Percent of total tyrosine-phosphorylated spectral counts for all OATP1B1 tyrosine residues after ... more Percent of total tyrosine-phosphorylated spectral counts for all OATP1B1 tyrosine residues after exposure to DMSO (vehicle) or nilotinib (10 μM). Three concentrated OATP1B1 protein extracts were purified by immunoprecipitation and analyzed by LC-MS analysis, per treatment group. *denotes P < 0.05 vs vehicle or wild-type and all values represent mean {plus minus} SEM. Data is represented by 3 independent experiments.
Oxaliplatin-induced peripheral neurotoxicity (OIPN) is a debilitating side effect that afflicts a... more Oxaliplatin-induced peripheral neurotoxicity (OIPN) is a debilitating side effect that afflicts approximately 90% of patients that is initiated by OCT2-dependent uptake of oxaliplatin in dorsal root ganglion (DRG) neurons. The antidepressant drug duloxetine has been used to treat OIPN, although its usefulness in preventing this side effect remains unclear. We hypothesized that duloxetine has OCT2-inhibitory properties and can be used as an adjunct to oxaliplatin-based regimens to prevent OIPN. Transport studies were performed in cells stably transfected with mouse or human OCT2 and in isolated mouse DRG neurons ex vivo. Wild-type and OCT2-deficient mice were used to assess effects of duloxetine on hallmarks of OIPN, endogenous OCT2 biomarkers, and the pharmacokinetics of oxaliplatin, and the translational feasibility of a duloxetine-oxaliplatin combination was evaluated in various models of colorectal cancer. We found that duloxetine potently inhibited the OCT2-mediated transport of...
The membrane transport of many cationic prescription drugs depends on facilitated transport by or... more 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 ...
A simple, rapid, and sensitive LC-MS/MS method for determining concentrations of the anticancer a... more 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 [ 2 H 3 ]-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 (r 2 > 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 interindividual pharmacokinetic variability associated with vincristine-induced peripheral neuropathy.
Organic cation transporter 1 (OCT1) is a transporter that regulates the hepatic uptake and subseq... more 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 sugg...
Proceedings of the National Academy of Sciences, 2021
Significance Understanding the initiating mechanisms of cardiac injury associated with doxorubici... more Significance Understanding the initiating mechanisms of cardiac injury associated with doxorubicin is fundamental to the development of novel preventative strategies. Evidence suggests that expression of membrane transporters contributes to the initial accumulation of doxorubicin in cardiomyocytes and, subsequently, cellular injury. Using patient-derived cardiomyocytes, we identified a candidate membrane transporter contributing to this debilitating phenotype and performed validation studies using engineered cell-based and animal models to demonstrate that targeting this transport mechanism can afford cardio-protection without compromising the anticancer properties of doxorubicin. Our findings provide insights that addresses an unmet therapeutic need in cancer patients receiving doxorubicin-based treatment.
Basic & Clinical Pharmacology & Toxicology, 2021
Membrane transporters play a key role in determining the pharmacokinetic profile, therapeutic saf... more 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.
Purpose: OATP1B1 (SLCO1B1) is the most abundant and pharmacologically relevant uptake transporter... more 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 non...
MBL has provided consultation regarding chemotherapy-induced peripheral neuropathy to PledPharma ... more MBL has provided consultation regarding chemotherapy-induced peripheral neuropathy to PledPharma and Disarm Therapeutics. CLL has provided consultation regarding chemotherapy-induced peripheral neuropathy to PledPharma, Disarm Therapeutics, Asahi Kasei, and Metys Pharmaceuticals.
Background Dofetilide is a delayed rectifier potassium channel inhibitor used to treat patients w... more Background Dofetilide is a delayed rectifier potassium channel inhibitor used to treat patients with atrial fibrillation and flutter. The clinical use of this drug is associated with increases in QTc interval, which can exacerbate cardiac arrhythmias. The elimination of dofetilide is predominantly mediated by renal tubular secretion, yet the mechanism by which this occurs remains poorly understood. Previously reported drug-drug interaction (DDI) studies of dofetilide with cimetidine, ketoconazole, and megestrol suggest the involvement of organic cation transporters. Here, we investigate the contribution of organic cation transporter 2 (OCT2; SLC22A2) and multidrug and toxin extrusion protein 1 (MATE1; SLC47A1) to the pharmacokinetics (PK) of dofetilide in examine these mechanisms in the drug’s elimination and its narrow therapeutic window.
Expert Opinion on Drug Metabolism & Toxicology, 2020
Introduction.-Membrane transporters are integral components to the maintenance of cellular integr... more Introduction.-Membrane transporters are integral components 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 to 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.
Journal of Pharmaceutical and Biomedical Analysis, 2019
A novel method using UPLC with tandem mass-spectrometric detection (UPLC-MS/MS) with positive ele... more 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 (R 2 ≥ 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 3.00-7.10%, 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 pharmacokinetics (PK) represents the absorption, distribution, metabolism, and elimination... more 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.
Percent of total tyrosine-phosphorylated spectral counts for all OATP1B1 tyrosine residues after ... more Percent of total tyrosine-phosphorylated spectral counts for all OATP1B1 tyrosine residues after exposure to DMSO (vehicle) or nilotinib (10 μM). Three concentrated OATP1B1 protein extracts were purified by immunoprecipitation and analyzed by LC-MS analysis, per treatment group. *denotes P < 0.05 vs vehicle or wild-type and all values represent mean {plus minus} SEM. Data is represented by 3 independent experiments.
Oxaliplatin-induced peripheral neurotoxicity (OIPN) is a debilitating side effect that afflicts a... more Oxaliplatin-induced peripheral neurotoxicity (OIPN) is a debilitating side effect that afflicts approximately 90% of patients that is initiated by OCT2-dependent uptake of oxaliplatin in dorsal root ganglion (DRG) neurons. The antidepressant drug duloxetine has been used to treat OIPN, although its usefulness in preventing this side effect remains unclear. We hypothesized that duloxetine has OCT2-inhibitory properties and can be used as an adjunct to oxaliplatin-based regimens to prevent OIPN. Transport studies were performed in cells stably transfected with mouse or human OCT2 and in isolated mouse DRG neurons ex vivo. Wild-type and OCT2-deficient mice were used to assess effects of duloxetine on hallmarks of OIPN, endogenous OCT2 biomarkers, and the pharmacokinetics of oxaliplatin, and the translational feasibility of a duloxetine-oxaliplatin combination was evaluated in various models of colorectal cancer. We found that duloxetine potently inhibited the OCT2-mediated transport of...
The membrane transport of many cationic prescription drugs depends on facilitated transport by or... more 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 ...
A simple, rapid, and sensitive LC-MS/MS method for determining concentrations of the anticancer a... more 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 [ 2 H 3 ]-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 (r 2 > 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 interindividual pharmacokinetic variability associated with vincristine-induced peripheral neuropathy.
Organic cation transporter 1 (OCT1) is a transporter that regulates the hepatic uptake and subseq... more 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 sugg...
Proceedings of the National Academy of Sciences, 2021
Significance Understanding the initiating mechanisms of cardiac injury associated with doxorubici... more Significance Understanding the initiating mechanisms of cardiac injury associated with doxorubicin is fundamental to the development of novel preventative strategies. Evidence suggests that expression of membrane transporters contributes to the initial accumulation of doxorubicin in cardiomyocytes and, subsequently, cellular injury. Using patient-derived cardiomyocytes, we identified a candidate membrane transporter contributing to this debilitating phenotype and performed validation studies using engineered cell-based and animal models to demonstrate that targeting this transport mechanism can afford cardio-protection without compromising the anticancer properties of doxorubicin. Our findings provide insights that addresses an unmet therapeutic need in cancer patients receiving doxorubicin-based treatment.
Basic & Clinical Pharmacology & Toxicology, 2021
Membrane transporters play a key role in determining the pharmacokinetic profile, therapeutic saf... more 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.
Purpose: OATP1B1 (SLCO1B1) is the most abundant and pharmacologically relevant uptake transporter... more 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 non...
MBL has provided consultation regarding chemotherapy-induced peripheral neuropathy to PledPharma ... more MBL has provided consultation regarding chemotherapy-induced peripheral neuropathy to PledPharma and Disarm Therapeutics. CLL has provided consultation regarding chemotherapy-induced peripheral neuropathy to PledPharma, Disarm Therapeutics, Asahi Kasei, and Metys Pharmaceuticals.
Background Dofetilide is a delayed rectifier potassium channel inhibitor used to treat patients w... more Background Dofetilide is a delayed rectifier potassium channel inhibitor used to treat patients with atrial fibrillation and flutter. The clinical use of this drug is associated with increases in QTc interval, which can exacerbate cardiac arrhythmias. The elimination of dofetilide is predominantly mediated by renal tubular secretion, yet the mechanism by which this occurs remains poorly understood. Previously reported drug-drug interaction (DDI) studies of dofetilide with cimetidine, ketoconazole, and megestrol suggest the involvement of organic cation transporters. Here, we investigate the contribution of organic cation transporter 2 (OCT2; SLC22A2) and multidrug and toxin extrusion protein 1 (MATE1; SLC47A1) to the pharmacokinetics (PK) of dofetilide in examine these mechanisms in the drug’s elimination and its narrow therapeutic window.
Expert Opinion on Drug Metabolism & Toxicology, 2020
Introduction.-Membrane transporters are integral components to the maintenance of cellular integr... more Introduction.-Membrane transporters are integral components 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 to 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.
Journal of Pharmaceutical and Biomedical Analysis, 2019
A novel method using UPLC with tandem mass-spectrometric detection (UPLC-MS/MS) with positive ele... more 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 (R 2 ≥ 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 3.00-7.10%, 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 pharmacokinetics (PK) represents the absorption, distribution, metabolism, and elimination... more 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.
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Papers by Erfan Uddin