Novel Essential Oil-Based Thiosemicarbazone Compounds as Potential Fungicides in Controlling Postharvest Diseases of Citrus Fruit.

To develop novel fungicides for controlling postharvest fungal diseases in citrus fruits, 12 essential oil (EO)-based thiosemicarbazones compounds, termed hydrazine-carbothioamide, were prepared according to the condensation method. In vitro assays showed that compound 13j exhibited the strongest antifungal activity (minimum inhibitory concentration [MIC] = minimum fungicidal concentration [MFC] = 0.0125 mg/mL) against Penicillium digitatum. An in vivo study revealed that 5 × MFC of compound 13j can effectively mitigate the green mold incidence of citrus fruit inoculated with P. digitatum, as well as fruit rot during natural storage, at a level comparable to that of the chemical fungicide prochloraz. Throughout this process, fruit quality was maintained. The hemolysis assay showed that these thiosemicarbazone compounds have good biocompatibility and that their safety is comparable to that of prochloraz. The antifungal activity of compound 13j was attributed to membrane damage, as confirmed using scanning electron microscopy (SEM), Calcofluor white (CFW) staining, propidium iodide (PI) staining, Fourier transform-infrared (FT-IR) spectroscopy, optical density (OD)260, and relative conductivity assays. Collectively, our results indicate that compound 13j can be used as an antifungal agent to control the postharvest decay of citrus fruits.

Identification of human-specific amino acid residues governing atenolol transport via organic cation transporter 2.

Organic cation transporter 2 (OCT2/SLC22A2) is predominantly localized on the basolateral membranes of renal tubular epithelial cells and plays a crucial role in the renal secretion of various cationic drugs. Although variations in substrate selectivity among renal organic cation transport systems across species have been reported, the characteristics of OCT2 remain unclear. In this study, we demonstrated that atenolol, a ß1-selective adrenergic antagonist, is transported almost exclusively by human OCT2, contrasting with OCT2s from other selected species. Using chimeric constructs between human OCT2 (hOCT2) and the highly homologous monkey OCT2 (monOCT2), along with site-directed mutagenesis, we identified non-conserved amino acids Val8, Ala31, Ala34, Tyr222, Tyr245, Ala270, Ile394, and Leu503 as pivotal for hOCT2-mediated atenolol transport. Kinetic analysis revealed that atenolol was transported by hOCT2 with a 12-fold lower affinity than MPP+, a typical OCT2 substrate. The inhibitory effect of atenolol on MPP+ transport was 6200-fold lower than that observed for MPP+ on atenolol transport. Additionally, we observed weaker inhibitory effects on MPP+ transport compared to atenolol transport with ten different OCT2 substrates. Altogether, this study suggests that eight hOCT2-specific amino acids constitute the low-affinity recognition site for atenolol transport, indicating differences in OCT2-mediated drug elimination between humans and highly homologous monkeys. Our findings underscore the importance of understanding species-specific differences in drug transport mechanisms, shedding light on potential variations in drug disposition and aiding in drug development.

Effect of Erdafitinib on the Pharmacokinetics of Midazolam and Metformin in Patients With Advanced Solid Tumors Harboring FGFR Gene Alterations.

Erdafitinib, an oral pan-FGFR inhibitor, is used in locally advanced or metastatic urothelial carcinoma for adults with FGFR3 genetic alterations and whose disease progressed following prior systemic therapy. This drug-drug interaction substudy evaluated the effect of erdafitinib on the pharmacokinetics of midazolam (cytochrome P450 3A4 substrate), and metformin (organic cation transporter 2 substrate). Twenty-five patients with advanced solid tumors harboring FGFR gene alterations received pretreatment with single doses of midazolam and metformin, followed by a daily dose of erdafitinib. Drug-drug interaction assessments were performed at erdafitinib steady state following coadministration of single doses of midazolam and metformin, respectively. Geometric mean ratios for maximum plasma concentration and area under the plasma concentration-time curve (AUC) from time 0 to the last measurable concentration, and AUC from time 0 to infinity were estimated using linear mixed-effects models (90% confidence interval within 80%-125% indicated no interaction). The 90% confidence intervals of geometric mean ratios for maximum plasma concentration, AUC from time 0 to the last measurable concentration, and AUC from time 0 to infinity of midazolam (86.3%, 88.5%, and 82.1%), 1-OH midazolam (99.8%, 97.4%, and 101.5%), and metformin (108.7%, 119.0%, and 113.9%) were either contained or slightly outside the 80%-125% interval and not considered clinically meaningful. Adverse events were consistent with the known erdafitinib safety profile; no new safety signals emerged. Thus, repeated dosing of erdafitinib had no clinically meaningful effect on the pharmacokinetics of midazolam or metformin.

Evaluation of In Vitro Metabolism- and Transporter-Based Drug Interactions with Sunscreen Active Ingredients.

PURPOSE: The aim of this study was to examine the ability of sunscreen active ingredients to inhibit in vitro drug metabolism via cytochrome P450 (CYP) enzymes and drug uptake transporters. METHODS: Metabolism assays with human liver microsomes were conducted for CYP2C9, CYP2D6 and CYP3A4 using probe substrates warfarin, bufuralol and midazolam, respectively. Uptake transporter assays with transfected cell lines were conducted for OAT3, OCT2 and OATP1B1 with probe substrates estrone-3-sulfate, metformin and rosuvastatin, respectively. Six sunscreen active ingredients, avobenzone, enzacamene, oxybenzone, octinoxate, trolamine, and homosalate, were evaluated up to their aqueous solubility limits in the assays. RESULTS: None of the sunscreen active ingredients inhibited CYP2D6 or CYP3A4 activities in the microsomes at concentration ranges up to tenfold higher than their known clinical total plasma levels. Only enzacamene, oxybenzone and trolamine were found to be inhibitory to CYP2C9 activity with IC50 values of 14.76, 22.46 and 154.7 µM, respectively. Avobenzone, enzacamene, homosalate and octinoxate were not inhibitory to the uptake transporters at the evaluated concentrations. Oxybenzone was inhibitory to OAT3 and OCT2 with IC50 values of 39.93 and 42.77 µM, respectively. Trolamine also inhibited uptake in OAT3 and OCT2 transfected cells with IC50 values of 448.1 and 1376 µM, respectively. CONCLUSIONS: Although enzacamene, oxybenzone and trolamine inhibited CYP2C9 and the renal transporters OAT3 and OCT2 in vitro, their IC50 values exceeded total plasma levels found in clinical studies. Therefore, it is unlikely that these sunscreen active ingredients in sunscreen products will inhibit the metabolism or transport of co-administered drugs in consumers.

Susceptibility of mouse cochlear hair cells to cisplatin ototoxicity largely depends on sensory mechanoelectrical transduction channels both Ex Vivo and In Vivo.

Cisplatin, a highly effective chemotherapeutic drug for various human cancers, induces irreversible sensorineural hearing loss as a side effect. Currently there are no highly effective clinical strategies for the prevention of cisplatin-induced ototoxicity. Previous studies have indicated that short-term cisplatin ototoxicity primarily affects the outer hair cells of the cochlea. Therefore, preventing the entry of cisplatin into hair cells may be a promising strategy to prevent cisplatin ototoxicity. This study aimed to investigate the entry route of cisplatin into mouse cochlear hair cells. The competitive inhibitor of organic cation transporter 2 (OCT2), cimetidine, and the sensory mechanoelectrical transduction (MET) channel blocker benzamil, demonstrated a protective effect against cisplatin toxicity in hair cells in cochlear explants. Sensory MET-deficient hair cells explanted from Tmc1Δ;Tmc2Δ mice were resistant to cisplatin toxicity. Cimetidine showed an additive protective effect against cisplatin toxicity in sensory MET-deficient hair cells. However, in the apical turn, cimetidine, benzamil, or genetic ablation of sensory MET channels showed limited protective effects, implying the presence of other entry routes for cisplatin to enter the hair cells in the apical turn. Systemic administration of cimetidine failed to protect cochlear hair cells from ototoxicity caused by systemically administered cisplatin. Notably, outer hair cells in MET-deficient mice exhibited no apparent deterioration after systemic administration of cisplatin, whereas the outer hair cells in wild-type mice showed remarkable deterioration. The susceptibility of mouse cochlear hair cells to cisplatin ototoxicity largely depends on the sensory MET channel both ex vivo and in vivo. This result justifies the development of new pharmaceuticals, such as a specific antagonists for sensory MET channels or custom-designed cisplatin analogs which are impermeable to sensory MET channels.

Deletion of the mRNA endonuclease Regnase-1 promotes NK cell anti-tumor activity via OCT2-dependent transcription of Ifng.

Limited infiltration and activity of natural killer (NK) and T cells within the tumor microenvironment (TME) correlate with poor immunotherapy responses. Here, we examined the role of the endonuclease Regnase-1 on NK cell anti-tumor activity. NK cell-specific deletion of Regnase-1 (Reg1ΔNK) augmented cytolytic activity and interferon-gamma (IFN-γ) production in vitro and increased intra-tumoral accumulation of Reg1ΔNK-NK cells in vivo, reducing tumor growth dependent on IFN-γ. Transcriptional changes in Reg1ΔNK-NK cells included elevated IFN-γ expression, cytolytic effectors, and the chemokine receptor CXCR6. IFN-γ induced expression of the CXCR6 ligand CXCL16 on myeloid cells, promoting further recruitment of Reg1ΔNK-NK cells. Mechanistically, Regnase-1 deletion increased its targets, the transcriptional regulators OCT2 and IκBζ, following interleukin (IL)-12 and IL-18 stimulation, and the resulting OCT2-IκBζ-NF-κB complex induced Ifng transcription. Silencing Regnase-1 in human NK cells increased the expression of IFNG and POU2F2. Our findings highlight NK cell dysfunction in the TME and propose that targeting Regnase-1 could augment active NK cell persistence for cancer immunotherapy.

IκBζ is a dual-use coactivator of NF-κB and POU transcription factors.

OCA-B, OCA-T1, and OCA-T2 belong to a family of coactivators that bind to POU transcription factors (TFs) to regulate gene expression in immune cells. Here, we identify IκBζ (encoded by the NFKBIZ gene) as an additional coactivator of POU TFs. Although originally discovered as an inducible regulator of NF-κB, we show here that IκBζ shares a microhomology with OCA proteins and uses this segment to bind to POU TFs and octamer-motif-containing DNA. Our functional experiments suggest that IκBζ requires its interaction with POU TFs to coactivate immune-related genes. This finding is reinforced by epigenomic analysis of MYD88L265P-mutant lymphoma cells, which revealed colocalization of IκBζ with the POU TF OCT2 and NF-κB:p50 at hundreds of DNA elements harboring octamer and κB motifs. These results suggest that IκBζ is a transcriptional coactivator that can amplify and integrate the output of NF-κB and POU TFs at inducible genes in immune cells.

Identification and characterization of an endogenous biomarker of the renal vectorial transport (OCT2-MATE1).

The renal tubular organic cation transporter 2 (OCT2) and multidrug and toxin extrusion protein 1 (MATE1) mediate the vectorial elimination of many drugs and toxins from the kidney, and endogenous biomarkers for vectorial transport (OCT2-MATE1) would allow more accurate drug dosing and help to characterize drug-drug interactions and toxicity. Human serum uptake in OCT2-overexpressing cells and metabolomics analysis were carried out. Potential biomarkers were verified in vitro and in vivo. The specificity of biomarkers was validated in renal transporter overexpressing cells and the sensitivity was investigated by Km . The results showed that the uptake of thiamine, histamine, and 5-hydroxytryptamine was significantly increased in OCT2-overexpressing cells. In vitro assays confirmed that thiamine, histamine, and 5-hydroxytryptamine were substrates of both OCT2 and MATE1. In vivo measurements indicated that the serum thiamine level was increased significantly in the presence of the rOCT2 inhibitor cimetidine, and the level in renal tissue was increased significantly by the rMATE1 inhibitor pyrimethamine. There were no significant changes in the uptake or efflux of thiamine in cell lines overexpressed OAT1, OAT2, OAT3, MRP4, organic anion transporting polypeptide 4C1, P-gp, peptide transporter 2, urate transporter 1, and OAT4. The Km for thiamine with OCT2 and MATE1 were 71.2 and 10.8 µM, respectively. In addition, the cumulative excretion of thiamine at 2 and 4 h was strongly correlated with metformin excretion (R2  > 0.6). Thus, thiamine is preferentially secreted by the OCT2 and MATE1 in renal tubules and can provide a reference value for evaluating the function of the renal tubular OCT2-MATE1.

Effect of 7-ketocholesterol incorporation on substrate binding affinity and turnover rate of the organic cation transporter 2 (OCT2).

The organic cation transporter 2 (OCT2) is pivotal in the renal elimination of several positively charged molecules. OCT2 mode of transport is profoundly influenced by the level of membrane cholesterol. The aim of this study was to investigate the effect of oxidized cholesterol on OCT2 transport activity in human embryonic kidney 293 cells stably transfected with OCT2 (OCT2-HEK293) and in primary renal proximal tubular epithelial cells (RPTEC). Cholesterol was exchanged with 7-ketocholesterol, the main product of cholesterol auto-oxidation, by exposing cells to sterol-saturated methyl-ß-cyclodextrin (mßcd). After a 30 min-exposure, approximately 50% of the endogenous cholesterol was replaced by 7-ketocholesterol without significant changes in total sterol level. In the presence of 7-ketocholesterol, [3H]1-methyl-4-phenylpyridinium (MPP+) uptake was significantly reduced in both cell lines. 7-ketocholesterol incorporation did not affect lipid raft integrity, nor OCT2 surface expression and spatial organization. The inhibitory effect of 7-ketocholesterol on MPP+ uptake was abolished by the presence of MPP+ in the trans-compartment. In the presence of 7-ketocholesterol, both Kt and Vmax of MPP+ influx decreased. Molecular docking using OCT2 structure in outward occluded conformation showed overlapping poses and similar binding energies between cholesterol and 7-ketocholesterol. The thermal stability of OCT2 was not changed when cholesterol was replaced with 7-ketocholesterol. We conclude that 7-ketocholesterol confers a higher rigidity to the carrier by reducing its conformational entropy, arguably as a result of changes in plasma membrane physical properties, thereby facilitating the achievement of a higher affinity state at the expense of the mobility and overall cycling rate of the transporter.

Transcriptional Coactivator BOB1 (OBF1, OCA-B) Modulates the Specificity of DNA Recognition by the POU-Domain Factors OCT1 and OCT2 in a Monomeric Configuration.

BOB1, a mammalian lymphocyte-specific transcriptional coactivator of the transcription factors OCT1 and OCT2 (OCT1/2), plays important roles in normal immune responses, autoimmunity, and hematologic malignancies. The issue of a DNA sequence preference change imposed by BOB1 was raised more than two decades ago but remains unresolved. In this paper, using the EMSA-SELEX-Seq approach, we have reassessed the intrinsic ability of BOB1 to modulate the specificity of DNA recognition by OCT1 and OCT2. Our results have reaffirmed previous conclusions regarding BOB1 selectivity towards the dimer configuration of OCT1/2. However, they suggest that the monomeric configuration of these factors, assembled on the classical octamer ATGCAAAT and related motifs, are the primary targets of BOB1. Our data further specify the DNA sequence preference imposed by BOB1 and predict the probability of ternary complex formation. These results provide an additional insight into the action of BOB1-an essential immune regulator and a promising molecular target for the treatment of autoimmune diseases and hematologic malignancies.

Thioarylation of 6-Amino-2,3,6-trideoxy-d-manno-oct-2-ulosonic Acid (IminoKdo): Access to 3,6-Disubstituted Picolinates and Mechanistic Insights.

In this work, we present a metal-free coupling protocol for the regio- and stereoselective C3-thioarylation of 6-amino-2,3,6-trideoxy-d-manno-oct-2-ulosonic acid (iminoKdo). The developed procedure enables the coupling of electron-rich, electron-deficient, and hindered arylthiols, providing a series of C3-modified iminoKdo derivatives in moderate to good yields. Elucidation of active species through controlled experimental studies and time-lapse 31 P NMR analysis provides insights into the reaction mechanism. We demonstrate that, following a tandem Staudinger/aza-Wittig reaction of an azido-containing keto ester, an inseparable equimolar mixture of imine/enamine is formed. The enamine then undergoes a Stork-like nucleophilic attack with the in situ-formed disulfide reagent, resulting in the formation of the coupling products. Additionally, we describe a rarely reported acid-promoted aromatization of the C3-thioarylated iminoKdo skeleton into 3,6-disubstituted picolinates, which are reminiscent of dichotomines.

Biosynthesis of the Inner Core of Bordetella pertussis Lipopolysaccharides: Effect of Mutations on LPS Structure, Cell Division, and Toll-like Receptor 4 Activation.

Previously developed whole-cell vaccines against Bordetella pertussis, the causative agent of whooping cough, appeared to be too reactogenic due to their endotoxin content. Reduction in endotoxicity can generally be achieved through structural modifications in the lipid A moiety of lipopolysaccharides (LPS). In this study, we found that dephosphorylation of lipid A in B. pertussis through the heterologous production of the phosphatase LpxE from Francisella novicida did, unexpectedly, not affect Toll-like receptor 4 (TLR4)-stimulating activity. We then focused on the inner core of LPS, whose synthesis has so far not been studied in B. pertussis. The kdtA and kdkA genes, responsible for the incorporation of a single 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) residue in the inner core and its phosphorylation, respectively, appeared to be essential. However, the Kdo-bound phosphate could be replaced by a second Kdo after the heterologous production of Escherichia coli kdtA. This structural change in the inner core affected outer-core and lipid A structures and also bacterial physiology, as reflected in cell filamentation and a switch in virulence phase. Furthermore, the eptB gene responsible for the non-stoichiometric substitution of Kdo-bound phosphate with phosphoethanolamine was identified and inactivated. Interestingly, the constructed inner-core modifications affected TLR4-stimulating activity. Whereas endotoxicity studies generally focus on the lipid A moiety, our data demonstrate that structural changes in the inner core can also affect TLR4-stimulating activity.

Escape from oncogene-induced senescence is controlled by POU2F2 and memorized by chromatin scars.

Although oncogene-induced senescence (OIS) is a potent tumor-suppressor mechanism, recent studies revealed that cells could escape from OIS with features of transformed cells. However, the mechanisms that promote OIS escape remain unclear, and evidence of post-senescent cells in human cancers is missing. Here, we unravel the regulatory mechanisms underlying OIS escape using dynamic multidimensional profiling. We demonstrate a critical role for AP1 and POU2F2 transcription factors in escape from OIS and identify senescence-associated chromatin scars (SACSs) as an epigenetic memory of OIS detectable during colorectal cancer progression. POU2F2 levels are already elevated in precancerous lesions and as cells escape from OIS, and its expression and binding activity to cis-regulatory elements are associated with decreased patient survival. Our results support a model in which POU2F2 exploits a precoded enhancer landscape necessary for senescence escape and reveal POU2F2 and SACS gene signatures as valuable biomarkers with diagnostic and prognostic potential.

Mechanism and linkage specificities of the dual retaining ß-Kdo glycosyltransferase modules of KpsC from bacterial capsule biosynthesis.

KpsC is a dual-module glycosyltransferase (GT) essential for "group 2" capsular polysaccharide biosynthesis in Escherichia coli and other Gram-negative pathogens. Capsules are vital virulence determinants in high-profile pathogens, making KpsC a viable target for intervention with small-molecule therapeutic inhibitors. Inhibitor development can be facilitated by understanding the mechanism of the target enzyme. Two separate GT modules in KpsC transfer 3-deoxy-ß-d-manno-oct-2-ulosonic acid (ß-Kdo) from cytidine-5'-monophospho-ß-Kdo donor to a glycolipid acceptor. The N-terminal and C-terminal modules add alternating Kdo residues with ß-(2→4) and ß-(2→7) linkages, respectively, generating a conserved oligosaccharide core that is further glycosylated to produce diverse capsule structures. KpsC is a retaining GT, which retains the donor anomeric carbon stereochemistry. Retaining GTs typically use an SNi (substitution nucleophilic internal return) mechanism, but recent studies with WbbB, a retaining ß-Kdo GT distantly related to KpsC, strongly suggest that this enzyme uses an alternative double-displacement mechanism. Based on the formation of covalent adducts with Kdo identified here by mass spectrometry and X-ray crystallography, we determined that catalytically important active site residues are conserved in WbbB and KpsC, suggesting a shared double-displacement mechanism. Additional crystal structures and biochemical experiments revealed the acceptor binding mode of the ß-(2→4)-Kdo transferase module and demonstrated that acceptor recognition (and therefore linkage specificity) is conferred solely by the N-terminal α/ß domain of each GT module. Finally, an Alphafold model provided insight into organization of the modules and a C-terminal membrane-anchoring region. Altogether, we identified key structural and mechanistic elements providing a foundation for targeting KpsC.

Augmented nephroprotective effect of liraglutide and rabeprazole via inhibition of OCT2 transporter in cisplatin-induced nephrotoxicity in rats.

AIMS: Cisplatin, a widely used anticancer treatment, has a marked nephrotoxic effect. This nephrotoxic effect is linked to the triggering of oxidative stress, inflammation, activation of mitogen-activated protein kinase (MAPK) pathway as well as apoptosis. The purpose of the present research was to examine the possible ameliorative effect of liraglutide and/or rabeprazole on cisplatin-induced nephrotoxicity in rats and to underline the potential molecular pathways involved. MAIN METHODS: Rats were divided into five groups: Control, cisplatin, liraglutide (200 µg/kg/day, i.p), rabeprazole (10 mg/kg/day, orally) and liraglutide + rabeprazole combination groups. All treatments were given for 7 days. Cisplatin was given as a single dose (7 mg/kg, i.p) at day 4 to induce nephrotoxicity in all groups except the control group. KEY FINDINGS: Treatment with liraglutide and/or rabeprazole prior to cisplatin maintained the function and morphology of kidney via decreasing cisplatin renal uptake by significant inhibition of OCT2. Besides, they showed a significant increase in GLP-1 receptor expression. Liraglutide and/or rabeprazole significantly attenuated the levels of TNF-α. ICAM, NF-κB, and downregulated MAPK pathway proteins such as JNK, and ERK1/2. Moreover, they maintained oxidant antioxidant balance by decreasing MDA level and increasing GSH level and CAT activity. Additionally, liraglutide and/or rabeprazole exhibited antiapoptotic effect evidenced by the decreased caspase-3 level and Bax expression and the increased Bcl-2 expression. SIGNIFICANCE: The current study showed that both liraglutide and rabeprazole exerted a nephroprotective effect against cisplatin-induced renal toxicity in rats. Interestingly, co-treatment with both drugs showed an augmented effect.

MATE1 expression in the cochlea and its potential involvement in cisplatin cellular uptake and ototoxicity.

BACKGROUND: Cisplatin appears to enter the cochlear cells through the organic cation transporter 2 (OCT2). There is recent evidence that multidrug and toxin extrusion protein 1 (MATE1) is involved in cisplatin-induced nephrotoxicity. Its presence and role in the ear are unknown. AIMS/OBJECTIVES: Evaluate the presence and localization of MATE1, and determine the localization of OCT2, in the cochlea. Evaluate cisplatin uptake with regard to MATE1 and OCT2 expression. MATERIAL AND METHODS: Murine cochlear explants and paraffin-embedded cochleae were evaluated with immunohistochemistry for OCT2 and MATE1. Explant cultures were also treated with Texas Red cisplatin to determine their cellular uptake. RESULTS: MATE1 is present in the cochlea. Most intense labeling of MATE1 and OCT2 was seen in the outer hair cells (OHCs) and pillar cells, respectively. Both transporters were observed in the spiral ganglion neurons and stria vascularis. Expression levels of OCT2 and MATE1 decreased following cisplatin exposure. Texas Red cisplatin staining was strong in OHCs and pillar cells. CONCLUSIONS AND SIGNIFICANCE: To the best of our knowledge, this is the first study demonstrating the presence and localization of MATE1 in the cochlea. OCT2 labeling was seen in pillar cells. Consistently, OHCs and pillar cells uptake Texas Red cisplatin.

Complex components of Shengmai formula interact with organic cation transporter 2 (OCT2) in MDCK cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Shengmai formula (SMF) is a well-known Chinese herbal compound preparation, which is utilized extensively for the treatment of myocardial ischemia, arrhythmia and other life-threatening conditions. Our previous researches have shown that some of the active ingredients in SMF can interact with organic anion transport polypeptide 1B1 (OATP1B1), breast cancer resistance protein (BCRP) and organic anion transporter 1 (OAT1), etc. Organic cation transporter 2 (OCT2) is a highly expressed uptake transporter in the kidney, and its interaction with the major active components in SMF remains unclear. AIM OF THE STUDY: We purposed to explore OCT2-mediated interactions and compatibility mechanisms of the main active compounds in SMF. MATERIALS AND METHODS: Fifteen active ingredients of SMF, including ginsenoside Rb1, Rd, Re, Rg1, Rf, Ro and Rc, methylophiopogonanone A and B, ophiopogonin D and D', schizandrin A and B, schizandrol A and B, were selected to investigate OCT2-mediated interactions in Madin-Darby cacine kidney (MDCK) cells stably expressing OCT2. RESULTS: Among the above 15 main active components, only ginsenosides Rd, Re and schizandrin B could significantly inhibit the uptake of 4-(4-(dimethylamino)styryl)-N-methyl pyridiniumiodide (ASP+), a classical substrate of OCT2. Ginsenoside Rb1 and methylophiopogonanone A can be transported by MDCK-OCT2 cells, and their uptake was significantly reduced when OCT2 inhibitor decynium-22 was added. Ginsenoside Rd could remarkably reduce the uptake of methylophiopogonanone A and ginsenoside Rb1 by OCT2, ginsenoside Re only decreased the uptake of ginsenoside Rb1, while schizandrin B had no effect on the uptake of both. CONCLUSIONS: OCT2 mediates the interaction of the major active components in SMF. Ginsenosides Rd, Re and schizandrin B are the potential inhibitors of OCT2, while ginsenosides Rb1 and methylophiopogonanone A are the potential substrates of OCT2. There is an OCT2-mediated compatibility mechanism among these active ingredients of SMF.

OCT2 expression in histiocytoses.

Diagnosis of histiocytosis can be difficult and one of the biggest challenges is to distinguish between reactive and neoplastic histiocytes on histology alone. Recently, OCT2 nuclear expression was reported in Rosai-Dorfman disease (RDD). Our purpose was to expand the testing of OCT2 on a broader variety of sporadic or H syndrome-related histiocytoses. Cases of histiocytoses were retrieved from the files of Ambroise Paré Pathology Department. All slides and molecular analyses were reviewed, and staining was completed with immunohistochemistry for OCT2. A total of 156 samples from different localizations were tested. Among sporadic cases, 52 patients had RDD, and 10 patients had mixed histiocytosis combining RDD with Erdheim Chester disease (ECD, n = 8), Langerhans cell histiocytosis (LCH, n = 2) or juvenile xanthogranuloma (JXG, n = 1). All these patients were positive for OCT2 in RDD characteristic histiocytes. Twenty-three patients had ECD and all but two (91% - 21/23) were negative for OCT2. By contrast, OCT2 was positive in 11/27 (41%) LCH and 6/16 (38%) JXG. Among the 10 samples of H syndrome-associated histiocytosis, 3 had typical RDD histology, 6 had unclassified histiocytosis, and one had mixed RDD-LCH; all were positive for OCT2. On 16 samples of granulomatous lymphadenitis, OCT2 was negative in epithelioid histiocytes. Our study shows that OCT2 has a sensitivity of 100% for RDD cases and mixed histiocytoses with an RDD component. It is negative in 92% of ECD but expressed in at least 38% of LCH, JXG, and C group histiocytoses. Finally, OCT2 is positive in all H syndrome-related histiocytoses, independent of their histology.

Renal tubular transporter-mediated interactions between mirogabalin and cimetidine in rats.

Cimetidine at a clinical dosage decreased the renal clearance (CLr) of mirogabalin in humans by inhibition of renal secretion. Mirogabalin is a substrate of human OAT1/3, OCT2, MATE1 and/or MATE2-K. To clarify the mechanism behind the above interaction, it was investigated whether cimetidine inhibits the process of mirogabalin uptake at the basolateral side or the process of its efflux at the apical side in rat kidney in vivo.Cimetidine was administered to rats by a constant infusion to achieve an unbound plasma concentration of 7.0 µM and examine its effect on the renal disposition of [14C]metformin, [3H]p-aminohippuric acid (PAH), and [14C]mirogabalin.Cimetidine significantly induced the intrarenal accumulation of radioactivity (Kp, kidney) and decreased the renal clearance (CLr) of [14C]mirogabalin. These effects resulted in significantly decreased total clearance (CLt). Kp, kidney, and CLr of [14C]metformin, except CLt, were also affected, but no parameters of [3H]PAH were affected by cimetidine.These findings clarified that an unbound plasma concentration of cimetidine of 7.0 µM inhibited the apical efflux not the basolateral uptake of [14C]mirogabalin in rat kidney, suggesting that mirogabalin/cimetidine interaction was caused by inhibiting the apical efflux transporter, human MATE1 and/or MATE2-K, not the basolateral uptake transporter, human OCT2, in the kidney.