Pump P-glycoprotein Research Articles

Role of Natural Phytoconstituents as a Potential Bioenhancer of Anti-Cancer and Anti-Microbial Agents: Spotlight on the Mechanism of Action, Clinical Studies and Patents

A drug design strategy with reduced side effects and economic feasibility is desirable for fatal diseases. Increasing the bioavailability of a drug using a bioenhancer is a smart strategy. Herbal/natural bioenhancers with no probable side effects are an ideal choice to enhance the pharmacokinetics of a therapeutic drug synergistically. The mechanism of bioenhancers relies on the retention of the drug molecule in the cell without causing any changes in the metabolic activity. Most of the herbal bioenhancers achieve this feat by inhibiting metabolic enzymes such as cytochrome P450 and Uridine 5′-diphospho-glucuronosyltransferase. The efflux pump p-glycoprotein, responsible for removal of xenobiotics, is also inhibited by herbal/natural bioenhancers. The increased bioavailability because of the higher Cmax and tmax of chemotherapeutics or anti-infectious agents such as rifampicin can result in a lower drug dosage regimen. The reduction in drug dosage is directly linked to fewer side effects and economic viability. Further, there is a significant effort in clinical trials to incorporate bioenhancers in drug regimens for cancer. The role of herbal/natural bioenhancers and their potential to augment the bioavailability of therapeutics used in cancer and infectious diseases, with a focus on the mechanisms of action, clinical studies and patents, have been summarized in this review article.

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

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. 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. 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. Collectively, these data demonstrate that Myo5b is necessary for the apical delivery of P-glycoprotein.

Complex formation of ML324, the histone demethylase inhibitor, with essential metal ions: Relationship between solution chemistry and anticancer activity

N-(3-(dimethylamino)propyl-4-(8-hydroxyquinolin-6-yl)benzamide (ML324, HL) is a potent inhibitor of the iron-containing histone demethylase KDM4, a recognized potential target of cancer therapeutics. Herein, we report the proton dissociation and complex formation processes of ML324 with essential metal ions such as Fe(II), Fe(III), Cu(II) and Zn(II) using UV–visible, fluorescence, electron paramagnetic resonance and 1H NMR spectroscopic methods. The electrochemical behaviour of the copper and iron complexes was characterized by cyclic voltammetry and spectroelectrochemistry. The solid phase structure of ML324 analysed by X-ray crystallography is also provided. Based on the solution equilibrium data, ML324 is present in solution in H2L+ form with a protonated dimethylammonium moiety at pH 7.4, and this (N,O) donor bearing ligand forms mono and bis complexes with all the studied metal ions and the tris-ligand species is also observed with Fe(III). At pH 7.4 the metal binding ability of ML324 follows the order: Fe(II) < Zn(II) < Cu(II) < Fe(III). Complexation with iron resulted in a negative redox potential (E'1/2 = −145 mV vs. NHE), further suggesting that the ligand has a preference for Fe(III) over Fe(II). ML324 was tested for its anticancer activity in chemosensitive and resistant human cancer cells overexpressing the efflux pump P-glycoprotein. ML324 exerted similar activity in all tested cells (IC50 = 1.9–3.6 μM). Co-incubation and complexation of the compound with Cu(II) and Zn(II) had no impact on the cytotoxicity of ML324, whereas Fe(III) decreased the toxicity in a concentration-dependent manner, and this effect was more pronounced in the multidrug resistant cells.

Vitexin along with verapamil downregulates efflux pump P-glycoprotein in macrophages and potentiate M1 to M2 switching via TLR4-NF-κB-TNFR2 pathway in lipopolysaccharide treated mice

The lipopolysaccharide, a microbial toxin, is one of the major causative agents of sepsis. P-gp expression and its functions are altered during inflammation. LPS has been known to impair the functions of P-gp, an efflux transporter. But the effect of LPS on P-gp expression in murine peritoneal macrophages is poorly understood. Molecular docking studies reveal that vitexin is a potent substrate and verapamil a potent inhibitor of P-gp. In the present experimental study, the curative potential of vitexin as a fruit component and verapamil treated as a control inhibitor of P-gp was examined in a murine LPS sepsis model. The effects of vitexin and verapamil on P-gp expression in macrophages correlating with changes in macrophage polarization and associated functional responses during LPS induced sepsis were studied. Peritoneal macrophages of LPS (10 mg/kg body weight) challenged mice exhibited elevated levels of H2O2, superoxide, and NO in parallel with lower antioxidant activity. LPS treatment increased P-gp expression through increased TLR4/expression. However, LPS challenged mice treated with vitexin (5 mg/kg body weight) + verapamil (5 mg/kg body weight) showed higher anti-oxidant enzyme activity (SOD, CAT and GRx) resulting in reduced oxidative stress. This combination treatment also elevated TNFR2, concomitant with down-regulation of TLR4, NF-κB and P-gp expression in murine peritoneal macrophages, resulting in a switch from M1 to M2 polarisation of macrophages and reduced inflammatory responses. In conclusion, combined vitexin and verapamil treatment could be used as a promising therapy to regulate P-gp expression and protection against LPS mediated sepsis and inflammatory damages.

BCMA-Targeted Bortezomib Nanotherapy (BCMA-BTZ-Nps) Targets Tumor, Enhances Therapeutic Efficacy, Triggers Immunogenic Cell Death, Overcomes Drug Resistance, and Reduces Off-Target Toxicity in Multiple Myeloma (MM)

Bortezomib (BTZ) is a standard-of-care treatment for multiple myeloma (MM). In addition to its direct anti-MM effects, we recently reported that BTZ triggers immunogenic cell death associated with improved outcome. However, adverse effects limit its long-term clinical use. To improve its therapeutic index, we encapsulated bortezomib in PEG-PLGA nanoparticles linked with antibodies that target B-cell maturation antigen (BCMA-BTZ-NPs) to specifically target MM cells. BCMA-BTZ-NPs showed significantly increased cellular internalization into wild-type (WT) H929 cells after 6h (99.2±0.7%) than non-targeted NPs (57.7±0.8%) (p&amp;lt;0.0001), with a time-dependent increase in internalization. Importantly, cellular internalization of the BCMA-BTZ-NPs in BCMA-KO H929 cells (15.9±2.5%) was significantly depleted (p&amp;lt;0.0001). BCMA-BTZ-NPs showed target-specific cytotoxicity against MM cell lines and primary tumor cells from MM patients. Furthermore, BCMA-BTZ-NPs induced cell death more efficiently than non-targeted nanoparticles or free BTZ, triggering potent mitochondrial depolarization assessed by JC1 staining in MM.1S (79.06±3.7% vs. 57.4±1.9% for BCMA-BTZ-NPs vs non-targeted NPs, p&amp;lt;0.01); and H929 cells (47.23±1.9% vs. 31.26±0.7% for BCMA-BTZ-NPs vs. non-targeted NPs, p&amp;lt;0.001). This was associated with enhanced activation of caspases 8, 9 and 3, followed by apoptosis. We also found that BCMA-BTZ-NPs were still effective in BTZ-resistant MM cell lines (36.66±1.8% residual chymotryptic activity in RPMI-Dox40 [resistant] and 40.29±1.0% in MM.1S [sensitive] cells), whereas free BTZ showed no inhibitory effect (100.01±4.6% residual chymotryptic activity in RPMI-Dox40), likely due to BCMA-BTZ-NPs entering the cell through receptor-mediated uptake which averts acquired BTZ resistance based on the drug pump P-glycoprotein (PgP). Importantly, BCMA-BTZ-NPs also enhanced immunogenic cell death (ICD) and activated the autophagic pathway more than free BTZ, evidenced by increased calreticulin cell surface exposure (free BTZ: 45.13±3.7% vs BCMA-BTZ-NP: 71.5±3.4%,) and induction of T-cell proliferation in both MM.1S (free BTZ: 36.5±2.6% vs. BCMA-BTZ-NP: 65.2±2.3%, p&amp;lt;0.001) and AMO-1 cells (free BTZ: 37.03±1.3% vs. BCMA-BTZ-NP: 66.33±3.3%, p&amp;lt;0.001). Of note, increased levels of TNFα, Granzyme B, and IFNγ were also present in BCMA-BTZ-NPs treated MM cells with DC (Dendritic cells) and T cells co-culture supernatants. Finally, we evaluated the in vivo target selectivity and therapeutic efficacy of BCMA-BTZ-NPs against human MM cells in a murine xenograft model. BCMA-BTZ-NPs selectively accumulated in the tumor more efficiently than non-targeted BTZ-NPs and were associated with more efficient tumor reduction and host survival. In summary, our study indicates that BCMA-BTZ nanotherapy accumulates at the tumor site, enhances therapeutic efficacy, triggers immunogenic cell death, overcomes drug resistance, and reduces off-target toxicity. These results provide the framework for clinical evaluation of BCMA-BTZ-NPs to increase the therapeutic index of BTZ and improve patient outcome in MM.

Oral paclitaxel and dostarlimab with or without trastuzumab in early-stage, high-risk breast cancer: Results from the neoadjuvant ISPY 2 TRIAL.

LBA612 Background: I-SPY2 is a multicenter, phase 2 trial using response-adaptive randomization within biomarker subtypes including MammaPrint (MP) status to evaluate novel neoadjuvant agents in high-risk breast cancer. The primary endpoint is pathologic complete response (pCR). Oral Paclitaxel and encequidar (OPE) is an oral combination of paclitaxel (P) with a p-glycoprotein pump inhibitor, encequidar. Dostarlimab (D) is an intravenous (IV) PD-1 inhibitor. Methods: Women with tumors ≥ 2.5cm and MP high risk cancers (MP1 = MP high; MP2 = MP ultra-high) were treated starting Oct 5, 2020. Treatment included OPE (Oral P 205mg/m2 + encequidar 12.9mg) on days 1-3 weekly x 12 and D 500 mg IV given q 3 weeks x 4, followed by doxorubicin/cyclophosphamide (AC) q 2-3 weeks x 4. Patients with HER2+ disease received IV weekly trastuzumab (T) during the first 12 weeks. The control arm was weekly IV P x 12 with or without trastuzumab followed by AC q 2-3 weeks x 4. OPE + D was eligible to graduate [85% chance of success in a 300-person phase 3 neoadjuvant trial with a pCR endpoint] in any of the pre-defined signatures. Results: 113 (78 HR+HER2-, 17 HR-HER2- and 18 HER2+ patients) received OPE + D +/- T. The control arm included 388 historical controls (201 HR+Her2-, 156 HR-HER2-, 31 HER2+). 77 patients (70 HR+ and 7 HR-) were MP1 and 36 patients (24 HR+ and 12 HR-) were MP2. Safety events of note for OPE + D versus IV P include increased rates of nausea (85% vs. 72%) diarrhea (77% vs. 41%). There was no significant difference in rates of neutropenia (23% vs.17%). Peripheral neuropathy (37% vs. 64%) and alopecia (59% vs. 66%) were significantly decreased. Immune related adverse events (irAEs) were lower than expected. Conclusions: Although both OPE and D have both been shown to have efficacy in other settings, combination therapy with OPE + D did not graduate in any of the predefined subtypes. In the HR+ signature where we would not expect a benefit of D, we see equal response to OPE with decreased rates of peripheral neuropathy and alopecia, which suggest this oral agent may be an attractive alternative to IV P in this subgroup and is under consideration in ISPY 2.2. We did not observe the expected improvement in pCR rates seen with PD-1 inhibitors in the HR- or MP2 subtypes (over P alone historic control). In addition, the irAEs were less than expected. Together these findings suggest interference of OPE with D. A potential mechanism of interference could be change in microbiome with the use of OPE vs. IV P, as the microbiome is known to influence the efficacy of immunotherapy. The source of interference is being investigated. Clinical trial information: NCT01042379 . [Table: see text]

Oral paclitaxel, carboplatin, and dostarlimab (OPE/Cb/D) without and with trastuzumab in early-stage, high-risk breast cancer: Results from the neoadjuvant I-SPY 2 TRIAL.

LBA520 Background: I-SPY 2 is a multicenter trial using response-adaptive randomization within biomarker subtypes including MammaPrint (MP) risk to evaluate novel neoadjuvant agents in high-risk breast cancer. Oral paclitaxel and encequidar (OPE) is an oral combination of paclitaxel (P) with a p-glycoprotein pump inhibitor, encequidar, to enhance gastrointestinal (GI) absorption. Dostarlimab (D) is an anti-PD-1 monoclonal antibody. Methods: Women with tumors ≥ 2.5cm and MP high-risk cancers were screened and treated starting Oct 5, 2020. Treatment included Oral Paclitaxel 205mg/m2 and encequidar 12.9 mg on days 1-3, Carboplatin (Cb) AUC 1.5 on day 1 weekly x 12, and Dostarlimab (D) 500 mg every 3 weeks x 4, followed by doxorubicin/ cyclophosphamide (AC) every 2-3 weeks x 4. The control arm was IV P weekly x 12 followed by AC every 2-3 weeks x 4. For patients with HER2+ disease, weekly Trastuzumab (T) was administered during the first 12 weeks. The arm was eligible for graduation [&gt; 85% chance of success in a 300-person phase 3 neoadjuvant trial with a pathologic complete response (pCR) endpoint] in 10 predefined signatures. Results: 106 patients (44 HR+HER2-, 56 HR-HER2- (TN), 6 HER2+) received OPE/Cb/D ± T. The control arm included 388 historical controls (201 HR+HER2-, 156 TN, 31 HER2+). 22 patients (20 HR+, 2 HR-) in OPE/Cb/D were MP1 (MP high) and 84 patients (29 HR+, 55 HR-) were MP2 (MP ultra-high). OPE/Cb/D graduated in the TN signature and accrual was stopped. Conclusions: OPE/Cb/D graduated in the TN signature with a higher predicted pCR rate compared to control. OPE and D have been, individually, shown to have efficacy in other settings. The lower-than-expected pCR rate and lower irAEs with triplet therapy (taxane, platinum, immune checkpoint inhibitor [ICI]) in TN compared to prior IV chemo + ICI arms on I-SPY 2 suggests a possible interference of OPE with ICI. Changes in gut microbiome is being investigated as an explanation. Clinical trial information: NCT01042379 . [Table: see text]

Bortezomib Pharmacogenetic Biomarkers for the Treatment of Multiple Myeloma: Review and Future Perspectives.

Multiple myeloma (MM) is a hematological neoplasm for which different chemotherapy treatments are used with several drugs in combination. One of the most frequently used drugs for the treatment of MM is the proteasome inhibitor bortezomib. Patients treated with bortezomib are at increased risk for thrombocytopenia, neutropenia, gastrointestinal toxicities, peripheral neuropathy, infection, and fatigue. This drug is almost entirely metabolized by cytochrome CYP450 isoenzymes and transported by the efflux pump P-glycoprotein. Genes encoding both enzymes and transporters involved in the bortezomib pharmacokinetic pathway are highly polymorphic. The response to bortezomib and the incidence of adverse drug reactions (ADRs) vary among patients, which could be due to interindividual variations in these possible pharmacogenetic biomarkers. In this review, we compiled all pharmacogenetic information relevant to the treatment of MM with bortezomib. In addition, we discuss possible future perspectives and the analysis of potential pharmacogenetic markers that could influence the incidence of ADR and the toxicity of bortezomib. It would be a milestone in the field of targeted therapy for MM to relate potential biomarkers to the various effects of bortezomib on patients.

Abstract P1-13-12: Targeting Dynamin-related protein 1 for the management of taxane-resistant triple-negative breast cancer

Abstract The development of drug resistance is a primary cause of chemotherapy failure in the treatment of triple-negative breast cancer (TNBC). Some cancer cells are resistant to drugs with unrelated structures and mechanisms of action, a phenomenon known as multidrug resistance (MDR). Although taxanes such as docetaxel and paclitaxel are effective against non-metastatic and metastatic TNBC and other types of breast cancer, they eventually become ineffective due to development of drug resistance. Mitochondrial dynamics has gained significant attention as a means to treat MDR and non-MDR cancers. Mitochondrial dynamics determine the number, shape, and location of mitochondria within cells, which are fundamental to the health of the cell. Among its key components is the mitochondrial fission-promoting dynamin-related protein 1 (Drp1). There is mounting evidence indicating that both Drp1 and its phosphorylated form promote cancer survival, resistance to apoptosis, and chemoresistance. We observed increased phosphorylation of Drp1 at Ser616 as well as phosphorylation of its upstream kinase ERK1/2 in TNBC lines SUM159PT and its paclitaxel-resistant derivative that we developed called SUM159PT/PAC200 that displays more than an 80-fold increase in resistance to paclitaxel. Using CRISPR-Cas9 technology, we knocked out the gene encoding Drp1 in both lines. When Drp1 is genetically ablated, paclitaxel resistance in SUM159PT/PAC200 was significantly reversed. Furthermore, proliferation, migration, and invasion capabilities were decreased in both parental and paclitaxel-resistant Drp1 knockout lines. In SUM159PT/PAC200 Drp1-KO cells, a mesenchymal to epithelial transition (MET) was observed as indicated by downregulation of mesenchymal markers such as N-cadherin and -catenin, and upregulation of the epithelial marker ZO-1. Interestingly, Drp1 knockout halted the ability of SUM159PT/PAC200 to form colonies in soft agar. SUM159PT/PAC200 cells strongly express the MDR pump P-glycoprotein (ABCB1), to which paclitaxel is a known substrate, and genetically targeting Drp1 reduced the expression to almost undetectable levels. Taken together, these findings suggest that Drp1 is both a resistance factor and a key protein in breast cancer proliferation and dissemination, making it a promising actionable molecular target in the treatment of TNBC. Further studies are in progress to understand the role of Drp1 in the development and maintenance of MDR phenotype, proliferation, and invasion. Citation Format: David Terrero, Amit Tiwari, Dayanidhi Raman. Targeting Dynamin-related protein 1 for the management of taxane-resistant triple-negative breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-12.

An injectable, in situ forming and NIR-responsive hydrogel persistently reshaping tumor microenvironment for efficient melanoma therapy

BackgroundMelanoma is a highly aggressive form of skin cancer with increasing incidence and mortality rates. Chemotherapy, the primary treatment for melanoma, is limited by hypoxia-induced drug resistance and suppressed immune response at the tumor site. Modulating the tumor microenvironment (TME) to alleviate hypoxia and enhance immune response has shown promise in improving chemotherapy outcomes.MethodsIn this study, a novel injectable and in situ forming hydrogel named MD@SA was developed using manganese dioxide (MnO2) nanosheets pre-loaded with the chemotherapy drug doxorubicin (DOX) and mixed with sodium alginate (SA). The sustainable drug delivery, oxygen generation ability, and photothermal property of MD@SA hydrogel were characterized. The therapeutic efficacy of hydrogel was studied in B16F10 in vitro and B16F10 tumor-bearing mice in vivo. The immune effects on macrophages were analyzed by flow cytometry, real-time quantitative reverse transcription PCR, and immunofluorescence analyses.ResultsThe MD@SA hydrogel catalyzed the tumoral hydrogen peroxide (H2O2) into oxygen, reducing the hypoxic TME, down-regulating hypoxia-inducible factor-1 alpha (HIF-1α) and drug efflux pump P-glycoprotein (P-gp). The improved TME conditions enhanced the uptake of DOX by melanoma cells, enhancing its efficacy and facilitating the release of tumor antigens. Upon NIR irradiation, the photothermal effect of the hydrogel induced tumor apoptosis to expose more tumor antigens, thus re-educating the M2 type macrophage into the M1 phenotype. Consequently, the MD@SA hydrogel proposes an ability to constantly reverse the hypoxic and immune-inhibited TME, which eventually restrains cancer proliferation.ConclusionThe injectable and in situ forming MD@SA hydrogel represents a promising strategy for reshaping the TME in melanoma treatment. By elevating oxygen levels and activating the immune response, this hydrogel offers a synergistic approach for TME regulation nanomedicine.

Cutting Off H+ Leaks on the Inner Mitochondrial Membrane: A Proton Modulation Approach to Selectively Eradicate Cancer Stem Cells.

Cancer stem cells (CSCs) are associated with the invasion and metastatic relapse of various cancers. However, current cancer therapies are limited to targeting the bulk of primary tumor cells while remaining the CSCs untouched. Here, we report a new proton (H+) modulation approach to selectively eradicate CSCs via cutting off the H+ leaks on the inner mitochondrial membrane (IMM). Based on the fruit extract of Gardenia jasminoides, a multimodal molecule channel blocker with high biosafety, namely, Bo-Mt-Ge, is developed. Importantly, in this study, we successfully identify that mitochondrial uncoupling protein UCP2 is closely correlated with the stemness of CSCs, which may offer a new perspective for selective CSC drug discovery. Mechanistic studies show that Bo-Mt-Ge can specifically inhibit the UCP2 activities, decrease the H+ influx in the matrix, regulate the electrochemical gradient, and deplete the endogenous GSH, which synergistically constitute a unique MoA to active apoptotic CSC death. Intriguingly, Bo-Mt-Ge also counteracts the therapeutic resistance via a two-pronged tactic: drug efflux pump P-glycoprotein downregulation and antiapoptotic factor (e.g., Bcl-2) inhibition. With these merits, Bo-Mt-Ge proved to be one of the safest and most efficacious anti-CSC agents, with ca. 100-fold more potent than genipin alone in vitro and in vivo. This study offers new insights and promising solutions for future CSC therapies in the clinic.

Atomic force microscopy kymographs provide robust access to conformational transitions in membrane proteins

Membrane proteins play critical roles in many biochemical processes, ranging from the efflux of pharmaceutical agents used in the treatment of diseases to the translocation of polypeptides in secretion pathways. High-resolution structural methods have shed light on the conformational landscape of many of these proteins, but understanding is limited by the static nature the data they provide. Atomic force microscopy (AFM) is a powerful single molecule technique that provides structural information of active membrane proteins in real time and in physiologically relevant conditions. By disabling the slow axis scan during AFM imaging, one can acquire kymographs, which are graphical representations of spatial position over time. Here, we demonstrate the utility of rigorous kymograph analysis at the 100-ms time scale for two different membrane proteins in supported lipid bilayers: (1) proton-driven protein translocation factor SecDF, and (2) multi-drug resistance associated efflux pump P-glycoprotein (Pgp). We find that changing specific components of the systems, such as domain-specific deletions in SecDF or introduction of non-hydrolysable nucleotides in Pgp, reduces the measured conformational dynamics in predictable ways. We further probed the limitations of kymograph analysis, which have not been extensively studied. We simulated various experimental challenges associated with the method, including protein orientation, instrument noise, and drift. Despite the complex behavior of kymographs that result from these conditions, we show that robust information can be obtained including state transitions and dwell times. Lateral drift of 75% of the protein dimension leads to only a 12% probability of erroneous transition detection. Average dwell time error achieves a maximum value of 6% for identified states. Our results indicate that AFM kymographs are a useful method for probing real-time conformational dynamics of active membrane proteins.

A self-microemulsion enhances oral absorption of docetaxel by inhibiting P-glycoprotein and CYP metabolism.

Oral absorption of docetaxel was limited by drug efflux pump p-glycoprotein (P-gp) and cytochrome P450 enzyme (CYP 450). Therefore, co-loading agent that inhibits P-gp and CYP450 in self-nanoemulsifying drug delivery systems (SMEs) is considered a promising strategy for oral delivery of docetaxel. In this study, curcumin was selected as an inhibitor of P-gp and CYP450, and it was co-encapsuled in SMEs to improve the oral bioavailability of docetaxel. SMEs quickly dispersed in water within 20s, and the droplet size was 32.23 ± 2.21nm. The release rate of curcumin from DC-SMEs was higher than that of docetaxel in vitro. Compared with free docetaxel, SMEs significantly increased the permeability of docetaxel by 4.6 times. And competitive experiments showed that the increased permeability was the result of inhibition of p-gp. The half-life and oral bioavailabilty of DC-SMEs increased about 1.7 times and 1.6 times than docetaxel SMEs, which indicated that its good pharmacokinetic behavior was related to the restriction of hepatic first-pass metabolism. In conclusion, DC-SME was an ideal platform to facilitate oral delivery of docetaxel through inhibited P-gp and CYP 450.

ERBB and P-glycoprotein inhibitors break resistance in relapsed neuroblastoma models through P-glycoprotein.

Chemotherapy resistance is a persistent clinical problem in relapsed high-risk neuroblastomas. We tested a panel of 15 drugs for sensitization of neuroblastoma cells to the conventional chemotherapeutic vincristine, identifying tariquidar, an inhibitor of the transmembrane pump P-glycoprotein (P-gp/ABCB1), and the ERBB family inhibitor afatinib as the top resistance breakers. Both compounds were efficient in sensitizing neuroblastoma cells to vincristine in trypan blue exclusion assays and in inducing apoptotic cell death. The evaluation of ERBB signaling revealed no functional inhibition, that is, dephosphorylation of the downstream pathways upon afatinib treatment but direct off-target interference with P-gp function. Depletion of ABCB1, but not ERRB4, sensitized cells to vincristine treatment. P-gp inhibition substantially broke vincristine resistance invitro and invivo (zebrafish embryo xenograft). The analysis of gene expression datasets of more than 50 different neuroblastoma cell lines (primary and relapsed) and more than 160 neuroblastoma patient samples from the pediatric precision medicine platform INFORM (Individualized Therapy For Relapsed Malignancies in Childhood) confirmed a pivotal role of P-gp specifically in neuroblastoma resistance at relapse, while the ERBB family appears to play a minor part.

FREQUENCY OF MUTATIONS IN ABCB1 AND ABCB11 GENES IN PATIENTS WITH CHRONIC HEPATITIS C (HCV) IN RIO DE JANEIRO

The post-COVID-19 syndrome is characterized by the appearance of symptoms and sequelae up to four weeks after the acute symptoms of COVID-19 and can affect various systems, such as respiratory, hematological and hepatic. Cases of chronic fatigue, thromboembolism events, myalgia and the increase in enzymes associated with liver damage have been reported. It is known that individuals with chronic liver disease, cirrhosis and/or hepatocellular carcinoma are more likely to develop post-COVID-19 syndrome. In addition, mutations in the ABCB1 gene (G2677T, C3435T and C1236T) that encode the drug-export pump (P-glycoprotein) are associated with drug resistance and hepatotoxicity. The T1331C mutation in the ABCB11 gene encodes the bile salt-exporting pump and is associated with cholestasis. Therefore, these mutations may be an aggravating factor for the post-COVID-19 syndrome, especially in hepatocompromised individuals. The aim of this study was to assess the frequencies of mutations in the ABCB1 and ABCB11 genes and to monitor the post-COVID-19 syndrome in patients with liver disease. We analyzed 197 samples from patients with chronic hepatitis C from the Liver Disease Outpatient Clinic of the Gaffrée and Guinle University Hospital. Extraction and mutation analysis by qPCR was performed using TaqMan Assays. In the ABCB1 gene, the most frequent mutation was C3435T (rs1045642) in 13.7% (TT), in this group 40.1% were wild-type (CC) and 46.2% were heterozygous (CT). For the C1236T (rs1128503) the frequency of wild-type (CC) was 45.2%, of heterozygotes (CT) 43.6% and mutants (TT) 11.2%. The lowest frequency of mutations in the ABCB1 gene was G2677T (rs2032582) in 8.6% of patients, 52.8% are wild-type (GG) and 38.6% are heterozygous (GT). As for the T1331C (rs2287622) in the ABCB11 gene, most individuals were heterozygotes ‒ TC (51.8%), followed by wild-type ‒ TT (34.0%) and mutants ‒ CC (14.2%). This is the first study to evaluate the association of cases and severity of post-COVID-19 syndrome with mutations in the ABCB1 and ABCB11. So far, there are only reports in the literature of the association of this mutations with liver damage and resistance to drugs used in the treatment of COVID-19, such as lopinavir and dexamethasone. Therefore, the presence of these mutations could influence the presence of the post-COVID-19 syndrome. We observed a greater presence of heterozygous individuals than the other genotypes in the population studied. It is necessary to investigate its association with cases of post-COVID-19 syndrome in hepatocompromised patients, in order to establish the risks associated with the syndrome in these individuals and to understand the influence of these polymorphisms on the clinical picture and evolution of these individuals. CAPES and FAPERJ.

Abstract 5399: Synergistic combination nanomedicine of doxorubicin and oligo hyaluronic acid inhibits DNA damage repair and overcomes drug resistance in metastatic triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is a highly aggressive breast cancer subtype. Despite the early response to chemotherapy, high incidence of recurrence leads to short overall survival and poor prognosis. Native hyaluronic acid (HA) interacts with CD44 and receptors for hyaluronan mediated motility (RHAMM) overexpressed in TNBC mediating chemo-resistance and metastasis. In contrast, oligomeric HA (oHA) can disrupt HA-CD44/RHAMM interactions and attenuate oncogenic and pro-metastatic pathways, such as the mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAPK/ERK) pathway, reducing downstream DNA repair and drug resistance markers. Our previous work demonstrated that oHA can exert synergistic anti-tumor and anti-metastatic effects when combined with doxorubicin (DOX) and co-loaded in an integrin-targeted iRGD-modified nanoparticle system (iRGD-DOX-oHA-PLNs). This study aims to investigate the inhibitory effect of iRGD-DOX-oHA-PLNs on both DNA single-strand break (SSB) and double-strand break (DSB) repair proteins and drug efflux pumps responsible for multidrug resistance to enhance DOX efficacy in breast cancer gene 1 (BRCA1) mutant and non-mutant TNBC. Methods: The cytotoxicity of DOX, oHA and their combinations in free solution or in nanoparticles was evaluated by clonogenic assay in human MDA-MB-231-luc-D3H2LN and MDA-MB-436 (BRCA1 mutant) TNBC cells. The in vitro expression of a DNA DSB marker, DNA repair markers, and drug efflux pump P-glycoprotein (P-gp) were measured by Western blot. The in vivo expression level of BRCA1 and Rad51 in an orthotopic TNBC mouse model was determined by immunohistochemical staining. Results: The combination of DOX-oHA showed synergism against both BRCA1 mutant and non-mutant TNBC cells. The iRGD-DOX-oHA-PLNs induced great increases in DNA DSBs demonstrated by the highest γH2AX level compared to other treatment groups. These nanoparticles also showed inhibitory effects on the expression of both DNA SSB repair protein (poly (ADP-ribose) polymerase) and DNA DSB repair proteins (Rad50 and Rad51), contributing to the enhanced efficacy of chemotherapy. The immunohistochemical staining of tumor tissues indicated lower levels of BRCA1 and Rad51 after iRGD-DOX-oHA-PLN treatment than the formulation without oHA, attributable to the effect of intracellularly delivered oHA on limiting the MAPK signaling. Additionally, iRGD-DOX-oHA-PLNs reduced the expression of the drug efflux pump P-gp as compared to DOX treatment groups without oHA. Conclusion: The co-delivery of oHA and DOX in the iRGD-DOX-oHA-PLNs efficiently blocked DNA damage repair and down-regulated the drug efflux pump P-gp, thus improving the efficacy of DOX. Collectively, this nanoparticle system could be a promising option for metastatic TNBC treatment. Citation Format: Pei Zhi, Ibrahim Alradwan, Tian Zhang, HoYin Lip, Abdulmutalib Zetrini, Chunsheng He, Jeffery Henderson, Andrew Michael Rauth, Xiao Yu Wu. Synergistic combination nanomedicine of doxorubicin and oligo hyaluronic acid inhibits DNA damage repair and overcomes drug resistance in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5399.

Long Range Communication between the Drug-Binding Sites and Nucleotide Binding Domains of the Efflux Transporter ABCB1.

The ABC efflux pump P-glycoprotein (P-gp) transports a wide variety of drugs and is inhibited by others. Some drugs stimulate ATP hydrolysis at the nucleotide binding domains (NBDs) and are transported, others uncouple ATP hydrolysis and transport, and others inhibit ATP hydrolysis. The molecular basis for the different behavior of these drugs is not well understood despite the availability of several structural models of P-gp complexes with ligands bound. Hypothetically, ligands differentially alter the conformational dynamics of peptide segments that mediate the coupling between the drug binding sites and the NBDs. Here, we explore by hydrogen-deuterium exchange mass spectrometry the dynamic consequences of a classic substrate and inhibitor, vinblastine and zosuquidar, binding to mouse P-gp (mdr1a) in lipid nanodiscs. The dynamics of P-gp in nucleotide-free, pre-hydrolysis, and post-hydrolysis states in the presence of each drug reveal distinct mechanisms of ATPase stimulation and implications for transport. For both drugs, there are common regions affected in a similar manner, suggesting that particular networks are the key to stimulating ATP hydrolysis. However, drug binding effects diverge in the post-hydrolysis state, particularly in the intracellular helices (ICHs 3 and 4) and neighboring transmembrane helices. The local dynamics and conformational equilibria in this region are critical for the coupling of drug binding and ATP hydrolysis and are differentially modulated in the catalytic cycle.

Acyclovir Brain Disposition: Interactions with P-gp, Bcrp, Mrp2, and Oat3 at the Blood-Brain Barrier.

Acyclovir is effective in treating herpes simplex virus infections of the central nervous system. The purpose of this study was to investigate the interactions between acyclovir and the efflux pumps P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), multidrug resistance protein 2 (Mrp2), and organic anion transporter 3 (Oat3) at the blood-brain barrier (BBB). Acyclovir concentrations in the blood and brain were evaluated by microdialysis and high-performance liquid chromatography. Acyclovir pharmacokinetic parameters, including the area under the unbound blood concentration-time curve (AUCu,blood), the area under the unbound brain concentration-time curve (AUCu,brain), and the ratio of AUCu,brain to AUCu,blood (Kp.uu.brain), were evaluated in the presence and absence of elacridar (P-gp/Bcrp inhibitor, 7.5 mg/kg), tariquidar (P-gp/Bcrp inhibitor, 7.5 mg/kg), MK571 (Mrp2 inhibitor, 7.5 mg/kg), cyclosporine (P-gp/Bcrp/Mrp2 inhibitor, 25 mg/kg), and probenecid (Oat3 inhibitor, 50 mg/kg). The average AUCu,blood, AUCu,brain, and Kp.uu.brain in rats who received acyclovir (25 mg/kg, intravenous) alone were 1377.7 min·μg/ml, 435.4 min·μg/ml, and 31.6%, respectively. Probenecid drastically increased the AUCu,blood of acyclovir 1.73-fold, whereas coadministration with elacridar, tariquidar, MK571, and cyclosporine did not alter the blood pharmacokinetic parameters of acyclovir. Elacridar, tariquidar, MK571, cyclosporine, and probenecid significantly increased the AUCu,brain of acyclovir 1.51-, 1.54-, 1.47-, 1.95-, and 2.34-fold, respectively. Additionally, the Kp.uu.brain of acyclovir markedly increased 1.48-, 1.63-, 1.39-, 1.90-, and 1.35-fold following elacridar, tariquidar, MK571, cyclosporine, and probenecid administration, respectively. The present study demonstrated that P-gp, Bcrp, Mrp2, and Oat3 inhibition increased the penetration of acyclovir across the BBB, supporting the hypothesis that these efflux pumps restrict the distribution of acyclovir in the brain.

Changes in the blood-brain barrier properties in vitro after treatment with sera from breast cancer patients with primary cancer and visceral, bone or cerebral metastases.

2026 Background: The progression of brain metastases during breast cancer correlates with poor overall survival, but also with a reduced quality of life. During the metastatic progression of breast cancer, the key event for entry into the brain is the migration of cancer cells across the blood-brain barrier (BBB). To date, it is still controversial which serum factors play a role in cerebral expansion and what effects they have on the BBB. We hypothesize that sera from breast cancer patients with cerebral metastases contain unique factors that can affect BBB integrity. Methods: We used the CD34+ cells-derived human in vitro BBB model (brain-like endothelial cells, BLECs) in co-culture with brain pericytes, which was validated in our previous studies, to analyse the BBB properties after patient sera treatment. We used paracellular permeability measurements for fluorescein (400 Da), immunofluorescence staining, Western blot and mRNA analysis to examine the effects of patient sera on the properties of BBB in vitro. We collected serum samples from five patient cohorts (30 patient per group planned/150 in total/ current recruited patients 130): 1) healthy donors (current recruited patients- 23), 2) breast cancer patients with primary cancer (recruited patients- 30), 3) breast cancer patients with bone metastases (recruited patients- 29), 4) visceral metastases (recruited patients- 30), or 5) cerebral metastases (current recruited patients- 18). We then used 2% patient sera in cell culture medium to treat the cells for 24 hours. Results: Sera from breast cancer patients with cerebral and bone metastases led to a significant increase in the paracellular permeability for fluorescein. This could also be visualized by immunostaining cells with anti-claudin-5 antibody. Tight junction protein claudin-5 and occludin mRNA was reduced in BLECs, while mRNA of efflux pumps Breast cancer resistance protein (BCRP) and P-glycoprotein (P-GP) was induced in BLECs treated with serum from breast cancer patients with primary cancer, cerebral and visceral metastases. At the protein level, efflux pumps BCRP and P-GP were induced in cells treated with sera from breast cancer patients with cerebral metastases, while they were reduced in cells treated with sera from breast cancer patients with bone metastases. Conclusions: Sera from breast cancer patients with primary cancer and breast cancer metastases have an effect on the integrity of BBB in vitro. Reduced barrier properties of brain endothelial cells can contribute to the formation of cerebral metastases in advanced stages of breast cancer. Keywords: metastatic breast cancer, blood-brain barrier, in vitro models

Transport of Drugs and Endogenous Compounds Mediated by Human OCT1: Studies in Single- and Double-Transfected Cell Models.

Organic Cation Transporter 1 (OCT1, gene symbol: SLC22A1) is predominately expressed in human liver, localized in the basolateral membrane of hepatocytes and facilitates the uptake of endogenous compounds (e.g. serotonin, acetylcholine, thiamine), and widely prescribed drugs (e.g. metformin, fenoterol, morphine). Furthermore, exogenous compounds such as MPP+, ASP+ and Tetraethylammonium can be used as prototypic substrates to study the OCT1-mediated transport in vitro. Single-transfected cell lines recombinantly overexpressing OCT1 (e.g., HEK-OCT1) were established to study OCT1-mediated uptake and to evaluate transporter-mediated drug-drug interactions in vitro. Furthermore, double-transfected cell models simultaneously overexpressing basolaterally localized OCT1 together with an apically localized export protein have been established. Most of these cell models are based on polarized grown MDCK cells and can be used to analyze transcellular transport, mimicking the transport processes e.g. during the hepatobiliary elimination of drugs. Multidrug and toxin extrusion protein 1 (MATE1, gene symbol: SLC47A1) and the ATP-driven efflux pump P-glycoprotein (P-gp, gene symbol: ABCB1) are both expressed in the canalicular membrane of human hepatocytes and are described as transporters of organic cations. OCT1 and MATE1 have an overlapping substrate spectrum, indicating an important interplay of both transport proteins during the hepatobiliary elimination of drugs. Due to the important role of OCT1 for the transport of endogenous compounds and drugs, in vitro cell systems are important for the determination of the substrate spectrum of OCT1, the understanding of the molecular mechanisms of polarized transport, and the investigation of potential drug-drug interactions. Therefore, the aim of this review article is to summarize the current knowledge on cell systems recombinantly overexpressing human OCT1.