Targeting a xenobiotic transporter to ameliorate vincristine-induced sensory neuropathy.

Vincristine is a widely used chemotherapeutic drug for the treatment of multiple malignant diseases that causes a dose-limiting peripheral neurotoxicity. There is no clinically effective preventative treatment for vincristine-induced sensory peripheral neurotoxicity (VIPN), and mechanistic details of this side effect remain poorly understood. We hypothesized that VIPN is dependent on transporter-mediated vincristine accumulation in dorsal root ganglion neurons. Using a xenobiotic transporter screen, we identified OATP1B3 as a neuronal transporter regulating the uptake of vincristine. In addition, genetic or pharmacological inhibition of the murine orthologue transporter OATP1B2 protected mice from various hallmarks of VIPN - including mechanical allodynia, thermal hyperalgesia, and changes in digital maximal action potential amplitudes and neuronal morphology - without negatively affecting plasma levels or antitumor effects of vincristine. Finally, we identified α-tocopherol from an untargeted metabolomics analysis as a circulating endogenous biomarker of neuronal OATP1B2 function, and it could serve as a companion diagnostic to guide dose selection of OATP1B-type transport modulators given in combination with vincristine to prevent VIPN. Collectively, our findings shed light on the fundamental basis of VIPN and provide a rationale for the clinical development of transporter inhibitors to prevent this debilitating side effect.

MATE1 Deficiency Exacerbates Dofetilide-Induced Proarrhythmia.

Dofetilide is a rapid delayed rectifier potassium current inhibitor widely used to prevent the recurrence of atrial fibrillation and flutter. The clinical use of this drug is associated with increases in QTc interval, which predispose patients to ventricular cardiac arrhythmias. The mechanisms involved in the disposition of dofetilide, including its movement in and out of cardiomyocytes, remain unknown. Using a xenobiotic transporter screen, we identified MATE1 (SLC47A1) as a transporter of dofetilide and found that genetic knockout or pharmacological inhibition of MATE1 in mice was associated with enhanced retention of dofetilide in cardiomyocytes and increased QTc prolongation. The urinary excretion of dofetilide was also dependent on the MATE1 genotype, and we found that this transport mechanism provides a mechanistic basis for previously recorded drug-drug interactions of dofetilide with various contraindicated drugs, including bictegravir, cimetidine, ketoconazole, and verapamil. The translational significance of these observations was examined with a physiologically-based pharmacokinetic model that adequately predicted the drug-drug interaction liabilities in humans. These findings support the thesis that MATE1 serves a conserved cardioprotective role by restricting excessive cellular accumulation and warrant caution against the concurrent administration of potent MATE1 inhibitors and cardiotoxic substrates with a narrow therapeutic window.

Contribution of membrane transporters to chemotherapy-induced cardiotoxicity.

Membrane transporters play a key role in determining the pharmacokinetic profile, therapeutic safety, and efficacy of many chemotherapeutic drugs by regulating cellular influx and efflux. Rapidly emerging evidence has shown that tissue-specific expression of transporters contributes to local drug accumulation and drug-drug interactions and that functional alterations in these transporters can directly influence an individual's susceptibility to drug-induced toxicity. Comprehending the complex mechanism of transporter function in regulating drug distribution in tissues, such as the heart, is necessary in order to acquire novel therapeutic strategies aimed at evading unwanted drug accumulation and toxicities and to ameliorate the safety of current therapeutic regimens. Here, we provide an overview of membrane transporters with a role in chemotherapy-induced cardiotoxicity and discuss novel strategies to improve therapeutic outcomes.

Intentional Modulation of Ibrutinib Pharmacokinetics through CYP3A Inhibition.

Ibrutinib (Imbruvica; PCI-32765) is an orally administered inhibitor of Bruton's tyrosine kinase that has transformed the treatment of B-cell malignancies. However, ibrutinib has very low oral bioavailability that contributes to significant variability in systemic exposure between patients, and this has the potential to affect both efficacy and toxicity. We hypothesized that the oral bioavailability of ibrutinib is limited by CYP3A isoform-mediated metabolism, and that this pathway can be inhibited to improve the pharmacokinetic properties of ibrutinib. Pharmacokinetic studies were performed in wild-type mice and mice genetically engineered to lack all CYP3A isoforms [CYP3A(-/-)] that received ibrutinib alone or in combination with CYP3A inhibitors cobicistat or ketoconazole. Computational modeling was performed to derive doses of ibrutinib that, when given after a CYP3A inhibitor, results in therapeutically-relevant drug levels. Deficiency of CYP3A in mice was associated with a ~10-fold increase in the area under the curve of ibrutinib. This result could be phenocopied by administration of cobicistat before ibrutinib in wild-type mice, but cobicistat did not influence levels of ibrutinib in CYP3A(-/-) mice. Population pharmacokinetic and prospectively validated physiologically-based pharmacokinetic models established preclinical and clinical doses of ibrutinib that could be given safely in combination with cobicistat without negatively affecting anti-leukemic properties. These findings signify a dominant role for CYP3A-mediated metabolism in the elimination of ibrutinib, and suggest a role for pharmacological inhibitors of this pathway to intentionally modulate the plasma levels and improve the therapeutic use of this clinically important agent.

In Vitro and In Vivo Inhibition of MATE1 by Tyrosine Kinase Inhibitors.

The membrane transport of many cationic prescription drugs depends on facilitated transport by organic cation transporters of which several members, including OCT2 (SLC22A2), are sensitive to inhibition by select tyrosine kinase inhibitors (TKIs). We hypothesized that TKIs may differentially interact with the renal transporter MATE1 (SLC47A1) and influence the elimination and toxicity of the MATE1 substrate oxaliplatin. Interactions with FDA-approved TKIs were evaluated in transfected HEK293 cells, and in vivo pharmacokinetic studies were performed in wild-type, MATE1-deficient, and OCT2/MATE1-deficient mice. Of 57 TKIs evaluated, 37 potently inhibited MATE1 function by >80% through a non-competitive, reversible, substrate-independent mechanism. The urinary excretion of oxaliplatin was reduced by about 2-fold in mice with a deficiency of MATE1 or both OCT2 and MATE1 (p < 0.05), without impacting markers of acute renal injury. In addition, genetic or pharmacological inhibition of MATE1 did not significantly alter plasma levels of oxaliplatin, suggesting that MATE1 inhibitors are unlikely to influence the safety or drug-drug interaction liability of oxaliplatin-based chemotherapy.

Gilteritinib Inhibits Glutamine Uptake and Utilization in FLT3-ITD-Positive AML.

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy associated with frequent relapse and poor overall survival. The tyrosine kinase inhibitor gilteritinib is approved for the treatment of relapse/refractory AML with FLT3 mutations, yet its mechanism of action is not completely understood. Here, we sought to identify additional therapeutic targets that can be exploited to enhance gilteritinib's antileukemic effect. Based on unbiased transcriptomic analyses, we identified the glutamine transporter SNAT1 (SLC38A1) as a novel target of gilteritinib that leads to impaired glutamine uptake and utilization within leukemic cells. Using metabolomics and metabolic flux analyses, we found that gilteritinib decreased glutamine metabolism through the TCA cycle and cellular levels of the oncometabolite 2-hydroxyglutarate. In addition, gilteritinib treatment was associated with decreased ATP production and glutathione synthesis and increased reactive oxygen species, resulting in cellular senescence. Finally, we found that the glutaminase inhibitor CB-839 enhanced antileukemic effect of gilteritinib in ex vivo studies using human primary FLT3-ITD-positive AML cells harboring mutations in the enzyme isocitrate dehydrogenase, which catalyzes the oxidative decarboxylation of isocitrate, producing α-ketoglutarate. Collectively, this work has identified a previously unrecognized, gilteritinib-sensitive metabolic pathway downstream of SLC38A1 that causes decreased glutaminolysis and disruption of redox homeostasis. These findings provide a rationale for the development and therapeutic exploration of targeted combinatorial treatment strategies for this subset of relapse/refractory AML.

Development, validation, and application of an LC-MS/MS method for the determination of the AXL/FLT3 inhibitor gilteritinib in mouse plasma.

A simple, fast and precise LC-MS/MS method for the quantitation of the tyrosine kinase inhibitor gilteritinib was developed and validated for micro-volumes of mouse plasma. The assay procedure involved a one-step extraction of gilteritinib and the internal standard [2H5]-gilteritinib with acetonitrile. An Accucore aQ column was used to separate analytes using a gradient elution delivered at a flow rate of 0.4 mL/min, and a total run time of 2.5 min. Validation studies with quality control samples processed on consecutive days revealed that values for intra-day and inter-day precision were <7.04%, with an accuracy of 101-108%. Linear responses were observed over the entire calibration curve range (up to 500 ng/mL), and the lower limit of quantification was 5 ng/mL. The developed method was successfully used to examine the pharmacokinetics of oral gilteritinib in wild-type mice and mice lacking the organic cation transporters OCT1, OCT2, and MATE1 to further understand mechanisms contributing to drug-drug interactions and causes of inter-individual pharmacokinetic variability.

Influence of YES1 Kinase and Tyrosine Phosphorylation on the Activity of OCT1.

Organic cation transporter 1 (OCT1) is a transporter that regulates the hepatic uptake and subsequent elimination of diverse cationic compounds. Although OCT1 has been involved in drug-drug interactions and causes pharmacokinetic variability of many prescription drugs, details of the molecular mechanisms that regulate the activity of OCT1 remain incompletely understood. Based on an unbiased phospho-proteomics screen, we identified OCT1 as a tyrosine-phosphorylated transporter, and functional validation studies using genetic and pharmacological approaches revealed that OCT1 is highly sensitive to small molecules that target the protein kinase YES1, such as dasatinib. In addition, we found that dasatinib can inhibit hepatic OCT1 function in mice as evidenced from its ability to modulate levels of isobutyryl L-carnitine, a hepatic OCT1 biomarker identified from a targeted metabolomics analysis. These findings provide novel insight into the post-translational regulation of OCT1 and suggest that caution is warranted with polypharmacy regimes involving the combined use of OCT1 substrates and kinase inhibitors that target YES1.

Rapid quantification of vincristine in mouse plasma using ESI-LC-MS/MS: Application to pharmacokinetic studies.

A simple, rapid, and sensitive LC-MS/MS method for determining concentrations of the anticancer alkaloid vincristine in micro volumes of mouse plasma was developed and validated in positive ion mode. Separation of vincristine and the internal standard [2H3]-vincristine was achieved on an Accucore aQ column with a gradient mobile phase delivered at a flow rate of 0.4 mL/min and a run time of 2.2 min. Calibration curves were linear (r2 > 0.99, n = 8) up to 250 ng/mL, with a lower limit of quantitation of 2.5 ng/mL. The matrix effect and extraction recovery for vincristine were ranging 108-110% and 88.4-107%, respectively. The intra-day and inter-day precision of quality controls tested at 3 different concentrations were always less than 15%, and accuracy ranged from 91.7 to 107%. The method was successfully applied to evaluate the pharmacokinetic profile of vincristine in wild-type and CYP3A-deficient mice in support of a project to provide mechanistic insight into drug-drug interactions and to identify sources of inter-individual pharmacokinetic variability associated with vincristine-induced peripheral neuropathy.

Regulation of OATP1B1 Function by Tyrosine Kinase-mediated Phosphorylation.

PURPOSE: OATP1B1 (SLCO1B1) is the most abundant and pharmacologically relevant uptake transporter in the liver and a key mediator of xenobiotic clearance. However, the regulatory mechanisms that determine OATP1B1 activity remain uncertain, and as a result, unexpected drug-drug interactions involving OATP1B1 substrates continue to be reported, including several involving tyrosine kinase inhibitors (TKI). EXPERIMENTAL DESIGN: OATP1B1-mediated activity in overexpressing HEK293 cells and hepatocytes was assessed in the presence of FDA-approved TKIs, while rosuvastatin pharmacokinetics in the presence of an OATP1B1 inhibiting TKI were measured in vivo. Tyrosine phosphorylation of OATP1B1 was determined by LC/MS-MS-based proteomics and transport function was measured following exposure to siRNAs targeting 779 different kinases. RESULTS: Twenty-nine of 46 FDA-approved TKIs studied significantly inhibit OATP1B1 function. Inhibition of OATP1B1 by TKIs, such as nilotinib, is predominantly noncompetitive, can increase systemic concentrations of rosuvastatin in vivo, and is associated with reduced phosphorylation of OATP1B1 at tyrosine residue 645. Using genetic screens and functional validation studies, the Src kinase LYN was identified as a potential regulator of OATP1B1 activity that is highly sensitive to inhibition by various TKIs at clinically relevant concentrations. CONCLUSIONS: A novel kinase-dependent posttranslational mechanism of OATP1B1 activation was identified and interference with this process by TKIs can influence the elimination of a broad range of xenobiotic substrates.

Targeting OCT3 attenuates doxorubicin-induced cardiac injury.

Doxorubicin is a commonly used anticancer agent that can cause debilitating and irreversible cardiac injury. The initiating mechanisms contributing to this side effect remain unknown, and current preventative strategies offer only modest protection. Using stem-cell-derived cardiomyocytes from patients receiving doxorubicin, we probed the transcriptomic landscape of solute carriers and identified organic cation transporter 3 (OCT3) (SLC22A3) as a critical transporter regulating the cardiac accumulation of doxorubicin. Functional validation studies in heterologous overexpression models confirmed that doxorubicin is transported into cardiomyocytes by OCT3 and that deficiency of OCT3 protected mice from acute and chronic doxorubicin-related changes in cardiovascular function and genetic pathways associated with cardiac damage. To provide proof-of-principle and demonstrate translational relevance of this transport mechanism, we identified several pharmacological inhibitors of OCT3, including nilotinib, and found that pharmacological targeting of OCT3 can also preserve cardiovascular function following treatment with doxorubicin without affecting its plasma levels or antitumor effects in multiple models of leukemia and breast cancer. Finally, we identified a previously unrecognized, OCT3-dependent pathway of doxorubicin-induced cardiotoxicity that results in a downstream signaling cascade involving the calcium-binding proteins S100A8 and S100A9. These collective findings not only shed light on the etiology of doxorubicin-induced cardiotoxicity, but also are of potential translational relevance and provide a rationale for the implementation of a targeted intervention strategy to prevent this debilitating side effect.

Neuronal uptake transporters contribute to oxaliplatin neurotoxicity in mice.

Peripheral neurotoxicity is a debilitating condition that afflicts up to 90% of patients with colorectal cancer receiving oxaliplatin-containing therapy. Although emerging evidence has highlighted the importance of various solute carriers to the toxicity of anticancer drugs, the contribution of these proteins to oxaliplatin-induced peripheral neurotoxicity remains controversial. Among candidate transporters investigated in genetically engineered mouse models, we provide evidence for a critical role of the organic cation transporter 2 (OCT2) in satellite glial cells in oxaliplatin-induced neurotoxicity, and demonstrate that targeting OCT2 using genetic and pharmacological approaches ameliorates acute and chronic forms of neurotoxicity. The relevance of this transport system was verified in transporter-deficient rats as a secondary model organism, and translational significance of preventive strategies was demonstrated in preclinical models of colorectal cancer. These studies suggest that pharmacological targeting of OCT2 could be exploited to afford neuroprotection in cancer patients requiring treatment with oxaliplatin.

Role of SLC transporters in toxicity induced by anticancer drugs.

INTRODUCTION: . Membrane transporters are integral to the maintenance of cellular integrity of all tissue and cell types. While transporters play an established role in the systemic pharmacokinetics of therapeutic drugs, tissue specific expression of uptake transporters can serve as an initiating mechanism that governs the accumulation and impact of cytotoxic drugs. AREAS COVERED: . This review provides an overview of organic cation transporters as determinants of chemotherapy-induced toxicities. We also provide insights into the recently updated FDA guidelines for in vitro drug interaction studies, with a particular focus on the class of tyrosine kinase inhibitors as perpetrators of transporter-mediated drug interactions. EXPERT OPINION: . Studies performed over the last few decades have highlighted the important role of basolateral uptake and apical efflux transporters in the pathophysiology of drug-induced organ damage. Increased understanding of the mechanisms that govern the accumulation of cytotoxic drugs has provided insights into the development of novel strategies to prevent debilitating toxicities. Furthermore, we argue that current regulatory guidelines provide inadequate recommendations for in vitro studies to identify substrates or inhibitors of drug transporters. Therefore, the translational and predictive power of FDA-approved drugs as modulators of transport function remains ambiguous and warrants further revision of the current guidelines.

Development and validation of a UPLC-MS/MS analytical method for dofetilide in mouse plasma and urine, and its application to pharmacokinetic study.

A novel method using UPLC with tandem mass-spectrometric detection (UPLC-MS/MS) with positive electrospray ionization was developed for the detection of the antiarrhythmic drug, dofetilide, in mouse plasma and urine. Protein precipitation was performed on 10 µL of plasma and 2 µL of urine samples using dofetilide-D4 as an internal standard, and separation of the analyte was accomplished on a C18 analytical column with the flow of 0.40 mL/min. Subsequently, the method was successfully applied to determine the pharmacokinetic parameters of dofetilide following oral and intravenous administration. The calibration curve was linear over the selected concentration range (R2 ≥ 0.99), with a lower limit of quantitation of 5 ng/mL. The intra-day and inter-day precisions, and accuracies obtained from a 5-day validation ranged from 3.00 to 7.10%, 3.80-7.20%, and 93.0-106% for plasma, and 3.50-9.00%, 3.70-10.0%, 87.0-106% for urine, while the recovery of dofetilide was 93.7% and 97.4% in plasma and urine, respectively. The observed pharmacokinetic profiles revealed that absorption is the rate-limiting step in dofetilide distribution and elimination. Pharmacokinetic studies illustrate that the absolute bioavailability of dofetilide in the FVB strain mice is 34.5%. The current developed method allows for accurate and precise quantification of dofetilide in micro-volumes of plasma and urine, and was found to be suitable for supporting in vivo pharmacokinetic studies.

Murine Pharmacokinetic Studies.

Murine pharmacokinetics (PK) represents the absorption, distribution, metabolism, and elimination of drugs from the body, which helps to guide clinical studies, ultimately resulting in more effective drug treatment. The purpose of this protocol is to describe a serial bleeding protocol, obtaining blood samples at six time points from single mouse to yield a complete PK profile. This protocol has proved to be rapid, highly repeatable, and relatively easy to acquire. Comparing with the conventional PK studies, this method not only dramatically reduces animal usage, but also decreases sample variation obtained from different animals.

CNS Depressant and Antinociceptive Effects of Different Fractions of Pandanus Foetidus Roxb. Leaf Extract in Mice.

BACKGROUND: Various parts of Pandanus foetidus Roxb. are used as traditional medicines. However, scientific reports concerning the effect of this plant on central nervous system (CNS) depression and analgesia are unavailable. This study investigated the CNS depressant and antinociceptive effects of Pandanus foetidus leaf extracts in a rodent model. METHODS: The sedative and anxiolytic activities of Pandanus foetidus extract (500 g) were tested using behavioural models of Swiss albino mice, and the analgesic activity was assessed by formalin-induced pain and tail immersion tests at 200 mg/kg body weight of the mice. The data were analysed by a one-way ANOVA, a repeated measure of ANOVA and a non-parametric test (Kruskal-Wallis test) using the SPSS software. Acute toxicity was tested using an established method. RESULTS: Compared with the aqueous fraction, the methanol, petroleum ether and chloroform fractions of the extract exhibited a more significant (P < 0.001) reduction of locomotor activity in the mice in the open field, hole-cross, and elevated plus maze (EPM). The methanol fraction maximized the duration of sleeping time caused by the thiopental sodium induction. The extract produced a significant step-down in pain, as shown by the paw licking time in the early and late phases of the formalin test. In the tail immersion test, the chloroform fraction maximally reduced the heat-induced analgesia. The extract was found to be non toxic. CONCLUSION: The methanol, petroleum ether, and chloroform fractions of P. foetidus have strong CNS depressant and antinociceptive effects and thus merit further pharmaceutical studies.

Evaluation of Antidiarrheal Activity of Methanolic Extract of Maranta arundinacea Linn. Leaves.

Diarrhea is one of the most common causes for thousands of deaths every year. Therefore, identification of new source of antidiarrheal drugs becomes one of the most prominent focuses in modern research. Our aim was to investigate the antidiarrheal and cytotoxic activities of methanolic extract of Maranta arundinacea linn. (MEMA) leaves in rats and brine shrimp, respectively. Antidiarrheal effect was evaluated by using castor oil-induced diarrhea, enteropooling, and gastrointestinal motility tests at 200 mg/kg and 400 mg/kg body weight in rats where the cytotoxic activity was justified using brine shrimp lethality bioassay at different concentrations of MEMA. The extract showed considerable antidiarrheal effect by inhibiting 42.67% and 57.75% of diarrheal episode at the doses of 200 and 400 mg/kg, respectively. MEMA also significantly (p < 0.01) reduced the castor oil-induced intestinal volume (2.14 ± 0.16 to 1.61 ± 0.12 mL) in enteropooling test as well as intestinal transit (33.00 to 43.36%) in GI motility test, compared to their respective control. These observed effects are comparable to that of standard drug loperamide (5 mg/kg). On the other hand, in brine shrimp lethality test after 24 h, surviving brine shrimp larvae were counted and LD50 was assessed. Result showed that MEMA was potent against brine shrimp with LD50 value of 420 µg/mL. So the highest dose of 400 µg/mL of MEMA was not toxic to mice. So these results indicate that bioactive compounds are present in methanolic extract of Maranta arundinacea leaves including significant antidiarrheal activity and could be accounted for pharmacological effects.

Investigation of in vivo neuropharmacological effect of Alpinia nigra leaf extract.

OBJECTIVE: To analyze in vivo neuro-pharmacological effects of Alpinia nigra as anxiety is a particular form of behavioral inhibition that occurs in response to novel environmental events. METHODS: In present study, the extract of Alpinia nigra was evaluated for its central nervous system depressant effect using mice behavioral models, such as hole cross, open field and thiopental sodium induced sleeping time tests for its sedative properties and an elevated plus-maze test for its anxiolytic potential, respectively. RESULTS: In anxiolytic study, the extract displayed increased percentage of entry into open arm at the dose of 400 and 200 mg/kg. The extract produced a significant (P<0.01) increase in sleeping duration and reduction of onset of sleep compared to sodium thiopental at both doses (200 and 400 mg/kg). The extract (200 and 400 mg/kg) also showed a dose-dependent suppression of motor activity and exploratory activity of the mice in both open field and hole cross test. CONCLUSION: This study demonstrates that the treated extract has significant central nervous system depressant effect. Further studies on active constituent of the extract can provide approaches for therapeutic intervention.