Browsing by Author "Masters, Andrea R."
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Item Chiral Plasma Pharmacokinetics and Urinary Excretion of Bupropion and Metabolites in Healthy Volunteers(ASPET, 2016-08) Masters, Andrea R.; Gufford, Brandon T.; Lu, Jessica Bo Li; Metzger, Ingrid F.; Jones, David R.; Desta, Zeruesenay; Medicine, School of MedicineBupropion, widely used as an antidepressant and smoking cessation aid, undergoes complex metabolism to yield numerous metabolites with unique disposition, effect, and drug–drug interactions (DDIs) in humans. The stereoselective plasma and urinary pharmacokinetics of bupropion and its metabolites were evaluated to understand their potential contributions to bupropion effects. Healthy human volunteers (n = 15) were administered a single oral dose of racemic bupropion (100 mg), which was followed by collection of plasma and urine samples and determination of bupropion and metabolite concentrations using novel liquid chromatography–tandem mass spectrometry assays. Time-dependent, elimination rate–limited, stereoselective pharmacokinetics were observed for all bupropion metabolites. Area under the plasma concentration-time curve from zero to infinity ratios were on average approximately 65, 6, 6, and 4 and Cmax ratios were approximately 35, 6, 3, and 0.5 for (2R,3R)-/(2S,3S)-hydroxybupropion, R-/S-bupropion, (1S,2R)-/(1R,2S)-erythrohydrobupropion, and (1R,2R)-/(1S,2S)-threohydrobupropion, respectively. The R-/S-bupropion and (1R,2R)-/(1S,2S)-threohydrobupropion ratios are likely indicative of higher presystemic metabolism of S- versus R-bupropion by carbonyl reductases. Interestingly, the apparent renal clearance of (2S,3S)-hydroxybupropion was almost 10-fold higher than that of (2R,3R)-hydroxybupropion. The prediction of steady-state pharmacokinetics demonstrated differential stereospecific accumulation [partial area under the plasma concentration-time curve after the final simulated bupropion dose (300–312 hours) from 185 to 37,447 nM⋅h] and elimination [terminal half-life of approximately 7–46 hours] of bupropion metabolites, which may explain observed stereoselective differences in bupropion effect and DDI risk with CYP2D6 at steady state. Further elucidation of bupropion and metabolite disposition suggests that bupropion is not a reliable in vivo marker of CYP2B6 activity. In summary, to our knowledge, this is the first comprehensive report to provide novel insight into mechanisms underlying bupropion disposition by detailing the stereoselective pharmacokinetics of individual bupropion metabolites, which will enhance clinical understanding of bupropion’s effects and DDIs with CYP2D6.Item Development of Liposomal Gemcitabine with High Drug Loading Capacity(ACS Publications, 2019-05-28) Tamam, Hassan; Park, Jinho; Gadalla, Hytham H.; Masters, Andrea R.; Abdel-Aleem, Jelan A.; Abdelrahman, Sayed I.; Abdelrahman, Aly A.; Lyle, L. Tiffany; Yeo, Yoon; Medicine, School of MedicineLiposomes are widely used for systemic delivery of chemotherapeutic agents to reduce their nonspecific side effects. Gemcitabine (Gem) makes a great candidate for liposomal encapsulation due to the short half-life and nonspecific side effects; however, it has been difficult to achieve liposomal Gem with high drug loading capacity. Remote loading, which uses a transmembrane pH gradient to induce an influx of drug and locks the drug in the core as a sulfate complex, does not serve Gem as efficiently as doxorubicin (Dox) due to the low pKa value of Gem. Existing studies have attempted to improve Gem loading capacity in liposomes by employing lipophilic Gem derivatives or creating a high-concentration gradient for active loading into the hydrophilic cores (small volume loading). In this study, we combine the remote loading approach and small volume loading or hypertonic loading, a new approach to induce the influx of Gem into the preformed liposomes by high osmotic pressure, to achieve a Gem loading capacity of 9.4–10.3 wt % in contrast to 0.14–3.8 wt % of the conventional methods. Liposomal Gem showed a good stability during storage, sustained-release over 120 h in vitro, enhanced cellular uptake, and improved cytotoxicity as compared to free Gem. Liposomal Gem showed a synergistic effect with liposomal Dox on Huh7 hepatocellular carcinoma cells. A mixture of liposomal Gem and liposomal Dox delivered both drugs to the tumor more efficiently than a free drug mixture and showed a relatively good anti-tumor effect in a xenograft model of hepatocellular carcinoma. This study shows that bioactive liposomal Gem with high drug loading capacity can be produced by remote loading combined with additional approaches to increase drug influx into the liposomes.Item Influence of CYP2B6 Pharmacogenetics on Stereoselective Inhibition and Induction of Bupropion Metabolism by Efavirenz in Healthy Volunteers(American Society for Pharmacology and Experimental Therapeutics, 2022-07-07) Gufford, Brandon T.; Metzger, Ingrid F.; Bamfo, Nadia O.; Benson, Eric A.; Masters, Andrea R.; Lu, Jessica Bo Li; Desta, Zeruesenay; Medicine, School of MedicineWe investigated the acute and chronic effects of efavirenz, a widely used antiretroviral drug, and CYP2B6 genotypes on the disposition of racemic and stereoisomers of bupropion (BUP) and its active metabolites, 4-hydroxyBUP, threohydroBUP and erythrohydroBUP. The primary objective of this study was to test how multiple processes unique to the efavirenz-CYP2B6 genotype interaction influence the extent of efavirenz-mediated drug-drug interaction (DDI) with the CYP2B6 probe substrate BUP. In a three-phase, sequential, open-label study, healthy volunteers (N=53) were administered a single 100 mg oral dose of BUP alone (control phase), with a single 600 mg oral efavirenz dose (inhibition phase), and after 17-days pretreatment with efavirenz (600 mg/day) (induction phase). Compared to the control phase, we show for the first time that efavirenz significantly decreases and chronically increases the exposure of hydroxyBUP and its diastereomers, respectively, and these interactions were CYP2B6 genotype dependent. Chronic efavirenz enhances the elimination of racemic BUP and its enantiomers as well as of threo- and erythro-hydroBUP and their diastereomers, suggesting additional novel mechanisms underlying efavirenz interaction with BUP. The effects of efavirenz and genotypes were nonstereospecific. In conclusion, acute and chronic administration of efavirenz inhibits and induces CYP2B6 activity. Efavirenz-BUP interaction is complex involving time- and CYP2B6 genotype-dependent inhibition and induction of primary and secondary metabolic pathways. Our findings highlight important implications to the safety and efficacy of BUP, study design considerations for future efavirenz interactions, and individualized drug therapy based on CYP2B6 genotypes. Significance Statement: The effects of acute and chronic doses of efavirenz on the disposition of racemic and stereoisomers of BUP and its active metabolites were investigated in healthy volunteers. Efavirenz causes an acute inhibition, but chronic induction of CYP2B6 in a genotype dependent manner. Chronic efavirenz induces BUP reduction and the elimination of BUP active metabolites. Efavirenz's effects were non-stereospecific. These data reveal novel mechanisms underlying efavirenz DDI with BUP and provide important insights into time- and CYP2B6 genotype dependent DDIs.Item Pharmacokinetic modeling of R and S-Methadone and their metabolites to study the effects of various covariates in post-operative children(Wiley, 2021-10) Aruldhas, Blessed W.; Quinney, Sara K.; Overholser, Brian R.; Heathman, Michael A.; Masters, Andrea R.; Ly, Reynold C.; Gao, Hongyu; Packiasabapathy, Senthil; Sadhasivam, Senthilkumar; Anesthesia, School of MedicineMethadone is a synthetic opioid used as an analgesic and for the treatment of opioid abuse disorder. The analgesic dose in the pediatric population is not well-defined. The pharmacokinetics (PKs) of methadone is highly variable due to the variability in alpha-1 acid glycoprotein (AAG) and genotypic differences in drug-metabolizing enzymes. Additionally, the R and S enantiomers of methadone have unique PK and pharmacodynamic properties. This study aims to describe the PKs of R and S methadone and its metabolite 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in pediatric surgical patients and to identify sources of inter- and intra-individual variability. Children aged 8-17.9 years undergoing orthopedic surgeries received intravenous methadone 0.1 mg/kg intra-operatively followed by oral methadone 0.1 mg/kg postoperatively every 12 h. Pharmacokinetics of R and S methadone and EDDP were determined using liquid chromatography tandem mass spectrometry assays and the data were modeled using nonlinear mixed-effects modeling in NONMEM. R and S methadone PKs were well-described by two-compartment disposition models with first-order absorption and elimination. EDDP metabolites were described by one compartment disposition models with first order elimination. Clearance of both R and S methadone were allometrically scaled by bodyweight. CYP2B6 phenotype was a determinant of the clearance of both the enantiomers in an additive gene model. The intronic CYP3A4 single-nucleotide polymorphism (SNP) rs2246709 was associated with decreased clearance of R and S methadone. Concentrations of AAG and the SNP of AAG rs17650 independently increased the volume of distribution of both the enantiomers. The knowledge of these important covariates will aid in the optimal dosing of methadone in children.Item Pharmacokinetic, pharmacodynamic, and transcriptomic analysis of chronic levetiracetam treatment in 5XFAD mice: A MODEL-AD preclinical testing core study(Wiley, 2022-08-23) Onos, Kristen D.; Quinney, Sara K.; Jones, David R.; Masters, Andrea R.; Pandey, Ravi; Keezer, Kelly J.; Biesdorf, Carla; Metzger, Ingrid F.; Meyers, Jill A.; Peters, Johnathon; Persohn, Scott C.; McCarthy, Brian P.; Bedwell, Amanda A.; Figueiredo, Lucas L.; Cope, Zackary A.; Sasner, Michael; Howell, Gareth R.; Williams, Harriet M.; Oblak, Adrian L.; Lamb, Bruce T.; Carter, Gregory W.; Sukoff Rizzo, Stacey J.; Territo, Paul R.; Obstetrics and Gynecology, School of MedicineIntroduction: Hyperexcitability and epileptiform activity are commonplace in Alzheimer's disease (AD) patients and associated with impaired cognitive function. The anti-seizure drug levetiracetam (LEV) is currently being evaluated in clinical trials for ability to reduce epileptiform activity and improve cognitive function in AD. The purpose of our studies was to establish a pharmacokinetic/pharmacodynamic (PK/PD) relationship with LEV in an amyloidogenic mouse model of AD to enable predictive preclinical to clinical translation, using the rigorous preclinical testing pipeline of the Model Organism Development and Evaluation for Late-Onset Alzheimer's Disease Preclinical Testing Core. Methods: A multi-tier approach was applied that included quality assurance and quality control of the active pharmaceutical ingredient, PK/PD modeling, positron emission tomography/magnetic resonance imaging (PET/MRI), functional outcomes, and transcriptomics. 5XFAD mice were treated chronically with LEV for 3 months at doses in line with those allometrically scaled to the clinical dose range. Results: Pharmacokinetics of LEV demonstrated sex differences in Cmax, AUC0-∞, and CL/F, and a dose dependence in AUC0-∞. After chronic dosing at 10, 30, 56 mg/kg, PET/MRI tracer 18F-AV45, and 18F-fluorodeoxyglucose (18F-FDG) showed specific regional differences with treatment. LEV did not significantly improve cognitive outcomes. Transcriptomics performed by nanoString demonstrated drug- and dose-related changes in gene expression relevant to human brain regions and pathways congruent with changes in 18F-FDG uptake. Discussion: This study represents the first report of PK/PD assessment of LEV in 5XFAD mice. Overall, these results highlighted non-linear kinetics based on dose and sex. Plasma concentrations of the 10 mg/kg dose in 5XFAD overlapped with human plasma concentrations used for studies of mild cognitive impairment, while the 30 and 56 mg/kg doses were reflective of doses used to treat seizure activity. Post-treatment gene expression analysis demonstrated LEV dose-related changes in immune function and neuronal-signaling pathways relevant to human AD, and aligned with regional 18F-FDG uptake. Overall, this study highlights the importance of PK/PD relationships in preclinical studies to inform clinical study design. Highlights: Significant sex differences in pharmacokinetics of levetiracetam were observed in 5XFAD mice. Plasma concentrations of 10 mg/kg levetiracetam dose in 5XFAD overlapped with human plasma concentration used in the clinic. Drug- and dose-related differences in gene expression relevant to human brain regions and pathways were also similar to brain region-specific changes in 18F-fluorodeoxyglucose uptake.Item Pharmacokinetics of vaginal versus buccal misoprostol for labor induction at term(Wiley, 2022) Vorontsova, Yana; Haas, David M.; Flannery, Kathleen; Masters, Andrea R.; Silva, Larissa L.; Pierson, Rebecca C.; Yeley, Brittany; Hogg, Graham; Guise, David; Heathman, Michael; Quinney, Sara K.; Obstetrics and Gynecology, School of MedicineThe IMPROVE study (NCT02408315) compared the efficacy and safety of vaginal and buccal administration of misoprostol for full-term, uncomplicated labor induction. This report compares the pharmacokinetics of misoprostol between vaginal and buccal routes. Women greater than or equal to 14 years of age undergoing induction of labor greater than or equal to 37 weeks gestation without significant complications were randomized to vaginal or buccal misoprostol 25 μg followed by 50 μg doses every 4 h. Misoprostol acid concentrations were determined using liquid chromatography-tandem mass spectrometry for the first 8 h in a subgroup of participants. A population pharmacokinetic model was developed using NONMEM. Plasma concentrations (n = 469) from 47 women were fit to a one-compartment nonlinear clearance model. The absorption rate constant (ka ) was dependent on both route and dose of administration: buccal 25 μg 0.724 (95% confidence interval, 0.54-0.92) h-1 ; 50 μg 0.531 (0.37-0.63) h-1 ; vaginal 25 μg 0.507 (0. 2-1. 4) h-1 ; and 50 μg 0.246 (0.103-0.453) h-1 . Relative bioavailability for vaginal compared to buccal route was 2.4 (1.63-4.77). There was no effect of body mass index or age on apparent clearance 705 (431-1099) L/h or apparent volume of distribution 632 (343-1008) L. The area under the concentration-time curve to 4 h following the first 25 μg dose of misoprostol was 16.5 (15.4-17.5) pg h/ml for buccal and 34.3 (32.5-36.1) pg h/ml for vaginal administration. The rate of buccal absorption was two times faster than that of vaginal, whereas bioavailability of vaginal administration was 2.4 times higher than that of buccal. Decreased time to delivery observed with vaginal dosing may be due to higher exposure to misoprostol acid compared to buccal.Item Population model analysis of chiral inversion and degradation of bupropion enantiomers, and application to enantiomer specific fraction unbound determination in rat plasma and brain(Elsevier, 2021) Bhattacharya, Chandrali; Masters, Andrea R.; Bach, Christine; Stratford, Robert E., Jr.; Medicine, School of MedicinePharmacologic effects elicited by drugs most directly relate to their unbound concentrations. Measurement of binding in blood, plasma and target tissues are used to estimate these concentrations by determining the fraction of total concentration in a biological matrix that is not bound. In the case of attempting to estimate R- and S-bupropion concentrations in plasma and brain following racemic bupropion administration, reversible chiral inversion and irreversible degradation of the enantiomers were hypothesized to confound attempts at unbound fraction estimation. To address this possibility, a kinetic modeling approach was used to quantify inversion and degradation specific processes for each enantiomer from separate incubations of each enantiomer in the two matrices, and in pH 7.4 buffer, which is also used in binding experiments based on equilibrium dialysis. Modeling analyses indicated that chiral inversion kinetics were two to four-fold faster in plasma and brain than degradation, with only inversion observed in buffer. Inversion rate was faster for S-bupropion in the three media; whereas, degradation rates were similar for the two enantiomers in plasma and brain, with overall degradation in plasma approximately 2-fold higher than in brain homogenate. Incorporation of degradation and chiral inversion kinetic terms into a model to predict enantiomer-specific binding in plasma and brain revealed that, despite existence of these two processes, empirically derived estimates of fraction unbound were similar to model-derived values, leading to a firm conclusion that observed extent of plasma and brain binding are accurate largely because binding kinetics are faster than parallel degradation and chiral inversion processes.Item Preclinical assessments of the MEK inhibitor PD-0325901 in a mouse model of neurofibromatosis type 1.(Wiley, 2015-10) Jousma, Edwin; Rizvi, Tilat A.; Wu, Jianqiang; Janhofer, David; Dombi, Eva; Dunn, Richard S.; Kim, Mi-Ok; Masters, Andrea R.; Jones, David R.; Cripe, Timothy P.; Ratner, Nancy; Department of Medicine, IU School of MedicineBackground: Neurofibromatosis type 1 (NF1) is a genetic disorder that predisposes affected individuals to formation of benign neurofibromas, peripheral nerve tumors that can be associated with significant morbidity. Loss of the NF1 Ras-GAP protein causes increased Ras-GTP, and we previously found that inhibiting MEK signaling downstream of Ras can shrink established neurofibromas in a genetically engineered murine model. Procedures: We studied effects of MEK inhibition using 1.5 mg/kg/day PD-0325901 prior to neurofibroma onset in the Nf1 flox/flox;Dhh-Cre mouse model. We also treated mice with established tumors at 0.5 and 1.5 mg/kg/day dosees of PD-0325901. We monitored tumor volumes using MRI and volumetric measurements, and measured pharmacokinetic and pharmacodynamic endpoints. Results: Early administration significantly delayed neurofibroma development as compared to vehicle controls. When treatment was discontinued neurofibromas grew, but no rebound effect was observed and neurofibromas remained significantly smaller than controls. Low dose treatment of mice with PD-0325901 resulted in neurofibroma shrinkage equivalent to that observed at higher doses. Tumor cell proliferation decreased, although less than at higher doses with drug. Tumor blood vessels per area correlated with tumor shrinkage. Conclusions: Neurofibroma development was not prevented by MEK inhibition, beginning at 1 month of age, but tumor size was controlled by early treatment. Moreover, treatment with PD-0325901 at very low doses may shrink neurofibromas while minimizing toxicity. These studies highlight how genetically engineered mouse models can guide clinical trial design.Item Prenatal methadone exposure disrupts behavioral development and alters motor neuron intrinsic properties and local circuitry(eLife Sciences, 2021-03-16) Grecco, Gregory G.; Mork, Briana E.; Huang, Jui-Yen; Metzger, Corinne E.; Haggerty, David L.; Reeves, Kaitlin C.; Gao, Yong; Hoffman, Hunter; Katner, Simon N.; Masters, Andrea R.; Morris, Cameron W.; Newell, Erin A.; Engleman, Eric A.; Baucum, Anthony J.; Kim, Jiuen; Yamamoto, Bryan K.; Allen, Matthew R.; Wu, Yu-Chien; Lu, Hui-Chen; Sheets, Patrick L.; Atwood, Brady K.; Pharmacology and Toxicology, School of MedicineDespite the rising prevalence of methadone treatment in pregnant women with opioid use disorder, the effects of methadone on neurobehavioral development remain unclear. We developed a translational mouse model of prenatal methadone exposure (PME) that resembles the typical pattern of opioid use by pregnant women who first use oxycodone then switch to methadone maintenance pharmacotherapy, and subsequently become pregnant while maintained on methadone. We investigated the effects of PME on physical development, sensorimotor behavior, and motor neuron properties using a multidisciplinary approach of physical, biochemical, and behavioral assessments along with brain slice electrophysiology and in vivo magnetic resonance imaging. Methadone accumulated in the placenta and fetal brain, but methadone levels in offspring dropped rapidly at birth which was associated with symptoms and behaviors consistent with neonatal opioid withdrawal. PME produced substantial impairments in offspring physical growth, activity in an open field, and sensorimotor milestone acquisition. Furthermore, these behavioral alterations were associated with reduced neuronal density in the motor cortex and a disruption in motor neuron intrinsic properties and local circuit connectivity. The present study adds to the limited body of work examining PME by providing a comprehensive, translationally relevant characterization of how PME disrupts offspring physical and neurobehavioral development.Item SHIP1 therapeutic target enablement: Identification and evaluation of inhibitors for the treatment of late‐onset Alzheimer's disease(Wiley, 2023) Jesudason, Cynthia D.; Mason, Emily R.; Chu, Shaoyou; Oblak, Adrian L.; Javens-Wolfe, June; Moussaif, Mustapha; Durst, Greg; Hipskind, Philip; Beck, Daniel E.; Dong, Jiajun; Amarasinghe, Ovini; Zhang, Zhong-Yin; Hamdani, Adam K.; Singhal, Kratika; Mesecar, Andrew D.; Souza, Sarah; Jacobson, Marlene; Di Salvo, Jerry; Soni, Disha M.; Kandasamy, Murugesh; Masters, Andrea R.; Quinney, Sara K.; Doolen, Suzanne; Huhe, Hasi; Sukoff Rizzo, Stacey J.; Lamb, Bruce T.; Palkowitz, Alan D.; Richardson, Timothy I.; Medicine, School of MedicineIntroduction: The risk of developing Alzheimer's disease is associated with genes involved in microglial function. Inositol polyphosphate-5-phosphatase (INPP5D), which encodes Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1), is a risk gene expressed in microglia. Because SHIP1 binds receptor immunoreceptor tyrosine-based inhibitory motifs (ITIMs), competes with kinases, and converts PI(3,4,5)P3 to PI(3,4)P2, it is a negative regulator of microglia function. Validated inhibitors are needed to evaluate SHIP1 as a potential therapeutic target. Methods: We identified inhibitors and screened the enzymatic domain of SHIP1. A protein construct containing two domains was used to evaluate enzyme inhibitor potency and selectivity versus SHIP2. Inhibitors were tested against a construct containing all ordered domains of the human and mouse proteins. A cellular thermal shift assay (CETSA) provided evidence of target engagement in cells. Phospho-AKT levels provided further evidence of on-target pharmacology. A high-content imaging assay was used to study the pharmacology of SHIP1 inhibition while monitoring cell health. Physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties were evaluated to select a compound suitable for in vivo studies. Results: SHIP1 inhibitors displayed a remarkable array of activities and cellular pharmacology. Inhibitory potency was dependent on the protein construct used to assess enzymatic activity. Some inhibitors failed to engage the target in cells. Inhibitors that were active in the CETSA consistently destabilized the protein and reduced pAKT levels. Many SHIP1 inhibitors were cytotoxic either at high concentration due to cell stress or they potently induced cell death depending on the compound and cell type. One compound activated microglia, inducing phagocytosis at concentrations that did not result in significant cell death. A pharmacokinetic study demonstrated brain exposures in mice upon oral administration. Discussion: 3-((2,4-Dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine activated primary mouse microglia and demonstrated exposures in mouse brain upon oral dosing. Although this compound is our recommended chemical probe for investigating the pharmacology of SHIP1 inhibition at this time, further optimization is required for clinical studies. Highlights: Cellular thermal shift assay (CETSA) and signaling (pAKT) assays were developed to provide evidence of src homology 2 (SH2) domain-containing inositol phosphatase 1 (SHIP1) target engagement and on-target activity in cellular assays. A phenotypic high-content imaging assay with simultaneous measures of phagocytosis, cell number, and nuclear intensity was developed to explore cellular pharmacology and monitor cell health. SHIP1 inhibitors demonstrate a wide range of activity and cellular pharmacology, and many reported inhibitors are cytotoxic. The chemical probe 3-((2,4-dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine is recommended to explore SHIP1 pharmacology.