Efflux Transporter P-glycoprotein Research Articles

Polymorphism of the multidrug resistance 1 gene MDR1/ABCB1 C3435T and response to antiepileptic drug treatment in temporal lobe epilepsy

PurposeA synonymous C to T variant at position 3435 (c.3435C>T) is one common polymorphism of the multidrug resistant 1 (MDR1) gene, which encodes the major transmembrane efflux transporter P-glycoprotein. It has been suggested that this polymorphism, and more specifically the 3435CC genotype, may be associated with the response to antiepileptic drug treatment. Here we wished to examine the role of such a candidate variant in a cohort of 175 patients (98 women and 76 men; mean±SD age: 47.90±17.64) with temporal lobe epilepsy (TLE). MethodsPatients were classified according to whether they had drug-responsive (n=134) or drug-resistant (n=41) epilepsy. We also enrolled 175 healthy controls (93 women and 82 men; mean±SD age: 72.5±6.8), matched for sex and ethnicity. ResultsPatients and controls were genotyped for detection of the 3435C>T polymorphism, but the analysis showed no significant association between the CC genotype and the risk of drug-resistant epilepsy. ConclusionThese findings rule out the MDR1 c.3435C>T polymorphism having a major role or increasing the risk of drug-resistance suggesting a revision is required to determine the contribution of this polymorphism in predicting drug response in epilepsy.

Abstract 3682: Contribution of hepatic uptake transporters OATP1B1/OATP1B3 to the disposition of docetaxel

Abstract The antimicrotubular agent docetaxel is a widely used chemotherapeutic drug that has been approved for the treatment of multiple malignant diseases, including cancers of the breast, lung, prostate, stomach, and head and neck. Docetaxel is predominantly dependent on hepatic disposition as ∼75% is excreted in bile. While it is known that docetaxel is a substrate of the efflux transporter P-glycoprotein (MDR1; P-gp), less is known regarding transporters that could mediate its cellular uptake and hepatic clearance. Accordingly, screening an array of drug uptake transporters and kinetic analysis for radiolabeled docetaxel transport using a recombinant vaccinia-based method demonstrated that organic anion transporting polypeptides (OATPs), including OATP1A2, OATP1B1 and OATP1B3, were capable of transporting docetaxel in vitro. In addition, an assessment of a panel of single nucleotide polymorphisms (SNPs) in SLCO1B1 revealed that a number of OATP1B1 variants were associated with impaired docetaxel transport. To further evaluate docetaxel transport by OATP1B1 and OATP1B3, we generated MDCKII cells stably expressing MDR1, OATP1B1, OATP1B3, OATP1B1-MDR1, and OATP1B3-MDR1 proteins. Using a transwell system, we then studied intracellular concentrations and transcellular transport after administration of radiolabeled docetaxel to the basal compartment. The transport assay revealed that docetaxel was transported significantly into the apical compartment of double transfected MDCKII-OATP1B1-MDR1/OATP1B3-MDR1 cells compared to the single transfected cells without MDR1 expression up to 3 hours (p < 0.05). Meanwhile, intracellular accumulation of docetaxel was not different between control cells and MDCKII-OATP1B1 or MDCKII-OATP1B3 cells, but was significantly lower in cell lines expressing MDR1 than in cell lines without MDR1 expression (p < 0.05). Consequently, in line with lower intracellular values, higher amounts of docetaxel were found in the apical compartment of cell lines expressing MDR1. Subsequently, we performed in vivo docetaxel transport studies in wild-type and Slco1b2-/- mice (ortholog to hepatic OATP1B1/OATP1B3). There was ∼>5-fold higher plasma concentrations of docetaxel in Slco1b2-/- versus wild-type mice (p < 0.01). The liver-to-plasma ratio of docetaxel was ∼3-fold decreased in Slco1b2-/- mice compared to wild-type mice (p < 0.05). The plasma clearance of docetaxel in Slco1b2-/- mice was 83% lower than wild-type mice (p < 0.05). In conclusion, we demonstrate functional interplay between transporter-mediated uptake and export in docetaxel disposition, suggesting hepatic OATP1B1 and OATP1B3 are important to docetaxel disposition. Moreover, OATP1B1 variants may contribute to the interindividual variability in docetaxel disposition and response. Accordingly, these findings reveal important new insights into the relevance of hepatic OATPs to the clinical pharmacology of docetaxel. Citation Format: Hye Jeong Lee, Brenda F. Leake, Wendy Teft, Richard B. Kim, Richard H. Ho. Contribution of hepatic uptake transporters OATP1B1/OATP1B3 to the disposition of docetaxel. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3682. doi:10.1158/1538-7445.AM2014-3682

Abstract 4479: Influence of the blood-brain barrier on drug distribution and efficacy of PI3K/mTOR inhibitors

Abstract Drug delivery to glioblastoma multiforme (GBM) is influenced by the presence of blood-brain barrier (BBB). PI3K/mTOR/AKT is frequently dysregulated in GBM and some agents targeting this pathway have limited distribution across the BBB due to activity of efflux transporters p-glycoprotein (P-gp) and breast cancer resistance protein1 (BCRP1). GNE-317 and GDC-0980 are structurally related PI3K/mTOR inhibitors with the former molecule optimized for brain delivery. Here the influence of the BBB on tumor distribution and resultant efficacy was evaluated in flank and intracranial tumors. Pharmacokinetic-pharmacodynamic approaches were used in GBM10 (patient-derived) and U87 (cell line) orthotopic xenograft models to evaluate the influence of delivery on efficacy. We investigated the brain distribution of GNE-317 and GDC-0980 in tumor-bearing (GBM10 and U87) athymic nu/nu mice and non-tumor-bearing (FVBn) mice. BBB-integrity in both GBM10 and U87-models was evaluated using Texas-Red(TR)-linked 3kD dextran prior to euthanasia. GNE-317 and GDC-0980 were administered orally at 30mg/kg and 7.5mg/kg, respectively, for all studies. Brain distribution studies were performed in FVB-wild type (WT) and P-gp/Bcrp knockout mice (TKO) and brain and plasma exposures (area-under-curve, AUC) were determined. Comparison of tumor pharmacokinetic-pharmacodynamic endpoints at 1- and 6-hr post single oral dose will be reported. GBM10 showed heterogeneous disruption of BBB-integrity, while U87 tumors exhibited homogenous and uniform disruption of BBB by analysis of TR-dextran accumulation within orthotopic xenografts. The brain-to-plasma AUC0-tlast ratio in FVBn-wild type was ∼10-fold higher in GNE-317(0.75) vs. GDC-0980 (0.07). Survival studies in intracranial tumors with U87 demonstrated no difference in survival between the GNE-317 vs. GDC-0980 treated groups (median survival∼26 days (GNE-317), 34 days(GDC-0980), p=0.07), and both treatments significantly extended survival compared to vehicle control (p<0.0003, ∼12 days). Survival with GNE-317 in GBM10 xenografts (median survival 42 days vs. 35 days for control, p<0.0003) was greater than the GDC-0980 treated group (median survival 32 days vs. 35 days in control, p=0.79); both treatments were not significantly different from each other (p=0.08). To evaluate the relative efficacy in the complete absence of the BBB, the effects on tumor growth are being evaluated in flank tumor models. These studies demonstrate that efficacy of a PI3K/mTOR inhibitor with low brain penetrance (GDC-0980) is marginally inferior to the related brain penetrant GNE-317 in tumor models with a partially intact BBB. Since GBM patients have regions of intact BBB, these data suggest that the brain penetration of targeted agents may be a critical factor that determines treatment efficacy in patients. Citation Format: Rajneet K. Oberoi, William F. Elmquist, Rajendar Mittapalli, Jenny Pokorny, Jann Sarkaria. Influence of the blood-brain barrier on drug distribution and efficacy of PI3K/mTOR inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4479. doi:10.1158/1538-7445.AM2014-4479

Abstract LB-224: Identification of a binding site for the tyrosine kinase inhibitor Nilotinib on the human ABC drug transporter, P-glycoprotein as determined by 3D-QSAR, molecular docking and mutational mapping

Abstract Nilotinib (Tasigna®) is a novel tyrosine kinase inhibitor (TKI) that has been used to treat chronic phase chronic myeloid leukemia patients who develop resistance due to the emergence of point mutations within the BCR-ABL kinase domain. It is known to be transported by the ATP binding-cassette (ABC) drug efflux transporters P-glycoprotein (P-gp) and ABCG2, which not only alters the efficacy of nilotinib but might also result in lower bioavailability. Identification of a nilotinib binding conformation in the drug-binding pocket of these transporters is important to understand its interactions and can be used to design the next generation of TKIs that do not interact with ABC drug transporters. A structure-activity relationship (SAR)-based approach for nilotinib was used in this study to identify an initial pharmacophore interacting with P-gp. [125I]-Iodoarylazidoprazosin (IAAP) photolabeling and FACS-based transport of fluorescent substrates were carried out with 26 structural derivatives of nilotinib to evaluate their potency for inhibiting the binding of substrate and the transport function of P-gp. IC50 values from these assays were used for 3D pharmacophore modeling and quantitative structure-activity relationships (QSARs) to propose the spatial arrangement of chemical features that are essential for inhibitory activity of nilotinib towards P-gp. Molecular docking of nilotinib in a homology model of human P-gp was performed to identify interactions with the drug-binding pocket. This docked model was evaluated by mutational mapping studies replacing the Y307, M949 and A985 residues with Cysteine in the primary-binding site, which resulted in the loss of nilotinib's activity to inhibit substrate binding to P-gp. The orientation of nilotinib in this binding pocket was further substantiated by assessing the interaction of nilotinib's structural derivatives with P-gp using photoaffinity labeling, ATP hydrolysis and transport assays. These results demonstrate that the pyridine and pyrimidine rings in nilotinib play a key role in its interaction with the drug-binding pocket, while orientation of the imidazole ring determines the affinity of nilotinib for P-gp. These results for the first time identify the primary binding site of nilotinib in the drug-binding pocket of P-gp and can be exploited to design novel tyrosine kinase inhibitors that would not be recognized by ABC drug transporters. Citation Format: Suneet Shukla, Eduardo E. Chufan, Satyakam Singh, Amanda P. Skoumbourdis, Khyati Kapoor, Matthew B. Boxer, Damien Y. Duveau, Craig J. Thomas, Tanaji T. Talele, Suresh V. Ambudkar. Identification of a binding site for the tyrosine kinase inhibitor Nilotinib on the human ABC drug transporter, P-glycoprotein as determined by 3D-QSAR, molecular docking and mutational mapping. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-224. doi:10.1158/1538-7445.AM2014-LB-224

Abstract 754: Selective ALK inhibitor alectinib (CH5424802/RO5424802) with potent antitumor activity in models of crizotinib resistance, including intracranial metastases

Abstract Purpose: EML4-ALK has been implicated as a driver oncogene and a therapeutic target in non-small cell lung cancer (NSCLC). The clinical efficacy of the ALK inhibitor crizotinib has been demonstrated in ALK fusion-positive NSCLC; however, resistance to crizotinib certainly occurs through ALK secondary mutations in clinical use. In addition, brain metastases are frequent sites of initial failure in patients because crizotinib has poor penetration of the central nervous system. This study aimed to clarify the efficacy of ALK inhibitor alectinib (CH5424802/RO5424802) in models of crizotinib-resistant ALK mutant tumors and intracranial tumors. Experimental Design: The antitumor activity of alectinib was evaluated in subcutaneous xenograft tumor models of Ba/F3 cells expressing mutated EML4-ALK, and in intracranial tumor implantation models of EML4-ALK-positive NSCLC NCI-H2228. Results: Alectinib had kinase inhibitory activity against some ALK mutations, including G1269A, and blocked tumor growth driven by a mutant of EML4-ALK in vitro and in vivo. In intracranial tumor implantation mouse models, alectinib resulted in regression of NCI-H2228 tumor in brain and provided a survival benefit. Alectinib is not a substrate for P-glycoprotein efflux transporter in vitro and showed a high brain-to-plasma ratio in rat. Additionally, alectinib led to tumor size reduction in the subcutaneous NCI-H2228 xenograft tumor that had failed to regress fully during treatment with crizotinib. Conclusions: Alectinib was effective against most EML4-ALK mutations and showed potent efficacy against intracranial ALK-positive tumor. Thus, alectinib might provide therapeutic opportunities for crizotinib-treated patients with ALK secondary mutations and brain metastases. Citation Format: Tatsushi Kodama, Toshiyuki Tsukaguchi, Masami Hasegawa, Miyuki Yoshida, Kenji Takanashi, Osamu Kondoh, Hiroshi Sakamoto. Selective ALK inhibitor alectinib (CH5424802/RO5424802) with potent antitumor activity in models of crizotinib resistance, including intracranial metastases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 754. doi:10.1158/1538-7445.AM2014-754

P-glycoprotein inhibition by the agricultural pesticide propiconazole and its hydroxylated metabolites: Implications for pesticide–drug interactions

The human efflux transporter P-glycoprotein (P-gp, MDR1) functions as an important cellular defense system against a variety of xenobiotics; however, little information exists on whether environmental chemicals interact with P-gp. Conazoles provide a unique challenge to exposure assessment because of their use as both pesticides and drugs. Propiconazole is an agricultural pesticide undergoing evaluation by the U.S. Environmental Protection Agency’s Endocrine Disruptor Screening Program. In this study, the P-gp interaction of propiconazole and its hydroxylated metabolites were evaluated using MDR1-expressing membrane vesicles and NIH-3T3/MDR1 cells. Membrane vesicle assays demonstrated propiconazole (IC50,122.9μM) and its metabolites (IC50s, 350.8μM, 366.4μM, and 456.3μM) inhibited P-gp efflux of a probe substrate, with propiconazole demonstrating the strongest interaction. P-gp mediated transport of propiconazole in MDR1-expressed vesicles was not detected indicating propiconazole interacts with P-gp as an inhibitor rather than a substrate. In NIH-3T3/MDR1 cells, propiconazole (1 and 10μM) led to decreased cellular resistance (chemosensitization) to paclitaxel, a chemotherapeutic drug and known MDR1 substrate. Collectively, these results have pharmacokinetic and risk assessment implications as P-gp interaction may influence pesticide toxicity and the potential for pesticide–drug interactions.

Antitumor activity of the selective ALK inhibitor alectinib in models of intracranial metastases.

The clinical efficacy of the anaplastic lymphoma kinase (ALK) inhibitor crizotinib has been demonstrated in ALK fusion-positive non-small cell lung cancer (NSCLC); however, brain metastases are frequent sites of initial failure in patients due to poor penetration of the central nervous system by crizotinib. Here, we examined the efficacy of a selective ALK inhibitor alectinib/CH5424802 in preclinical models of intracranial tumors. We established intracranial tumor implantation mouse models of EML4-ALK-positive NSCLC NCI-H2228 and examined the antitumor activity of alectinib in this model. Plasma distribution and brain distribution of alectinib were examined by quantitative whole-body autoradiography administrating a single oral dose of (14)C-labeled alectinib to rats. The drug permeability of alectinib was evaluated in Caco-2 cell. Alectinib resulted in regression of NCI-H2228 tumor in mouse brain and provided a survival benefit. In a pharmacokinetic study using rats, alectinib showed a high brain-to-plasma ratio, and in an in vitro drug permeability study using Caco-2 cells, alectinib was not transported by P-glycoprotein efflux transporter that is a key factor in blood-brain barrier penetration. We established intracranial tumor implantation models of EML4-ALK-positive NSCLC. Alectinib showed potent efficacy against intracranial EML4-ALK-positive tumor. These results demonstrated that alectinib might provide therapeutic opportunities for crizotinib-treated patients with brain metastases.

In vivo P-glycoprotein function before and after epilepsy surgery.

Objectives:To study the functional activity of the multidrug efflux transporter P-glycoprotein (Pgp) at the blood-brain barrier of patients with temporal lobe epilepsy using (R)-[11C]verapamil (VPM)-PET before and after temporal lobe surgery to assess whether postoperative changes in seizure frequency and antiepileptic drug load are associated with changes in Pgp function.Methods:Seven patients with drug-resistant temporal lobe epilepsy underwent VPM-PET scans pre- and postsurgery. Patients were followed up for a median of 6 years (range 4–7) after surgery. Pgp immunoreactivity in surgically resected hippocampal specimens was determined with immunohistochemistry.Results:Optimal surgical outcome, defined as seizure freedom and withdrawal of antiepileptic drugs, was associated with higher temporal lobe Pgp function before surgery, higher Pgp-positive staining in surgically resected hippocampal specimens, and reduction in global Pgp function postoperatively, compared with nonoptimal surgery outcome.Conclusions:The data from our pilot study suggest that Pgp overactivity in epilepsy is dynamic, and complete seizure control and elimination of antiepileptic medication is associated with reversal of overactivity, although these findings will require confirmation in a larger patient cohort.

L-1416, a novel MDR reversing agent with possible reduced calcium antagonism

BackgroundMultidrug efflux transporter P-glycoprotein (P-gp) is highly expressed on membrane of tumor cells and supposed to be implicated in the resistance to tumor chemotherapy. However, currently none of P-gp inhibitors has been approved by Food and Drug Administration not only due to toxicity but also lack of efficacy in clinical trials. MethodsTo solve the problem, our lab synthesized a novel compound named 1416 [1-(2,6-dimethylphenoxy)-3,4-dimethoxyphenylethylamino) propane hydrochloride] with the hope of high P-gp inhibition and low side effects. Caco-2 cell monolayer and tumor bearing mice were used to evaluate the P-gp inhibition of 1416 in vitro and in vivo, respectively. One of its potential side effects, calcium antagonism was also evaluated. ResultsResults showed that 1416 showed a similar P-gp inhibition as verapamil in Caco-2 cell monolayer. No significant difference was observed in antitumor enhancement when the optical isomers of 1416 (D-1416 and L-1416) were co-administered with vinblastine. In calcium antagonism, L-1416 showed less calcium inhibition than both D-1416 and verapamil. ConclusionThe novel compound 1416 could significantly increase the antitumor effects of cytotoxic drugs and one of its optical isomers, L-1416, might be more promising due to its potential low calcium antagonism.

Setting-up an <em>In Vitro</em> Model of Rat Blood-brain Barrier (BBB): A Focus on BBB Impermeability and Receptor-mediated Transport

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

Setting-up an in vitro model of rat blood-brain barrier (BBB): a focus on BBB impermeability and receptor-mediated transport.

The blood brain barrier (BBB) specifically regulates molecular and cellular flux between the blood and the nervous tissue. Our aim was to develop and characterize a highly reproducible rat syngeneic in vitro model of the BBB using co-cultures of primary rat brain endothelial cells (RBEC) and astrocytes to study receptors involved in transcytosis across the endothelial cell monolayer. Astrocytes were isolated by mechanical dissection following trypsin digestion and were frozen for later co-culture. RBEC were isolated from 5-week-old rat cortices. The brains were cleaned of meninges and white matter, and mechanically dissociated following enzymatic digestion. Thereafter, the tissue homogenate was centrifuged in bovine serum albumin to separate vessel fragments from nervous tissue. The vessel fragments underwent a second enzymatic digestion to free endothelial cells from their extracellular matrix. The remaining contaminating cells such as pericytes were further eliminated by plating the microvessel fragments in puromycin-containing medium. They were then passaged onto filters for co-culture with astrocytes grown on the bottom of the wells. RBEC expressed high levels of tight junction (TJ) proteins such as occludin, claudin-5 and ZO-1 with a typical localization at the cell borders. The transendothelial electrical resistance (TEER) of brain endothelial monolayers, indicating the tightness of TJs reached 300 ohm·cm2 on average. The endothelial permeability coefficients (Pe) for lucifer yellow (LY) was highly reproducible with an average of 0.26 ± 0.11 x 10-3 cm/min. Brain endothelial cells organized in monolayers expressed the efflux transporter P-glycoprotein (P-gp), showed a polarized transport of rhodamine 123, a ligand for P-gp, and showed specific transport of transferrin-Cy3 and DiILDL across the endothelial cell monolayer. In conclusion, we provide a protocol for setting up an in vitro BBB model that is highly reproducible due to the quality assurance methods, and that is suitable for research on BBB transporters and receptors.

P-gp and taxanes.

In the past years, the development of oral formulations of the taxane anticancer drugs paclitaxel (Taxol®) and docetaxel (Taxotere®) has been the focus of preclinical and clinical research in our groups. A major limitation in the concept of oral administration of paclitaxel and docetaxel is their low oral availability [1]. Paclitaxel and docetaxel have poor aqueous solubility and upon oral administration, intestinal uptake is seriously hampered by drug efflux through the active efflux transporter P-glycoprotein (P-gp/MDR1/ABCB1) and by drug metabolism via Cytochrome P450 (CYP) 3A [2,3]. Several studies by our groups have shown that the oral bioavailability of paclitaxel and docetaxel can be enhanced by 1) oral administration of a solid dispersion of these taxanes [4], and 2) combining taxanes with P-gp inhibitors or CYP3A4 inhibitors to reduce intestinal and hepatic drug efflux transport and drug metabolism [1]. Importantly, since P-gp is not only expressed in tissues like intestine, liver, and kidney, but also at the blood-brain barrier (BBB) where it keeps its substrates out of the brain [5], co-administration of orally administered taxanes with P-gp inhibitors might also result in increased brain penetration of the taxanes.

Uptake of compounds that selectively kill multidrug-resistant cells: the copper transporter SLC31A1 (CTR1) increases cellular accumulation of the thiosemicarbazone NSC73306.

Acquired drug resistance in cancer continues to be a challenge in cancer therapy, in part due to overexpression of the drug efflux transporter P-glycoprotein (P-gp, MDR1, ABCB1). NSC73306 is a thiosemicarbazone compound that displays greater toxicity against cells expressing functional P-gp than against other cells. Here, we investigate the cellular uptake of NSC73306, and examine its interaction with P-gp and copper transporter 1 (CTR1, SLC31A1). Overexpression of P-gp sensitizes LLC-PK1 cells to NSC73306. Cisplatin (IC50 = 77 μM), cyclosporin A (IC50 = 500 μM), and verapamil (IC50 = 700 μM) inhibited cellular accumulation of [3H]NSC73306. Cellular hypertoxicity of NSC73306 to P-gp-expressing cells was inhibited by cisplatin in a dose-dependent manner. Cells transiently expressing the cisplatin uptake transporter CTR1 (SLC31A1) showed increased [3H]NSC73306 accumulation. In contrast, CTR1 knockdown decreased [3H]NSC73306 accumulation. The presence of NSC73306 reduced CTR1 levels, similar to the negative feedback of CTR1 levels by copper or cisplatin. Surprisingly, although cisplatin is a substrate of CTR1, we found that CTR1 protein was overexpressed in high-level cisplatin-resistant KB-CP20 and BEL7404-CP20 cell lines. We confirmed that the CTR1 protein was functional, as uptake of NSC73306 was increased in KB-CP20 cells compared to their drug-sensitive parental cells, and downregulation of CTR1 in KB-CP20 cells reduced [3H]NSC73306 accumulation. These results suggest that NSC73306 is a transport substrate of CTR1.

Characterization of a novel brain barrier ex vivo insect-based P-glycoprotein screening model.

In earlier studies insects were proposed as suitable models for vertebrate blood–brain barrier (BBB) permeability prediction and useful in early drug discovery. Here we provide transcriptome and functional data demonstrating the presence of a P-glycoprotein (Pgp) efflux transporter in the brain barrier of the desert locust (Schistocerca gregaria). In an in vivo study on the locust, we found an increased uptake of the two well-known Pgp substrates, rhodamine 123 and loperamide after co-administration with the Pgp inhibitors cyclosporine A or verapamil. Furthermore, ex vivo studies on isolated locust brains demonstrated differences in permeation of high and low permeability compounds. The vertebrate Pgp inhibitor verapamil did not affect the uptake of passively diffusing compounds but significantly increased the brain uptake of Pgp substrates in the ex vivo model. In addition, studies at 2°C and 30°C showed differences in brain uptake between Pgp-effluxed and passively diffusing compounds. The transcriptome data show a high degree of sequence identity of the locust Pgp transporter protein sequences to the human Pgp sequence (37%), as well as the presence of conserved domains. As in vertebrates, the locust brain–barrier function is morphologically confined to one specific cell layer and by using a whole-brain ex vivo drug exposure technique our locust model may retain the major cues that maintain and modulate the physiological function of the brain barrier. We show that the locust model has the potential to act as a robust and convenient model for assessing BBB permeability in early drug discovery.

Evaluation of in vitro absorption, distribution, metabolism, and excretion (ADME) properties of mitragynine, 7-hydroxymitragynine, and mitraphylline.

Mitragyna speciosa (kratom) is a popular herb in Southeast Asia, which is traditionally used to treat withdrawal symptoms associated with opiate addiction. Mitragynine, 7-hydroxymitragynine, and mitraphylline are reported to be the central nervous system active alkaloids which bind to the opiate receptors. Mitraphylline is also present in the bark of Uncaria tomentosa (cat's claw). Several therapeutic properties have been reported for these compounds but limited information is available on the absorption and distribution properties. This study focuses on evaluating the absorption, distribution, metabolism, and excretion (ADME) properties of these compounds and their effect on major efflux transporter P-glycoprotein, using in vitro methods. Quantitative analysis was performed by the Q-TOF LC-MS system. Mitragynine was unstable in simulated gastric fluid with 26 % degradation but stable in simulated intestinal fluid. 7-Hydroxymitragynine degraded up to 27 % in simulated gastric fluid, which could account for its conversion to mitragynine (23 %), while only 6 % degradation was seen in simulated intestinal fluid. Mitraphylline was stable in simulated gastric fluid but unstable in simulated intestinal fluid (13.6 % degradation). Mitragynine and 7-hydroxymitragynine showed moderate permeability across Caco-2 and MDR-MDCK monolayers with no significant efflux. However, mitraphylline was subjected to efflux mediated by P-glycoprotein in both Caco-2 and MDR-MDCK monolayers. Mitragynine was found to be metabolically stable in both human liver microsomes and S9 fractions. In contrast, both 7-hydroxymitragynine and mitraphylline were metabolized by human liver microsomes with half-lives of 24 and 50 min, respectively. All three compounds exhibited high plasma protein binding (> 90 %) determined by equilibrium dialysis. Mitragynine and 7-hydroxymitragynine inhibited P-glycoprotein with EC50 values of 18.2 ± 3.6 µM and 32.4 ± 1.9 µM, respectively, determined by the calcein-AM fluorescent assay, while no inhibition was seen with mitraphylline. These data indicate the possibility of a drug interaction if mitragynine and 7-hydroxymitragynine are coadministered with drugs that are P-glycoprotein substrates.

Identification of a functional homolog of the mammalian CYP3A4 in locusts.

Insects have been proposed as a new tool in early drug development. It was recently demonstrated that locusts have an efflux transporter localized in the blood-brain barrier (BBB) that is functionally similar to the mammalian P-glycoprotein efflux transporter. Two insect BBB models have been put forward, an ex vivo model and an in vivo model. To use the in vivo model it is necessary to fully characterize the locust as an entire organism with regards to metabolic pathways and excretion rate. In the present study, we have characterized the locust metabolism of terfenadine, a compound that in humans is specific to the cytochrome P450 enzyme 3A4. Using high-resolution mass spectrometry coupled to ultra-high-performance liquid chromatography, we have detected metabolites identical to human metabolites of terfenadine. The formation of human metabolites in locusts was inhibited by ketoconazole, a mammalian CYP3A4 inhibitor, suggesting that the enzyme responsible for the human metabolite formation in locusts is functionally similar to human CYP3A4. Besides the human metabolites of terfenadine, additional metabolites were formed in locusts. These were tentatively identified as phosphate and glucose conjugates. In conclusion, not only may locusts be a model useful for determining BBB permeation, but possibly insects could be used in metabolism investigation. However, extensive characterization of the insect model is necessary to determine its applicability.

Multilayer Spheroids To Quantify Drug Uptake and Diffusion in 3D

There is a need for new quantitative in vitro models of drug uptake and diffusion to help assess drug toxicity/efficacy as well as new more predictive models for drug discovery. We report a three-dimensional (3D) multilayer spheroid model and a new algorithm to quantitatively study uptake and inward diffusion of fluorescent calcein via gap junction intercellular communication (GJIC). When incubated with calcein-AM, a substrate of the efflux transporter P-glycoprotein (Pgp), spheroids from a variety of cell types accumulated calcein over time. Accumulation decreased in spheroids overexpressing Pgp (HEK-MDR) and was increased in the presence of Pgp inhibitors (verapamil, loperamide, cyclosporin A). Inward diffusion of calcein was negligible in spheroids that lacked GJIC (OVCAR-3, SK-OV-3) and was reduced in the presence of an inhibitor of GJIC (carbenoxolone). In addition to inhibiting Pgp, verapamil and loperamide, but not cyclosporin A, inhibited inward diffusion of calcein, suggesting that they also inhibit GJIC. The dose response curves of verapamil’s inhibition of Pgp and GJIC were similar (IC50: 8 μM). The method is amenable to many different cell types and may serve as a quantitative 3D model that more accurately replicates in vivo barriers to drug uptake and diffusion.

Modulation of Blood–Brain Barrier Permeability in Mice Using Synthetic E-Cadherin Peptide

The present work characterizes the effects of synthetic E-cadherin peptide (HAV) on blood–brain barrier (BBB) integrity using various techniques including magnetic resonance imaging (MRI) and near-infrared fluorescent imaging (NIRF). The permeability of small molecular weight permeability marker gadolinium diethylenetriaminepentaacetate (Gd-DTPA) contrast agent, the large molecular weight permeability marker, IRDye 800CW PEG, and the P-glycoprotein (P-gp) efflux transporter contrast agent, rhodamine 800 (R800), were examined in the presence and absence of HAV peptide. The results consistently demonstrated that systemic iv administration of HAV peptide resulted in a reversible disruption of BBB integrity and enhanced the accumulation of all the dyes examined. The magnitude of increase ranged from 2-fold to 5-fold depending on the size and the properties of the permeability markers. The time frame for BBB disruption with HAV peptide was rapid, occurring within 3–6 min following injection of the peptide. Furthermore, modulation of BBB permeability was reversible with the barrier integrity being restored within 60 min of the injection. The increased BBB permeability observed following HAV peptide administration was not attributable to changes in cerebral blood flow. These studies support the potential use of cadherin peptides to rapidly and reversibly modulate BBB permeability of a variety of therapeutic agents.

P-glycoprotein Modulates Morphine Uptake into the CNS: A Role for the Non-steroidal Anti-inflammatory Drug Diclofenac

Our laboratory has previously demonstrated that peripheral inflammatory pain (PIP), induced by subcutaneous plantar injection of λ-carrageenan, results in increased expression and activity of the ATP-dependent efflux transporter P-glycoprotein (P-gp) that is endogenously expressed at the blood-brain barrier (BBB). The result of increased P-gp functional expression was a significant reduction in CNS uptake of morphine and, subsequently, reduced morphine analgesic efficacy. A major concern in the treatment of acute pain/inflammation is the potential for drug-drug interactions resulting from P-gp induction by therapeutic agents co-administered with opioids. Such effects on P-gp activity can profoundly modulate CNS distribution of opioid analgesics and alter analgesic efficacy. In this study, we examined the ability of diclofenac, a non-steroidal anti-inflammatory drug (NSAID) that is commonly administered in conjunction with the opioids during pain therapy, to alter BBB transport of morphine via P-gp and whether such changes in P-gp morphine transport could alter morphine analgesic efficacy. Administration of diclofenac reduced paw edema and thermal hyperalgesia in rats subjected to PIP, which is consistent with the known mechanism of action of this NSAID. Western blot analysis demonstrated an increase in P-gp expression in rat brain microvessels not only following PIP induction but also after diclofenac treatment alone. Additionally, in situ brain perfusion studies showed that both PIP and diclofenac treatment alone increased P-gp efflux activity resulting in decreased morphine brain uptake. Critically, morphine analgesia was significantly reduced in animals pretreated with diclofenac (3 h), as compared to animals administered diclofenac and morphine concurrently. These novel findings suggest that administration of diclofenac and P-gp substrate opioids during pain pharmacotherapy may result in a clinically significant drug-drug interaction.

Coadministration of P-Glycoprotein Modulators on Loperamide Pharmacokinetics and Brain Distribution

The efflux transporter P-glycoprotein, expressed at high levels at the blood-brain barrier, exerts a profound effect on the disposition of various therapeutic compounds in the brain. A rapid and efficient modulation of this efflux transporter could enhance the distribution of its substrates and thereby improve central nervous system pharmacotherapies. This study explored the impact of the intravenous coadministration of two P-glycoprotein modulators, tariquidar and elacridar, on the pharmacokinetics and brain distribution of loperamide, a P-glycoprotein substrate probe, in rats. After 1 hour postdosing, tariquidar and elacridar, both at a dose of 1.0 mg/kg, increased loperamide levels in the brain by 2.3- and 3.5-fold, respectively. However, the concurrent administration of both P-glycoprotein modulators, each at a dose of 0.5 mg/kg, increased loperamide levels in the brain by 5.8-fold and resulted in the most pronounced opioid-induced clinical signs. This phenomenon may be the result of a combined noncompetitive modulation by tariquidar and elacridar. Besides, the simultaneous administration of elacridar and tariquidar did not significantly modify the pharmacokinetic parameters of loperamide. This observation potentially allows the concurrent use of low but therapeutic doses of P-gp modulators to achieve full inhibitory effects.