RESUMO
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.
Assuntos
Metformina , Proteínas de Transporte de Cátions Orgânicos , Animais , Cães , Humanos , Transportador 2 de Cátion Orgânico/genética , Transportador 2 de Cátion Orgânico/metabolismo , Proteínas de Transporte de Cátions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Metformina/metabolismo , Interações Medicamentosas , Rim/metabolismoRESUMO
Heart failure (HF) is a chronic disease affecting 1%-2% of the global population.123I-labeled meta-iodobenzylguanidine (mIBG) is US Food and Drug Administration-approved for cardiac imaging and prognosis risk assessment in patients with HF. As a norepinephrine analog, mIBG is believed to be transported into adrenergic nerve terminals by the neuronal norepinephrine transporter (NET) and hence image sympathetic innervation of the myocardium. We previously showed that mIBG is an excellent substrate of organic cation transporter 3 (OCT3), an extraneuronal transporter expressed in cardiomyocytes. Here, we evaluated the in vivo impact of Oct3 on mIBG disposition and tissue distribution using Oct3 knockout mice. Oct3 +/+ and Oct3 -/- mice were administered with mIBG intravenously, and mIBG plasma pharmacokinetics and tissue exposures were determined. In Oct3 +/+ mice, mIBG exhibited extensive accumulation in multiple tissues (heart, salivary gland, liver, and adrenal gland). No difference was observed in overall plasma exposure between Oct3 +/+ and Oct3 -/- mice. Strikingly, cardiac mIBG was depleted in Oct3 -/- mice, resulting in 83% reduction in overall cardiac exposure (AUC0-24 h: 12.7 vs. 2.1 µg × h/g). mIBG tissue exposure (AUC0-24 h) was also reduced by 66%, 36%, and 31% in skeletal muscle, salivary gland, and lung, respectively, in Oct3 -/- mice. Our data demonstrated that Oct3 is the primary transporter responsible for cardiac mIBG uptake in vivo and suggested that cardiac mIBG imaging mainly measures OCT3 activity in cardiomyocytes but not NET-mediated uptake in adrenergic nerve endings. Our findings challenge the current paradigm in interpreting cardiac mIBG imaging results and suggest OCT3 as a potential genetic risk marker for HF prognosis. SIGNIFICANCE STATEMENT: 123I-labeled meta-iodobenzylguanidine is used for cardiac imaging and risk assessment in heart failure patients. Contrary to the current belief that meta-iodobenzylguanidine (mIBG) tracks cardiac sympathetic innervation due to its uptake by the neuronal norepinephrine transporter, the authors demonstrated that cardiac mIBG uptake is mediated by the extraneuronal transporter Oct3. Their findings warrant a re-evaluation of the scientific rationale behind cardiac mIBG scan and further suggest organic cation transporter 3 as a risk factor for disease progression in heart failure patients.
Assuntos
3-Iodobenzilguanidina , Camundongos Knockout , Miocárdio , Fator 3 de Transcrição de Octâmero , Animais , Camundongos , 3-Iodobenzilguanidina/farmacocinética , 3-Iodobenzilguanidina/metabolismo , Miocárdio/metabolismo , Distribuição Tecidual , Masculino , Fator 3 de Transcrição de Octâmero/metabolismo , Compostos Radiofarmacêuticos/farmacocinética , Coração/diagnóstico por imagem , Coração/inervação , Camundongos Endogâmicos C57BL , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/diagnóstico por imagem , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismoRESUMO
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.
Assuntos
Compostos de Benzalcônio , Proteínas de Transporte de Cátions Orgânicos , Animais , Cães , Humanos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Distribuição Tecidual , Linhagem Celular , Células Madin Darby de Rim Canino , Transportador 2 de Cátion Orgânico/metabolismoRESUMO
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.
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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.