Inhibition Of Efflux Transporters Research Articles

Ibrutinib disrupts blood-tumor barrier integrity and prolongs survival in rodent glioma model

In malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-tumor barrier (BTB). Ibrutinib, FDA-approved lymphoma agent, inhibits Bruton tyrosine kinase (BTK) and has previously been shown to independently impair aortic endothelial adhesion and increase rodent glioma model survival in combination with cytotoxic therapy. Yet additional research is required to understand ibrutinib’s effect on BTB function. In this study, we detail baseline BTK expression in glioma cells and its surrounding vasculature, then measure endothelial junctional expression/function changes with varied ibrutinib doses in vitro. Rat glioma cells and rodent glioma models were treated with ibrutinib alone (1–10 µM and 25 mg/kg) and in combination with doxil (10–100 µM and 3 mg/kg) to assess additive effects on viability, drug concentrations, tumor volume, endothelial junctional expression and survival. We found that ibrutinib, in a dose-dependent manner, decreased brain endothelial cell–cell adhesion over 24 h, without affecting endothelial cell viability (p < 0.005). Expression of tight junction gene and protein expression was decreased maximally 4 h after administration, along with inhibition of efflux transporter, ABCB1, activity. We demonstrated an additive effect of ibrutinib with doxil on rat glioma cells, as seen by a significant reduction in cell viability (p < 0.001) and increased CNS doxil concentration in the brain (56 ng/mL doxil alone vs. 74.6 ng/mL combination, p < 0.05). Finally, Ibrutinib, combined with doxil, prolonged median survival in rodent glioma models (27 vs. 16 days, p < 0.0001) with brain imaging showing a − 53% versus − 75% volume change with doxil alone versus combination therapy (p < 0.05). These findings indicate ibrutinib’s ability to increase brain endothelial permeability via junctional disruption and efflux inhibition, to increase BTB drug entry and prolong rodent glioma model survival. Our results motivate the need to identify other BTB modifiers, all with the intent of improving survival and reducing systemic toxicities.

266 Combination Drug Therapy in H3 K27-Altered Diffuse Midline Glioma Enhances Tumor Retention and Treatment Efficacy

INTRODUCTION: Diffuse midline gliomas (DMG) with H3 K27 alterations are uniformly lethal tumors for which no therapy has been found to be effective. One reason for our therapy failure is the blood-brain barrier (BBB), which severely restricts uptake and accumulation of systemically administered drugs in the brain. Convection-enhanced delivery (CED) has emerged as a technique that circumvents the BBB by directly infusing therapeutics into the tumor parenchyma through generation of a mechanical pressure gradient, allowing for homogenous drug distribution at high local concentrations with minimal systemic toxicity. However, efflux transport proteins expressed by the BBB and the tumor, including P-gp and BCRP, rapidly clear the majority of drugs given by CED, severely limiting its therapeutic potential. METHODS: A panel of FDA-approved drugs (ponatinib, topotecan, tretinoin) were evaluated alone and in combination with the known efflux transporter inhibitor everolimus. RESULTS: A high degree of formal synergy was observed in multiple DMG cell lines in vitro, which was attributed to everolimus’ ability to inhibit mTOR signaling and P-gp/BCRP. In vivo, drugs that were expected to penetrate the BBB and be less likely to undergo active efflux persisted at the site of infusion the longest following CED. Concurrent dosing with everolimus further enhanced this effect, which was particularly apparent with the dual P-gp/BCRP substrate ponatinib. CONCLUSIONS: The proclivity of many drugs to be rapidly cleared from the brain by efflux transporters generates a vulnerability that can be exploited for the development of novel therapeutic regimens in DMG, such as with concomitant dosing of P-gp/BCRP-inhibiting drugs when using CED.

Evaluation of ABT-751, a novel anti-mitotic agent able to overcome multi-drug resistance, in melanoma cells

PurposeDrug efflux transporter associated multi-drug resistance (MDR) is a potential limitation in the use of taxane chemotherapies for the treatment of metastatic melanoma. ABT-751 is an orally bioavailable microtubule-binding agent capable of overcoming MDR and proposed as an alternative to taxane-based therapies.MethodsThis study compares ABT-751 to taxanes in vitro, utilizing seven melanoma cell line models, publicly available gene expression and drug sensitivity databases, a lung cancer cell line model of MDR drug efflux transporter overexpression (DLKP-A), and drug efflux transporter ATPase assays.ResultsMelanoma cell lines exhibit a low but variable protein and RNA expression of drug efflux transporters P-gp, BCRP, and MDR3. Expression of P-gp and MDR3 correlates with sensitivity to taxanes, but not to ABT-751. The anti-proliferative IC50 profile of ABT-751 was higher than the taxanes docetaxel and paclitaxel in the melanoma cell line panel, but fell within clinically achievable parameters. ABT-751 IC50 was not impacted by P-gp-overexpression in DKLP-A cells, which display strong resistance to the P-gp substrate taxanes compared to DLKP parental controls. The addition of ABT-751 to paclitaxel treatment significantly decreased cell proliferation, suggesting some reversal of MDR. ATPase activity assays suggest that ABT-751 is a potential BCRP substrate, with the ability to inhibit P-gp ATPase activity.ConclusionOur study confirms that ABT-751 is active against melanoma cell lines and models of MDR at physiologically relevant concentrations, it inhibits P-gp ATPase activity, and it may be a BCRP and/or MDR3 substrate. ABT-751 warrants further investigation alone or in tandem with other drug efflux transporter inhibitors for hard-to-treat MDR melanoma.

Pharmacokinetics and Absorption, Distribution, Metabolism and Excretion of RGLS4326 in Mouse and Monkey, an Anti-miR-17 Oligonucleotide for the Treatment of Polycystic Kidney Disease.

RGLS4326 is a short oligonucleotide inhibitor of microRNA-17 (miR-17) that preferentially distributes to the kidney and displaces miR-17 from translationally active polysomes. Here, we present pharmacokinetics and absorption, distribution, metabolism, and excretion properties of RGLS4326 from mice and monkeys. RGLS4326 was absorbed rapidly after subcutaneous administration, distributed extensively to the kidney and liver, with preferential distribution to the kidney, and cleared rapidly from plasma by tissue uptake and renal excretion. Plasma exposure increased in a dose-proportional manner with no notable accumulation after repeat doses. Plasma protein binding of RGLS4326 across all species tested was between 79% and 96%. RGLS4326 predominantly distributed to the kidney with a long half-life (t1/2; t1/2 ranged from 8-11 days) and no marked (≤twofold) accumulation in kidney and liver after repeat doses. RGLS4326 was minimally metabolized by nucleases, not cytochrome P450 (P450) isozymes, across species and underwent sequential hydrolysis from both 3' and 5' ends to produce chain-shortened metabolites. There were no human unique metabolites observed. Renal excretion was the major route of elimination of RGLS4326, and a significant fraction (50%-79%) of the dose was recovered intact in the urine of mice and monkeys across all dose levels. RGLS4326 is not a substrate, inhibitor, or inducer of P450 isozymes, and it is not a substrate or inhibitor of uptake and most efflux transporters. Thus, RGLS4326 exhibits low potential of mediating drug-drug interactions involving P450 isozymes and drug transporters. SIGNIFICANCE STATEMENT: Pharmacokinetics (PK) and absorption, distribution, metabolism, and excretion (ADME) properties of RGLS4326 were characterized in vivo and in vitro. RGLS4326 shows similar PK and ADME properties across mice and monkeys in vivo and across human and animal matrices in vitro. Subcutaneous administration results in preferential exposure of RGLS4326 to the intended target organ (kidney) to drive maximum target engagement. These studies support the interpretation of toxicology and efficacy studies and help characterize the disposition of RGLS4326 in humans.

Inhibition of efflux transporters by poly ADP-ribose polymerase inhibitors.

Poly ADP-ribose polymerase (PARP) inhibitors have been approved for the treatment of various cancers. They share a similar mechanism of action but have differences in pharmacokinetic characteristics and potential for drug-drug interactions (DDI). This study evaluated the potential ATP-binding cassette transporter-mediated interactions between PARP inhibitors (niraparib, olaparib and rucaparib) and statins (atorvastatin and rosuvastatin). We studied the inhibitory activity of PARP inhibitors on breast cancer resistance protein (BCRP), multidrug resistance-associated protein 3 (MRP3) and P-glycoprotein (P-gp) using vesicular transport assays and determined the concentrations required for 50% inhibition (IC50 ). Then, we predicted the increase of statin exposure followed by the administration of PARP inhibitors using a mechanistic static model. Rucaparib was the strongest inhibitor of BCRP-mediated rosuvastatin transport (IC50 13.7 μM), followed by niraparib (42.6 μM) and olaparib (216 μM). PARP inhibitors did not affect MRP3. While niraparib appeared to inhibit P-gp, the inhibition showed large variability. The inhibition of intestinal BCRP by rucaparib, niraparib and olaparib was predicted to elevate rosuvastatin exposure by 52%, 37% and 24%, respectively. The interactions between PARP inhibitors and rosuvastatin are probably of minor clinical significance alone, but combined with other predisposing factors, they may increase the risk of rosuvastatin-associated adverse effects.

The inhibitor preincubation effect is universal to SLC transporter assays and is only partially eliminated in the presence of extracellular protein.

Variation in the methodology of in vitro transporter inhibition assays causes wide divergence in reported IC50/Ki data. Notably, although potentiation of transporter inhibition by preincubation (PTIP) has been described, current guidelines do not specifically recommend inhibitor preincubation, only encourage sponsors to follow emerging literature. To clarify how generally preincubation should be considered in transporter inhibition studies, and whether PTIP can be solely explained by protein binding of the respective inhibitors, we performed in vitro inhibition assays on SLC and ABC transporters scarcely or not covered in prior research, and examined the effect of extracellular protein in preincubation and washout experiments. In SLC assays without extracellular protein, a 30-minute preincubation caused significant >2-fold change of IC50 in 21/33 transporter-inhibitor combinations involving 19 evolutionarily disparate transporters. The preincubation effect correlated with inhibitor properties like protein binding and aqueous solubility. In vesicular transport assays of MDR1, BCRP, MRP2, and BSEP, sizable PTIP was observed for only 2/23 combinations, and preincubation was practically inconsequential in BCRP or MDR1 monolayer assays. In SLC assays, PTIP partly persisted in the presence of 5% albumin, indicating that the absence of extracellular protein does not fully explain PTIP. The presence of protein, however, complicated the interpretation of results. Overall, while preincubating without protein may overpredict inhibitory potency, adding protein compromises clarity, and omitting preincubation altogether may miss clinically relevant inhibitors. Therefore, we propose that protein-free preincubation should be considered in all SLC inhibition assays. ABC inhibition seems less commonly affected by preincubation, but conclusions require further investigation. Significance Statement Drugs may inhibit transporter proteins in the body, which may precipitate drug interactions. In vitro transporter inhibition assays help predict such drug interactions. Some inhibitors act more potently when preincubated with the transporter prior to the assay. Here we argue that this effect is not a mere in vitro artefact due to the lack of plasma proteins and should be considered in all uptake inhibition assays to model the worst-case scenario. Preincubation in efflux transporter inhibition assays is likely dispensable.

Synthesis and Preclinical Positron Emission Tomography Imaging of the p38 MAPK Inhibitor [11C]Talmapimod: Effects of Drug Efflux and Sex Differences.

Stress-activated kinases are targets of interest in neurodegenerative disease due to their involvement in inflammatory signaling and synaptic dysfunction. The p38α kinase has shown clinical and preclinical promise as a druggable target in several neurodegenerative conditions. We report the radiosynthesis and evaluation of the first positron emission tomography (PET) radiotracer for imaging MAPK p38α/β through radiolabeling of the inhibitor talmapimod (SCIO-469) with carbon-11. [11C]Talmapimod was reliably synthesized by carbon-11 methylation with non-decay corrected radiochemical yields of 3.1 ± 0.7%, molar activities of 38.9 ± 13 GBq/μmol, and >95% radiochemical purity (n = 20). Preclinical PET imaging in rodents revealed a low baseline brain uptake and retention with standardized uptake values (SUV) of ∼0.2 over 90 min; however, pretreatment with the P-glycoprotein (P-gp) drug efflux transporter inhibitor elacridar enabled [11C]talmapimod to pass the blood-brain barrier (>1.0 SUV) with distinct sex differences in washout kinetics. Blocking studies with a structurally dissimilar p38α/β inhibitor, neflamapimod (VX-745), and displacement imaging studies with talmapimod were attempted in elacridar-pretreated rodents, but neither compound displaced radiotracer uptake in the brain of either sex. Ex vivo radiometabolite analysis revealed substantial differences in the composition of radioactive species present in blood plasma but not in brain homogenates at 40 min post radiotracer injection. Digital autoradiography in fresh-frozen rodent brain tissue confirmed that the radiotracer signal was largely non-displaceable in vitro, where self-blocking and blocking with neflamapimod marginally decreased the total signal by 12.9 ± 8.8% and 2.66 ± 2.1% in C57bl/6 healthy controls and 29.3 ± 2.7% and 26.7 ± 12% in Tg2576 rodent brains, respectively. An MDCK-MDR1 assay suggests that talmapimod is likely to suffer from drug efflux in humans as well as rodents. Future efforts should focus on radiolabeling p38 inhibitors from other structural classes to avoid P-gp efflux and non-displaceable binding.

Studies on the interaction of five triazole fungicides with human renal transporters in cells

The widespread use of triazole fungicides in agricultural production poses a potential risk to human health. This study investigates the interaction of five triazole fungicides, i.e., tebuconazole, triticonazole, hexaconazole, penconazole, and uniconazole with human renal transporters, including OAT1, OAT3, OCT2, OCTN1, OCTN2, MATE1, MATE2-K, MRP2, MDR1, and BCRP, using transgenic cell models. For uptake transporters, triticonazole was the substrate of OAT1 and OAT3 and the inhibitor of OCT2. Tebuconazole and penconazole inhibited OCTN2 (100 μM), while tebuconazole, triticonazole, hexaconazole, penconazole, and uniconazole inhibited MATE1 (100 μM). Tebuconazole and hexaconazole inhibited MATE2-K (100 μM). All five triazole fungicides were not substrates or strong inhibitors of MRP2, MDR1, and BCRP efflux transporters. Penconazole inhibited OCT2 with IC50 = 1.12 μM. Penconazole and uniconazole inhibited MATE1 with IC50 = 0.94 μM and 0.87 μM. Tebuconazole and hexaconazole inhibited MATE2-K with IC50 = 0.96 μM and 1.04 μM, indicating that triazole fungicides may inhibit renal drug transporter activity at low concentrations. Triticonazole was transported by OAT1 and OAT3, and the Km values of triticonazole were 5.81 ± 1.75 and 47.35 ± 14.27, respectively. Tebuconazole and uniconazole were transported by OAT3, and the Km values of tebuconazole and uniconazole were 30.28 ± 7.18 and 87.61 ± 31.70, respectively, which may induce nephrotoxicity.

Semi-Synthetic Ecdysteroid 6-Oxime Derivatives of 20-Hydroxyecdysone Possess Anti-Cryptococcal Activity

Cryptococcosis, a life-threatening fungal infection, frequently occurs in patients suffering from AIDS. The treatment of the disease is hampered by the limited number of the effective drugs and the increasing resistance; therefore, to find new active substances is needed. As meningitis is the most serious infection affecting the AIDS patients, effective drugs have to be capable of entering to the central nervous system. Ecdysteroids are natural bioactive molecules with considerable anabolic activity and without toxic side effects on humans. The aim of this work was to study the anti-cryptococcal activity of a natural ecdysteroid, 20E, and its three semi-synthetic derivatives obtained by structural modification of the original molecule. We established the minimum inhibitory concentration of the compounds with microdilution method and demonstrated their fungicidal activity by flow cytometry and cultivation of the drug-treated cells. The interaction of the compounds with each other and efflux transporter inhibitors was assessed by checkerboard titration method. Two derivatives, 20E-EOx and 20E-ZOx, inhibited the growth of Cryptococcus neoformans with minimum inhibitory concentration 2 mg/mL and 1 mg/mL, respectively; both compounds possess fungicidal effect. A combination of the ecdysteroids with each other and verapamil resulted in additive interaction. This study confirmed that structural modification of an originally non-antimicrobial molecule can enhance its effectiveness.

Genotoxicity of pyrrolizidine alkaloids in metabolically inactive human cervical cancer HeLa cells co-cultured with human hepatoma HepG2 cells

Pyrrolizidine alkaloids (PAs) are secondary plant metabolites, which can be found as contaminant in various foods and herbal products. Several PAs can cause hepatotoxicity and liver cancer via damaging hepatic sinusoidal endothelial cells (HSECs) after hepatic metabolization. HSECs themselves do not express the required metabolic enzymes for activation of PAs. Here we applied a co-culture model to mimic the in vivo hepatic environment and to study PA-induced effects on not metabolically active neighbour cells. In this co-culture model, bioactivation of PA was enabled by metabolically capable human hepatoma cells HepG2, which excrete the toxic and mutagenic pyrrole metabolites. The human cervical epithelial HeLa cells tagged with H2B-GFP were utilized as non-metabolically active neighbours because they can be identified easily based on their green fluorescence in the co-culture. The PAs europine, riddelliine and lasiocarpine induced micronuclei in HepG2 cells, and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Metabolic inhibition of cytochrome P450 enzymes with ketoconazole abrogated micronucleus formation. The efflux transporter inhibitors verapamil and benzbromarone reduced micronucleus formation in the co-culture model. Furthermore, mitotic disturbances as an additional genotoxic mechanism of action were observed in HepG2 cells and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Overall, we were able to show that PAs were activated by HepG2 cells and the metabolites induced genomic damage in co-cultured HeLa cells.

Intracellular Metabolomics Identifies Efflux Transporter Inhibitors in a Routine Caco-2 Cell Permeability Assay—Biological Implications

Caco-2 screens are routinely used in laboratories to measure the permeability of compounds and can identify substrates of efflux transporters. In this study, we hypothesized that efflux transporter inhibition of a compound can be predicted by an intracellular metabolic signature in Caco-2 cells in the assay used to test intestinal permeability. Using selective inhibitors and transporter knock-out (KO) cells and a targeted Liquid Chromatography tandem Mass Spectrometry (LC-MS) method, we identified 11 metabolites increased in cells with depleted P-glycoprotein (Pgp) activity. Four metabolites were altered with Breast Cancer Resistance (BCRP) inhibition and nine metabolites were identified in the Multidrug Drug Resistance Protein 2 (MRP2) signature. A scoring system was created that could discriminate among the three transporters and validated with additional inhibitors. Pgp and MRP2 substrates did not score as inhibitors. In contrast, BCRP substrates and inhibitors showed a similar intracellular metabolomic signature. Network analysis of signature metabolites led us to investigate changes of enzymes in one-carbon metabolism (folate and methionine cycles). Our data shows that methylenetetrahydrofolate reductase (MTHFR) protein levels increased with Pgp inhibition and Thymidylate synthase (TS) protein levels were reduced with Pgp and MRP2 inhibition. In addition, the methionine cycle is also affected by both Pgp and MRP2 inhibition. In summary, we demonstrated that the routine Caco-2 assay has the potential to identify efflux transporter inhibitors in parallel with substrates in the assays currently used in many DMPK laboratories and that inhibition of efflux transporters has biological consequences.

The effects of cyclosporine A or activated charcoal co-administration on the pharmacokinetics of enrofloxacin in chickens

The study aimed to investigate the possible role of efflux transporter proteins in the pharmacokinetics of enrofloxacin (ENR) in broilers in the model of co-administration of activated charcoal (AC) or cyclosporine A (CsA). The concentrations of enrofloxacin and its metabolite ciprofloxacin were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) and population approach was used for pharmacokinetic analysis. It was found that body weight has a significant effect on the volume of distribution in the central compartment and on the systemic clearance. Oral AC increased the systemic clearance of intravenously administered ENR suggesting some role of enterohepatic recirculation. For orally administered ENR, CsA increased the area under the curve which can be explained by the inhibition of efflux transporters. Metabolism of the antibacterial drug was not affected by cyclosporine. The data suggest a role of efflux transporter proteins in the pharmacokinetics of drugs in chickens and drug-drug interactions have to be considered when substrates and modulators of these transporters are co-administered.

Uptake and transport of Laportea bulbifera extract in Caco-2 cell model

Laportea bulbifera extract is effective in resisting inflammation and shows a good therapeutic effect on rheumatoid arthritis in rats. However, the absorption characteristics of active components in L. bulbifera extract in Caco-2 cells are still unclear, which limits the in-depth development of L. bulbifera resources. The purpose of this study was to investigate the absorption and transport mechanism of the active components of L. bulbifera extract in the Caco-2 cell model and explore the effects of different factors(concentration, time, pH value, temperature, and efflux transporter inhibitor) on its uptake and transport. The results showed that L. bulbifera extract at the concentration of 2.0-8.0 mg·mL~(-1) showed no toxicity to Caco-2 cells. The uptake and transport of L. bulbifera extract in the Caco-2 cell model were concentration-dependent and time-dependent. The main absorption mechanism was passive diffusion, and acidic condition(pH 5.0-6.0) and 37 ℃ were more favorable for drug absorption. P_(app)&gt;1.0×10~(-6 )cm·s~(-1) of each component indicated that L. bulbifera was a moderately absorbed drug. P-gp, MRP2, and BCRP were not involved in its uptake and transport.

Mechanisms of Transplacental Transfer of (‐)‐∆ 9‐Tetrahydrocannabinol (THC) in the Human Dual Cotyledon, Dual Perfusion, Placenta Model

Background(‐)‐∆ 9‐Tetrahydrocannabinol (THC) is the most abundant psychoactive compound in marijuana. With legalization of marijuana, its use has increased including by pregnant women. The latter raises concerns of fetal toxicity which is likely driven by fetal THC exposure. Interestingly, in the catheterized maternal‐fetal macaque model, fetal THC exposure is lower than its corresponding maternal exposure (fetal/maternal AUC0‐inf ratio of 0.37). This difference is supported by sparse human THC umbilical vein/maternal plasma concentrations at term. The most likely explanation for these observations is that THC is effluxed by P‐glycoprotein (P‐gp) which is highly expressed in the human placenta. THC oral pharmacokinetics studies in P‐gp knock‐out mice support the hypothesis that P‐gp can transport THC. Therefore, the objective of our study was to investigate if THC is effluxed in human placentae by estimating the maternal to fetal clearance (m‐f‐CL) and fetal to maternal clearance (f‐m‐CL) using the dual cotyledon, dual perfusion, placenta model.MethodsTerm placentae were utilized from uncomplicated, unlabored, cesarean deliveries with no known history of tobacco or drug use. Two cotyledons from each placenta were perfused at physiological rates (m: 10 mL/min; f: 4 mL/min) for 2‐4 hours in a single‐pass mode with oxygenated HBSS buffer containing 0.2% BSA, 2000 U/L sodium heparin and 5 mg/mL gentamicin. To determine m‐f CL, the maternal perfusate also contained 5 μM THC, a P‐gp transporter probe (1 or 10 μM saquinavir), and a passive diffusion probe (20 μg/mL antipyrine) and effluent from the fetal vein and intervillous space were collected at multiple timepoints. To determine f‐m‐CL, the fetal perfusate of the second cotyledon contained the aforementioned drugs. All perfusions were conducted with (n = 7) and without (n = 5) a pan efflux transporter inhibitor, 4 μM valspodar. THC concentrations in the effluent were quantified using LC‐MS/MS. THC CL indices, i.e. m‐f‐CL and f‐m‐CL, normalized to the corresponding antipyrine CL, were compared in the absence and presence of valspodar.ResultsTHC m‐f‐CL index, 0.40 ± 0.15, was not significantly different from the f‐m‐CL index, 0.54 ± 0.13 (p = 0.31; two‐tailed Wilcoxon signed rank test), suggesting that THC passively diffuses across the placental barrier. Surprisingly, in the presence of valspodar, THC m‐f‐CL index, 0.24 ± 0.14, was significantly lower than the corresponding f‐m‐CL index, 0.44 ± 0.14 (p = 0.016; two‐tailed Wilcoxon signed rank test). The reason for this difference is unknown.ConclusionsBecause THC passively crosses the human placenta, changes in activity of placental transporters (e.g. drugs that inhibit/induce placental transporters or pharmacogenetics) should not alter fetal THC exposure. Further, mechanisms other than P‐gp efflux (e.g. binding to placental tissue) need to be explored to explain the fetal and maternal THC exposure of less than unity detailed in the introduction.

Novel Bruton's tyrosine kinase inhibitor remibrutinib: Assessment of drug-drug interaction potential as a perpetrator of cytochrome P450 enzymes and drug transporters and the impact of covalent binding on possible drug interactions

Pharmacokinetic drug-drug interactions (DDIs) are investigated to ensure safety for patients receiving concomitant medications. Here, we present a strategy to characterise the DDI potential of remibrutinib, as an inhibitor of drug-metabolising enzymes and drug transporters, and as an inducer. Initial in vitro studies were performed, followed by a biomarker-based assessment of induction in a first in human study, concluded by a clinical study to verify initial results. Remibrutinib is a covalent inhibitor of Bruton's Tyrosine kinase (BTKi) carrying a reactive acrylamide moiety (warhead), thus the potential contribution of covalent binding (off-target) to observed interactions was investigated as this could lead to prolonged and more potent drug interactions. DDI assessment was focused on the putative inhibition of key metabolic enzymes (Cytochrome P450, CYP), drug transporters and a potential effect on oral contraceptives (OC) by induction of enzymes that are involved in their clearance (CYP3A4). The impact of covalent binding was assessed by synthesising an identical reference molecule but with an inactivated warhead. An interaction potential of limited clinical relevance was revealed for remibrutinib for CYP enzymes and drug transporters. The reactive warhead of remibrutinib had no impact on CYP enzyme and transporter inhibition, including time-dependent inhibition of CYP3A4, but may increase the induction potential of remibrutinib. Observed inhibition of metabolic enzymes indicated that remibrutinib is a weak inhibitor of CYP3A4 and CYP2C9 and is not a clinically relevant inhibitor of uptake and efflux transporters, except for intestinal P-glycoprotein and breast cancer resistance protein inhibition. OC may be safely administered and are effective when given with pharmacologically relevant doses of remibrutinib.

Exploration of novel phthalazinone derivatives as potential efflux transporter inhibitors for reversing multidrug resistance and improving the oral absorption of paclitaxel

Chemotherapy is an important means of cancer treatment. However, overexpression of efflux transporters (including but not limited to P-gp and BCRP) can lead to resistance to cancer chemotherapy. Multiple-target inhibitors of efflux transporter can be overcome the resistance and improve the oral bioavailability of chemotherapy drugs. Therefore, we designed and synthesized a series of phthalazinone ring derivatives (1-20) with different aromatic heterocycles substituents on the amide bond for dual inhibition of P-gp and BCRP. Most target compounds significantly increased the accumulation of P-gp substrates in the chemo-resistant cancer cell lines by inhibiting the efflux of transporters. Compound 19 in particular showed stronger MDR reversal compared to Gefitinib and Verapamil, and comparable to that of the BCRP inhibitor Ko143. In addition, compound 19 improved intestinal absorption of paclitaxel (PTX) and enhanced the bioavailability of the orally administered drug in vivo.

Drug-Herb Interactions among Thai Herbs and Anticancer Drugs: A Scoping Review.

More than half of Thai patients with cancer take herbal preparations while receiving anticancer therapy. There is no systematic or scoping review on interactions between anticancer drugs and Thai herbs, although several research articles have that Thai herbs inhibit cytochrome P450 (CYP) or efflux transporter. Therefore, we gathered and integrated information related to the interactions between anticancer drugs and Thai herbs. Fifty-two anticancer drugs from the 2020 Thailand National List of Essential Medicines and 75 herbs from the 2020 Thai Herbal Pharmacopoeia were selected to determine potential anticancer drug–herb interactions. The pharmacological profiles of the selected anticancer drugs were reviewed and matched with the herbal pharmacological activities to determine possible interactions. A large number of potential anticancer drug–herb interactions were found; the majority involved CYP inhibition. Efflux transporter inhibition and enzyme induction were also found, which could interfere with the pharmacokinetic profiles of anticancer drugs. However, there is limited knowledge on the pharmacodynamic interactions between anticancer drugs and Thai herbs. Therefore, further research is warranted. Information regarding interactions between anticancer drugs and Thai herbs should provide as a useful resource to healthcare professionals in daily practice. It could enable the prediction of possible anticancer drug–herb interactions and could be used to optimize cancer therapy outcomes.

Inhibitory Effect of 5,7-Dimethoxyfl avone On Rosuvastatin Uptake From The Apical Membrane Of Caco-2 Cells

5,7-Dimethoxyfl avone (5,7-DMF) is a natural polymethoxyfl avone, and acts as an inhibitor of ABC efflux transporters (BCRP, MRP2 and/ or P-gp), and rosuvastatin is taken up via OATP2B1 and secreted by BCRP and MRP2. In this study, we investigated the effect of 5,7-DMF on the transport of rosuvastatin through the apical membrane and the accumulation in Caco-2 cells. Furthermore, we investigated whether the rosuvastatin accumulation is mediated by monocarboxylate transporter 1 (MCT1), in addition to OATP2B1. Coincubation with 5,7- DMF significantly increased the cellular accumulation of rosuvastatin from the apical membranes of Caco-2 cells cultured on the plastic dish. Coincubation with Ko-143 (a BCRP inhibitor) or MK-571 (an MRP inhibitor) significantly increased the rosuvastatin accumulation, whereas coincubation with verapamil (a P-gp inhibitor) did not. Coincubation with benzoic acid or pravastatin, which are substrates of both OATP2B1 and MCT1, signifi cantly decreased the rosuvastatin accumulation, whereas coincubation with estron-3-sulfate or sulfobromophthalein, which are substrates of both OATP2B1 and ABC efflux transporter, did not decrease and increased the rosuvastatin accumulation, respectively. On the other hand, the transcellular transport of rosuvastatin from basolateral to apical side (B-to-A transport) was markedly higher than that from apical to basolateral side (A-to-B transport). Coincubation with 5,7-DMF from the apical side significantly increased the A-to-B transport and the accumulation of rosuvastatin, whereas that from the basolateral side significantly decreased the B-to-A transport of rosuvastatin with increases in the accumulation. These results suggest that 5,7-DMF may increase the rosuvastatin accumulation as a result of the inhibition of rosuvastatin efflux mediated by BCRP and MRP2, and the rosuvastatin transport through the apical membrane may be mediated by not only OATB2B1 but also MCT1.

Transplacental Pharmacokinetic Model of Digoxin Based on Ex Vivo Human Placental Perfusion Study.

Digoxin is used as first-line therapy to treat fetal supraventricular tachycardia; however, because of the narrow therapeutic window, it is essential to estimate digoxin exposure in the fetus. The data from ex vivo human placental perfusion study are used to predict in vivo fetal exposure noninvasively, but the ex vivo fetal-to-maternal concentration (F:M) ratios observed in digoxin perfusion studies were much lower than those in vivo. In the present study, we developed a human transplacental pharmacokinetic model of digoxin using previously reported ex vivo human placental perfusion data. The model consists of maternal intervillous, fetal capillary, non-perfused tissue, and syncytiotrophoblast compartments, with multidrug resistance protein (MDR) 1 and influx transporter at the microvillous membrane (MVM) and influx and efflux transporters at the basal plasma membrane (BM). The model-predicted F:M ratio was 0.66, which is consistent with the mean in vivo value of 0.77 (95% confidence interval: 0.64-0.91). The time to achieve the steady state from the ex vivo perfusion study was estimated as 1,500 minutes, which is considerably longer than the reported ex vivo experimental durations, and this difference is considered to account for the inconsistency between ex vivo and in vivo F:M ratios. Reported digoxin concentrations in a drug-drug interaction study with MDR1 inhibitors quinidine and verapamil were consistent with the profiles simulated by our model incorporating inhibition of efflux transporter at the BM in addition to MVM. Our modeling and simulation approach should be a powerful tool to predict fetal exposure and DDIs in human placenta. SIGNIFICANCE STATEMENT: We developed a human transplacental pharmacokinetic model of digoxin based on ex vivo human placental perfusion studies in order to resolve inconsistencies between reported ex vivo and in vivo fetal-to-maternal concentration ratios. The model successfully predicted the in vivo fetal exposure to digoxin and the drug-drug interactions of digoxin and P-glycoprotein/multidrug resistance protein 1 inhibitors in human placenta.

Dysregulation of cholesterol homeostasis in human lung cancer tissue and tumour-associated macrophages.

BackgroundBased on reports on elevated cholesterol levels in cancer cells, strategies to lower cholesterol synthesis have been suggested as an antitumour strategy. However, cholesterol depletion has also been shown to induce tumour-promoting actions in tumour-associated macrophages (TAMs).MethodsWe performed lipidomic and transcriptomic analyses of human lung cancer material. To assess whether the TAM phenotype is shaped by secreted factors produced by tumour cells, primary human monocyte-derived macrophages were polarized towards a TAM-like phenotype using tumour cell-conditioned medium.FindingsLipidomic analysis of lung adenocarcinoma (n=29) and adjacent non-tumour tissues (n=22) revealed a significant accumulation of free cholesterol and cholesteryl esters within the tumour tissue. In contrast, cholesterol levels were reduced in TAMs isolated from lung adenocarcinoma tissues when compared with alveolar macrophages (AMs) obtained from adjacent non-tumour tissues. Bulk-RNA-Seq revealed that genes involved in cholesterol biosynthesis and metabolism were downregulated in TAMs, while cholesterol efflux transporters were upregulated. In vitro polarized TAM-like macrophages showed an attenuated lipogenic gene expression signature and exhibited lower cholesterol levels compared with non-polarized macrophages. A genome-wide comparison by bulk RNA-Seq confirmed a high similarity of ex vivo TAMs and in vitro TAM-like macrophages. Modulation of intracellular cholesterol levels by either starving, cholesterol depletion, or efflux transporter inhibition indicated that cholesterol distinctly shapes macrophage gene expression.InterpretationOur data show an opposite dysregulation of cholesterol homeostasis in tumour tissue vs. TAMs. Polarization of in vitro differentiated macrophages by tumour cell-conditioned medium recapitulates key features of ex vivo TAMs.