Temperature Self-Limited Intelligent Thermo-chemotherapeutic Lipid Nanosystem for P-gp Reversal Time Window Matched Pulse Treatment of MDR Tumor.

Mild photothermal therapy (PTT) shows the potential for chemosensitization by tumor-localized P-glycoprotein (P-gp) modulation. However, conventional mild PTT struggles with real-time uniform temperature control, obscuring the temperature-performance relationship and resulting in thermal damage. Besides, the time-performance relationship and the underlying mechanism of mild PTT-mediated P-gp reversal remains elusive. Herein, we developed a temperature self-limiting lipid nanosystem (RFE@PD) that integrated a reversible organic heat generator (metal-phenolic complexes) and metal chelator (deferiprone, DFP) encapsulated phase change material. Upon NIR irradiation, RFE@PD released DFP for blocking ligand-metal charge transfer to self-limit temperature below 45 °C, and rapidly reduced P-gp within 3 h via Ubiquitin-proteasome degradation. Consequently, the DOX·HCl-loaded thermo-chemotherapeutic lipid nanosystem (RFE@PD-DOX) led to dramatically improved drug accumulation and 5-fold chemosensitization in MCF-7/ADR tumor models by synchronizing P-gp reversal and drug pulse liberation, achieving a tumor inhibition ratio of 82.42%. This lipid nanosystem integrated with "intrinsic temperature-control" and "temperature-responsive pulse release" casts new light on MDR tumor therapy.

Modelling the maternal-fetal interface: An in vitro approach to investigate nutrient and drug transport across the human placenta.

The placenta plays a critical role in maternal-fetal nutrient transport and fetal protection against drugs. Creating physiological in vitro models to study these processes is crucial, but technically challenging. This study introduces an efficient cell model that mimics the human placental barrier using co-cultures of primary trophoblasts and primary human umbilical vein endothelial cells (HUVEC) on a Transwell®-based system. Monolayer formation was examined over 7 days by determining transepithelial electrical resistance (TEER), permeability of Lucifer yellow (LY) and inulin, localization of transport proteins at the trophoblast membrane (immunofluorescence), and syncytialization markers (RT-qPCR/ELISA). We analysed diffusion-based (caffeine/antipyrine) and transport-based (leucine/Rhodamine-123) processes to study the transfer of physiologically relevant compounds. The latter relies on the adequate localization and function of the amino-acid transporter LAT1 and the drug transporter P-glycoprotein (P-gp) which were studied by immunofluorescence microscopy and application of respective inhibitors (2-Amino-2-norbornanecarboxylic acid (BCH) for LAT1; cyclosporine-A for P-gp). The formation of functional monolayer(s) was confirmed by increasing TEER values, low LY transfer rates, minimal inulin leakage, and appropriate expression/release of syncytialization markers. These results were supported by microscopic monitoring of monolayer formation. LAT1 was identified on the apical and basal sides of the trophoblast monolayer, while P-gp was apically localized. Transport assays confirmed the inhibition of LAT1 by BCH, reducing both intracellular leucine levels and leucine transport to the basal compartment. Inhibiting P-gp with cyclosporine-A increased intracellular Rhodamine-123 concentrations. Our in vitro model mimics key aspects of the human placental barrier. It represents a powerful tool to study nutrient and drug transport mechanisms across the placenta, assisting in evaluating safer pregnancy therapies.

Expression of GnT-III decreases chemoresistance via negatively regulating P-glycoprotein expression: Involvement of the TNFR2-NF-κB signaling pathway.

The phenomenon of multidrug resistance (MDR) is called chemoresistance with respect to the treatment of cancer, and it continues to be a major challenge. The role of N-glycosylation in chemoresistance, however, remains poorly understood. Here, we established a traditional model for adriamycin resistance in K562 cells, which are also known as K562/adriamycin-resistant (ADR) cells. Lectin blot, mass spectrometry, and RT-PCR analysis showed that the expression levels of N-acetylglucosaminyltransferase III (GnT-III) mRNA and its products, bisected N-glycans, are significantly decreased in K562/ADR cells, compared with the levels in parent K562 cells. By contrast, the expression levels of both P-glycoprotein (P-gp) and its intracellular key regulator, NF-κB signaling, are significantly increased in K562/ADR cells. These upregulations were sufficiently suppressed by the overexpression of GnT-III in K562/ADR cells. We found that the expression of GnT-III consistently decreased chemoresistance for doxorubicin and dasatinib, as well as activation of the NF-κB pathway by tumor necrosis factor (TNF) α, which binds to two structurally distinct glycoproteins, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), on the cell surface. Interestingly, our immunoprecipitation analysis revealed that only TNFR2, but not TNFR1, contains bisected N-glycans. The lack of GnT-III strongly induced TNFR2's autotrimerization without ligand stimulation, which was rescued by the overexpression of GnT-III in K562/ADR cells. Furthermore, the deficiency of TNFR2 suppressed P-gp expression while it increased GnT-III expression. Taken together, these results clearly show that GnT-III negatively regulates chemoresistance via the suppression of P-gp expression, which is regulated by the TNFR2-NF/κB signaling pathway.

Modulation of Multidrug Resistance Transporters by Food Components and Dietary Supplements: Implications for Cancer Therapy Efficacy and Safety.

The aim of this review is to explore how diet and dietary supplements influence the activity of key multidrug resistance (MDR) transporters-MRP2, BCRP, and P-gp. These transporters play a crucial role in drug efflux from cancer cells and significantly affect chemotherapy outcomes. This review focuses on how dietary phytochemicals, such as catechins and quercetin, impact the expression and function of these transporters. Both in vitro and in vivo experiments were examined to assess changes in drug bioavailability and intracellular drug accumulation. The findings show that certain dietary components-such as catechins, flavonoids, resveratrol, curcumin, terpenoids, sterols, and alkaloids-can either inhibit or induce MDR transporter activity, thus influencing the effectiveness of chemotherapy. These results highlight the importance of understanding diet-drug interactions in cancer therapy to improve treatment outcomes and reduce side effects. In conclusion, dietary modifications and supplements should be carefully considered in cancer treatment plans to optimize therapeutic efficacy.

Identification and characterization of endogenous biomarkers for hepatic vectorial transport (OATP1B3-P-gp) function using metabolomics with serum pharmacology.

The organic anion-transporting polypeptide 1B3 and P-glycoprotein (P-gp) provide efficient directional transport (OATP1B3-P-gp) from the blood to the bile that serves as a key determinant of hepatic disposition of the drug. Unfortunately, there is still a lack of effective means to evaluate the disposal ability mediated by transporters. The present study was designed to identify a suitable endogenous biomarker for the assessment of OATP1B3-P-gp function in the liver. We established stably transfected HEK293T-OATP1B3 and HEK293T-P-gp cell lines. Results showed that azelaic acid (AzA) was an endogenous substrate for OATP1B3 and P-gp using serum pharmacology combined with metabolomics. There is a good correlation between the serum concentration of AzA and probe drugs of rOATP1B3 and rP-gp when rats were treated with their inhibitors. Importantly, after 5-fluorouracil-induced rat liver injury, the relative mRNA level and expression of rOATP1B3 and rP-gp were markedly down-regulated in the liver, and the serum concentration of AzA was significantly increased. These observations suggest that AzA is an endogenous substrate of both OATP1B3 and P-gp, and may serve as a potential endogenous biomarker for the assessment of the function of OATP1B3-P-gp for the prediction of changes in the pharmacokinetics of drugs transported by OATP1B3-P-gp in liver disease states.

BI-2865, a pan-KRAS inhibitor, reverses the P-glycoprotein induced multidrug resistance in vitro and in vivo.

BACKGROUND: Multidrug resistance (MDR) limits successful cancer chemotherapy. P-glycoprotein (P-gp), BCRP and MRP1 are the key triggers of MDR. Unfortunately, no MDR modulator was approved by FDA to date. Here, we will investigate the effect of BI-2865, a pan-KRAS inhibitor, on reversing MDR induced by P-gp, BCRP and MRP1 in vitro and in vivo, and its reversal mechanisms will be explored. METHODS: The cytotoxicity of BI-2865 and its MDR removal effect in vitro were tested by MTT assays, and the corresponding reversal function in vivo was assessed through the P-gp mediated KBv200 xenografts in mice. BI-2865 induced alterations of drug discharge and reservation in cells were estimated by experiments of Flow cytometry with fluorescent doxorubicin, and the chemo-drug accumulation in xenografts' tumor were analyzed through LC-MS. Mechanisms of BI-2865 inhibiting P-gp substrate's efflux were analyzed through the vanadate-sensitive ATPase assay, [125I]-IAAP-photolabeling assay and computer molecular docking. The effects of BI-2865 on P-gp expression and KRAS-downstream signaling were detected via Western blotting, Flow cytometry and/or qRT-PCR. Subcellular localization of P-gp was visualized by Immunofluorescence. RESULTS: We found BI-2865 notably fortified response of P-gp-driven MDR cancer cells to the administration of chemo-drugs including paclitaxel, vincristine and doxorubicin, while such an effect was not observed in their parental sensitive cells and BCRP or MRP1-driven MDR cells. Importantly, the mice vivo combination study has verified that BI-2865 effectively improved the anti-tumor action of paclitaxel without toxic injury. In mechanism, BI-2865 prompted doxorubicin accumulating in carcinoma cells by directly blocking the efflux function of P-gp, which more specifically, was achieved by BI-2865 competitively binding to the drug-binding sites of P-gp. What's more, at the effective MDR reversal concentrations, BI-2865 neither varied the expression and location of P-gp nor reduced its downstream AKT or ERK1/2 signaling activity. CONCLUSIONS: This study uncovered a new application of BI-2865 as a MDR modulator, which might be used to effectively, safely and specifically improve chemotherapeutic efficacy in the clinical P-gp mediated MDR refractory cancers.

The placenta in fetal death: molecular evidence of dysregulation of inflammatory, proliferative, and fetal protective pathways.

BACKGROUND: It is estimated that over 2 million cases of fetal death occur worldwide every year, but, despite the high incidence, several basic and clinical characteristics of this disorder are still unclear. Placenta is suggested to play a central role in fetal death. Placenta produces hormones, cytokines and growth factors that modulate functions of the placental-maternal unit. Fetal death has been correlated with impaired secretion of some of these regulatory factors. OBJECTIVE: The aim of the present study was to evaluate, in placentas collected from fetal death, the gene expression of inflammatory, proliferative and protective factors. STUDY DESIGN: Cases of fetal death in singleton pregnancy were retrospectively selected, excluding pregnancies complicated by fetal anomalies, gestational diabetes, intrauterine growth restriction and moderate to severe maternal diseases. A group of placentas collected from healthy singleton term pregnancies were used as controls. Groups were compared regarding maternal and gestational age, fetal sex and birthweight. Placental messenger RNA expression of inflammatory (interleukin 6), proliferative (activin A, transforming growth factor ß1) and regulatory (vascular endothelial growth factor, vascular endothelial growth factor receptor 2, ATP-binding cassette transporters (ABC) ABCB1 and ABCG2, sphingosine 1-phosphate signaling pathway) markers was conducted using real-time polymerase chain reaction. Statistical analysis and graphical representation of the data were performed using the GraphPad Prism 5 software. For the statistical analysis, Student's t test was used, and P values<.05 were considered significant. RESULTS: Placental mRNA expression of interleukin 6 and vascular endothelial growth factor receptor 2 resulted significantly higher in the fetal death group compared to controls (P<.01), while activin A, ABCB1, and ABCG2 expression resulted significantly lower (P<.01). A significant alteration in the sphingosine 1-phosphate signaling pathway was found in the fetal death group, with an increased expression of the specific receptor isoforms sphingosine 1-phosphate receptor 1, 3, and 4 (sphingosine 1-phosphate1, sphingosine 1-phosphate3, sphingosine 1-phosphate4) and of sphingosine kinase 2, 1 of the enzyme isoforms responsible for sphingosine 1-phosphate synthesis (P<.01). CONCLUSION: The present study confirmed a significantly increased expression of placental interleukin 6 and vascular endothelial growth factor receptor 2 mRNA, and for the first time showed an increased expression of sphingosine 1-phosphate receptors and sphingosine kinase 2 as well as a decreased expression of activin A and of selected ATP-binding cassette transporters, suggesting that multiple inflammatory and protective factors are deranged in placenta of fetal death.

Effect of Apolipoprotein E isoforms on the Abundance and Function of P-glycoprotein in Human Brain Microvascular Endothelial Cells.

BACKGROUND: Individuals with Alzheimer's disease (AD) often require many medications; however, these medications are dosed using regimens recommended for individuals without AD. This is despite reduced abundance and function of P-glycoprotein (P-gp) at the blood-brain barrier (BBB) in AD, which can impact brain exposure of drugs. The fundamental mechanisms leading to reduced P-gp abundance in sporadic AD remain unknown; however, it is known that the apolipoprotein E (apoE) gene has the strongest genetic link to sporadic AD development, and apoE isoforms can differentially alter BBB function. The aim of this study was to assess if apoE affects P-gp abundance and function in an isoform-dependent manner using a human cerebral microvascular endothelial cell (hCMEC/D3) model. METHODS: This study assessed the impact of apoE isoforms on P-gp abundance (by western blot) and function (by rhodamine 123 (R123) uptake) in hCMEC/D3 cells. Cells were exposed to recombinant apoE3 and apoE4 at 2 - 10 µg/mL over 24 - 72 hours. hCMEC/D3 cells were also exposed for 72 hours to astrocyte-conditioned media (ACM) from astrocytes expressing humanised apoE isoforms. RESULTS: P-gp abundance in hCMEC/D3 cells was not altered by recombinant apoE4 relative to recombinant apoE3, nor did ACM containing human apoE isoforms alter P-gp abundance. R123 accumulation in hCMEC/D3 cells was also unchanged with recombinant apoE isoform treatments, suggesting no change to P-gp function, despite both abundance and function being altered by positive controls SR12813 (5 µM) and PSC 833 (5 µM), respectively. CONCLUSIONS: Different apoE isoforms have no direct influence on P-gp abundance or function within this model, and further in vivo studies would be required to address whether P-gp abundance or function are reduced in sporadic AD in an apoE isoform-specific manner.

Recent developments of P-glycoprotein inhibitors and its structure-activity relationship (SAR) studies.

P-glycoprotein (P-gp) over-expression is a key factor in multi-drug resistance (MDR), which is a major factor in the failure of cancer treatment. P-gp inhibitors have been demonstrated to have powerful pharmacological properties and may be used as a therapeutic approach to overcome the MDR in cancer cells. Combining clinical investigations with biochemical and computational research may potentially lead to a clearer understanding of the pharmacological properties and the mechanisms of action of these P-gp inhibitors. The task of turning these discoveries into effective therapeutic candidates for a variety of malignancies, including resistant and metastatic kinds, falls on medicinal chemists. A variety of P-gp inhibitors with great potency, high selectivity, and minimal toxicity have been identified in recent years. The latest advances in drug design, characterization, structure-activity relationship (SAR) research, and modes of action of newly synthesized, powerful small molecules P-gp inhibitors over the previous ten years are highlighted in this review. P-gp transporter over-expression has been linked to MDR, therefore the development of P-gp inhibitors will expand our understanding of the processes and functions of P-gp-mediated drug efflux, which will be helpful for drug discovery and clinical cancer therapies.

Design, synthesis, and ex vivo anti-drug resistant cervical cancer activity of novel molecularly targeted chalcone derivatives.

Chemotherapy toxicity and tumor multidrug resistance remain the main reasons for clinical treatment failure in cervical cancer. In this study, 79 novel chalcone derivatives were designed and synthesized using the principle of active substructure splicing with the parent nucleus of licorice chalcone as the lead compound and VEGFR-2 and P-gp as the target of action and their potentials for anticervical cancer activity were preliminarily evaluated. The results showed that the IC50 values of candidate compound B20 against HeLa and HeLa/DDP cells were 3.66 ± 0.10 and 4.35 ± 0.21 µΜ, respectively, with a resistance index (RI) of 1.18, which was significantly higher than that of the positive drug cisplatin (IC50:13.60 ± 1.63, 100.03 ± 7.94 µΜ, RI:7.36). In addition, B20 showed significant inhibitory activity against VEGFR-2 kinase and P-gp-mediated rhodamine 123 efflux, as well as the ability to inhibit the phosphorylation of VEGFR-2 and downstream PI3K/AKT signaling pathway proteins, inducing apoptosis, blocking cells in the S-phase, and inhibiting invasive migration and tubule generation by HUVEC cells. Acceptable safety was demonstrated in acute toxicity tests when B20 was at 200 mg/kg. In the nude mouse HeLa/DDP cell xenograft tumor model, the inhibition rate of transplanted tumors was 39.2 % and 79.2 % when B20 was at 10 and 20 mg/kg, respectively. These results suggest that B20 is a potent VEGFR-2 and P-gp inhibitor with active potential for treating cisplatin-resistant cervical cancer.

A novel FAK-degrading PROTAC molecule exhibited both anti-tumor activities and efficient MDR reversal effects.

FAK (focal adhesion kinase) is widely involved in cancer growth and drug resistance development. Thus, FAK inhibition has emerged as an effective strategy for tumor treatment both as a monotherapy or in combination with other treatments. But the current FAK inhibitors mainly concentrate on its kinase activity, overlooking the potential significance of FAK scaffold proteins. In this study we employed the PROTAC technology, and designed a novel PROTAC molecule F2 targeting FAK based on the FAK inhibitor IN10018. F2 exhibited potent inhibitory activities against 4T1, MDA-MB-231, MDA-MB-468 and MDA-MB-435 cells with IC50 values of 0.73, 1.09, 5.84 and 3.05 µM, respectively. On the other hand, F2 also remarkably reversed the multidrug resistance (MDR) in HCT8/T, A549/T and MCF-7/ADR cells. Both the effects of F2 were stronger than the FAK inhibitor IN10018. To our knowledge, F2 was the first reported FAK-targeted PROTAC molecule exhibiting reversing effects on chemotherapeutic drug resistance, and its highest reversal fold could reach 158 times. The anti-tumor and MDR-reversing effects of F2 might be based on its inhibition on AKT (protein kinase B, PKB) and ERK (extracellular signal-regulated kinase) signaling pathways, as well as its impact on EMT (epithelial-mesenchymal transition). Furthermore, we found that F2 could reduce the protein level of P-gp in HCT8/T cells, thereby contributing to reverse drug resistance from another perspective. Our results will boost confidence in future research focusing on targeting FAK and encourage further investigation of PROTAC with potent in vivo effects.

Lack of CYP3A4 protein induction despite mRNA induction in primary hepatocytes exposed to rifabutin as a possible explanation for its low interaction risk in vivo.

Rifampicin is a strong inducer of cytochrome P450 (CYP3A4) and P-glycoprotein (P-gp/ABCB1), leading to profound drug-drug interactions. In contrast, the chemically related rifabutin does not show such pronounced induction properties in vivo. The aim of our study was to conduct a comprehensive analysis of the different induction potentials of rifampicin and rifabutin in primary human hepatocytes and to analyze the mechanism of potential differences. Therefore, we evaluated CYP3A4/ABCB1 mRNA expression (polymerase chain reaction), CYP3A4/P-gp protein expression (immunoaffinity-liquid chromatography-mass spectrometry, IA-LC-MS/MS), CYP3A4 activity (testosterone hydroxylation), and considered intracellular drug uptake after treatment with increasing rifamycin concentrations (0.01-10 µM). Furthermore, rifamycin effects on the protein levels of CYP2C8, CYP2C9, and CYP2C19 were analyzed (IA-LC-MS/MS). Mechanistic analysis included the evaluation of possible suicide CYP3A4 inhibition (IC50 shift assay) and drug impact on translational efficiency (cell-free luminescence assays). Rifabutin accumulated 6- to 15-fold higher in hepatocytes than rifampicin, but induced CYP3A4 mRNA comparably to rifampicin (e. g. rifampicin 61-fold vs. rifabutin 44-fold, 72 h). While rifampicin for example enhanced protein (10 µM: 21-fold) and activity levels considerably (53-fold), rifabutin only slightly increased CYP3A4 protein expression (10 µM: 3.3-fold) or activity (11-fold) compared to rifampicin after 72 h. Both rifamycins similarly influenced expression of other eliminating proteins. A potential CYP3A4 suicide inhibition by a specific rifabutin metabolite or disruption of ribosome function were excluded experimentally. In conclusion, the lack of protein enhancement, could explain rifabutin's weaker induction-related drug-drug interaction risk in vivo.

Assessment of drug-drug interaction of dapagliflozin with LCZ696 based on an LC-MS/MS method.

The co-administration of dapagliflozin (DPF) and sacubitril/valsartan (LCZ696) has emerged as a promising therapeutic approach for managing heart failure. Given that DPF and LCZ696 are substrates for P-glycoprotein, there is a plausible potential for drug-drug interactions when administered concomitantly. To investigate the pharmacokinetic changes when these drugs are co-administered, we have established and validated a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method capable of simultaneously detecting DPF, LBQ657 (the active metabolite of sacubitril) and valsartan in rat plasma. This method has demonstrated selectivity, sensitivity, and accuracy. Drug-drug interactions were examined by the LC-MS/MS method. The mechanisms were investigated using everted intestinal sac models and Caco-2 cells. The results showed that DPF significantly increased the area under the curve (AUC(0-t)) (3,563.3 ± 651.7 vs. 7,146.5 ± 1,714.9 h µg/L) of LBQ657 (the active metabolite of sacubitril) and the AUC(0-t) (24,022.4 ± 6,774.3 vs. 55,728.3 ± 32,446.3 h µg/L) of valsartan after oral co-administration. Dapagliflozin significantly increased the amount of LBQ657 and valsartan in intestinal sacs by 1- and 1.25-fold at 2.25 h. Caco-2 cell uptake studies confirmed that P-glycoprotein is the transporter involved in this interaction. This finding enhances the understanding of drug-drug interactions in the treatment of heart failure and provides a guidence for clinical therapy.

P-glycoprotein-mediated herb-drug interaction evaluation between Tenacissoside G and paclitaxel.

P-glycoprotein (P-gp)-mediated herb-drug interactions (HDIs) may impact drug efficacy and safety. Tenacissoside G (Tsd-G), a major active component of Marsdenia tenacissima, exhibits anticancer activity. To analyze the effect of Tsd-G on the pharmacokinetics of paclitaxel (PTX), researchers selected 30 Sprague-Dawley (SD) rats, randomized into a solvent control group, a verapamil positive control group, and 20, 40, and 60 mg/kg Tsd-G groups. After seven consecutive days of intraperitoneal injection of verapamil or Tsd-G, a single dose of 6 mg/kg PTX was injected intravenously. Plasma samples were collected at different time points, and proteins were precipitated using a methanol-acetonitrile solution. An ultrahigh-performance liquid chromatography-tandem mass spectrometry method was developed, with docetaxel as an internal standard, and quantified using positive ion multiple reaction monitoring (MRM) mode. This analytical method's specificity, accuracy, precision, recovery, matrix effect, and sample stability meet the requirements for biological sample determination. After Tsd-G administration in rats, the mean residence time of PTX was significantly prolonged. And Tsd-G can stably bind to P-gp by forming hydrogen bonds and inhibiting the expression of P-gp in rat liver. Although the metabolites of PTX were not detected in this study, the above results still indicate the existence of HDIs between Tsd-G and PTX, and P-gp may be the main target to mediate HDIs.

Dual Inhibitors of P-gp and Carbonic Anhydrase XII (hCA XII) against Tumor Multidrug Resistance with Piperazine Scaffold.

A new series of piperazine derivatives were synthesized and studied with the aim of obtaining dual inhibitors of P-glycoprotein (P-gp) and carbonic anhydrase XII (hCA XII) to synergistically overcome the P-gp-mediated multidrug resistance (MDR) in cancer cells expressing the two proteins, P-gp and hCA XII. Indeed, these hybrid compounds contain both P-gp and hCA XII binding groups on the two nitrogen atoms of the heterocyclic ring. All compounds showed good inhibitory activity on each protein (P-gp and hCA XII) studied individually, and many of them showed a synergistic effect in the resistant HT29/DOX and A549/DOX cell lines which overexpress both the target proteins. In particular, compound 33 displayed the best activity by enhancing the cytotoxicity and intracellular accumulation of doxorubicin in HT29/DOX and A549/DOX cells, thus resulting as promising P-gp-mediated MDR reverser with a synergistic mechanism. Furthermore, compounds 13, 27 and 32 induced collateral sensitivity (CS) in MDR cells, as they were more cytotoxic in resistant cells than in the sensitive ones; their CS mechanisms were extensively investigated.

Experimental and Computational Methods to Assess Central Nervous System Penetration of Small Molecules.

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

Studies on pharmacokinetic properties and intestinal absorption mechanism of sanguinarine chloride: in vivo and in situ.

Sanguinarine (SAN) is an alkaloid with multiple biological activities, mainly extracted from Sanguinaria canadensis or Macleaya cordata. The low bioavailability of SAN limits its utilization. At present, the nature and mechanism of SAN intestinal absorption are still unclear. The pharmacokinetics, single-pass intestinal perfusion test (SPIP), and equilibrium solubility test of SAN in rats were studied. The absorption of SAN at 20, 40, and 80 mg/L in different intestinal segments was investigated, and verapamil hydrochloride (P-gp inhibitor), celecoxib (MPR2 inhibitor), and ko143 (BCRP inhibitor) were further used to determine the effect of efflux transporter proteins on SAN absorption. The equilibrium solubility of SAN in three buffer solutions (pH 1.2, 4.5 and 6.8) was investigated. The oral pharmacokinetic results in rats showed that SAN was rapidly absorbed (Tmax=0.5 h), widely distributed (Vz/F = 134 L/kg), rapidly metabolized (CL = 30 L/h/kg), and had bimodal phenomena. SPIP experiments showed that P-gp protein could significantly affect the effective permeability coefficient (Peff) and apparent absorption rate constant (Ka) of SAN. Equilibrium solubility test results show that SAN has the best solubility at pH 4.5. In conclusion, SAN is a substrate of P-gp, and its transport modes include efflux protein transport, passive transport and active transport.

Molecular Heterogeneity of the Brain Endothelium.

The blood-brain barrier (BBB) is part of a neurovascular structure located in the brain's micro vessels, that is essential to maintain brain homeostasis, but prevents the brain uptake of most drugs. Because of its importance in neuro-pharmacotherapy, the BBB has been the subject of extensive research since its discovery over 100 years ago. Major advances in understanding the structure and function of the barrier have been made. Drugs are re-designed to cross the BBB. However, despite these efforts, overcoming the BBB efficiently to treat brain diseases safely remains challenging. The majority of BBB research studies focus on the BBB as a homogenous structure throughout the different brain regions. However, this simplification may lead to an inadequate understanding of the BBB function with significant therapeutic consequences. From this perspective, we analyzed the gene and protein expression profiles of the BBB in the micro vessels from the brains of mice that were isolated from two different brain regions, namely the cortex and the hippocampus. The expression profile of the inter-endothelial junctional protein (claudin-5), three ABC transporters (P-glycoprotein, Bcrp and Mrp-1), and three BBB receptors (lrp-1, TRF and GLUT-1) were analyzed. Our gene and protein analysis showed that the brain endothelium in the hippocampus exhibits different expression profiles compared to the brain cortex. Specifically, brain endothelial cells (BECs) of the hippocampus express higher gene levels of abcb1, abcg2, lrp1, and slc2a1 compared to the BECs of the cortex regions with a trend of increase for claudin-5, while BECs of the cortex express higher gene levels of abcc1 and trf compared to the hippocampus. At the protein levels, the P-gp expression was found to be significantly higher in the hippocampus compared to the cortex, while TRF was found to be up-regulated in the cortex. These data suggest that the structure and function of the BBB are not homogeneous, and imply that drugs are not delivered similarly among the different brain regions. Appreciation of the BBB heterogeneity by future research programs is thus critical for efficient drug delivery and the treatment of brain diseases.

Infection and disruption of placental multidrug resistance (MDR) transporters: Implications for fetal drug exposure.

P-glycoprotein (P-gp, encoded by the ABCB1 gene) and breast cancer resistance protein (BCRP/ABCG2) are efflux multidrug resistance (MDR) transporters localized at the syncytiotrophoblast barrier of the placenta and protect the conceptus from drug and toxin exposure throughout pregnancy. Infection is an important modulator of MDR expression and function. This review comprehensively examines the effect of infection on the MDR transporters, P-gp and BCRP in the placenta. Infection PAMPs such as bacterial lipopolysaccharide (LPS) and viral polyinosinic-polycytidylic acid (poly I:C) and single-stranded (ss)RNA, as well as infection with Zika virus (ZIKV), Plasmodium berghei ANKA (modeling malaria in pregnancy - MiP) and polymicrobial infection of intrauterine tissues (chorioamnionitis) all modulate placental P-gp and BCRP at the levels of mRNA, protein and or function; with specific responses varying according to gestational age, trophoblast type and species (human vs. mice). Furthermore, we describe the expression and localization profile of Toll-like receptor (TLR) proteins of the innate immune system at the maternal-fetal interface, aiming to better understand how infective agents modulate placental MDR. We also highlight important gaps in the field and propose future research directions. We conclude that alterations in placental MDR expression and function induced by infective agents may not only alter the intrauterine biodistribution of important MDR substrates such as drugs, toxins, hormones, cytokines, chemokines and waste metabolites, but also impact normal placentation and adversely affect pregnancy outcome and maternal/neonatal health.

In vitro and in vivo analyses on anti-NSCLC activity of apatinib: rediscovery of a new drug target V600E mutation.

BACKGROUND: Apatinib (YN968D1) is the first small-molecule-targeting drug with anti-tumor activity created in China for the treatment of advanced gastric cancer (GC) and hepatocellular carcinoma (HCC). It showed significant variation in the efficacy for treating cancers, including advanced non-squamous non-small-cell lung cancer (NSCLC). Whether its efficacy could be optimized by subgrouping patients with certain genetic variation remains elusive. METHODS: Here, we firstly used kinase screening to identify any possible target of apatinib against 138 kinases. The effects of apatinib on proliferation rates, cell cycle, cell apoptosis, and cell migration on cancer cell lines were analyzed; the in vitro potential pathways of apatinib on cancer cell lines were screened. The effect of apatinib on mouse cancer models in vivo was also analyzed. RESULTS: Based on HCC364 cells with BRAF V600E mutation, we have shown that apatinib could inhibit their growth, migration, cell cycle, and induce their apoptosis. Based on mice with transplanted HCC364 cells, we have also shown that apatinib could inhibit the tumor growth. Based on immunohistochemistry, we have demonstrated that apatinib could suppress the phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase and extracellular regulated protein kinases. This may account at least part of the apatinib's inhibitory effect on HCC364 cancer cells. CONCLUSIONS: BRAF V600E protein kinase is a target of apatinib by kinase screening. We have demonstrated that apatinib can effectively inhibit tumor cells with BRAF V600E mutation by in vitro and in vivo experiments. Our results have demonstrated that targeting BRAF V600E mutation, apatinib appears to be effective and safe for treating NSCLC and possibly other cancers with the same mutation.