Upregulation Of P-glycoprotein Research Articles

Collaborative CuMn diatomic nanozyme to boost nanocatalytic/mild photothermal/chemo-therapy through overcoming therapeutic resistance

The combined mild-temperature photothermal/chemo-therapy has emerged as a promising approach to complement traditional tumor treatment and enhance therapeutic effects while reduce side effects. However, therapeutic resistance caused by up-regulation of heat shock proteins (HSPs) and P-glycoprotein (P-gp) drastically compromises the synergetic therapeutic effect. Herein, we develop a multifunctional CuMn dual-atom nanozyme loaded with the chemotherapeutic agent doxorubicin (DOX@CuMn-DAzymes) for P-gp and HSPs simultaneous silencing induced synergistic trimodal therapy. The resulting CuMn double-atom pairs with significant synergistic effects can efficiently catalyze cascade reactions of catalase (CAT)-like and oxidase (OXD)-like with endogenous H2O2 as the initial reactant to produce highly reactive singlet oxygen (1O2) under the trigger of acidic tumor microenvironment, destroying the P-gp and HSPs in tumor cells. Benefitting from their black polyporous nanocarbon support, DOX@CuMn-DAzymes not only show superior 1064 nm laser triggered photothermal performance, but also possess high DOX encapsulation efficiency and dual pH- and NIR-responsive DOX release properties. Moreover, in vitro and in vivo study results demonstrate that DOX@CuMn-DAzymes effectively overcome therapeutic resistance by boosting intracellular 1O2 level to simultaneously silence P-gp and HSPs, thereby achieving an impressive synergetic therapeutic efficacy.

Salmonella-mediated blood‒brain barrier penetration, tumor homing and tumor microenvironment regulation for enhanced chemo/bacterial glioma therapy.

Chemotherapy is an important adjuvant treatment of glioma, while the efficacy is far from satisfactory, due not only to the biological barriers of blood‒brain barrier (BBB) and blood‒tumor barrier (BTB) but also to the intrinsic resistance of glioma cells via multiple survival mechanisms such as up-regulation of P-glycoprotein (P-gp). To address these limitations, we report a bacteria-based drug delivery strategy for BBB/BTB transportation, glioma targeting, and chemo-sensitization. Bacteria selectively colonized into hypoxic tumor region and modulated tumor microenvironment, including macrophages repolarization and neutrophils infiltration. Specifically, tumor migration of neutrophils was employed as hitchhiking delivery of doxorubicin (DOX)-loaded bacterial outer membrane vesicles (OMVs/DOX). By virtue of the surface pathogen-associated molecular patterns derived from native bacteria, OMVs/DOX could be selectively recognized by neutrophils, thus facilitating glioma targeted delivery of drug with significantly enhanced tumor accumulation by 18-fold as compared to the classical passive targeting effect. Moreover, the P-gp expression on tumor cells was silenced by bacteria type III secretion effector to sensitize the efficacy of DOX, resulting in complete tumor eradication with 100% survival of all treated mice. In addition, the colonized bacteria were finally cleared by anti-bacterial activity of DOX to minimize the potential infection risk, and cardiotoxicity of DOX was also avoided, achieving excellent compatibility. This work provides an efficient trans-BBB/BTB drug delivery strategy via cell hitchhiking for enhanced glioma therapy.

Establishment and Characterization of Single and Triple‐Agent Resistant Osteosarcoma Cell Lines

Two human osteosarcoma cell lines (MG-63 and HOS-143B) are developed into drug-resistant models using a short-term drug exposure and recovery in drug-free media. Cisplatin, doxorubicin, and methotrexate are used as single agents and in triple combination. The highest level of resistance to cisplatin is observed in MG-63/CISR8, doxorubicin in HOS-143B/DOXR8, and methotrexate in HOS-143B/MTXR8. The MG-63/TRIR8 and HOS-143B/TRIR8 triple-resistance models show lower levels of resistance to combination treatment and are not resistant to the drugs individually. Apoptosis assays suggest that the resistance in MG-63/TRIR8 isfrom cisplatin and methotrexate and not doxorubicin. In contrast, the resistance in HOS-143B/TRIR8 is from doxorubicin and methotrexate instead of cisplatin. Upregulation of P-glycoprotein is seen in all resistant models except those developed with single-agent methotrexate. However, P-glycoprotein is not causing resistance in all cell lines as the inhibitor elacridar only reverses the resistance of doxorubicin on MG-63/DOXR8 and HOS-143B/TRIR8. The migration of the MG-63 resistant models is significantly increased, their invasion rate tends to increase, and RT-PCR shows a switch from epithelial to mesenchymal gene signaling. In contrast, a significant decrease in migration is seen in HOS-143B resistant models with their invasion rate tending to decrease and a switch from mesenchymal to epithelial gene signaling.

Updated Analyses: Safety and Efficacy of Gemtuzumab Ozogamicin and Venetoclax in Patients with Relapsed or Refractory CD33+ Acute Myeloid Leukemia: A Phase Ib/II Study

Background: Results of therapy for relapsed/refractory (R/R) AML patients (pts) remain poor, with few surviving >2 years. Refractoriness to treatment may be due to the resistance of quiescent leukemia stem cells (LSCs) to conventional chemotherapy. Gemtuzumab Ozogamicin (GO), a monoclonal antibody linked to the cytotoxin calicheamicin, targets CD33, an antigen with variable expression on LSC. However, decreased blast CD33 expression, p-glycoprotein upregulation and decreased mitochondrial apoptosis are recognized mechanism of resistance to GO (Linenberger et al Leukemia 2005; 19:176-82). GO-induced apoptosis requires Bax and Bak effectors and is opposed by the anti-apoptotic proteins BCL-2 and BCL-XL. Thus, Venetoclax (VEN), a BH3 mimetic with high affinity for BCL-2 should facilitate GO-mediated LSC apoptosis. In addition, LSC, due to their reliance on oxidative phosphorylation (Lagadinou, Cell Stem Cell 2013, 12:329-41) are also uniquely sensitive to BCL-2 inhibition. Thus, we hypothesized a potential synergy between these drugs in targeting LSC in R/R AML. Study design: Big Ten Cancer Research Consortium (BTCRC)-AML17-113 is a single arm, open-label, multi-center, dose-escalation phase Ib/II study of the combination of GO and escalating doses of VEN in R/R AML pts (18-75y), using a 3+3 design. Correlative studies include pre-treatment AML blast CD33 expression and BH3 profiling, minimal residual disease (MRD) measurements at post-therapy time points using digital drop PCR technology, and quality of life (QOL) assessments (EORTC QLQ-C30, FACT-Fatigue). Methods: The primary endpoint is to determine the maximum tolerated dose (MTD) of VEN with GO. Secondary endpoints include overall response rate (ORR), anti-leukemic activity, characterization of treatment emergent adverse events (TEAEs), and estimates of relapse free survival (RFS), event free survival (EFS), and overall survival (OS). Eligibility: ECOG 0-2, adequate organ function, CD33+ in ≥20% AML blasts, ≤2 lines of prior therapy, no prior use of GO, no history of veno-occlusive disease (VOD), no BMT within 2 months, no CNS disease or HIV. Induction: 3-day VEN ramp-up to the target dose of 200 (cohort #1), 400 (#2), or 600 (#3) mg daily x 28d, with GO 3mg/m2 infused d1, 4, and 7. Consolidation/Maintenance: If CR/CRi achieved, pts proceed to BMT if applicable. If no BMT, or if partial response (PR), pts are consolidated with VEN at the prescribed dose x 28d with GO 3mg/m2 given d1 and 4 (Cycle 2). If BMT not applicable, and in CR/CRi, VEN alone is then given as maintenance in cycles 3+ until disease progression or toxicity. Updated Analysis: Trial design was previously described by Arain, S et al. JCO 2020, 15:suppl. TPS7566. Cohort#3 is completing accrual with the last of 6 patients currently undergoing induction treatment. Enrollment n=12 pts. Barriers to accrual have been the COVID-19 pandemic and widespread use of VEN/HMA by referring community oncologists. The study has been amended to allow for prior use of VEN containing regimens. SAE: Respiratory failure gr 3 (n=1), febrile neutropenia (FN) gr 3/4 (n=3), Anemia/rectal pain gr 3/4 (n=1). TEAE:(%;max grade (gr)): thrombocytopenia and anemia (both 60%;gr 4); neutropenia (40%;gr 4); pruritus (30%;gr 3); FN (20%;gr 4), weight loss, diarrhea , headache, constipation (all 20%;gr 1-2). Outcomes: None of the 6 pts in cohorts #1 and #2 responded to treatment. One pt developed VOD gr 4, related to the use of total marrow irradiation conditioning regimen post-induction with GO/VEN. In cohort #3, 3 patients are in CR (one post-HSCT), 2 are undergoing induction and 1 has progressed. Conclusion: The updated analysis of this study reveals a well tolerated drug combination with low toxicities, demonstrating responses in cohort#3 (VEN 600 mg). We plan to expand accrual to this cohort adding 6-12 pts. This trial is registered at clinicaltrials.gov as NCT040768.

THE DANGER OF DIGOXIN: TOXICITY WITHIN THE "THERAPEUTIC” RANGE

THE DANGER OF DIGOXIN: TOXICITY WITHIN THE "THERAPEUTIC” RANGE

Antibiotics-induced disruption of gut microbiota increases systemic exposure of clopidogrel active metabolite in type 2 diabetic rats.

Gut microbiota play an important role in the pathophysiology of type 2 diabetic mellitus (T2DM) and biodisposition of drugs. Our previous study demonstrated that T2DM rats had the decreased plasma exposure of clopidogrel active metabolite (Clop-AM) due to upregulation of P-glycoprotein (P-gp). However, whether the change to clopidogrel (Clop) disposition under T2DM condition is associated with gut microbiota needs to be elucidated. In the study, we used an antibiotic cocktail consisting of ampicillin, vancomycin, metronidazole, and neomycin to disrupt gut microbiota and observed their influence on pharmacokinetic profiles of Clop-AM. Antibiotic administration markedly alleviated T2DM rats' phenotype including hyperglycemia, insulin resistance, oxidative stress, inflammation, hyperlipidemia, and liver dysfunction. Meanwhile, treatment with antibiotics significantly reversed the reduced systemic exposure of Clop-AM in T2DM rats relative to control rats, which was associated with the decreased intestinal P-gp level that might promote Clop absorption, resulting in more Clop transformation to Clop-AM. Fecal microbiome analysis exhibited a serious disruption of gut microbiota after antibiotic treatment with the sharply reduced microbial load and the altered microbial composition. Interestingly, an in vitro study showed that antibiotics had no influence on P-gp mRNA leve in SW480 cells, suggesting the microbiome disruption, not the direct role of antibiotics on P-gp expression, contributes to the altered P-gp level and Clop disposition in T2DM rats. The findings add new insights into the potential impact of gut microbiota on Clop biodisposition. Significance Statement 1.Antibiotics increase systemic exposure of Clop-AM in T2DM rats, which is associated with the downregulation of P-gp level.2.Antibiotics-induced disruption of gut microbiota, not direct effect of antibiotics on P-gp and CYPs expression, contributes to the altered Clop disposition.3.Antibiotics also alleviate T2DM phenotype including hyperglycemia, hyperlipidemia, insulin resistance, liver dysfunction and inflammation.

MiRNA-1246 in extracellular vesicles secreted from metastatic tumor induces drug resistance in tumor endothelial cells

Tumor endothelial cells (TECs) reportedly exhibit altered phenotypes. We have demonstrated that TECs acquire drug resistance with the upregulation of P-glycoprotein (P-gp, ABCB1), contrary to traditional assumptions. Furthermore, P-gp expression was higher in TECs of highly metastatic tumors than in those of low metastatic tumors. However, the detailed mechanism of differential P-gp expression in TECs remains unclear. miRNA was identified in highly metastatic tumor extracellular vesicles (EVs) and the roles of miRNA in endothelial cell resistance were analyzed in vitro and in vivo. In the present study, we found that treatment of highly metastatic tumor-conditioned medium induced resistance to 5-fluorouracil (5-FU) with interleukin-6 (IL-6) upregulation in endothelial cells (ECs). Among the soluble factors secreted from highly metastatic tumors, we focused on EVs and determined that miR-1246 was contained at a higher level in highly metastatic tumor EVs than in low metastatic tumor EVs. Furthermore, miR-1246 was transported via the EVs into ECs and induced IL-6 expression. Upregulated IL-6 induced resistance to 5-FU with STAT3 and Akt activation in ECs in an autocrine manner. These results suggested that highly metastatic tumors induce drug resistance in ECs by transporting miR-1246 through EVs.

Digoxin absorption decreased independently of P-gp activity in rats with irinotecan-induced gastrointestinal damage

BackgroundIrinotecan (CPT-11) is clinically known to cause severe diarrhea and gastrointestinal damage. Recently, we have reported that CPT-11-induced gastrointestinal damage is associated with the upregulation of intestinal P-glycoprotein (P-gp) expression and decreased absorption of its substrate, dabigatran etexilate (DABE), using a rat model. However, the P-gp activity or its contribution to the decreased absorption remains unclear. The aim of this study was to quantitatively evaluate how P-gp activity changes in rats with CPT-11-induced gastrointestinal damage, as assessed by the absorption of digoxin (DGX), a typical P-gp substrate.MethodsMale Sprague-Dawley rats were intravenously administered CPT-11 at a dose of 60 mg/kg/day for 4 days to induce gastrointestinal damage. Then, the rats were administered DGX orally (40 μg/kg), after some of them were orally administered clarithromycin (CAM; 10 mg/kg), a P-gp inhibitor. DGX (30 μg/kg) was administered intravenously to determine the bioavailability (BA). The rats’ DGX plasma concentration profiles were determined using LC-MS/MS.ResultsCPT-11 treatment decreased the maximum concentration (Cmax) and area under the plasma concentration-time curve (AUCpo) of DGX, which does not contradict to the DABE study. Although in the CPT-11-treated group the BA of DGX was significantly decreased to 40% of the control value, CAM did not affect the BA of DGX in the CPT-11-treated group.ConclusionsIncreased P-gp expression in rats with CPT-11-induced gastrointestinal damage is not necessarily associated with increased P-gp activity or contribution to the drug absorption in vivo. The decreased DGX absorption observed in this study might be attributable to other factors, such as a reduction in the absorptive surface area of the gastrointestinal tract.

Changes in digoxin pharmacokinetics associated with hepatic P-glycoprotein upregulation in rats with non-alcoholic fatty liver disease.

Upregulation of hepatic P-glycoprotein (P-gp) expression has been reported in patients with non-alcoholic fatty liver disease (NAFLD) and rodent models thereof. Here, we explored the changes hepatic P-gp expression and activity in a NAFLD rat model and the effects thereof on the pharmacokinetics of digoxin (a probe substrate of P-gp). Rats were fed a 1% (w/w) orotic acid-containing diet for 20days to induce NAFLD; control rats received a normal diet. P-gp expression and biliary digoxin excretion were examined. The pharmacokinetics of digoxin were evaluated after it had been administered intravenously (10μg·kg-1 ) and orally (200μg·kg-1 ) to control and NAFLD rats. The total areas under the plasma concentration-time curves (AUCs) of digoxin after intravenous and oral administration were significantly smaller (by 39.1% and 73.0%, respectively) in NAFLD rats because of faster biliary digoxin excretion, reflecting elevations of hepatic P-gp expression and activity. Notably, the steady-state volume of distribution rose by 98.2%, while extent of oral bioavailability fell by 55.5% in NAFLD rats. This is the first study to report digoxin pharmacokinetic changes caused by hepatic P-gp upregulation in NAFLD. Further studies are needed to explore the clinical impact of enhanced P-gp-mediated biliary excretion on pharmacotherapies using P-gp substrates in patients with NAFLD.

Overexpression of P-glycoprotein and MRP-1 are pharmacogenomic biomarkers to determine steroid resistant phenotype in childhood idiopathic nephrotic syndrome.

Steroid remains the keystone therapy for Idiopathic Nephrotic Syndrome (NS). Besides genetic factors and histological changes, pharmacogenomic factors also affect the steroid response. The upregulation of P-glycoprotein (P-gp) and Multidrug resistance-associated protein 1 (MRP-1) modulate the pharmacokinetics of steroids and may contribute to steroid resistance. Flow-cytometric analysis of P-gp, MRP-1 expression and functional activity on peripheral blood mononuclear cells (PBMCs) was carried out in steroid-sensitive nephrotic syndrome (SSNS) (n = 171, male 103, mean age = 8.54 ± 4.3); and steroid-resistant nephrotic syndrome (SRNS) (n = 83, male 43, mean age = 7.43 ± 4.6) patients. The genotypings of MDR-1 gene were carried out using PCR-RFLP. We observed that the percentage expression of P-gp (10.01 ± 2.09 and 3.79 ± 1.13, p < 0.001); and MRP-1 (15.91 ± 3.99 and 7.40 ± 2.33, p < 0.001) on lymphocyte gated population were significantly higher in SRNS than that of SSNS. The functional activity of P-gp and MRP-1 was also significantly escalated in SRNS as compared to SSNS (68.10 ± 13.35 and 28.93 ± 7.57, p < 0.001); (72.13 ± 8.34 and 31.56 ± 8.65, p < 0.001) respectively. AUC-ROC curve analysis revealed that P-gp and MRP-1 expression with a cut-off value of 7.13% and 9.62% predicted SRNS with the sensitivity of 90% and 80.7%; and specificity 90% and 80%, respectively. Moreover, MDR-1 homozygous mutant TT+AA for G2677T/A (rs2032582) was significantly associated with SRNS (p = 0.025, OR = 2.86 CI = 1.14-7.14). The expression of P-gp (9.68 ± 4.99 v/s 5.88 ± 3.38, p = 0.002) was significantly higher in the patients of homozygous mutant alleles compared to wildtype GG. The increased expression and functionality of P-gp and MRP-1 contribute to steroid resistance, and MDR-1 homozygous mutant G2677T/A promotes steroid resistance by inducing P-gp expression in NS.

Cystine supplementation rebalances the redox homeostasis of microenvironment in non-small cell lung cancer cells and reverses their resistance to docetaxel.

Continuous docetaxel (DTX) treatment of non-small cell lung cancer induces development of drug resistance, but the mechanism is poorly understood. In this study we performed metabolomics analysis to characterize the metabolic patterns of sensitive and resistant A549 non-small cell lung cancer cells (A549/DTX cells). We showed that the sensitive and resistant A549 cells exhibited distinct metabolic phenotypes: the resistant cells were characterized by an altered microenvironment of redox homeostasis with reduced glutathione and elevated reactive oxygen species (ROS). DTX induction reprogrammed the metabolic phenotype of the sensitive cells, which acquired a phenotype similar to that of the resistant cells: it reduced cystine influx, inhibited glutathione biosynthesis, increased ROS and decreased glutathione/glutathione disulfide (GSH/GSSG); the genes involved in glutathione biosynthesis were dramatically depressed. Addition of the ROS-inducing agent Rosup (25, 50 μg/mL) significantly increased P-glycoprotein expression and reduced intracellular DTX in the sensitive A549 cells, which ultimately acquired a phenotype similar to that of the resistant cells. Supplementation of cystine (1.0 mM) significantly increased GSH synthesis, rebalanced the redox homeostasis of A549/DTX cells, and reversed DTX-induced upregulation of P-glycoprotein, and it markedly improved the effects of DTX and inhibited the growth of A549/DTX in vitro and in vivo. These results suggest that microenvironmental redox homeostasis plays a key role in the acquired resistance of A549 cancer cells to DTX. The enhancement of GSH synthesis by supplementary cystine is a promising strategy to reverse the resistance of tumor cells and has potential for translation in the clinic.

Reduced cytotoxicity in doxorubicin-exposed HepG2 cells pretreated with menthol due to upregulation of P-glycoprotein.

The aim of the present study was to examine changes in the expression and activity of P-glycoprotein (P-gp) in human hepatocellular carcinoma HepG2 cells after exposure to menthol, and their relationship to the cytotoxicity of and apoptotic responses to doxorubicin (DOX), a substrate of P-gp, in the cells. The expression of P-gp in HepG2 cells was significantly increased by menthol treatment. Intracellular accumulation of DOX in HepG2 cells was significantly lower in the menthol-treated group than in the control group, but this phenomenon was abolished in the presence of verapamil. Decreased cell viability by DOX was significantly attenuated by 24-h menthol treatment prior to DOX exposure, which coincided with the changes in mRNA expression of Bcl-xl and caspase-3. These results demonstrate that menthol causes hepatocellular carcinoma cells to acquire resistance to DOX by increasing its efflux through the upregulation of P-gp.

Curcumin induces chemosensitization to doxorubicin in Duke's type B coloadenocarcinoma cell line.

Cancer cells require higher levels of ATP for their sustained growth, proliferation, and chemoresistance. Mitochondrial matrix protein, C1qbp is upregulated in colon cancer cell lines. It protects the mitochondria from oxidative stress, by inhibiting the Membrane Permeability Transition (MPT) pore and providing uninterrupted synthesis of ATP. This intracellular interaction of C1qbp could be involved in chemoresistance development. Natural chemosensitizing agent, curcumin has been used in the treatment of multiple cancers. In this current study, we elucidate the role of C1qbp during curcumin induced chemosensitization to doxorubicin resistant colon cancercells. The possible interaction between C1qbp and curcumin was determined using bioinformatics tools-AutoDock, SYBYL, and PyMol. Intracellular doxorubicin accumulation by fluorimetry and dead cell count was carried out to determine development of chemoresistance. Effect of curcumin treatment and cytotoxicity was measured by MTT and lactate dehydrogenase release. Morphological analysis by phase contrast microscopy and colony forming ability by colonogenic assay were alsoperformed. Inaddition,Cox-2 could mediate P-glycoprotein upregulation via phosphorylation of c-Jun.Thus, the gene level expression of P-glycoprotein and Cox-2 was alsoinvestigated using PCR. Through molecular docking we identified possible interaction between curcumin and C1qbp. We observed development of chemoresistance upon 6th day treatment. Concentration dependent alleviation of chemoresistance development by curcumin was confirmed and wasfound to reducegene level expression ofP-glycoprotein and Cox-2. Hence, curcumin could interact directly with C1qbp protein and this interaction could contribute to the chemosensiting effect to doxorubicin in colon cancer cells.

Potentiation of anticonvulsant effect of phenytoin by celecoxib against maximum electroshock induced convulsions in albino rats

Background: Epilepsy, a chronic neurological disorder affects more than 1% of world population. Despite the availability of a number of antiepileptics, refractoriness to them exists in approximately one third of cases worldwide. Induction of cycloxygenase and increased levels of proinflammatory meditators are seen in epilepsy. P-glycoprotein upregulation due to phenytoin was found to contribute to its pumping out of cell, leading to refractoriness to phenytoin therapy. Also, cycloxygenase-2 inhibitors were found to prevent P-glycoprotein upregulation. Since cycloxygenase-2 inhibition decreases levels of proinflammatory cytokines responsible for neuroinflammation, this study aims to evaluate anticonvulsant effect of celecoxib and also to investigate whether it potentiates the anticonvulsant effect of phenytoin.Methods: Maximum electroshock seizures (MES) were induced in Albino rats using electroconvulsiometer to evaluate tonic convulsions, identified by tonic hind limb extension (THLE) in rats. A delay in onset of THLE and a reduction in duration of THLE were taken as deciding parameters to ascertain anticonvulsive activity. Rats randomly divided into groups, received pretreatment with celecoxib at 3 doses (10, 20, 40 mg/kg), phenytoin (6.25 mg/kg), phenytoin (12.5 mg/kg) and combination of phenytoin (6.25 mg/kg) with celecoxib (ED50, i.e. 20 mg/kg), before inducing MES seizures and findings compared to control group.Results: Celecoxib (20 and 40 mg/kg) showed significant anticonvulsant effect by MES test. Also, its combination with phenytoin caused significant decrease in the duration of THLE when compared to phenytoin alone at the same dose.Conclusions: The results of this study indicate that celecoxib potentiates the anticonvulsant effect of phenytoin.

Type 2 diabetes mellitus decreases systemic exposure of clopidogrel active metabolite through upregulation of P-glycoprotein in rats

Patients with diabetic mellitus tend to have a poor response to clopidogrel (Clop) due to reduced generation of active metabolite (Clop-AM). However, the underlying mechanism is not elucidated. A type 2 diabetic mellitus (T2DM) rat model was established by combining high-fat diet feeding and low-dose streptozotocin (STZ) injection. The reduced Clop-AM exposure was observed in T2DM rats after oral administration of Clop. However, in vitro liver microsomes incubated with Clop exhibited increased Clop-AM levels in T2DM rats due to a significant decrease in carboxylesterase (CES)1 expression and activity and a significant increase in the expression or activity of CYP1A2 and CYP3A. Interestingly, different from oral administration, the significantly increased Cmax of Clop-AM was observed in T2DM rats after intravenous injection, with no difference in AUC0-t and t1/2 values between the two strains. Meanwhile, in situ single -pass intestinal perfusion study showed lower absorption rate constant (Ka) and effective apparent permeability values (Peff) of Clop in T2DM rats than in control rats. It is explained by the increased expression or function of P-glycoprotein (P-gp) and pregnane X receptor (PXR) in duodenum and jejunum of T2DM rats. Moreover, the decreased Clop-AM level in T2DM rats was eliminated by the pretreatment of cyclosporin A, a P-gp inhibitor. It suggests that intestinal absorption, not hepatic metabolism is responsible for the reduced Clop-AM exposure in T2DM rats. P-gp might be the key factor causing the reduction of Clop absorption, consequently making less Clop available for Clop-AM formation.

Safety and efficacy of gemtuzumab ozogamicin and venetoclax in patients with relapsed or refractory CD33+ acute myeloid leukemia: A phase Ib study.

TPS7566 Background: AML is predominantly a disease of the elderly, yet outcomes remain dismal, especially for relapsed/refractory (R/R) AML patients (pts). Gemtuzumab Ozogamicin (GO) is a monoclonal antibody targeting CD33–commonly expressed on AML blasts, and, critically, AML stem cells (LSC)–that is linked to the cytotoxin calicheamicin. Recognized mechanisms of GO resistance include decreased (or aberrant) blast CD33 expression, upregulation of p-glycoprotein (re-exports calicheamicin), and decreased mitochondrial apoptosis. GO-induced apoptosis depends on pro-apoptotic Bax and Bak and is inhibited by overexpression of anti-apoptotic BCL-2 and BCL-XL. Venetoclax (VEN) is a BH3 mimetic, binding BCL-2, dislodging its binding to Bak/Bax, thus facilitating apoptosis. LSC uniquely overexpress BCL-2, however VEN resistance develops rapidly. Hypothesis: VEN targeting of BCL-2 proteins that protect LSC from GO-induced apoptosis will synergistically increase GO efficacy. Correlative studies include pre-treatment AML blast BH3 profiling and CD33 expression (&amp; sequencing for isoforms), MRD measurement at post-therapy timepoints using digital drop PCR technology, and quality of life assessments (EORTC QLQ-C30, FACT-Fatigue). Methods: Single arm, open-label, multi-center (BTCRC), dose-escalation phase Ib study of combination of VEN and GO in R/R AML pts (18-75y), using a 3+3 design. Major eligibility: ECOG 0-2, adequate organ function, CD33+ in ≥ 20% AML blasts, ≤2 lines of prior therapy, no prior use of GO or VEN, no previous VOD, no BMT within 2 months, no CNS disease, and no history of HIV. Induction: 3-day VEN ramp-up to the target dose of 200 (cohort i), 400 (ii), or 600 (iii) mg daily x 28 d, with GO 3mg/m2 infused d 1, 4, and 7. If CR/CRi achieved, pts proceed to BMT if applicable, otherwise, if in CR/CRi (provided ANC &gt; 1000, plts 100K) or PR (regardless of counts), they are consolidated with VEN at the prescribed dose x 28d and GO 3mg/m2 on days 1 and 4 (Cycle 2). If BMT not applicable, and then in CR/CRi or PR (as above), proceed to VEN alone as Maintenance in cycles 3+ until progression or toxicity. The primary endpoint is MTD of VEN with GO. Secondary endpoints include ORR, anti-leukemic activity, characterization of AEs, and estimates of RFS, EFS, and OS. This study is currently open and has to date enrolled 2 pts. Clinical trial information: NCT04070768 .

Immuno-metabolic impact of the multiple sclerosis patients\u2019 sera on endothelial cells of the blood-brain barrier

BackgroundMultiple sclerosis (MS) is an autoimmune disease which results from the invasion of the brain by activated immune cells across the endothelial cells (ECs) of the blood-brain barrier (BBB), due to loss of immune self-tolerance. Many reports define the metabolic profile of immune cells in MS, however little is known about the metabolism of the BBB ECs during the disease. We aim to determine whether circulating factors in MS induce metabolic alterations of the BBB ECs compared to a healthy state, which can be linked with disruption of BBB integrity and subsequent immune cell extravasation.Methods and resultsIn this report, we used an in vitro model to study the effect of sera from naïve-to-treatment, relapsing-remitting MS (RRMS) patients on the human brain microvascular endothelium, comparing effects to age/sex-matched healthy donor (HD) sera. Our data show that RRMS serum components affect brain endothelial cells by impairing intercellular tightness through the down-modulation of occludin and VE-cadherin, and facilitating immune cell extravasation through upregulation of intercellular adhesion molecules (ICAM-1) and P-glycoprotein (P-gp). At a metabolic level, the treatment of the endothelial cells with RRMS sera reduced their glycolytic activity (measured through the extracellular acidification rate-ECAR) and oxygen consumption rate (oxidative phosphorylation rate-OCR). Such changes were associated with the down-modulation of endothelial glucose transporter 1 (GLUT-1) expression and by altered mitochondrial membrane potential. Higher level of reactive oxygen species released from the endothelial cells treated with RRMS sera indicate a pro-inflammatory status of the cells together with the higher expression of ICAM-1, endothelial cell cytoskeleton perturbation (stress fibres) as well as disruption of the cytoskeleton signal transduction MSK1/2 and β-catenin phosphorylation.ConclusionsOur data suggest that circulating factors present in RRMS patient serum induce physiological and biochemical alterations to the BBB, namely reducing expression of essential tightness regulators, as well as reduced engagement of glycolysis and alteration of mitochondrial potential. As these last changes have been linked with alterations in nutrient usage and metabolic function in immune cells; we propose that the BBB endothelium of MS patients may similarly undergo metabolic dysregulation, leading to enhanced permeability and increased disease susceptibility.

Pulchinenosides from Pulsatilla Chinensis Increase P-Glycoprotein Activity and Induce P-Glycoprotein Expression.

Five pulchinenosides (pulchinenoside B3, pulchinenoside BD, pulchinenoside B7, pulchinenoside B10, and pulchinenoside B11) isolated from Pulsatilla chinensis (Bge) Regel saponins extract exhibited strong antitumor activities but poor gastrointestinal absorption properties. The enteric induction of P-glycoprotein (P-gp) is understood to restrict the oral bioavailability of some pharmaceutical compounds and lead to adverse drug reactions. Therefore, the present investigation was intended to delineate the impacts of pulchinenosides on cellular P-gp function and expression using Sf9 membrane vesicles and LS180 cells as a surrogate of human intestinal epithelial cells. Preliminary cytotoxic studies showed that 10 μM was an acceptable concentration for cytotoxicity and antiproliferation studies for all pulchinenosides using the alamarBlue assay. The cell cycle of LS180 cells detected by flow cytometry was not significantly influenced after 48 hours of coincubation with 10 μM of pulchinenosides. In the presence of pulchinenosides, the ATP-dependent transport of N-methyl-quinidine mediated by P-glycoprotein was stimulated significantly. The upregulation of P-glycoprotein and mRNA levels was found by Western blot and real-time PCR analysis in LS180 cells. Parallel changes indicate that all pulchinenosides are exposed to pulchinenosides-mediated transcriptional regulation. In conclusion, pulchinenosides could induce P-glycoprotein expression and directly increase its functional activity.

Involvement of hypoxia-inducible factor-1 alpha in the upregulation of P-glycoprotein in refractory epilepsy.

To explore the involvement of hypoxia-inducible factor-1 alpha (HIF-1α) in the upregulation of P-glycoprotein (P-gp) in refractory epilepsy. Brain tissue specimens were collected and analyzed for expression of HIF-1α and P-gp using an immunohistochemical (IHC) staining method in both refractory epilepsy group and control group. Correlation between HIF-1α and P-gp expression level in refractory epilepsy group was analyzed. Then, a hypoxia cell model was established by simulating the nerve cell hypoxic microenvironment in the human U251 cell line using cobalt chloride (CoCl2). Western blot analysis was used to detect expression levels of HIF-1α and P-gp in the hypoxic cell model. Finally, expression of HIF-1α and P-gp was detected using real-time quantitative PCR and Western blot, respectively, after U251 hypoxic model cells were infected with HIF-1α siRNA. IHC scores of HIF-1α and P-gp in refractory epilepsy group were significantly higher than that in control group. In addition, the expression of HIF-1α was positively correlated with the expression of P-gp in refractory epilepsy group. Expression levels of HIF-1α and P-gp in U251 cells cultured with 250 µmol/L CoCl2 for 48 hours were significantly higher than that in controls. After transfection with siRNA targeting HIF-1α, expressions of HIF-1α and P-gp at mRNA and protein level were decreased, respectively, in the hypoxia cell model. HIF-1α may be involved in the upregulation of P-gp in refractory epilepsy through inducement of P-gp expression. Therefore, activation of the HIF-1α/P-gp pathway is one hypothesis proposed to explain the pathogenesis of refractory epilepsy.

Enhanced Reactive Oxygen Species Generation by Mitochondria Targeting of Anticancer Drug To Overcome Tumor Multidrug Resistance.

As a major clinical tumor chemotherapeutic burden, multidrug resistance (MDR) is often a result of up-regulation of P-glycoprotein (P-gp), which strongly enhances anticancer drug efflux. The excess mitochondrial reactive oxygen species (ROS) could not onlyinhibit the function of P-gp through insufficient adenosine triphosphate supply but also cause apoptosis in MDR cells. Here, we designed a mitochondria targeting nanoparticulate system (GNPs-P-Dox-GA) for overcoming MDR through enhanced ROS generation, where increased cellular uptake as well as mitochondria accumulation were both realized by glycyrrhetinic acid (GA). First, doxorubicin was conjugated with GA (GA-Dox) and then grafted onto a N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer backbone via hydrazone bond (P-Dox-GA). The obtained P-Dox-GA was subsequently attached to the surface of gelatin nanoparticles (GNPs). As gelatin is a substrate of tumor extracellular metal matrix protease-2 (MMP2), GNPs-P-Dox-GA nanoparticles could be degraded and release small size P-Dox-GA to facilitate tumor tissue penetration. After P-Dox-GA internalized by tumor cells under GA mediation, Dox-GA detached from HPMA copolymer through hydrolysis of hydrazone bond and then efficiently delivered to mitochondria. Compared to non-GA modified carriers, GNPs-P-Dox-GA exhibited increased cellular uptake nearly 4-fold and mitochondria distribution 8.8-fold, and increased ROS production level nearly 3-fold, significantly decreased efflux rate (55% compared with Dox group) in drug resistant HepG2/ADR cells, and then led to improved in vitro antitumor efficiency in HepG2/ADR cells (IC50 only 19.5% of unmodified ones) as well as exciting in vivo antitumor efficiency on HepG2/ADR heterotopic tumor nude mice (1.75-fold higher tumor growth inhibition rate than free drug).