Search Results (25)

Background/Objectives: Predicting whether a compound is subject to active transport is crucial in drug development. We propose a simple threshold for passive membrane permeability (P_m), derived from the cell’s energy limitation, to identify compounds unlikely to be actively effluxed. Results: By considering fundamental cellular energy constraints, our approach provides a mechanistic rationale for why compounds with very high passive permeability in combination with low applied concentration will not undergo active efflux. This moves beyond the empirical observation (such as in previous systems that associate fast-permeating, poorly soluble compounds with low transporter activity) by grounding the prediction in the cell’s energetic limitations. For MDCK (Madin–Darby canine kidney) cells, this threshold—normalized to the applied compound concentration (C_ext)—was determined to be P_m×C_ext = 10^−1.7 cm/s×µM. Methods: To derive this threshold, we conducted an extensive analysis of literature-reported efflux ratios (ERs) in MDCKII cells overexpressing efflux transporters (MDR1, BCRP, MRP2; 294 datapoints across 136 unique compounds). Concentration-dependent measurements for Amprenavir, Eletriptan, Loperamide, and Quinidine—chosen because these borderline compounds exhibited the highest P_m×C_ext while still being actively effluxed—enabled the most accurate determination of the threshold. Literature ER values were re-evaluated through the experimental determination of reliable P_m values, as well as newly measured ER values with MDCK efflux assays. Conclusions: The results of these assays and the re-evaluation allowed us to reclassify all but three outliers (compounds with ER > 2.5 and log(P_m×C_ext) > −1.7). In contrast, more than 60% of the compounds analyzed without significant ER values (123 compounds) fell above the threshold, in strong agreement with our theory of an energy limitation to active transport. This permeability threshold thus provides a simple and broadly applicable criterion to identify compounds for which active efflux is energetically not feasible and may serve as a practical tool for early drug discovery and optimization, pending further validation in practical applications. Full article

Reversing the multiple drug resistance (MDR) arising from the overexpression of the efflux transporters often fails mainly due to the high toxicity or the poor water solubility of the inhibitors of these transporters. Here, we demonstrate the delivery of an inhibitor targeting three ABC transporters (ABCB1, ABCC1 and ABCG2) directly to the cell membrane using membrane-fusing vehicles (MFVs). Three different transfected MDCK II cell lines, along with parental cells, were used to investigate the inhibitory effect of cyclosporine A (CsA) in solution versus direct delivery to the cell membrane. CsA-loaded MFVs successfully reversed MDR for all three investigated efflux transporters at significantly lower concentrations compared with CsA in solution. Results showed a 15-fold decrease in the IC₅₀ value for ABCB1, a 7-fold decrease for ABCC1 and an 11-fold decrease for ABCG2. We observed binding site specificity for ABCB1 and ABCG2 transporters. Lower concentrations of empty MFVs along with CsA contribute to the inhibition of Hoechst 33342 efflux. However, higher concentrations of CsA along with the high amount of MFVs activated transport via the H-binding site. This supports the conclusion that MFVs can be useful beyond their role as delivery systems and also help to elucidate differences between these transporters and their binding sites. Full article

In CNS drug discovery, the estimation of brain exposure to lead compounds is critical for their optimization. Compounds need to cross the blood–brain barrier (BBB) to reach the pharmacological targets in the CNS. The BBB is a complex system involving passive and active mechanisms of transport and efflux transporters such as P-glycoproteins (P-gp) and breast cancer resistance protein (BCRP), which play an essential role in CNS penetration of small molecules. Several in vivo, in vitro, and in silico methods are available to estimate human brain penetration. Preclinical species are used as in vivo models to understand unbound brain exposure by deriving the Kp,uu parameter and the brain/plasma ratio of exposure corrected with the plasma and brain free fraction. The MDCK-mdr1 (Madin Darby canine kidney cells transfected with the MDR1 gene encoding for the human P-gp) assay is the commonly used in vitro assay to estimate compound permeability and human efflux. The in silico methods to predict brain exposure, such as CNS MPO, CNS BBB scores, and various machine learning models, help save costs and speed up compound discovery and optimization at all stages. These methods enable the screening of virtual compounds, building of a CNS penetrable compounds library, and optimization of lead molecules for CNS penetration. Therefore, it is crucial to understand the reliability and ability of these methods to predict CNS penetration. We review the in silico, in vitro, and in vivo data and their correlation with each other, as well as assess published experimental and computational approaches to predict the BBB penetrability of compounds. Full article

The human P-glycoprotein (P-gp), a transporter responsible for multidrug resistance, is present in the plasma membrane’s raft and non-raft domains. One specific conformation of P-gp that binds to the monoclonal antibody UIC2 is primarily associated with raft domains and displays heightened internalization in cells overexpressing P-gp, such as in NIH-3T3 MDR1 cells. Our primary objective was to investigate whether the trafficking of this particular P-gp conformer is dependent on cholesterol levels. Surprisingly, depleting cholesterol using cyclodextrin resulted in an unexpected increase in the proportion of raft-associated P-gp within the cell membrane, as determined by UIC2-reactive P-gp. This increase appears to be a compensatory response to cholesterol loss from the plasma membrane, whereby cholesterol-rich raft micro-domains are delivered to the cell surface through an augmented exocytosis process. Furthermore, this exocytotic event is found to be part of a complex trafficking mechanism involving lysosomal exocytosis, which contributes to membrane repair after cholesterol reduction induced by cyclodextrin treatment. Notably, cells overexpressing P-gp demonstrated higher total cellular cholesterol levels, an increased abundance of stable lysosomes, and more effective membrane repair following cholesterol modifications. These modifications encompassed exocytotic events that involved the transport of P-gp-carrying rafts. Importantly, the enhanced membrane repair capability resulted in a durable phenotype for MDR1 expressing cells, as evidenced by significantly improved viabilities of multidrug-resistant Pgp-overexpressing immortal NIH-3T3 MDR1 and MDCK-MDR1 cells compared to their parents when subjected to cholesterol alterations. Full article

Currently, the mechanisms involved in drug access to the central nervous system (CNS) are not completely elucidated, and research efforts to understand the behaviour of the therapeutic agents to access the blood–brain barrier continue with the utmost importance. The aim of this work was the creation and validation of a new in vitro model capable of predicting the in vivo permeability across the blood–brain barrier in the presence of glioblastoma. The selected in vitro method was a cell co-culture model of epithelial cell lines (MDCK and MDCK-MDR1) with a glioblastoma cell line (U87-MG). Several drugs were tested (letrozole, gemcitabine, methotrexate and ganciclovir). Comparison of the proposed in vitro model, MDCK and MDCK-MDR1 co-cultured with U87-MG, and in vivo studies showed a great predictability for each cell line, with R2 values of 0.8917 and 0.8296, respectively. Therefore, both cells lines (MDCK and MDCK-MDR1) are valid for predicting the access of drugs to the CNS in the presence of glioblastoma. Full article

The efficacy and pharmacokinetics of the biologically active components in Anemarrhenae rhizoma (AR) would be affected by the interaction of P-glycoprotein(P-gp) and effective components in AR. However, little is known about the interaction between them. The goal of this research was to examine the transmembrane absorption of timosaponin AIII(TAIII), timosaponin BII(TBII), sarsasapogenin (SSG), mangiferin(MGF), neomangiferin(NMGF), isomangiferin(IMGF), and baohuosideI(BHI) in AR and their interaction with P-gp. Seven effective components in AR(TAIII, TBII, SSG, MGF, NMGF, IMGF, and BHI) were investigated, and MDCK-MDR1 cells were used as the transport cell model. CCK-8 assays, bidirectional transport assays, and Rhodamine-123 (Rh-123) transport assays were determined in the MDCK-MDR1 cells. LC/MS was applied to the quantitative analysis of TAIII, TBII, MGF, NMGF, IMGF, SSG, and BHI in transport samples. The efflux ratio of MGF, TAIII, TBII, and BHI was greater than 2 and significantly descended with the co-administration of Verapamil, indicating MGF, TAIII, TBII, and BHI as the substrates of P-gp. The efflux ratio of the seven effective components in the extracts (10 mg/mL) of AR decreased from 3.00~1.08 to 1.92~0.48. Compared to the efflux ratio of Rh-123 in the control group (2.46), the efflux ratios of Rh-123 were 1.22, 1.27, 1.25, 1.09, 1.31, and 1.47 by the addition of TAIII, TBII, MGF, IMGF, NMGF, and BHI, respectively, while the efflux ratio of Rh-123 with the co-administration of SSG had no statistical difference compared to the control group. These results indicated that MGF, TAIII, TBII, and BHI could be the substrates of P-gp. TAIII, TBII, MGF, IMGF, NMGF, and BHI show the effect of inhibiting P-gp function, respectively. These findings provide important basic pharmacological data to assist the therapeutic development of AR constituents and extracts. Full article

The main aim of this work is the biopharmaceutical characterization of a new hybrid benzodiazepine-dihydropyridine derivative, JM-20, derived with potent anti-ischemic and neuroprotective effects. In this study, the pKa and the pH-solubility profile were experimentally determined. Additionally, effective intestinal permeability was measured using three in vitro epithelial cell lines (MDCK, MDCK-MDR1 and Caco-2) and an in situ closed-loop intestinal perfusion technique. The results indicate that JM-20 is more soluble at acidic pH (9.18 ± 0.16); however, the Dose number (Do) was greater than 1, suggesting that it is a low-solubility compound. The permeability values obtained with in vitro cell lines as well as with the in situ perfusion method show that JM-20 is a highly permeable compound (Caco-2 value 3.8 × 10⁻⁵). The presence of an absorption carrier-mediated transport mechanism was also demonstrated, as well as the efflux effect of P-glycoprotein on the permeability values. Finally, JM-20 was provisionally classified as class 2 according to the biopharmaceutical classification system (BCS) due to its high intestinal permeability and low solubility. The potential good oral absorption of this compound could be limited by its solubility. Full article

One of the main obstacles in neurological disease treatment is the presence of the blood–brain barrier. New predictive high-throughput screening tools are essential to avoid costly failures in the advanced phases of development and to contribute to the 3 Rs policy. The objective of this work was to jointly develop a new in vitro system coupled with a physiological-based pharmacokinetic (PBPK) model able to predict brain concentration levels of different drugs in rats. Data from in vitro tests with three different cells lines (MDCK, MDCK-MDR1 and hCMEC/D3) were used together with PK parameters and three scaling factors for adjusting the model predictions to the brain and plasma profiles of six model drugs. Later, preliminary quantitative structure–property relationships (QSPRs) were constructed between the scaling factors and the lipophilicity of drugs. The predictability of the model was evaluated by internal validation. It was concluded that the PBPK model, incorporating the barrier resistance to transport, the disposition within the brain and the drug–brain binding combined with MDCK data, provided the best predictions for passive diffusion and carrier-mediated transported drugs, while in the other cell lines, active transport influence can bias predictions. Full article

Isobavachalcone (IBC) is an active substance from the medicinal plant Psoralea corylifolia. This prenylated chalcone was reported to possess antioxidative, anti-inflammatory, antibacterial, and anticancer activities. Multidrug resistance (MDR) associated with the over-expression of the transporters of vast substrate specificity such as ABCB1 (P-glycoprotein) belongs to the main causes of cancer chemotherapy failure. The cytotoxic, MDR reversing, and ABCB1-inhibiting potency of isobavachalcone was studied in two cellular models: human colorectal adenocarcinoma HT29 cell line and its resistant counterpart HT29/Dx in which doxorubicin resistance was induced by prolonged drug treatment, and the variant of MDCK cells transfected with the human gene encoding ABCB1. Because MDR modulators are frequently membrane-active substances, the interaction of isobavachalcone with model phosphatidylcholine bilayers was studied by means of differential scanning calorimetry. Molecular modeling was employed to characterize the process of membrane permeation by isobavachalcone. IBC interacted with ABCB1 transporter, being a substrate and/or competitive inhibitor of ABCB1. Moreover, IBC intercalated into model membranes, significantly affecting the parameters of their main phospholipid phase transition. It was concluded that isobavachalcone interfered both with the lipid phase of cellular membrane and with ABCB1 transporter, and for this reason, its activity in MDR cancer cells was presumptively beneficial. Full article

Multidrug resistance (MDR) is the main obstacle to current chemotherapy and it is mainly due to the overexpression of some efflux transporters such as MRP1. One of the most studied strategies to overcome MDR has been the inhibition of MDR pumps through small molecules, but its translation into the clinic unfortunately failed. Recently, a phenomenon called collateral sensitivity (CS) emerged as a new strategy to hamper MDR acting as a synthetic lethality, where the genetic changes developed upon the acquisition of resistance towards a specific agent are followed by the development of hypersensitivity towards a second agent. Among our library of sigma ligands acting as MDR modulators, we identified three compounds, F397, F400, and F421, acting as CS-promoting agents. We deepened their CS mechanisms in the “pure” model of MRP1-expressing cells (MDCK-MRP1) and in MRP1-expressing/drug resistant non-small cell lung cancer cells (A549/DX). The in vitro results demonstrated that (i) the three ligands are highly cytotoxic for MRP1-expressing cells; (ii) their effect is MRP1-mediated; (iii) they increase the cytotoxicity induced by cis-Pt, the therapeutic agent commonly used in the treatment of lung tumors; and (iv) their effect is ROS-mediated. Moreover, a preclinical in vivo study performed in lung tumor xenografts confirms the in vitro findings, making the three CS-promoting agents candidates for a novel therapeutic approach in lung resistant tumors. Full article

P-glycoprotein affects the transport of numerous drugs including chemotherapeutic drugs vincristine sulfate (VCR) and docetaxel (DTX), and is one of the main causes for multidrug resistance. Our previous studies have shown that oxypeucedanin (OPD) can enhance the intestinal transit of puerarin and VCR. However, the underlying mechanism is unclear. This study investigated the potential mechanism by which OPD improves P-gp-mediated drug transport. Molecular docking was performed to predict the binding force between OPD and P-gp and the contribution of OPD on P-gp activity. We observed the effect of OPD on the transport of VCR in MDCK-MDR1 cell monolayer and also measured the plasma pharmacokinetic parameters of DTX in the presence and absence of OPD by LC-MS/MS. Moreover, we further investigated the reversal mechanism of OPD on P-gp-mediated drug transport by determining the intracellular accumulation of Rhodamine-123 (Rh123) and P-gp ATPase activity as well as protein expression and mRNA level of P-gp. Our molecular docking results revealed that the binding force between OPD and P-gp was much lower than that between P-gp and verapamil (a P-gp substrate). The transport study in vitro indicated that OPD increased the flux of VCR across MDCK-MDR1 cell monolayer. The in vivo pharmacokinetic parameters data showed OPD increased the absorption of DTX. OPD activated P-gp ATPase activity and enhanced intracellular accumulation of Rh123 in MDCK-MDR1 cells. Western blotting and qRT-PCR outcomes indicated that OPD suppressed P-gp protein expression as well as downregulated P-gp mRNA level. Thus, OPD reverse P-gp-mediated drug transport via inhibition of P-gp activity and P-gp protein expression as well as downregulation of P-gp mRNA level. Our results suggest that OPD could reverse P-gp-mediated drug resistance in tumor cells. Full article

Seven medicinal plants popularly used for treating malaria in West Africa were selected to assess herb-drug interaction potential through a series of in vitro methods. Fluorescent cytochrome P450 (CYP) assays were conducted using the recombinant CYP enzymes for CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 to assess the effect of the methanolic extracts on the metabolic activity of CYPs. Secondly, the inhibitory effect of the extracts was evaluated on P-glycoproteins (P-gp) using calcein-AM, a fluorescent substrate, in MDCK-II and hMDR1-MDCK-II cells. The inhibition of P-gp activity was determined as a reflection of increase in calcein-AM uptake. Additionally, the enzyme induction potential of the extracts was assessed through the modulation of PXR activity in HepG2 cells transiently transfected with pSG5-PXR and PCR5 plasmid DNA. Significant inhibition of CYP activity (IC₅₀ < 10 µg/mL) was observed with the following herbs: A. muricata [CYP2C9, 3A4 and CYP2D6]; M. indica [CYP2C9]; M. charantia [CYP2C9 and CYP2C19]; P. amarus [CYP2C19, CYP2C9 and CYP3A4]; T. diversifolia [CYP2C19 and CYP3A4]. Extracts of four herbs (P. amarus, M. charantia, T. diversifolia and A. muricata) exhibited significant inhibition of P-gp with IC₅₀ values (µg/mL) of 17 ± 1, 16 ± 0.4, 26 ± 1, and 24 ± 1, respectively. In addition, four herbs (A. mexicana, M. charantia, P. amarus and T. diversifolia) showed a >two-fold increase in induction in PXR activity. These findings suggest that these herbs may be capable of eliciting herb-drug interactions if consumed in high quantities with concomitant use of conventional therapies. Full article

Uncariae Ramulus Cum Uncis (URCU) is a widely used traditional Chinese medicine, and is reported to have various central nervous system effects. Alkaloids have been demonstrated to be the predominant pharmacological active components of URCU. In order to evaluate the blood-brain barrier (BBB) permeability and transport mechanism of six typical indole alkaloids from URCU, the MDCK-pHaMDR cell monolayer model was used as an in vitro surrogate model for BBB. The samples were analyzed by high-performance liquid chromatography, and the apparent permeability coefficients (P_app) were calculated. Among the six alkaloids, isorhynchophylline (2), isocorynoxeine (4), hirsutine (5) and hirsuteine (6) showed high permeability, with P_app values at 10⁻⁵ cm/s level in bidirectional transport. For rhynchophylline (1) and corynoxeine (3), they showed moderate permeability, with P_app values from the apical (AP) side to the basolateral (BL) side at 10⁻⁶ cm/s level and efflux ratio (P_{app BL→AP}/P_{app AP→BL}) above 2. The time- and concentration-dependency experiments indicated that the main mechanism for 2, 4, 5 and 6 through BBB was passive diffusion. The efflux mechanism involved in the transports of compounds 1 and 3 could be reduced significantly by verapamil, and molecular docking screening also showed that 1 and 3 had strong bindings to P-glycoprotein. This study provides useful information for predicting the BBB permeability for 1–6, as well as better understanding of their central nervous system pharmacological activities. Full article

In our study, we attempted to investigate the influences of P-glycoprotein (P-gp) on DL0410, a novel synthetic molecule for Alzheimer’s disease (AD) treatment, for intestinal absorption and blood-brain barrier permeability in vitro and related binding mechanisms in silico. Caco-2, MDCK, and MDCK-MDR1 cells were utilized for transport studies, and homology modelling of human P-gp was built for further docking study to uncover the binding mode of DL0410. The results showed that the apparent permeability (Papp) value of DL0410 was approximately 1 × 10⁻⁶ cm/s, indicating the low permeability of DL0410. With the presence of verapamil, the directional transport of DL0410 disappeared in Caco-2 and MDCK-MDR1 cells, suggesting that DL0410 should be a substrate of P-gp, which was also confirmed by P-gp ATPase assay. In addition, DL0410 could competitively inhibit the transport of Rho123, a P-gp known substrate. According to molecular docking, we also found that DL0410 could bind to the drug binding pocket (DBP), but not the nucleotide binding domain (NBD). In conclusion, DL0410 was a substrate as well as a competitive inhibitor of P-gp, and P-gp had a remarkable impact on the intestine and brain permeability of DL0410, which is of significance for drug research and development. Full article

P-glycoprotein (P-gp) affects the transport of many drugs; including puerarin and vincristine. Our previous study demonstrated that imperatorin increased the intestinal absorption of puerarin and vincristine by inhibiting P-gp-mediated drug efflux. However; the underlying mechanism was not known. The present study investigated the mechanism by which imperatorin promotes P-gp-mediated drug transport. We used molecular docking to predict the binding force between imperatorin and P-gp and the effect of imperatorin on P-gp activity. P-gp efflux activity and P-gp ATPase activity were measured using a rhodamine 123 (Rh-123) accumulation assay and a Pgp-Glo™ assay; respectively. The fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to assess cellular membrane fluidity in MDCK-MDR1 cells. Western blotting was used to analyze the effect of imperatorin on P-gp expression; and P-gp mRNA levels were assessed by qRT-PCR. Molecular docking results demonstrated that the binding force between imperatorin and P-gp was much weaker than the force between P-gp and verapamil (a P-gp substrate). Imperatorin activated P-gp ATPase activity; which had a role in the inhibition of P-gp activity. Imperatorin promoted Rh-123 accumulation in MDCK-MDR1 cells and decreased cellular membrane fluidity. Western blotting demonstrated that imperatorin inhibited P-gp expression; and qRT-PCR revealed that imperatorin down-regulated P-gp (MDR1) gene expression. Imperatorin decreased P-gp-mediated drug efflux by inhibiting P-gp activity and the expression of P-gp mRNA and protein. Our results suggest that imperatorin could down-regulate P-gp expression to overcome multidrug resistance in tumors. Full article