In vitro models to evaluate multidrug resistance in cancer cells: Biochemical and morphological techniques and pharmacological strategies.

The overexpression of ATP-binding cassette (ABC) transporters contributes to the failure of chemotherapies and symbolizes a great challenge in oncology, associated with the adaptation of tumor cells to anticancer drugs such that these transporters become less effective, a mechanism known as multidrug resistance (MDR). The aim of this review is to present the most widely used methodologies for induction and comprehension of in vitro models for detection of multidrug-resistant (MDR) modulators or inhibitors, including biochemical and morphological techniques for chemosensitivity studies. The overexpression of MDR proteins, predominantly, the subfamily glycoprotein-1 (P-gp or ABCB1) multidrug resistance, multidrug resistance-associated protein 1 (MRP1 or ABCCC1), multidrug resistance-associated protein 2 (MRP2 or ABCC2) and cancer resistance protein (ABCG2), in chemotherapy-exposed cancer lines have been established/investigated by several techniques. Amongst these techniques, the most used are (i) colorimetric/fluorescent indirect bioassays, (ii) rhodamine and efflux analysis, (iii) release of 3,30-diethyloxacarbocyanine iodide by fluorescence microscopy and flow cytometry to measure P-gp function and other ABC transporters, (iv) exclusion of calcein-acetoxymethylester, (v) ATPase assays to distinguish types of interaction with ABC transporters, (vi) morphology to detail phenotypic characteristics in transformed cells, (vii) molecular testing of resistance-related proteins (RT-qPCR) and (viii) 2D and 3D models, (ix) organoids, and (x) microfluidic technology. Then, in vitro models for detecting chemotherapy MDR cells to assess innovative therapies to modulate or inhibit tumor cell growth and overcome clinical resistance. It is noteworthy that different therapies including anti-miRNAs, antibody-drug conjugates (to natural products), and epigenetic modifications were also considered as promising alternatives, since currently no anti-MDR therapies are able to improve patient quality of life. Therefore, there is also urgency for new clinical markers of resistance to more reliably reflect in vivo effectiveness of novel antitumor drugs.

Lymphatic Targeting of Cilnidipine by Designing and Developing a Nanostructured Lipid Carrier Drug Delivery System.

OBJECTIVE: The objective of current research is to design, develop, and optimize a cilnidipine (CLN) nanostructured lipid carrier-based drug delivery system (NLC) for the effective treatment of Hypertension (HT). SIGNIFICANCE: Oral administration of CLN NLC containing Glyceryl monostearate (GMS) as a solid and Isopropyl myristate (IPM) as a liquid lipid may show remarkable lymphatic uptake through payer patches. METHODS: The emulsification probe sonication technique was used followed by optimization using 32 factorial designs. RESULTS: The optimized batch showed a mean particle size of 115.4 ± 0.22 nm with encapsulation efficiency of 98.32 ± 0.23%, polydispersity index (PDI) of 0.342 ± 0.03, and zeta potential (ζ) was -60.5 ± 0.24 which indicate excellent physical stability. In vitro studies showed a controlled release of CLN NLCs. Pharmacokinetics studies determined the Cmax of NLCs (373.47 ± 15.1) indicates 2.3-fold enhancement compared with plain drug (160.64 ± 7.63). Pharmacodynamic studies indicated that CLN NLCs were maintaining systolic blood pressure in a controlled manner without any signs of side effects. CONCLUSION: CLN NLCs significantly improved lymphatic delivery and proved to be effective in the treatment and management of hypertension. It has been proved that CLN NLCs are found to be better than any traditional CLN dosage form due to enhancement in solubility, absorption, bioavailability, intestinal permeability, avoidance of first-pass metabolism, P-glycoprotein efflux and reduction in dose-related side effects, achievement of controlled and sustained release action.

Bisecting GlcNAc modification reverses the chemoresistance via attenuating the function of P-gp.

Rationale: Chemoresistance is a key factor contributing to the failure of anti-breast cancer chemotherapy. Although abnormal glycosylation is closely correlated with breast cancer progression, the function of glycoconjugates in chemoresistance remains poorly understood. Methods: Levels and regulatory roles of bisecting N-acetylglucosamine (GlcNAc) in chemoresistant breast cancer cells were determined in vitro and in vivo. Glycoproteomics guided identification of site-specific bisecting GlcNAc on P-glycoprotein (P-gp). Co-immunoprecipitation coupled mass spectrometry (Co-IP-MS) and proximity labelling MS identified the interactome of P-gp, and the biological function of site-specific bisecting GlcNAc was investigated by site/truncation mutation and structural simulations. Results: Bisecting GlcNAc levels were reduced in chemoresistant breast cancer cells, accompanied by an enhanced expression of P-gp. Enhanced bisecting GlcNAc effectively reversed chemoresistance. Mechanical study revealed that bisecting GlcNAc impaired the association between Ezrin and P-gp, leading to a decreased expression of membrane P-gp. Bisecting GlcNAc suppressed VPS4A-mediated P-gp recruitment into microvesicles, and chemoresistance transmission. Structural dynamics analysis suggested that bisecting GlcNAc at Asn494 introduced structural constraints that rigidified the conformation and suppressed the activity of P-gp. Conclusion: Our findings highlight the crucial role of bisecting GlcNAc in chemoresistance and suggest the possibility of reversing chemoresistance by modulating the specific glycosylation in breast cancer therapy.

A Molecular Dynamic Simulation, Structural Analysis, and Ex Vivo Insights into the P-glycoprotein-Mediated Interactions of Dietary Polyphenols with Cyclin-dependent Kinase Inhibitors: A Potential Strategy to Counteract Drug Efflux.

INTRODUCTION: P-glycoprotein, an ATP-dependent efflux transporter, plays a crucial role in eliminating cellular toxins and affects the intracellular concentration and bioavailability of CDK 4/6 inhibitors. Moreover, dietary flavonoids are natural bio-enhancers that can effectively inhibit the efflux function of these transporters. Therefore, this study aimed to assess the impact of dietary polyphenols on the inhibition of P-glycoprotein and the subsequent efflux of CDK inhibitors palbociclib and ribociclib. METHODS: A molecular docking approach was implemented to evaluate the binding interaction characteristics of CDK4/6 inhibitors in the presence of dietary polyphenols at the ATP binding site. Furthermore, the stability of the complexes was evaluated in two conformations of P-glycoprotein, followed by an ex vivo everted gut sac experiment. RESULTS: The findings demonstrated that the binding of curcumin and quercetin with high affinity (-51.63 and 47.16 Kcal/mol) to ATP binding sites of P-glycoprotein-palbociclib and ribociclib inward conformation complexes resulted in good stability of complex and minimal fluctuation throughout the course of the simulation. It was evident from the everted gut sac ex vivo study that the presence of 100µM of curcumin resulted in an increase of 1.77 and 4.20-fold in the intestinal transit of palbociclib and ribociclib, respectively. CONCLUSION: The study emphasizes the significance of curcumin and quercetin as inhibitors of P-glycoprotein, demonstrating their potential to decrease the efflux of palbociclib and ribociclib, consequently contributing to their bioavailability enhancement.

A Revolutionary Approach for Combating Efflux Transporter-mediated Resistant Epilepsy: Advanced Drug Delivery Systems.

Epilepsy is a persistent neurological condition that affects 60 million individuals globally, with recurrent spontaneous seizures affecting 80% of patients. Antiepileptic drugs (AEDs) are the main course of therapy for approximately 65% of epileptic patients, and the remaining 35% develop resistance to medication, which leads to Drug-Resistant Epilepsy (DRE). DRE continues to be an important challenge in clinical epileptology. There are several theories that attempt to explain the neurological causes of pharmacoresistance in epilepsy. The theory that has been studied the most is the transporter hypothesis. Therefore, it is believed that upregulation of multidrug efflux transporters at the blood-brain barrier (BBB), such as P-glycoprotein (P-gp), which extrudes AEDs from their target location, is the major cause, leading to pharmacoresistance in epilepsy. The most effective strategies for managing this DRE are peripheral and central inhibition of P-gp and maintaining an effective concentration of the drug in the brain parenchyma. Presently, no medicinal product that inhibits P-gp is being used in clinical practice. In this review, several innovative and promising treatment methods, including gene therapy, intracranial injections, Pgp inhibitors, nanocarriers, and precision medicine, are discussed. The primary goal of this work is to review the P-gp transporter, its substrates, and the latest novel treatment methods for the management of DRE.

Proteomic Dynamics of Multidrug Resistance Mechanisms in Lucena 1 Cell Line.

The Lucena 1 cell line, derived from the human chronic myeloid leukemia cell line K562 under selective pressure of vincristine supplementation, exhibits multidrug resistance (MDR). This study aims to explore and elucidate the underlying mechanisms driving MDR in the Lucena 1 cell line. A proteomic analysis comparing K562 and Lucena 1 revealed qualitative differences, with a focus on the ATP-dependent efflux pump, Translocase ABCB1, a key contributor to drug resistance. Tubulin analysis identified two unique isoforms, Tubulin beta 8B and alpha chain-like 3, exclusive to Lucena 1, potentially influencing resistance mechanisms. Additionally, the association of Rap1A and Krit1 in cytoskeletal regulation and the presence of STAT1, linked to the urea cycle and tumor development, offered insights into Lucena 1's distinctive biology. The increased expression of carbonic anhydrase I suggested a role in pH regulation. The discovery of COP9, a tumor suppressor targeting p53, further highlighted the Lucena 1 complex molecular landscape. This study offers new insights into the MDR phenotype and its multifactorial consequences in cellular pathways. Thus, unraveling the mechanisms of MDR holds promise for innovating cancer models and antitumor targeted strategies, since inhibiting the P-glycoprotein (P-gp)/ABCB1 protein is not always an effective approach given the associated treatment toxicity.

Synthesis, Preclinical Toxicity, and Biodistribution of [18F]AVT-011 to Assess the P-Glycoprotein Function.

Introduction: Many studies have reported the role of P-glycoprotein (Pgp) in chemoresistance in various pathological conditions such as cancer and neurodegenerative diseases, such as Alzheimer's. In this study, we are reporting the high-performance liquid chromatography (HPLC)-based purification of fluorine-18 [18F]AVT-011 and its preclinical evaluation. Methods: AVT-011 was labeled with 18F using the nucleophilic substitution method by heating the reaction mixture at 110°C for 10 min, followed by purification using preparative HPLC and C18ec cartridge. The in vitro cell uptake study was carried out in U87 cells with and without an inhibitor. The preclinical toxicity was carried out in CD1 mice in three groups, including control, AVT-011 treated, and [18F]AVT-011 treated. The biodistribution study was done in CD1 mice (n = 12) after intravenous injection of 4-6 MBq [18F]AVT-011, and mice were sacrificed at various time intervals. A dose of 3.7 ± 0.7 MBq of [18F]AVT-011 was injected intravenously in the healthy Swiss albino mice, and the whole-body micro-positron emission tomography was acquired at 0-, 30-, 60-, and 120-min postinjection. Results: The radiochemical purity of [18F]AVT-011 was 97 ± 1.5% as evaluated by radio-HPLC with a yield of 14 ± 2% and was stable up to 95% under in vitro conditions in blood and in vivo conditions up to 4 h. The in vitro cell uptake study showed a significant difference in control (27.4 ± 2.1%) and blocked U987 cells (73.2 ± 3.2%) after incubation of 120 min. The tissue distribution in mice showed the highest uptake in the liver (17.3 ± 2.4%), kidneys (16.6 ± 3.1%), lungs (10.4 ± 2.9%), and spleen (5.6 ± 0.8%) at 15 min, and the activity was washed out with time. The radioactivity cleared through the hepatorenal pathway. The animal imaging study also demonstrates a similar biodistribution pattern. Conclusions: [18F]AVT-011 showed higher specific activity than the cartridge-based method but showed similar biological activity.

The pharmacokinetics of dabigatran in a rat model of hyperlipidaemia induced by poloxamer 407.

Various pharmacokinetic changes have been reported in experimental hyperlipidemic (HL) animal models. To evaluate whether P-glycoprotein (P-gp) activity was affected in HL rats, we assessed the pharmacokinetics of dabigatran after oral administration of dabigatran etexilate (DABE); this is a dabigatran prodrug and a well-known P-gp substrate.HL and control rats exhibited similar area under the plasma concentration-time curve (AUC), total body clearance (CL), and steady state volume of distribution (Vss) values following intravenous administration of dabigatran (1 mg/kg). This suggested that the distribution and elimination of dabigatran were similar in control and HL rats.The hepatic and intestinal P-gp protein levels did not differ significantly between control and HL rats. The dabigatran AUC and extent of absolute oral bioavailability (F) values were similar in control and HL rats following oral administration of DABE (10 mg/kg as dabigatran). Therefore, there was no apparent change in intestinal P-gp activity in HL rats compared to control rats.This study revealed no significant change in P-gp expression or activity in the intestine or liver of HL rats, and similar pharmacokinetics of dabigatran. Hyperlipidaemia may not directly affect the oral absorption of P-gp substrate drugs.

Differential Effect of Simulated Microgravity on the Cellular Uptake of Small Molecules.

The space environment can affect the function of all physiological systems, including the properties of cell membranes. Our goal in this study was to explore the effect of simulated microgravity (SMG) on the cellular uptake of small molecules based on reported microgravity-induced changes in membrane properties. SMG was applied to cultured cells using a random-positioning machine for up to three hours. We assessed the cellular accumulation of compounds representing substrates of uptake and efflux transporters, and of compounds not shown to be transported by membrane carriers. Exposure to SMG led to an increase of up to 60% (p < 0.01) in the cellular uptake of efflux transporter substrates, whereas a glucose transporter substrate showed a decrease of 20% (p < 0.05). The uptake of the cathepsin activity-based probe GB123 (MW, 1198 g/mol) was also enhanced (1.3-fold, p < 0.05). Cellular emission of molecules larger than ~3000 g/mol was reduced by up to 50% in SMG (p < 0.05). Our findings suggest that short-term exposure to SMG could differentially affect drug distribution across membranes. Longer exposure to microgravity, e.g., during spaceflight, may have distinct effects on the cellular uptake of small molecules.

P-glycoprotein (P-gp)-driven cancer drug resistance: biological profile, non-coding RNAs, drugs and nanomodulators.

Drug resistance has compromised the efficacy of chemotherapy. The dysregulation of drug transporters including P-glycoprotein (P-gp) can mediate drug resistance through drug efflux. In this review, we highlight the role of P-gp in cancer drug resistance and the related molecular pathways, including phosphoinositide 3-kinase (PI3K)-Akt, phosphatase and tensin homolog (PTEN) and nuclear factor-κB (NF-κB), along with non-coding RNAs (ncRNAs). Extracellular vesicles secreted by the cells can transport ncRNAs and other proteins to change P-gp activity in cancer drug resistance. P-gp requires ATP to function, and the induction of mitochondrial dysfunction or inhibition of glutamine metabolism can impair P-gp function, thus increasing chemosensitivity. Phytochemicals, small molecules and nanoparticles have been introduced as P-gp inhibitors to increase drug sensitivity in human cancers.

Plant-Based Products Originating from Serbia That Affect P-glycoprotein Activity.

Our review paper evaluates the impact of plant-based products, primarily derived from plants from Serbia, on P-glycoprotein (P-gp) activity and their potential in modulating drug resistance in cancer therapy. We focus on the role and regulation of P-gp in cellular physiology and its significance in addressing multidrug resistance in cancer therapy. Additionally, we discuss the modulation of P-gp activity by 55 natural product drugs, including derivatives for some of them, based on our team's research findings since 2011. Specifically, we prospect into sesquiterpenoids from the genera Artemisia, Curcuma, Ferula, Inula, Petasites, and Celastrus; diterpenoids from the genera Salvia and Euphorbia; chalcones from the genera Piper, Glycyrrhiza, Cullen, Artemisia, and Humulus; riccardins from the genera Lunularia, Monoclea, Dumortiera, Plagiochila, and Primula; and diarylheptanoids from the genera Alnus and Curcuma. Through comprehensive analysis, we aim to highlight the potential of natural products mainly identified in plants from Serbia in influencing P-gp activity and overcoming drug resistance in cancer therapy, while also providing insights into future perspectives in this field.

Borneol promotes berberine-induced cardioprotection in a rat model of myocardial ischemia/reperfusion injury via inhibiting P-glycoprotein expression.

Berberine is reported to protect the heart against ischemia/reperfusion (I/R) injury, although efficacy is limited by low bioavailability. This study aims to determine whether borneol, a classic guiding drug, can enhance the cardioprotection induced by berberine and to clarify the underlying mechanisms involving P-glycoprotein (P-gp) in the heart. Adult male Sprague Dawley rats were gavaged with berberine (200 mg/kg) with or without borneol (100 mg/kg) for 7 consecutive days. A rat model of myocardial I/R injury was established by 30 min left coronary artery occlusion followed with 120 min reperfusion. The arrhythmia score, cardiac enzyme content, and myocardial infarct size were determined following reperfusion. Heart tissues were collected for Western blot and immunofluorescence analyses to measure the protein expression levels of Bcl-2, Bax, and P-gp. The results showed that administration of berberine protected the heart against I/R injury, as demonstrated by lower arrhythmia scores, serum cTnI contents, myocardial infarct size, and cardiomyocytes apoptosis. Moreover, borneol substantially enhanced the cardioprotective effects of berberine. Western blot and immunofluorescence analyses showed that both berberine and I/R injury did not alter P-gp expression in heart. In contrast, borneol combined with berberine significantly reduced P-gp levels by 43.4% (P = 0.0240). Interestingly, treatment with borneol alone decreased P-gp levels, but did not protect against myocardial I/R injury. These findings suggest that borneol, as an adjuvant drug, improved the cardioprotective effects of berberine by inhibiting P-gp expression in heart. Borneol combined with berberine administration provides a new strategy to protect the heart against I/R injury.

Identification of NanoLuciferase Substrates Transported by Human ABCB1 and ABCG2 and their Zebrafish Homologs at the Blood-Brain Barrier.

ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier (BBB) impede delivery of therapeutic agents to the brain, including agents to treat neurodegenerative diseases and primary and metastatic brain cancers. Two transporters, P-glycoprotein (P-gp, ABCB1) and ABCG2, are highly expressed at the BBB and are responsible for the efflux of numerous clinically useful chemotherapeutic agents, including irinotecan, paclitaxel, and doxorubicin. Based on a previous mouse model, we have generated transgenic zebrafish where expression of NanoLuciferase (NanoLuc) is controlled by the promoter of glial fibrillary acidic protein, leading to expression in zebrafish glia. To identify agents that disrupt the BBB including inhibitors of ABCB1 and ABCG2, we identified NanoLuc substrates that are also transported by P-gp, ABCG2, and their zebrafish homologs. These substrates will elevate the amount of bioluminescent light produced in the transgenic zebrafish with BBB disrpution. We transfected HEK-293 cells with both NanoLuc and human ABCB1 or ABCG2, or their zebrafish homologs Abcb4 and Abcg2a, which are functionally homologous to human P-gp and ABCG2, respectively, and expressed at the zebrafish BBB. We evaluated the brightness of ten NanoLuc substrates, then screened the eight brightest for their ability to be effluxed by the ABC transporters. We identified one ABCB1 substrate, two Abcb4 substrates, six ABCG2 substrates, and four Abcg2a substrates. These data will aid in the development of a transgenic zebrafish model of the BBB to identify novel BBB disruptors and should prove useful in the development of other animal models that use NanoLuc as a reporter. Significance Statement The ATP-Binding Cassette (ABC) transporters ABCB1 and ABCG2 at the blood-brain barrier (BBB) hinder pharmacological treatment of brain-related diseases. Consequently, there is a need for tools to identify BBB disruptors. We conducted a screen of ten NanoLuciferase substrates, identifying the brightest and those that were transported by human and zebrafish ABC transporters at the BBB. This work supports and complements our development of a transgenic zebrafish model, in which NanoLuciferase is expressed within glial cells, enabling detection of BBB disruption.

The Drug Transporter P-Glycoprotein and Its Impact on Ceramide Metabolism-An Unconventional Ally in Cancer Treatment.

The tumor-suppressor sphingolipid ceramide is recognized as a key participant in the cytotoxic mechanism of action of many types of chemotherapy drugs, including anthracyclines, Vinca alkaloids, the podophyllotoxin etoposide, taxanes, and the platinum drug oxaliplatin. These drugs can activate de novo synthesis of ceramide or stimulate the production of ceramide via sphingomyelinases to limit cancer cell survival. On the contrary, dysfunctional sphingolipid metabolism, a prominent factor in cancer survival and therapy resistance, blunts the anticancer properties of ceramide-orchestrated cell death pathways, especially apoptosis. Although P-glycoprotein (P-gp) is famous for its role in chemotherapy resistance, herein, we propose alternate interpretations and discuss the capacity of this multidrug transporter as a "ceramide neutralizer", an unwelcome event, highlighting yet another facet of P-gp's versatility in drug resistance. We introduce sphingolipid metabolism and its dysfunctional regulation in cancer, present a summary of factors that contribute to chemotherapy resistance, explain how P-gp "neutralizes" ceramide by hastening its glycosylation, and consider therapeutic applications of the P-gp-ceramide connection in the treatment of cancer.

The role of efflux transporters in cytotoxicity and intracellular concentration of chlorpyrifos and chlorpyrifos oxon in human cell lines.

In this study, we investigated the role of two efflux transporters, p-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), in the cytotoxicity and intracellular accumulation of the organophosphate pesticide chlorpyrifos (CPF) and its active metabolite, CPF-oxon (CPFO), in a human-derived liver cell line (HepG2) and kidney epithelial cell line (HK-2). The cytotoxicity to CPF and CPFO differed between cell lines where HK-2 had lower IC50 values which could be attributed to lower basal expression and inducibility of metabolizing enzymes, transporters, and nuclear receptors in HK-2 cells. In HepG2 cells, co-exposure of CPF with a specific inhibitor of either P-gp or BCRP enhanced the cytotoxicity of CPF while co-exposure of CPFO with VRP enhanced the cytotoxicity of CPFO, suggesting the role of these transporters in the elimination CPF and CPFO. Inhibition of efflux transporters did not affect the cytotoxicity of CPF and CPFO in HK-2 cells. Co-incubation of CPF with P-gp and BCRP inhibitors increased the intracellular concentration of CPF in HepG2 cells suggesting that both transporters play a role in limiting the cellular accumulation of CPF in HepG2 cells. Our results provide evidence that inhibition of efflux transporters can enhance CPF-induced toxicity through enhanced cellular accumulation and raises additional questions regarding how pesticide-transporter interactions may influence toxicity of mixtures containing pesticides and other environmental chemicals.

Myosin Vb Traffics P-Glycoprotein to the Apical Membrane of Intestinal Epithelial Cells.

BACKGROUND & AIMS: The xenobiotic efflux pump P-glycoprotein is highly expressed on the apical membrane of the gastrointestinal tract, where it regulates the levels of intracellular substrates. P-glycoprotein is altered in disease, but the mechanisms that regulate the levels of P-glycoprotein are still being explored. The molecular motor myosin Vb (Myo5b) traffics diverse cargo to the apical membrane of intestinal epithelial cells. We hypothesized that Myo5b was responsible for the delivery of P-glycoprotein to the apical membrane of enterocytes. METHODS: We used multiple murine models that lack functional Myo5b or the myosin binding partner Rab11a to analyze P-glycoprotein localization. Pig and human tissue were analyzed to determine P-glycoprotein localization in the setting of MYO5B mutations. Intestinal organoids were used to examine P-glycoprotein trafficking and to assay P-glycoprotein function when MYO5 is inhibited. RESULTS: In mice lacking Myo5b or the binding partner Rab11a, P-glycoprotein was improperly trafficked and had decreased presence in the brush border of enterocytes. Immunostaining of a pig model lacking functional Myo5b and human biopsies from a patient with an inactivating mutation in Myo5b also showed altered localization of intestinal P-glycoprotein. Human intestinal organoids expressing the motorless MYO5B tail domain had colocalization with P-glycoprotein, confirming that P-glycoprotein was trafficked by MYO5B in human enterocytes. Inhibition of MYO5 in human intestinal cell lines and organoids resulted in decreased P-glycoprotein capacity. Additionally, inhibition of MYO5 in human colon cancer cells diminished P-glycoprotein activity and increased cell death in response to a chemotherapeutic drug. CONCLUSIONS: Collectively, these data demonstrate that Myo5b is necessary for the apical delivery of P-glycoprotein.

Isoorientin Alleviates DSS-Treated Acute Colitis in Mice by Regulating Intestinal Epithelial P-Glycoprotein (P-gp) Expression.

Isoorientin (ISO) is a naturally occurring flavonoid with diverse functional properties that mitigate the risk of diseases stemming from oxidation, inflammation, and cancer cell proliferation. P-glycoprotein (P-gp) is a vital component of the intestinal epithelium and may play a role in the onset of intestinal inflammatory conditions, such as inflammatory bowel disease (IBD). Recent studies have suggested that short-chain fatty acids (SCFAs) and secondary bile acids (SBAs) produced by the gut microbiota stimulate the increase of P-gp expression, alleviating excessive inflammation and thereby preservation of intestinal homeostasis. ISO has been shown to improve colon health and modulate the gut microbiota. In this study, we aimed to explore whether ISO can modulate the microbes and their metabolites to influence P-gp expression to alleviate IBD. First, the impact of ISO on dextran sulfate sodium (DSS)-treated colitis in mice was investigated. Second, 16S rRNA gene sequencing was conducted. The present study indicated that ISO mitigated the symptoms and pathological damage associated with DSS-treated colitis in mice. Western blot analysis revealed ISO upregulated P-gp in colon tissues, suggesting the critical role of P-gp protein in intestinal epithelial cells. 16S microbial diversity sequencing revealed ISO restored the richness and variety of intestinal microorganisms in colitis-bearing mice and enriched SCFA-producing bacteria, such as Lachnospiraceae_NK4A136_group. The experiments also revealed that the ISO fecal microbiota transplantation (FMT) inoculation of DSS-treated mice had similarly beneficial results. FMT mice showed a reduction in colitis symptoms, which was more pronounced in ISO-FMT than in CON-FMT mice. Meanwhile, ISO-FMT expanded the abundance of beneficial microorganisms, increased the expression of metabolites, such as SCFAs and total SBAs, and significantly upregulated the expression of P-gp protein. In addition, Spearman's correlation analysis demonstrated a positive correlation between the production of SCFAs and SBAs and the expression of P-gp. The present study identified that ISO increases the expression of P-gp in the intestinal epithelium by regulating intestinal microorganisms and their metabolites, which maintains colonic homeostasis, improves the integrity of the colonic epithelium, and alleviates colitis.

Mutational analysis reveals the importance of residues of the access tunnel inhibitor site to human P-glycoprotein (ABCB1)-mediated transport.

Human P-glycoprotein (P-gp) utilizes energy from ATP hydrolysis for the efflux of chemically dissimilar amphipathic small molecules and plays an important role in the development of resistance to chemotherapeutic agents in most cancers. Efforts to overcome drug resistance have focused on inhibiting P-gp-mediated drug efflux. Understanding the features distinguishing P-gp inhibitors from substrates is critical. Cryo-electron microscopy has revealed distinct binding patterns, emphasizing the role of the L-site or access tunnel in inhibition. We substituted 5-9 residues of the L-site with alanine to investigate whether the binding of a second inhibitor molecule to the L-site is required for inhibiting drug efflux. We reveal, for the first time, that mutations in the L-site affect the drug efflux activity of P-gp, despite their distance from the substrate-binding pocket (SBP). Surprisingly, after the mutations were introduced, inhibitors such as tariquidar and zosuquidar still inhibited drug efflux by mutant P-gps. Communication between the transmembrane helices (TMHs) and nucleotide-binding domains (NBDs) was evaluated using the ATPase assay, revealing distinct modulation patterns by inhibitors for the mutants, with zosuquidar exhibiting substrate-like stimulation of ATPase. Furthermore, L-site mutations abolished ATP-dependent thermal stabilization. In silico molecular docking studies corroborated the altered inhibitor binding due to mutations in the L-site residues, shedding light on their critical role in substrate transport and inhibitor interactions with P-gp. These findings suggest that inhibitors bind either to the SBP alone, and/or to alternate site(s) when the L-site is disabled by mutagenesis.

The metabolic activation of and platelet response to vicagrel vary with P-glycoprotein deficiency, rather than P-glycoprotein inhibition, in mice.

This study aimed to determine changes in the hydrolysis of vicagrel, a substrate drug of arylacetamide deacetylase (Aadac) and carboxylesterase 2 (Ces2), in P-glycoprotein (P-gp)-deficient or P-gp-inhibited mice and to elucidate the mechanisms involved.Male wild-type (WT) and P-gp knock-out (KO) mice were used to investigate the systemic exposure of vicagrel thiol active metabolite H4 and platelet response to vicagrel, and the mRNA and protein expression levels of intestinal Aadac and Ces2. Moreover, WT mice were administered vicagrel alone or in combination with elacridar (a potent P-gp inhibitor) to determine drug-drug interactions.Compared with WT mice, P-gp KO mice exhibited significant increases in the systemic exposure of H4, the protein expression levels of intestinal Aadac and Ces2, and inhibition of ADP-induced platelet aggregation by vicagrel. Further, the H4 exposure was positively correlated with intestinal Aadac protein expression levels but did not vary with short-term inhibition of P-gp efflux activity by elacridar.P-gp-deficient mice, rather than elacridar-treated mice, exhibited significant upregulation of intestinal Aadac and Ces2 and thus, enhanced metabolic activation of and platelet response to vicagrel, suggesting that the metabolic activation of vicagrel may vary with P-gp deficiency, not P-gp inhibition, in mice.

Innovative Approaches to Optimize Clinical Transporter Drug-Drug Interaction Studies.

Of the 450 cell membrane transporters responsible for shuttling substrates, nutrients, hormones, neurotransmitters, antioxidants, and signaling molecules, approximately nine are associated with clinically relevant drug-drug interactions (DDIs) due to their role in drug and metabolite transport. Therefore, a clinical study evaluating potential transporter DDIs is recommended if an investigational product is intestinally absorbed, undergoes renal or hepatic elimination, or is suspected to either be a transporter substrate or perpetrator. However, many of the transporter substrates and inhibitors administered during a DDI study also affect cytochrome P450 (CYP) activity, which can complicate data interpretation. To overcome these challenges, the assessment of endogenous biomarkers can help elucidate the mechanism of complex DDIs when multiple transporters or CYPs may be involved. This perspective article will highlight how creative study designs are currently being utilized to address complex transporter DDIs and the role of physiology-based -pharmacokinetic (PBPK) models can play.