Jessica K. Roberts

Transient Receptor Potential Vanilloid-1 (TRPV1) Is a Mediator of Lung Toxicity for Coal Fly Ash Particulate Material

Environmental particulate matter (PM) pollutants adversely affect human health, but the molecular basis is poorly understood. The ion channel transient receptor potential vanilloid-1 (TRPV1) has been implicated as a sensor for environmental PM and a mediator of adverse events in the respiratory tract. The objectives of this study were to determine whether TRPV1 can distinguish chemically and physically unique PM that represents important sources of air pollution; to elucidate the molecular basis of TRPV1 activation by PM; and to ascertain the contributions of TRPV1 to human lung cell and mouse lung tissue responses exposed to an insoluble PM agonist, coal fly ash (CFA1). The major findings of this study are that TRPV1 is activated by some, but not all of the prototype PM materials evaluated, with rank-ordered responses of CFA1 > diesel exhaust PM > crystalline silica; TRP melastatin-8 is also robustly activated by CFA1, whereas other TRP channels expressed by airway sensory neurons and lung epithelial cells that may also be activated by CFA1, including TRPs ankyrin 1 (A1), canonical 4α (C4α), M2, V2, V3, and V4, were either slightly (TRPA1) or not activated by CFA1; activation of TRPV1 by CFA1 occurs via cell surface interactions between the solid components of CFA1 and specific amino acid residues of TRPV1 that are localized in the putative pore-loop region; and activation of TRPV1 by CFA1 is not exclusive in mouse lungs but represents a pathway by which CFA1 affects the expression of selected genes in lung epithelial cells and airway tissue.

Contributions of TRPV1, endovanilloids, and endoplasmic reticulum stress in lung cell death in vitro and lung injury

Endogenous agonists of transient receptor potential vanilloid-1 (TRPV1) (endovanilloids) are implicated as mediators of lung injury during inflammation. This study tested the hypothesis that endovanilloids produced following lipopolysaccharide (LPS) treatment activate TRPV1 and cause endoplasmic reticulum stress/GADD153 expression in lung cells, representing a mechanistic component of lung injury. The TRPV1 agonist nonivamide induced GADD153 expression and caused cytotoxicity in immortalized and primary human bronchial, bronchiolar/alveolar, and microvascular endothelial cells, proportional to TRPV1 mRNA expression. In CF-1 mice, Trpv1 mRNA was most abundant in the alveoli, and intratracheal nonivamide treatment promoted Gadd153 expression in the alveolar region. Treatment of CF-1 mice with LPS increased Gadd153 in the lung, lactate dehydrogenase (LDH) in bronchoalveolar lavage (BAL) fluid, and lung wet-to-dry weight ratio. Cotreating mice with LPS and the TRPV1 antagonist LJO-328 reduced Gadd153 induction and LDH in BAL but did not inhibit increases in lung wet-to-dry ratio. In Trpv1(-/-) mice treated with LPS, Gadd153 induction and LDH in BAL were reduced relative to wild-type mice, and the wet-to-dry weight ratios of lungs from both wild-type and Trpv1(-/-) mice decreased. Organic extracts of blood collected from LPS-treated mice were more cytotoxic to TRPV1-overexpressing cells compared with BEAS-2B cells and extracts from control mice, however, most pure endovanilloids did not produce cytotoxicity in a characteristic TRPV1-dependent manner. Collectively, these data indicate a role for TRPV1, and endogenous TRPV1 agonists, in ER stress and cytotoxicity in lung cells but demonstrate that ER stress and cytotoxicity are not essential for pulmonary edema.

Metabolic Pathways of Inhaled Glucocorticoids by the CYP3A Enzymes

Asthma is one of the most prevalent diseases in the world, for which the mainstay treatment has been inhaled glucocorticoids (GCs). Despite the widespread use of these drugs, approximately 30% of asthma sufferers exhibit some degree of steroid insensitivity or are refractory to inhaled GCs. One hypothesis to explain this phenomenon is interpatient variability in the clearance of these compounds. The objective of this research is to determine how metabolism of GCs by the CYP3A family of enzymes could affect their effectiveness in asthmatic patients. In this work, the metabolism of four frequently prescribed inhaled GCs, triamcinolone acetonide, flunisolide, budesonide, and fluticasone propionate, by the CYP3A family of enzymes was studied to identify differences in their rates of clearance and to identify their metabolites. Both interenzyme and interdrug variability in rates of metabolism and metabolic fate were observed. CYP3A4 was the most efficient metabolic catalyst for all the compounds, and CYP3A7 had the slowest rates. CYP3A5, which is particularly relevant to GC metabolism in the lungs, was also shown to efficiently metabolize triamcinolone acetonide, budesonide, and fluticasone propionate. In contrast, flunisolide was only metabolized via CYP3A4, with no significant turnover by CYP3A5 or CYP3A7. Common metabolites included 6β-hydroxylation and ∆6-dehydrogenation for triamcinolone acetonide, budesonide, and flunisolide. The structure of ∆6-flunisolide was unambiguously established by NMR analysis. Metabolism also occurred on the D-ring substituents, including the 21-carboxy metabolites for triamcinolone acetonide and flunisolide. The novel metabolite 21-nortriamcinolone acetonide was also identified by liquid chromatography–mass spectrometry and NMR analysis.

Metabolism of Beclomethasone Dipropionate by Cytochrome P450 3A Enzymes

Inhaled glucocorticoids, such as beclomethasone dipropionate (BDP), are the mainstay treatment of asthma. However, ∼30% of patients exhibit little to no benefit from treatment. It has been postulated that glucocorticoid resistance, or insensitivity, is attributable to individual differences in glucocorticoid receptor-mediated processes. It is possible that variations in cytochrome P450 3A enzyme-mediated metabolism of BDP may contribute to this phenomenon. This hypothesis was explored by evaluating the contributions of CYP3A4, 3A5, 3A7, and esterase enzymes in the metabolism of BDP in vitro and relating metabolism to changes in CYP3A enzyme mRNA expression via the glucocorticoid receptor in lung and liver cells. CYP3A4 and CYP3A5 metabolized BDP via hydroxylation ([M4] and [M6]) and dehydrogenation ([M5]) at similar rates; CYP3A7 did not metabolize BDP. A new metabolite [M6], formed by the combined action of esterases and CYP3A4 hydroxylation, was also characterized. To validate the results observed using microsomes and recombinant enzymes, studies were also conducted using A549 lung and DPX2 liver cells. Both liver and lung cells produced esterase-dependent metabolites [M1–M3], with [M1] correlating with CYP3A5 mRNA induction in A549 cells. Liver cells produced both hydroxylated and dehydrogenated metabolites [M4, M5, and M6], but lung cells produced only the dehydrogenated metabolite [M5]. These studies show that CYP3A4 and CYP3A5 metabolize BDP to inactive metabolites and suggest that differences in the expression or function of these enzymes in the lung and/or liver could influence BDP disposition in humans.

Darbepoetin administration to neonates undergoing cooling for encephalopathy: a safety and pharmacokinetic trial

Background:Despite therapeutic hypothermia, neonates with encephalopathy (NE) have high rates of death or disability. Darbepoetin alfa (Darbe) has comparable biological activity to erythropoietin, but has extended circulating half-life (t1/2). Our aim was to determine Darbe safety and pharmacokinetics as adjunctive therapy to hypothermia.Study design:Thirty infants (n = 10/arm) ≥36 wk gestation undergoing therapeutic hypothermia for NE were randomized to receive placebo, Darbe low dose (2 μg/kg), or high dose (10 μg/kg) given intravenously within 12 h of birth (first dose/hypothermia condition) and at 7 d (second dose/normothermia condition). Adverse events were documented for 1 mo. Serum samples were obtained to characterize Darbe pharmacokinetics.Results:Adverse events (hypotension, altered liver and renal function, seizures, and death) were similar to placebo and historical controls. Following the first Darbe dose at 2 and 10 μg/kg, t1/2 was 24 and 32 h, and the area under the curve (AUCinf) was 26,555 and 180,886 h*mU/ml*, respectively. In addition, clearance was not significantly different between the doses (0.05 and 0.04 l/h). At 7 d, t1/2 was 26 and 35 h, and AUCinf was 10,790 and 56,233 h*mU/ml*, respectively (*P < 0.01).Conclusion:Darbe combined with hypothermia has similar safety profile to placebo with pharmacokinetics sufficient for weekly administration.

Optimal design in pediatric pharmacokinetic and pharmacodynamic clinical studies

It is not trivial to conduct clinical trials with pediatric participants. Ethical, logistical, and financial considerations add to the complexity of pediatric studies. Optimal design theory allows investigators the opportunity to apply mathematical optimization algorithms to define how to structure their data collection to answer focused research questions. These techniques can be used to determine an optimal sample size, optimal sample times, and the number of samples required for pharmacokinetic and pharmacodynamic studies. The aim of this review is to demonstrate how to determine optimal sample size, optimal sample times, and the number of samples required from each patient by presenting specific examples using optimal design tools. Additionally, this review aims to discuss the relative usefulness of sparse vs rich data. This review is intended to educate the clinician, as well as the basic research scientist, whom plan on conducting a pharmacokinetic/pharmacodynamic clinical trial in pediatric patients.

Use of Modeling and Simulation in the Design and Conduct of Pediatric Clinical Trials and the Optimization of Individualized Dosing Regimens

Mathematical models of drug action and disease progression can inform pediatric pharmacotherapy. In this tutorial, we explore the key issues that differentiate pediatric from adult pharmacokinetic (PK) / pharmacodynamic (PD) studies, describe methods to calculate the number of participants to be enrolled and the optimal times at which blood samples should be collected, and therapeutic drug monitoring methods for individualizing pharmacotherapy. The development of pediatric‐specific drug dosing dashboards is also highlighted, with an emphasis on clinical‐relevance and ease of use.

Vancomycin pharmacokinetic models: informing the clinical management of drug-resistant bacterial infections

This review aims to critically evaluate the pharmacokinetic literature describing the use of vancomycin in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) infections. Guidelines recommend that trough concentrations be used to guide vancomycin dosing for the treatment of MRSA infections; however, numerous in vitro, animal model and clinical studies have demonstrated that the therapeutic effectiveness of vancomycin is best described by the area under the concentration versus time curve (AUC) divided by the minimum inhibitory concentration (MIC) of the infecting organism (AUC/MIC). Among patients with lower respiratory tract infections, an AUC/MIC ≥400 was associated with a superior clinical and bacteriological response. Similarly, patients with MRSA bacteremia who achieved an Etest AUC/MIC ≥320 within 48 h were 50% less likely to experience treatment failure. For other patient populations and different clinical syndromes (e.g., children, the elderly, patients with osteomyelitis, etc.), pharmacokinetic/pharmacodynamic studies and prospective clinical trials are needed to establish appropriate therapeutic targets.

Pharmacokinetics of Budesonide Administered with Surfactant in Premature Lambs: Implications for Neonatal Clinical Trials.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of premature human infants, which may persist through adulthood. Airway inflammation has been firmly established in the pathogenesis of BPD. Previous studies to reduce airway inflammation with high-dose dexamethasone demonstrated adverse neurological outcomes, despite lower incidences of BPD. Instillation of budesonide and surfactant can facilitate early extubation and reduce the incidence of BPD and death among very low birth weight infants. However, the pharmacokinetics of budesonide and its distribution into the lung and brain are unknown. Therefore, 5 premature lambs were administered 0.25 mg/kg budesonide, with surfactant as the vehicle. Plasma and tissue samples were taken from the lambs for measurement of budesonide, 16α- hydroxy prednisolone, and budesonide palmitate using LC/MS/MS. Peak plasma budesonide concentrations were inversely correlated with the oxygenation index (correlation coefficient of -0.75). plasma budesonide concentrations were extremely low (~10% of expected) for two lambs that had high oxygenation indices and were excluded from further analyses. For the remaining 5 premature lambs, a non-compartmental analysis demonstrated an AUCinf of 148.77 ± 28.16 h*μg/L, half-life of 4.76 ± 1.79 h, and Cmax of 46.17 ± 17.71 µg/L. Using population pharmacokinetic methods, a onecompartment model with exponential residual error and first-order absorption adequately described the data. The apparent clearance and apparent volume of distribution of budesonide were estimated at 6.29 L/h (1.99 L/h/kg) and 29.1 L (9.2 L/kg), respectively. Budesonide and budesonide palmitate, but not 16α-hydroxy prednisolone, were detected in lung tissue. In this study, budesonide and its metabolites were not detected in the brain, which suggests that intratracheal instillation suggests that after local pulmonary deposition, there is no evidence of budesonide accumulation in the central nervous system. Overall, these results show that peak plasma budesonide concentrations are inversely correlated with the oxygenation index and that lung-specific delivery of budesonide avoids accumulation of budesonide in the brain.

A propensity-matched cohort study of vancomycin-associated nephrotoxicity in neonates

Background The incidence of nephrotoxicity among vancomycin-treated neonates has been reported to range from 2% to 20%. These widely varying estimates have led to confusion and controversy regarding the safety of vancomycin among neonates. Objective Evaluate the incidence of nephrotoxicity among neonates receiving vancomycin concomitantly with gentamicin. Design Retrospective observational cohort study using propensity score matching to provide covariate balance between neonates who did or did not receive vancomycin based on factors known to be related to the development of renal dysfunction. Setting Hospitals (n=22) throughout the Intermountain West, including a quaternary care childrens hospital. Patients Neonates ≤44 postmenstrual weeks (median gestational age: 31 (IQR 28–36) weeks) receiving intravenous gentamicin with or without exposure to vancomycin from January 2006 to December 2012. Main outcome measures Nephrotoxicity based on the modified Acute Kidney Injury Network criteria for acute kidney injury (AKI) or serum creatinine concentration ≥1.5 mg/dL persisting for ≥48 h. Results The final cohort was comprised of 1066 neonates (533 receiving vancomycin and gentamicin vs 533 receiving gentamicin). In a propensity score-matched cohort that was well balanced across 16 covariates, AKI was not associated with vancomycin use (16 neonates receiving vancomycin vs 7 controls experienced AKI; OR 1.5; 95% CI 0.6 to 4.0). However, the presence of a patent ductus arteriosus, concomitant non-steroidal anti-inflammatory drug use, ≥1 positive blood cultures, low birth weight and higher severity of illness and risk of mortality scores were associated with an increased risk of nephrotoxicity. Conclusions These results corroborate several earlier reports and much anecdotal evidence describing the infrequent occurrence of nephrotoxicity in neonates receiving concomitant vancomycin and gentamicin.