An accurate haplotyping method using multiplex pyrosequencing with AS-PCR to detect ABCB1 haplotypes associated with rivaroxaban-derived hemorrhagic events.

In clinical practice, owing to the comprehensive genetic insights they offer, haplotypes have attracted greater attention than individual single nucleotide polymorphisms (SNPs). Due to the long distances across SNP locations, detecting the haplotype using genomic DNA is challenging. Current haplotyping methods are either expensive and labor-intensive (high-throughput DNA sequencing), or haplotyping a single clinical sample (computational approach) is impossible. Herein, we propose using mRNA as a haplotyping target to minimize the distance among SNPs and employing allele-specific PCR (AS-PCR) to pick up a desired haplotype, followed by multiplex pyrosequencing to type the alleles at the SNP location of interest. AS-PCR was improved by combining an additional 3'-phosphorylated modified probe to achieve the specific separation of two closely similar templates. Only the sample with more than two heterozygotes needs to be haplotyped; therefore, we propose a stratification strategy to screen the samples for further haplotyping. This method was evaluated by associating ABCB1 haplotypes with the rivaroxaban-derived side effect in a cohort of 505 patients with nephrotic syndrome, focusing on the SNPs of ABCB1: rs1236C > T, rs2677G > T/A, and rs3435C > T. We successfully identified five bleeding-related haplotypes: rs1236T-rs2677T-rs3435T, rs1236C-rs2677G-rs3435T, rs1236T-rs2677G-rs3435C, rs1236C-rs2677G-rs3435C, and rs1236T-rs2677T-rs3435C. We compared the results with those from the conventional computational algorithm PHASE and observed that PHASE results dismissed the impact of rs1236C-rs2677G-rs3435C and rs1236C-rs2677G-rs3435T on bleeding risk and erroneously suggested a false positive association of rs1236C-rs2677A-rs3435T with increased bleeding risk.

Structural insights into brassinosteroid export mediated by the Arabidopsis ABC transporter ABCB1.

Brassinosteroids (BRs) are steroidal phytohormones indispensable for plant growth, development, and responses to environmental stresses. The export of bioactive BRs to the apoplast is essential for BR signalling initiation, which requires binding of BR molecule to the extracellular domains of the plasma membrane-localized receptor complex. We have previously shown that the Arabidopsis thaliana ATP-binding cassette (ABC) transporter, ABCB19, functions as a BR exporter, and together with its close homologue, ABCB1, positively regulate BR signalling. Here, we demonstrate that ABCB1 is another BR transporter. The ATP hydrolysis activity of ABCB1 was stimulated by bioactive BRs, and its transport activity was confirmed in proteoliposomes and protoplasts. Structures of ABCB1 in substrate-unbound (apo), brassinolide (BL)-bound, and ATP plus BL-bound states were determined. In the BL-bound structure, BL was bound to the hydrophobic cavity formed by the transmembrane domain, and triggered local conformational changes. Together, our data provide additional insights into the ABC transporter-mediated BR export.

Involvement of everolimus­induced ABCB1 downregulation in drug­drug interactions.

Everolimus is an oral mammalian target of rapamycin (mTOR) inhibitor used in cancer chemotherapy and transplantation. Due to its therapeutic properties, everolimus has been used long-term in clinical practice. Drug interactions with everolimus during gastrointestinal absorption can alter the oral bioavailability of everolimus and/or concomitant drugs. However, the effects of everolimus on gastrointestinal absorption remain unknown. The present study assessed the impact of continuous exposure to everolimus on expression and function of the ATP-binding cassette (ABC) transporter ABCB1 and ABCG2 using a Caco-2 intestinal cell model. Caco-2 subline, Caco/EV, was established by continuously exposing Caco-2 cells to 1 µM everolimus. Cell viability was evaluated using WST-1 assay. mRNA levels were measured by reverse transcription-quantitative PCR. Transport activity of ABCB1 was evaluated through the cellular accumulation of Rhodamin 123, a substrate for ABCB1. The half-maximal inhibitory concentration (IC50) values for everolimus in Caco-2 and Caco/EV cells were 0.31 and 4.33 µM, respectively, indicating 14-fold resistance in Caco/EV cells. Sensitivity to paclitaxel and 7-ethyl-10-hydroxycamptothecin, which are substrates for ABCB1 and ABCG2, respectively, was enhanced in Caco/EV, but not in Caco-2 cells. The IC50 values of cisplatin were comparable in both cell lines. Furthermore, mRNA expression levels of ABCB1 and ABCG2 were lower in Caco/EV cells than in Caco-2 cells, and the cellular accumulation of Rhodamine 123 was significantly higher in Caco/EV cells. These findings demonstrated that continuous exposure to everolimus suppressed the expression and function of ABCB1 and ABCG2, suggesting potential drug-drug interactions via the suppression of ABCB1 and ABCG2 in the intestinal tract.

Intracellular TAS2Rs act as a gatekeeper for the excretion of harmful substances via ABCB1 in keratinocytes.

Bitter taste receptors (TAS2Rs) are not only expressed in the oral cavity but also in skin. Extraoral TAS2Rs are thought to be involved in non-taste perception and tissue-specific functions. Keratinocytes that express TAS2Rs in the skin provide a first-line defense against external threats. However, the functional roles of these receptors in host defense remain unclear. Here, we demonstrated the sensory role of intracellularly located TAS2Rs against toxic substances in keratinocytes. Although many G protein-coupled receptors elicit signals from the surface, TAS2Rs were found to localize intracellularly, possibly to the ER, in human keratinocytes and HaCaT cells. TAS2R38, one of the TAS2R members, activated the Gα12/13/RhoA/ROCK/p38 MAP kinase/NF-κB pathway upon stimulation by phenylthiocarbamide (PTC), an agonist for this receptor, leading to the production of ABC transporters, such as ABCB1, in these cells. Notably, treatment with bitter compounds, such as PTC and saccharin, induced the upregulation of ABCB1 in HaCaT cells. Mechanistically, intracellular TAS2R38 and its downstream signaling Gα12/13/RhoA/ROCK/p38 MAP kinase/NF-κB pathway were identified to be responsible for the above effect. Pretreatment with PTC prevented the accumulation of rhodamine 123 because of its excretion via ABCB1. Furthermore, pretreatment with PTC or saccharin counteracted the effect of the toxic compound, diphenhydramine, and pretreated HaCaT cells were found to proliferate faster than untreated cells. This anti-toxic effect was suppressed by treatment with verapamil, an ABCB1 inhibitor, indicating that enhanced ABCB1 helps clear toxic substances. Altogether, harmless activators of TAS2Rs may be promising drugs that enhance the excretion of toxic substances from the human skin.

Bleeding Events Associated with Rivaroxaban Therapy in Naive Patients with Nonvalvular Atrial Fibrillation: A Longitudinal Study from a Genetic Perspective with INR Follow-Up.

Background and Objectives: Rivaroxaban is a direct-acting anticoagulant used to prevent stroke in patients with atrial fibrillation. Rivaroxaban is a substrate for P-glycoprotein, which is encoded by the ABCB1 gene. Rivaroxaban is also metabolized by the CYP3A5 gene. Therefore, the current study is carried out to study the effects of polymorphisms in the ABCB1 and CYP3A5 genes, which may affect the plasma levels of rivaroxaban, with subsequent clinical outcomes (bleeding events) associated with the therapy. Materials and Methods: The study was conducted on 66 naive patients with atrial fibrillation treated with rivaroxaban. Blood samples of rivaroxaban were taken at 3 h and after 1 month following the administration of the drug to measure plasma levels. The blood level of rivaroxaban was measured with an HPLC-UV detector. Sanger sequencing was used to find polymorphisms in the targeted genes. Coagulation parameters were measured at 3 h and after 1 month of administration of rivaroxaban. Frequencies of bleeding events were recorded throughout the one-month course of drug therapy. Results: The heterozygous and homozygous mutant genotypes of ABCB1 (rs2032582, rs1045642, rs1128503, and rs4148738) and CYP3A5 (rs776746) showed lower plasma concentrations as compared to the wild-type genotype. ABCB1 (rs2032582, rs1045642, rs1128503, and rs4148738) and CYP3A5 (rs776746) gene polymorphisms had a statistically significant impact on the plasma concentration of rivaroxaban among the heterozygous and homozygous mutant genotypes compared to the wild-type genotype. The heterozygous variant of ABCB1 and homozygous variant of CYP3A5 suffered more events of bleeding. Conclusions: It was concluded that ABCB1 (rs2032582, rs1045642, rs1128503, and rs4148738) and CYP3A5 (rs776746) gene polymorphisms had a significant impact on the plasma levels of rivaroxaban in patients treated for atrial fibrillation on day three as well as after one month of the therapy. The lowest plasma levels were observed in patients with a homozygous variant of ABCB1 (rs2032582, rs1045642, or rs4148738) along with the CYP3A5*1/*3 allele. The heterozygous variant of ABCB1 SNPs and homozygous variant of CYP3A5 SNPs suffered more events of bleeding.

Epertinib counteracts multidrug resistance in cancer cells by antagonizing the drug efflux function of ABCB1 and ABCG2.

A significant hurdle in cancer treatment arises from multidrug resistance (MDR), often due to overexpression of ATP-binding cassette (ABC) transporters like ABCB1 and/or ABCG2 in cancer cells. These transporters actively diminish the efficacy of cytotoxic drugs by facilitating ATP hydrolysis-dependent drug efflux and reducing intracellular drug accumulation in cancer cells. Addressing multidrug-resistant cancers poses a significant challenge due to the lack of approved treatments, prompting the exploration of alternative avenues like drug repurposing (also referred to as drug repositioning) of molecularly targeted agents to reverse MDR-mediated by ABCB1 and/or ABCG2 in multidrug-resistant cancer cells. Epertinib, a potent inhibitor of EGFR and HER2 currently in clinical trials for solid tumors, was investigated for its potential to resensitize ABCB1- and ABCG2-overexpressing multidrug-resistant cancer cells to chemotherapeutic agents. Our findings reveal that at sub-toxic, submicromolar concentrations, epertinib restores the sensitivity of multidrug-resistant cancer cells to cytotoxic drugs in a concentration-dependent manner. The results demonstrate that epertinib enhances drug-induced apoptosis in these cancer cells by impeding the drug-efflux function of ABCB1 and ABCG2 without altering their expression. ATPase activity and molecular docking were employed to reveal potential interaction sites between epertinib and the drug-binding pockets of ABCB1 and ABCG2. In summary, our study demonstrates an additional pharmacological capability of epertinib against the activity of ABCB1 and ABCG2. These findings suggest that incorporating epertinib into combination therapy could be advantageous for a specific patient subset with tumors exhibiting high levels of ABCB1 or ABCG2, warranting further exploration.

Molecular Docking and Pharmacokinetic Profiling of Nab-paclitaxel as Advanced Chemotherapeutic Agent Against HER-2 Positive Breast Cancer Patients.

OBJECTIVE: The main objective of the study is to explore the potential molecular benefits of Nab-paclitaxel as an effective advanced chemotherapeutic agent for HER2-positive breast cancer patients. Specifically, the study aims to assess Nab-paclitaxel as a potential drug candidate for breast cancer treatment. METHODS: This study used bioinformatics and cheminformatics to analyze the HER2 signaling pathway and its possible interactions with Nab-Paclitaxel. This involves using pharmacokinetic profiling software to evaluate its physicochemical properties, analyzing its potential impact on gene expression modulation, and assessing its binding affinity to the HER2 receptor through molecular docking. RESULT: The results indicate that the most favorable docking pose occurs between chain B of the HER-2 receptor and Paclitaxel, with a binding energy of -9.4 kcal/mol. Notably, a hydrogen bond is observed in ARG849, with 3.0 Angstrom (Å). Previous research highlights Paclitaxel's impact on breast cancer patients' genes, particularly the ABCB1 gene responsible for P-glycoprotein production, contributing to drug resistance in chemotherapy. Nab-paclitaxel exhibits potential ease of metabolism, as it minimally inhibits drug-metabolizing cytochrome P450 enzymes. Additionally, despite initial concerns related to drug-likeness parameters and molecular weight discrepancies, the pharmacokinetic profile of Nab-Paclitaxel suggests improvements in delivery facilitated by an albumin-supported nanoparticle delivery mechanism. CONCLUSION: The binding energy confirms the secure docking of ligands to receptors, suggesting the stability of the interaction between them. Nevertheless, prolonged administration of Paclitaxel poses the risk of inducing drug resistance, a significant factor contributing to treatment failure. This emphasizes the need to explore new candidate drug combinations or identify alternative drug-binding interaction sites. Such endeavors hold the potential to enhance the effectiveness of drug treatments and address challenges associated with prolonged Paclitaxel use.

Analysis of ABCB1 Gene Polymorphisms and Their Impact on Tacrolimus Blood Levels in Kidney Transplant Recipients.

Tacrolimus (Tc) is an immunosuppressant used in transplant patients, but its therapeutic range is narrow, making precise dosing essential. This study investigates the association of three single nucleotide polymorphisms (SNPs) (ABCB1 3435C>T, 1236C>T, 2677G>T/A) with Tc levels over time to gain better insights into their role in personalized medicine. We conducted the study over four distinct periods: 1-14 days, 15-30 days, 31-60 days, and beyond 60 days post-transplantation. The analysis included allele, genotype, haplotype, and diplotype frequencies of the three SNPs concerning Tc blood levels. Statistical significance was determined, and false discovery rate (PFDR) correction was applied where appropriate. Significant associations were found between the C (ABCB1 C1236T), A alleles (ABCB1 G2677T/A), the CAC haplotype and lower Tc levels. The CAC-TGT and TGT-TGT diplotypes significantly influence how patients metabolize the drug. The TGT haplotype and the AA genotype (ABCB1 G2677T/A) were associated with higher Tc levels, suggesting a long-term genetic influence. Genetic factors, specifically certain SNPs and diplotypes, significantly impact Tc blood levels, with their influence varying over time.

Synthesis and evaluation of novel tetrahydroisoquinoline-benzo[h]chromen-4-one conjugates as dual ABCB1/CYP1B1 inhibitors for overcoming MDR in cancer.

The emergence of multidrug resistance (MDR) in malignant tumors is one of the major threats encountered currently by many chemotherapeutic agents. Among the various mechanisms involved in drug resistance, P-glycoprotein (P-gp, ABCB1), a member of the ABC transporter family that significantly increases the efflux of various anticancer drugs from tumor cells, and the metabolic enzyme CYP1B1 are widely considered to be two critical targets for overcoming MDR. Unfortunately, no MDR modulator has been approved by the FDA to date. In this study, based on pharmacophore hybridization, bioisosteric and fragment-growing strategies, we designed and synthesized 11 novel tetrahydroisoquinoline-benzo[h]chromen-4-one conjugates as dual ABCB1/CYP1B1 inhibitors. Among them, the preferred compound A10 exhibited the best MDR reversal activity (IC50 = 0.25 µM, RF = 44.4) in SW620/AD300 cells, being comparable to one of the most potent third-generation P-gp inhibitors WK-X-34. In parallel, this dual ABCB1/CYP1B1 inhibitory effect drives compound A10 exhibiting prominent drug resistance reversal activity to doxorubicin (IC50 = 4.7 µM, RF = 13.7) in ABCB1/CYP1B1-overexpressing DOX-SW620/AD300-1B1 resistant cells, which is more potent than that of the CYP1B1 inhibitor ANF. Furthermore, although compound A2 possessed moderate ABCB1/CYP1B1 inhibitory activity, it showed considerable antiproliferative activity towards drug-resistant SW620/AD300 and MKN45-DDP-R cells, which may be partly related to the increase of PUMA expression to promote the apoptosis of the drug-resistant MKN45-DDP-R cells as confirmed by proteomics and western blot assay. These results indicated that the tetrahydroisoquinoline-benzo[h]chromen-4-one conjugates may provide a fundamental scaffold reference for further discovery of MDR reversal agents.

Mobocertinib antagonizes multidrug resistance in ABCB1- and ABCG2-overexpressing cancer cells: in vitro and in vivo studies.

Overexpression of ATP-binding cassette (ABC) transporters, particularly ABCB1 and ABCG2, strongly correlates with multidrug resistance (MDR), rendering cancer chemotherapy ineffective. Exploration and identification of novel inhibitors targeting ABCB1 and ABCG2 are necessary to overcome the related MDR. Mobocertinib is an approved EGFR/HER2 inhibitor for non-small cell lung cancer (NSCLC) with EGFR exon 20 insertion mutations. This study demonstrates that mobocertinib can potentially reverse ABCB1- and ABCG2-mediated MDR. Our findings indicate a strong interaction between mobocertinib and these two proteins, supported by its high binding affinity with ABCB1 and ABCG2 models. Through inhibiting the drug efflux function of ABCB1 and ABCG2, mobocertinib facilitates substrate drugs accumulation, thereby re-sensitizing substrate drugs in drug-resistant cancer cells. Additionally, mobocertinib inhibited the ATPase activity of ABCB1 and ABCG2 without changing the expression levels or subcellular localization. In the tumor-bearing mouse model, mobocertinib boosted the antitumor effect of paclitaxel and topotecan, resulting in tumor regression. In summary, our study uncovers a novel potential for repurposing mobocertinib as a dual inhibitor of ABCB1 and ABCG2, and suggests the combination of mobocertinib with substrate drugs as a strategy to counteract MDR.

MBD2 regulates the progression and chemoresistance of cholangiocarcinoma through interaction with WDR5.

BACKGROUND: Cholangiocarcinoma (CCA) is a highly malignant, rapidly progressing tumor of the bile duct. Owing to its chemoresistance, it always has an extremely poor prognosis. Therefore, detailed elucidation of the mechanisms of chemoresistance and identification of therapeutic targets are still needed. METHODS: We analyzed the expression of MBD2 (Methyl-CpG-binding domain 2) in CCA and normal bile duct tissues using the public database and immunohistochemistry (IHC). The roles of MBD2 in CCA cell proliferation, migration, and chemoresistance ability were validated through CCK-8, plate cloning assay, wound healing assays and xenograft mouse model. In addition, we constructed a primary CCA mouse model to further confirm the effect of MBD2. RNA-seq and co-IP-MS were used to identify the mechanisms by how MBD2 leads to chemoresistance. RESULTS: MBD2 was upregulated in CCA. It promoted the proliferation, migration and chemoresistance of CCA cells. Mechanistically, MBD2 directly interacted with WDR5, bound to the promoter of ABCB1, promoted the trimethylation of H3K4 in this region through KMT2A, and activated the expression of ABCB1. Knocking down WDR5 or KMT2A blocked the transcriptional activation of ABCB1 by MBD2. The molecular inhibitor MM-102 targeted the interaction of WDR5 with KMT2A. MM-102 inhibited the expression of ABCB1 in CCA cells and decreased the chemoresistance of CCA to cisplatin. CONCLUSION: MBD2 promotes the progression and chemoresistance of CCA through interactions with WDR5. MM-102 can effectively block this process and increase the sensitivity of CCA to cisplatin.

Gilteritinib reverses ABCB1-mediated multidrug resistance: Preclinical in vitro and animal investigations.

Multi-drug resistance (MDR) poses a significant challenge to cancer treatment. Targeting ATP-binding cassette subfamily B member 1 (ABCB1) is a viable strategy for overcoming MDR. This study examined the preclinical in vitro and animal studies that used gilteritinib, a FLT3 inhibitor that reverses ABCB1-mediated MDR. At nontoxic levels, gilteritinib significantly increased the susceptibility of cancer cells overexpressing ABCB1 to chemotherapeutic drugs. Furthermore, it impaired the development of drug-resistant cell colonies and 3D spheroids. Studies on the reversal mechanism have shown that gilteritinib can directly bind to the drug-binding site of ABCB1, inhibiting drug efflux activity. Consequently, the substrate's drug cytotoxicity increases in MDR cells. Furthermore, gilteritinib increased ATPase activity while leaving ABCB1 expression and subcellular distribution unchanged and inhibited AKT or ERK activation. Docking analysis indicated that Gilteritinib could interact with the drug-binding site of the ABCB1 transporter. In vivo studies have shown that gilteritinib improves the antitumor efficacy of paclitaxel in nude mice without obvious toxic effects. In conclusion, our preclinical investigations show that gilteritinib has the potential to successfully overcome ABCB1-mediated MDR in a clinical environment when combined with substrate medicines.

Zosuquidar Promotes Antitumor Immunity by Inducing Autophagic Degradation of PD-L1.

The intracellular distribution and transportation process are essential for maintaining PD-L1 (programmed death-ligand 1) expression, and intervening in this cellular process may provide promising therapeutic strategies. Here, through a cell-based high content screening, it is found that the ABCB1 (ATP binding cassette subfamily B member 1) modulator zosuquidar dramatically suppresses PD-L1 expression by triggering its autophagic degradation. Mechanistically, ABCB1 interacts with PD-L1 and impairs COP II-mediated PD-L1 transport from ER (endoplasmic reticulum) to Golgi apparatus. The treatment of zosuquidar enhances ABCB1-PD-L1 interaction and leads the ER retention of PD-L1, which is subsequently degraded in the SQSTM1-dependent selective autophagy pathway. In CT26 mouse model and a humanized xenograft mouse model, zosuquidar significantly suppresses tumor growth and accompanies by increased infiltration of cytotoxic T cells. In summary, this study indicates that ABCB1 serves as a negative regulator of PD-L1, and zosuquidar may act as a potential immunotherapy agent by triggering PD-L1 degradation in the early secretory pathway.

In vitro assessment of the risk of ABCB1-mediated drug-drug interaction between rivaroxaban and tacrolimus in human embryonic kidney 293 recombinant cell lines.

Background: In lung transplant patients, direct oral anticoagulants are often taken in combination with immunosuppressive drugs such as tacrolimus. Since tacrolimus is a substrate and inhibitor of the efflux protein ABCB1, also transporting direct oral anticoagulants, a possible drug-drug interaction mediated by competition for this transporter needs to be investigated. Objectives: To determine the in vitro effect of tacrolimus on ABCB1-mediated rivaroxaban transport in order to support clinician practice. Methods: Recombinant cell line models, based on human embryonic kidney 293 cells, were generated by a stable transfection process to overexpress ABCB1 or not (control cells). The impact of tacrolimus on ABCB1-mediated rivaroxaban transport was assessed by accumulation experiments. Results: ABCB1 expression decreased the cellular accumulation of rivaroxaban and tacrolimus at their respective clinically relevant concentrations when compared with control cells. This confirms the involvement of ABCB1 in the active transport of tacrolimus and rivaroxaban. However, tacrolimus had no significant influence on rivaroxaban disposition at those clinically relevant concentrations. Conclusion: Our study does not provide evidence for a possible interaction between tacrolimus and rivaroxaban when used together in practice.

Brain Exposure to the Macrocyclic ALK Inhibitor Zotizalkib is Restricted by ABCB1, and Its Plasma Disposition is Affected by Mouse Carboxylesterase 1c.

Zotizalkib (TPX-0131), a fourth-generation macrocyclic anaplastic lymphoma kinase (ALK) inhibitor, is designed to overcome resistance due to secondary ALK mutations in non-small cell lung cancer (NSCLC). We here evaluated the pharmacokinetic roles of the ABCB1 (P-gp/MDR1) and ABCG2 (BCRP) efflux transporters, OATP1 influx transporters and the metabolizing enzymes CES1 and CYP3A in plasma and tissue disposition of zotizalkib after oral administration in relevant mouse models. Zotizalkib was efficiently transported by hABCB1 in vitro. In vivo, a significant ∼9-fold higher brain-to-plasma ratio was observed in Abcb1a/b-/- and Abcb1a/b;Abcg2-/- compared to wild-type mice. No change in brain disposition was observed in Abcg2-/- mice, suggesting that mAbcb1a/b markedly restricts the brain accumulation of zotizalkib. ABCB1-mediated efflux of zotizalkib was completely inhibited by elacridar, a dual ABCB1/ABCG2 inhibitor, increasing brain exposure without any signs of acute CNS-related toxicities. In Oatp1a/b-/- mice, no marked changes in plasma exposure or tissue-to-plasma ratios were observed, indicating that zotizalkib is not a substantial in vivo substrate for mOatp1a/b. Zotizalkib may further be metabolized by CYP3A4 but only noticeably at low plasma concentrations. In Ces1-/- mice, a 2.5-fold lower plasma exposure was seen compared to wild-type, without alterations in tissue distribution. This suggests increased plasma retention of zotizalkib by binding to the abundant mouse plasma Ces1c. Notably, the hepatic expression of human CES1 did not affect zotizalkib plasma exposure or tissue distribution. The obtained pharmacokinetic insights may be useful for the further development and optimization of therapeutic efficacy and safety of zotizalkib and related compact macrocyclic ALK inhibitors.

Development of Natural-Product-Inspired ABCB1 Inhibitors Through Regioselective Tryptophan C3-Benzylation.

The emergence of drug resistance in cancer cells eventually causing relapse is a serious threat that demands new advances. Upregulation of the ATP-dependent binding cassette (ABC) transporters, such as ABCB1, significantly contributes to the emergence of drug resistance in cancer. Despite more than 30 years of therapeutic discovery, and several generations of inhibitors against P-gp, the search for effective agents that minimize toxicity to human cells, while maintaining efflux pump inhibition is still underway. Leads derived from natural product scaffolds are well-known to be effective in various therapeutic approaches. Inspired by the biosynthetic pathway to Nocardioazine A, a marine alkaloid known to inhibit the P-gp efflux pump in cancer cells, we devised a regioselective pathway to create structurally unique indole-C3-benzyl cyclo-L-Trp-L-Trp diketopiperazines (DKPs). Using bat cells as a model to derive effective ABCB1 inhibitors for targeting human P-gp efflux pumps, we have recently identified exo-C3-N-Dbn-Trp2 (13) as a lead ABCB1 inhibitor. This C3-benzylated lead inhibited ABCB1 better than Verapamil.[21] Additionally, C3-N-Dbn-Trp2 restored chemotherapy sensitivity in drug-resistant human cancer cells and had no adverse effect on cell proliferation in cell cultures. For a clearer structure-activity relationship, we developed a broader screen to test C3-functionalized pyrroloindolines as ABCB1 inhibitors and observed that C3-benzylation is outperforming respective isoprenylated derivatives. Results arising from the molecular docking studies indicate that the interactions at the access tunnel between ABCB1 and the inhibitor result in a powerful predictor for the efficacy of the inhibitor. Based on fluorescence-based assays, we conclude that the most efficacious inhibitor is the p-cyano-derived exo-C3-N-Dbn-Trp2 (33 a), closely followed by the p-nitro substituted analogue. By combining assay results with molecular docking studies, we further correlate that the predictions based on the inhibitor interactions at the access tunnel provide clues about the design of improved ABCB1 inhibitors. As it has been well documented that ABCB1 itself is powerfully engaged in multi-drug resistance, this work lays the foundation for the design of a new class of inhibitors based on the endogenous amino acid-derived cyclo-L-Trp-L-Trp DKP scaffold.

Gefitinib-Induced Severe Dermatological Adverse Reactions: A Case Report and Pharmacogenetic Profile.

Gefitinib is a selective inhibitor of the epidermal growth factor receptor that is used to treat advanced and metastatic non-small cell lung cancer (NSCLC). Dermatological adverse reactions are most commonly associated with gefitinib treatment. The cause of adverse reactions in individuals is multifactorial. Pharmacogenetics is an effective tool to detect such adverse reactions. This case report describes a female patient with NSCLC who was administered gefitinib at a dose of 250 mg/day. However, due to severe adverse dermatological reactions, the treatment was interrupted for 15 d and antibiotic therapy was administered to manage the skin rashes, maculopapular rashes, and hyperpigmentation. Treatment adherence was adequate, and no drug interactions were detected. A pharmacogenetic analysis revealed homozygosity in the ATP-binding cassette (ABC)-B1 rs1128503 (c.1236A>G), heterozygosity in ABCG2 rs2231142 (c.421G>T) and rs2622604 (c.-20+614T>C), and a non-functional variant of the cytochrome P450 family 3, subfamily A, member 5 (CYP3A5). The relationship between altered genetic variants and the presence of adverse reactions induced by gefitinib is still controversial. Overall, this case report highlights the importance of continuing to study pharmacogenetics as predictors of adverse drug reactions.

ATP-binding cassette transporter inhibitor potency and substrate drug affinity are critical determinants of successful drug delivery enhancement to the brain.

BACKGROUND: Pharmacotherapy for brain diseases is severely compromised by the blood-brain barrier (BBB). ABCB1 and ABCG2 are drug transporters that restrict drug entry into the brain and their inhibition can be used as a strategy to boost drug delivery and pharmacotherapy for brain diseases. METHODS: We employed elacridar and tariquidar in mice to explore the conditions for effective inhibition at the BBB. Abcg2;Abcb1a/b knockout (KO), Abcb1a/b KO, Abcg2 KO and wild-type (WT) mice received a 3 h i.p. infusion of a cocktail of 8 typical substrate drugs in combination with elacridar or tariquidar at a range of doses. Abcg2;Abcb1a/b KO mice were used as the reference for complete inhibition, while single KO mice were used to assess the potency to inhibit the remaining transporter. Brain and plasma drug levels were measured by LC-MS/MS. RESULTS: Complete inhibition of ABCB1 at the BBB is achieved when the elacridar plasma level reaches 1200 nM, whereas tariquidar requires at least 4000 nM. Inhibition of ABCG2 is more difficult. Elacridar inhibits ABCG2-mediated efflux of weak but not strong ABCG2 substrates. Strikingly, tariquidar does not enhance the brain uptake of any ABCG2-subtrate drug. Similarly, elacridar, but not tariquidar, was able to inhibit its own brain efflux in ABCG2-proficient mice. The plasma protein binding of elacridar and tariquidar was very high but similar in mouse and human plasma, facilitating the translation of mouse data to humans. CONCLUSIONS: This work shows that elacridar is an effective pharmacokinetic-enhancer for the brain delivery of ABCB1 and weaker ABCG2 substrate drugs when a plasma concentration of 1200 nM is exceeded.

Differing temperature dependencies of functional homologs zebrafish Abcb4 and human ABCB1.

The ATP binding cassette (ABC) transporters human ABCB1 and zebrafish (Danio rerio) Abcb4 are functionally homologous multixenobiotic/multidrug (MXR/MDR) efflux transporters that confer the efflux of a broad range of diverse chemical compounds from the cell. As ATPases, the transporters utilize the energy released by ATP cleavage for protein conformation changes and concomitant active transport of substrate compounds. The temperatures, at which human ABCB1 and zebrafish Abcb4 need to function, can substantially differ: Whereas the ambient temperature of human ABCB1, which is that of the human body, is constant, zebrafish Abcb4 has to be active in a wider temperature range as the body temperature of zebrafish can considerably vary, depending on the ambient water temperature (18°C-40°C). Here, we examined the effect of temperature on the ATPase activities of recombinant human ABCB1 and zebrafish Abcb4 generated with the baculovirus expression system. Incubation temperatures for enzyme reactions were set to 37°C and 27°C, corresponding to the human body temperature and the cultivation temperature of zebrafish in our lab, respectively. For stimulation and inhibition of zebrafish Abcb4 and human ABCB1 ATPase activities verapamil and cyclosporin A were added at different concentrations and 50% effect concentrations (EC50) were determined. The different temperatures had a stronger effect on the human ABCB1 than on the zebrafish Abcb4 ATPase: Differences between EC50 values for verapamil at 37°C and 27°C, respectively, were 1.8-fold for human ABCB1 but only 1.2-fold for zebrafish Abcb4. Activation energies (Ea) of basal and verapamil-stimulated ATPases, calculated based on the Arrhenius equation, were 2-fold (basal) and 1.5-fold (verapamil-stimulated) higher for human ABCB1 than for zebrafish Abcb4. The differences between zebrafish Abcb4 and human ABCB1 ATPases in temperature sensitivity and activation energy could be important for the comparison of the functional properties of the two transporter proteins in the context of pharmaco-/toxicokinetics. Related to this, our finding that at equal reaction conditions the zebrafish Abcb4 ATPase activity tended to be generally higher than that of human ABCB1 may also be important, as this may point to a higher substrate compound transport rate of Abcb4.

Inflammation alters the expression pattern of drug transporters during Caco-2 cell stimulation and azoxymethane-induced colon tumorigenesis.

Drug transporters play a pivotal role in modulating drug disposition and are subject to alterations under inflammatory conditions. This study aimed to elucidate the intricate expression patterns of drug transporters during both acute and chronic inflammation, which are closely linked to malignant transformation. To investigate acute inflammation, we employed an in vitro model by subjecting Caco-2 cells to various inflammatory stimuli (IL-1ß, TNF-α, or LPS) individually or in combination. The successful induction of inflammation was confirmed by robust increases in IL-6 and NO production. Notably, inflamed Caco-2 cells exhibited significantly diminished levels of ABCB1 and ABCG2, while the expression of ABCC2 was upregulated. For chronic inflammation induction in vivo, we employed the well-established AOM/DSS mouse model known for its association with colitis-driven tumorigenesis. Persistent inflammation was effectively monitored throughout the experiment via elevated IL-6 and NO levels. The sequential stages of tumorigenesis were confirmed through Ki-67 immunohistochemistry. Intriguingly, we observed gradual alterations in the expression patterns of the studied drug transporters during stepwise induction, with ABCB1, ABCG2, and ABCC1 showing downregulation and ABCC2 exhibiting upregulation. Immunohistochemistry further revealed dynamic changes in the expression of ABCB1 and ABCC2 during the induction cycles, closely paralleling the gradual increase in Ki-67 expression observed during the development of precancerous lesions. Collectively, our findings underscore the significant impact of inflammation on drug transporter expression, potentially influencing the process of malignant transformation of the colon.