs can be boosted in adults by sequential exposure to heterosubtypic viruses through natural infection or vaccination. However, little is known about if or how bNAbs are induced by vaccination in more immunologically naive children. Here, we describe the impact of repeated seasonal influenza vaccination and vaccine type on induction of bNAbs against group 1 influenza viruses in a pediatric cohort enrolled in randomized controlled trials of seasonal influenza vaccination. Repeated seasonal vaccination results in significant boosting of a durable bNAb response. Boosting of serological bNAb titers is comparable within inactivated and live attenuated (LAIV) vaccinees and declines with age. These data provide insights into vaccine-elicited bNAb induction in children, which have important implications for the design of universal influenza vaccine modalities in this critical population.

Clinical Applications and the Roles of Transporters in Disposition, Tumor Targeting, and Tissue Toxicity of meta-Iodobenzylguanidine (mIBG).

Transporters on the plasma membrane of tumor cells are promising molecular "Trojan horses" to deliver drugs and imaging agents into cancer cells. Radioiodine-labeled meta-iodobenzylguanidine (mIBG) is used as a diagnostic agent (123I-mIBG) and a targeted radiotherapy (131I-mIBG) for neuroendocrine cancers. mIBG enters cancer cells through the norepinephrine transporter (NET) where the radioactive decay of 131I causes DNA damage, cell death, and tumor necrosis. mIBG is predominantly eliminated unchanged by the kidney. Despite its selective uptake by neuroendocrine tumors, mIBG accumulates in several normal tissues and leads to tissue-specific radiation toxicities. Emerging evidences suggest that the polyspecific organic cation transporters play important roles in systemic disposition and tissue-specific uptake of mIBG. In particular, human organic cation transporter 2 (hOCT2) and toxin extrusion proteins 1 and 2-K (hMATE1/2-K) likely mediate renal secretion of mIBG whereas hOCT1 and hOCT3 may contribute to mIBG uptake into normal tissues such as the liver, salivary glands, and heart. This mini-review focuses on the clinical applications of mIBG in neuroendocrine cancers and the differential roles of NET, OCT and MATE transporters in mIBG disposition, response and toxicity. Understanding the molecular mechanisms governing mIBG transport in cancer and normal cells is a critical step for developing strategies to optimize the efficacy of 131I-mIBG while minimizing toxicity in normal tissues. Significance Statement Radiolabeled mIBG has been used as a diagnostic tool and as radiotherapy for neuroendocrine cancers and other diseases. NET, OCT and MATE transporters play differential roles in mIBG tumor targeting, systemic elimination, and accumulation in normal tissues. The clinical use of mIBG as a radiopharmaceutical in cancer diagnosis and treatment can be further improved by taking a holistic approach considering mIBG transporters in both cancer and normal tissues.

Live Tissue Imaging Reveals Distinct Transcellular Pathways for Organic Cations and Anions at the Blood-Cerebrospinal Fluid Barrier.

Formed by the choroid plexus epithelial (CPE) cells, the blood-cerebrospinal fluid barrier (BCSFB) plays an active role in removing drugs, toxins, and metabolic wastes from the brain. Several organic cation and anion transporters are expressed in the CPE cells, but how they functionally mediate transepithelial transport of organic cations and anions remain unclear. In this study, we visualized the transcellular transport of fluorescent organic cation and organic anion probes using live tissue imaging in freshly isolated mouse choroid plexuses (CPs). The cationic probe, 4-[4-(dimethylamino)phenyl]-1-methylpyridinium iodide (IDT307) was transported into CPE cells at the apical membrane and highly accumulated in mitochondria. Consistent with the lack of expression of organic cation efflux transporters, there was little efflux of IDT307 into the blood capillary space. Furthermore, IDT307 uptake and intracellular accumulation was attenuated by approximately 70% in CP tissues from mice with targeted deletion of the plasma membrane monoamine transporter (Pmat). In contrast, the anionic probe fluorescein-methotrexate (FL-MTX) was rapidly transported across the CPE cells into the capillary space with little intracellular accumulation. Rifampicin, an inhibitor of organic anion transporting polypeptides (OATPs), completely blocked FL-MTX uptake into the CPE cells whereas MK-571, a pan-inhibitor of multidrug resistance associated proteins (MRPs), abolished basolateral efflux of FL-MTX. In summary, our results suggest distinct transcellular transport pathways for organic cations and anions at the BCSFB and reveal a pivotal role of PMAT, OATP and MRP transporters in organic cation and anion transport at the blood-cerebrospinal fluid interface. SIGNIFICANCE STATEMENT: Live tissue imaging revealed that while organic cations are transported from the cerebrospinal fluid (CSF) into the choroid plexus epithelial cells by plasma membrane monoamine transporter without efflux into the blood, amphipathic anions in the CSF are efficiently transported across the BCSFB through the collaborated function of apical organic anion transporting polypeptides and basolateral multidrug resistance associated proteins. These findings contribute to a mechanistic understanding of the molecular and cellular pathways for choroid plexus clearance of solutes from the brain.

Multiple Transport Mechanisms Involved in the Intestinal Absorption of Metformin: Impact on the Nonlinear Absorption Kinetics.

The aim of this study was to investigate the contributions of multiple transport mechanisms to the intestinal absorption of metformin, focusing on OCT3, PMAT, THTR2, SERT and OCTN2. We also assessed the impact of these transporters on the nonlinear absorption of metformin. Uptake studies with MDCKII cells expressing OCT3, PMAT, THTR2 or SERT confirmed that metformin is a substrate of these transporters. Decynium22 strongly inhibited metformin uptake mediated by all the transporters. 7-Cyclopentyl inhibited OCT3- and THTR2-mediated uptake of metformin. AG835, thiamine and paroxetine specifically inhibited PMAT-, THTR2- and SERT-mediated uptake of metformin, respectively. Using these inhibitors, the relative contributions of OCT3, PMAT, THTR2, SERT, OCTN2 and others to the intestinal permeation of metformin across Caco-2 cells were estimated to be 9.77%, 9.68%, 22.2%, 1.52%, 0% and 0.66%, respectively. Concentration-dependent analysis of metformin uptake by Caco-2 cells revealed nonlinear kinetics with the similar Km(app) value to the value for THTR2. Further in situ absorption study demonstrated that rat intestinal permeability of metformin was significantly decreased in the presence of decynium22, 7-cyclopentyl and thiamine. The present study indicated that THTR2 is the major determinant of the nonlinear absorption of metformin, although multiple transport mechanisms contribute to its intestinal absorption.

Emergency Department Utilization Is Low After Outpatient Elective Rotator Cuff Repair.

INTRODUCTION: The purpose of this study was to establish emergency department (ED) utilization rate and reasons for presentation to the ED after outpatient rotator cuff repair (RCR) and determine preoperative predictors for these ED visits. METHODS: Patients who underwent outpatient RCR between 2014 and 2015 were retrospectively evaluated using the New York and Florida State Databases. The primary outcome was all-cause 7- and 30-day ED utilization rates. Reasons for presentation to the ED were recorded and stratified. Univariate and multivariate analyses were done to identify independent predictors of ED utilization. RESULTS: The 7- and 30-day ED visit rates were 3.2% and 5.0%, respectively. The most common cause for an ED visit after outpatient RCR at 7- and 30-days postoperatively were postoperative pain (29.0%) and GI complaints (16.3%), respectively. African American race (odds ratio [OD], 1.69; P < 0.001), Hispanic race (OD, 1.47; P = 0.005), and comorbid diagnoses of hypertension (OD, 1.51; P < 0.001), diabetes (OD, 1.58; P < 0.001), and/or schizophrenia (OD, 5.14; P < 0.001) were independent risk factors for an ED visit at up to 30 days postoperatively. Those with Medicare (OD, 2.01; P < 0.001) or Medicaid (OD, 2.61; P < 0.001) were more than twice as likely to present to the ED within 30 days than those with private health insurance. DISCUSSION: ED utilization after outpatient RCR is uncommon with postoperative pain as the most common chief concern for ED visits within the first 7 days and GI issues as the most common reason for ED encounters at up to 30 days postoperatively. Hypertension, diabetes, renal failure, liver disease, rheumatologic diseases, schizophrenia, depression, and Medicare and Medicaid insurance were independent predictors of ED encounters at up to 30 days postoperatively. Procedures done at freestanding surgery centers were protective against ED utilization. LEVEL OF EVIDENCE: Level III, Retrospective Cohort.

The distal femur trochlear groove appears to compensate for tibial deformity but not femoral deformity in an investigation of five-hundred and seventy-nine cadaveric skeletons.

BACKGROUND: The etiology of patellofemoral disorders is multifactorial. Preoperative patellofemoral anatomy evaluation is of great importance in patients undergoing surgery for patellofemoral disorders. Although anatomical risk factors of patellofemoral disorders have been thoroughly investigated in clinical and radiological studies, there are sparse data regarding the association between trochlear dysplasia with other anatomical parameters. This study sought to explore those associations using a large osteological collection. METHODS: Five-hundred and seventy-nine cadaveric skeletons were obtained from the Hamann-Todd osteological collection. Mechanical lateral distal femoral angle (mLDFA), medial proximal tibial angle (MPTA), femoral version, tibial torsion, lateral position of the tibial tubercle, and femoral length were modeled as predictors of medial and lateral flange height of the distal femur trochlea at various degrees of knee flexion. RESULTS: The average age and standard deviation for the 1158 lower extremities analyzed was 55.9 ± 10.2 years. There were 500 males (86%) and 79 females. Increasing MPTA or tibial valgus predicted increasing lateral femoral flange height at 0, 30, and 50 degrees of knee flexion [(standardized beta 0.111, p = 0.01), (standardized beta 0.129, p < 0.001), and (standardized beta 0.186, p < 0.001), respectively]. Increasing internal tibial torsion predicted increased medial flange height at 30 and 50° [(standardized beta - 0.114, p = 0.006), (standardized beta - 0.108, p = 0.006), respectively]. Increased femoral retroversion predicted increasing lateral flange height at 0 and 30 degrees [(standardized beta - 0.105, p = 0.005), (standardized beta - 0.098, p = 0.004), respectively]. CONCLUSIONS: To maintain the equilibrium necessary for effective patellar tracking, the depth of trochlear groove appears to effectively compensate for some of the anatomical parameters of the proximal tibia associated with patellofemoral disorders, and appears to be less influenced by femoral alignment. The clinical relevance of these findings warrants further investigation, and emphasizes the importance of carefully assessing the lower limb alignment in the management of patellofemoral disorders.