Search

Roxifiban is an esterprodrug that is hydrolyzed, after oral administration, to the active glycoprotein (GP) IIb/IIIa antagonist, XV459. The objectives of the study were to investigate the safety, tolerability, pharmacokinetics, and the time course of the pharmacologic response of XV459 in escalating doses of roxifiban and to assess the effect of age, loading dose of roxifiban, and aspirin pretreatment on XV459 pharmacokinetics, pharmacologic response, and safety profile in a five-part double-blind, placebo-controlled study. Healthy male volunteers (ages 18-46 years) received 7 (0.75-1.5 mg; n = 20) and 10 (0.75-1.0 mg; n = 8) multiple, oral, qd doses of roxifiban or placebo (n = 5). Healthy older male and female volunteers (ages 47-75 years) received roxifiban qd doses (0.5-0.75 mg; n = 8) or placebo (n = 3) for 7 days. Healthy male subjects (ages 18-46 years; n = 16) received a 1.5 or 1.0 mg loading dose either with or without pretreatment of 325 mg aspirin once daily for 3 days followed by single daily doses of 1.0 mg roxifiban for 6 days. Measurable plasma concentrations of XV459 appeared rapidly and were sustained throughout the dosing interval of 24 hours. The pharmacokinetics of XV459 were nonlinear. Systemic exposure of XV459 plateaued at the 1-mg dose level; plasma concentrations approached steady state in 4 to 6 days for doses greater than 1.0 mg. The time course of pharmacologic response as measured by the inhibition of platelet aggregation in response to an ex vivo 10 microM adenosine 5'-diphosphate (ADP) agonist correlated closely to the plasma concentration of XV459. Potent inhibition of ADP-induced platelet aggregation (IPA) persisted over the entire dosing interval. A clear dose response was achieved with roxifiban doses of 0.5 and 1.0 mg. For doses greater than 1.0 mg, a dose-proportional increase in IPA was not observed. Both the pharmacokinetics and pharmacologic response of XV459 exhibited low intraindividual variability (coefficient of variation [CV] < 15%) and higher interindividual variability (CV < 30%). Pretreatment with aspirin and/or a loading dose of 1.5 mg roxifiban had no significant effect on the pharmacokinetics and pharmacologic response of XV459. A dose-related increase in template bleeding time was observed at 1.25- and 1.5-mg doses of roxifiban, as compared to placebo. However, these bleeding time increases in the 1.25- and 1.5-mg dose groups were not significantly different from those at the lower dose groups. Overall, once-daily oral administration of roxifiban was fairly well tolerated and provided sustained systemic drug exposure and pharmacologic response over the entire administration interval. [ABSTRACT FROM AUTHOR]

Background: Mycobacterium tuberculosis (Mtb) strains H37Ra and H37Rv are commonly used to study new and re-evaluate old antituberculous agents with respect to their pharmacodynamic effects in vitro. The differences in membrane proteins and, in particular, differences in carrier proteins between Mtb H37Ra and Mtb H37Rv may have an impact on antibiotic potency. The question of whether H37Ra can be used as a reliable surrogate for H37Rv and clinical strains has not been addressed sufficiently. The purpose of this study is to provide a full comparison of susceptibility data of the most common antituberculosis (TB) agents against both Mtb strains., Methods: In addition to a literature review, in vitro checkerboard susceptibility study was conducted comparing the in vitro minimum inhibitory concentration (MIC) of 16 common antituberculous drugs against H37Ra and H37Rv. Heifets-Sanchez TB agar drug susceptibility plates were utilized., Results: Half of the antibiotics demonstrated similar growth inhibition against both strains, while slightly differing MIC values were found for 7 of 16 drugs. With the exception of rifampicin, no marked difference in MIC against H37Ra and H37Rv was observed., Conclusion: While neither the attenuated (H37Ra) nor the virulent strain (H37Rv) is a clinical strain, both strains predicted MICs of clinical isolates equally well, when comparing the current in vitro results to clinical susceptibility data in the literature. H37Ra comes with the benefits of lower experimental costs and less administrative barriers including the requirement of a biosafety Level III environment., Competing Interests: There are no conflicts of interest

Objectives: This study evaluated the in vitro pharmacodynamics of combinations of ceftazidime and the non-β-lactam β-lactamase inhibitor, avibactam, against ceftazidime-, piperacillin/tazobactam- and meropenem-multiresistant Pseudomonas aeruginosa by a quantitative time-kill method., Methods: MICs of ceftazidime plus 0-16 mg/L avibactam were determined against eight isolates of P. aeruginosa . Single-compartment, 24 h time-kill kinetics were investigated for three isolates at 0-16 mg/L avibactam with ceftazidime at 0.25-4-fold the MIC as measured at the respective avibactam concentration. Ceftazidime and avibactam concentrations were measured by LC-MS/MS during the time-kill kinetic studies to evaluate drug degradation., Results: Avibactam alone displayed no antimicrobial activity. MICs of ceftazidime decreased by 8-16-fold in the presence of avibactam at 4 mg/L. The changes in log 10 cfu/mL at both the 10 h and 24 h timepoints (versus 0 h) revealed bacterial killing at ≥1-fold MIC. Significantly higher concentrations of ceftazidime alone, as compared with those of ceftazidime in combination, were required to produce any given kill. Without avibactam, ceftazidime degradation was significant (defined as degradation t 1/2  <   24 h), with as little as 19%   ±   18% of the original concentration remaining at 8 h for the most resistant strain. In combination with avibactam, ceftazidime degradation at ≥ 1-fold MIC was negligible., Conclusion: The addition of avibactam protected ceftazidime from degradation in a dose-dependent manner and restored its cidal and static activity at concentrations in combination well below the MIC of ceftazidime alone., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with poor clinical outcomes and currently lacks effective treatment. Both the three-dimensional (3D) environment and the dynamic mechanical forces are very important factors in this metastatic cascade. However, traditional cell cultures fail to recapitulate this natural tumor microenvironment. Thus, in vivo-like models that can emulate the intraperitoneal environment are of obvious importance. In this study, a new microfluidic platform of the peritoneum was set up to mimic the situation of ovarian cancer spheroids in the peritoneal cavity during metastasis. Ovarian cancer spheroids generated under a non-adherent condition were cultured in microfluidic channels coated with peritoneal mesothelial cells subjected to physiologically relevant shear stress. In summary, this dynamic 3D ovarian cancer-mesothelium microfluidic platform can provide new knowledge on basic cancer biology and serve as a platform for potential drug screening and development.

Background and Objectives: Apatinib is an oral tyrosine kinase inhibitor approved in China for the treatment of patients with advanced metastatic gastric cancer. The approved dosing schedule is 850 mg once daily. The objective of this study was to develop a population pharmacokinetic (popPK) model of apatinib and determine factors that affect its pharmacokinetics., Methods: A popPK model for apatinib was developed using data from 106 individuals, including healthy volunteers and patients with malignant solid tumors. The potential influence of demographic, patient, and laboratory characteristics on oral apatinib pharmacokinetics were investigated in a covariate analysis. The extent of the impact of significant covariates on the exposure of apatinib was evaluated using simulations., Results: The final popPK model was a two-compartment model with mixed first- and zero-order absorption and first-order elimination. The population estimates of apparent clearance (CL/F) and apparent volume at steady-state were 57.8 L/h and 112.5 L, respectively. The non-linear dose proportionality in apatinib relative bioavailability was characterized by a sigmoidal maximum effect (E max ) equation wherein the midpoint dose for the decrease in bioavailability was 766 mg. Patients with advanced gastric cancer exhibited lower bioavailability. Cancer patients in general had lower CL/F than healthy volunteers. Simulation results indicated that apatinib exposure in various population groups were impacted by disease and laboratory characteristics., Conclusions: The increase in apatinib exposure was less than proportional to dose. The pharmacokinetics of apatinib in gastric cancer patients were significantly different from those in patients with other cancer types. Dosing of apatinib in various cancer subpopulations may require adjustments to optimize efficacy and benefits to patients.

Purpose: The relationship between incidences of neutropenia and 10-hydroxy-7-ethyl camptothecin (SN38) exposure was explored using SN38 pharmacokinetic and neutrophil count data from toxicology studies of etirinotecan pegol (EP) and irinotecan in beagle dogs., Methods: Dogs received four weekly intravenous infusions of either vehicle control (n = 22), EP (6, 15, 20, 25, 40/25 mg/kg; n = 3-9 dogs/dose group/sex; n = 48), or irinotecan (20 or 25 mg/kg n = 3-4 dogs/dose group/sex; n = 14). Blood samples were collected up to 50 days post-dose for characterization of SN38 pharmacokinetics. Two separate models were created describing SN38 concentration time profiles after either irinotecan or EP administrations to project the AUC 0-168h after Day 1 and Day 22 doses. The relationship between incidence of neutropenia and SN38 exposure was explored using logistic regression., Results: The incidence of neutropenia in dogs receiving weekly doses of irinotecan or EP was strongly correlated with maximum plasma SN38 concentration (C max ), but not SN38 area under the concentration-time curve (AUC). Neutropenia occurred in approximately 80% of dogs receiving irinotecan (mean SN38 C max of 13.5 and 26.3 ng/mL for 20 and 25 mg/kg, respectively). No neutropenia occurred in dogs receiving EP at doses up to and including 25 mg/kg (mean SN38 C max of 3.4 and 4.9 ng/mL for 20 and 25 mg/kg, respectively), despite 2.5-3.6 times greater SN38 AUC after EP compared to irinotecan at equivalent doses., Conclusions: EP administration avoids both high SN38 C max values and development of dose-limiting neutropenia observed after irinotecan, while maintaining greater and sustained SN38 exposure between doses., Competing Interests: All authors are employees and own stocks and stock options of Nektar Therapeutics. Ethical approval All procedures performed in the study was reviewed and approved by the Animal Care Committee of ITR Laboratories Canada, Inc. where the study was conducted. All animals were handled in accordance with the principles outlined in the Guide to the Care and Use of Experimental Animals and Guide for the Care and Use of Laboratory Animals.

Objectives: The combination of aztreonam and avibactam has been proposed for the treatment of infections caused by metallo-β-lactamase-producing Gram-negative organisms, given the stability of aztreonam against metallo-β-lactamases plus the broad coverage of avibactam against AmpC β-lactamases and ESBLs. This study aimed to evaluate the efficacy of the combination against four clinical isolates with defined but diverse β-lactamase profiles., Methods: The MICs of aztreonam were determined without and with avibactam (1, 2, 4, 8 and 16 mg/L). Using the MIC values, the static time-kill kinetic studies were designed to encompass aztreonam concentrations of 0.25, 0.5, 1, 2 and 4 times the MIC at the respective avibactam concentrations from 0 to 8 mg/L. Aztreonam and avibactam concentrations were determined by LC-MS/MS during the course of the time-kill kinetic studies to evaluate whether avibactam protects aztreonam from degradation., Results: Three of the four isolates had aztreonam MICs ≥128 mg/L in monotherapy. Dramatically increasing susceptibility associated with a decrease in aztreonam MIC was observed with increasing avibactam concentration. Against all isolates, the combinations resulted in greater killing with a much lower dose requirement for aztreonam. The resulting changes in base-10 logarithm of cfu/mL at both the 10 h and 24 h references (versus 0 h) were synergistic. In contrast, a significantly higher concentration of aztreonam in the monotherapy was required to produce the same kill as that in the combination therapy, due to rapid aztreonam degradation in two isolates., Conclusions: The aztreonam/avibactam combination protects aztreonam from hydrolysis and provides synergy in antimicrobial activity against multiple β-lactamase-expressing strains with a wide MIC range., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

KPC-producing Klebsiella pneumoniae causes serious infections associated with high death rates worldwide. Combination therapy consisting of fosfomycin and a carbapenem is better than monotherapy to combat multidrug-resistant microorganisms, but no dosages for the combination have been defined. The MICs of meropenem and fosfomycin were evaluated against 18 clinical isolates of KPC-2-producing K. pneumoniae The activities of combination antimicrobials were also determined by the checkerboard method. The MIC50 and MIC90 of each agent alone and in combination were challenged against short (1.5-h) or prolonged (3-h) infusion regimens of meropenem (1 g every 8 h [q8h], 1.5 g q6h, 2 g q8h) and fosfomycin (4 g q8h, 6 g q6h, 8 g q8h) by Monte Carlo simulation to evaluate the time above the MIC of the free drug concentration as a percentage of the dosing interval (fT>MIC). The monotherapy MIC50s and MIC90s were 32 and 256 mg/liter for meropenem and 64 and 512 mg/liter for fosfomycin, respectively. Antimicrobial combination increased bacterial susceptibility to 1/4 the MIC50s and to 1/8 to 1/16 the MIC90s of monotherapy. The antimicrobial combination demonstrated a synergistic effect for at least two-thirds of the isolates. In combination therapy, fosfomycin regimens of 6 g q6h and 8 g q8h as a 3-h infusion against the MIC50 and MIC90 had better chances of achieving ≥90% probability of target attainment (PTA) of 70% fT>MIC. Meropenem regimens of 1.5 g q6h and 2 g q8h in prolonged infusion can achieve close to 90% PTA of 40% fT>MIC for MIC50 but not MIC90 The significant reduction in the MIC values and the achievement of appropriate PTA demonstrated that regimens containing fosfomycin with meropenem can be effective against KPC-2-producing K. pneumoniae., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

One of greatest challenges to the successful treatment of cancer is drug resistance. An exciting approach is the eradication of cancer stem cells (CSCs). However, little is known about key signals regulating the formation and expansion of CSCs. Moreover, lack of a reliable predictive preclinical model has been a major obstacle to discover new cancer drugs and predict their clinical activity. Here, in ovarian cancer, a highly chemoresistant tumor that is rapidly fatal, we provide the first evidence demonstrating the causal involvement of mechanical stimulus in the CSC phenotype using a customizable microfluidic platform and three-dimensional spheroids, which most closely mimic tumor behavior. We found that ovarian cancer cells significantly acquired the expression of epithelial-to-mesenchymal transition and CSC markers and a remarkable chemoresistance to clinically relevant doses of frontline chemotherapeutic drugs cisplatin and paclitaxel when grown under fluid shear stress, which corroborates with the physiological attainable levels in the malignant ascites, but not under static condition. Furthermore, we uncovered a new link of microRNA-199a-3p, phosphatidylinositol 3-kinase/Akt, and multidrug transporter activation in shear stress-induced CSC enrichment. Our findings shed new light on the significance of hydrodynamics in cancer progression, emphasizing the need of a flow-informed framework in the development of therapeutics.

Objectives: Gentamicin is widely used in end-stage renal disease (ESRD) patients for the treatment of infections. The goal of this study was to find the most reasonable dosing regimen for gentamicin in ESRD patients receiving haemodialysis., Methods: The in vitro antimicrobial activity of gentamicin was evaluated by static and dynamic time-kill experiments against three bacterial strains of MSSA, MRSA and Pseudomonas aeruginosa. A semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model was established afterwards, allowing the characterization of the antibacterial effect of gentamicin in the human body. The model was utilized to assess dosing regimens of gentamicin in ESRD patients receiving haemodialysis, taking both efficacy and safety into account., Results: The PK/PD model was capable of describing the bacterial response to gentamicin exposure in all three strains. Simulation based on the PK/PD model showed that pre-dialysis and post-dialysis dosing would bring comparable benefit to the ESRD patient regardless of whether the PK/PD target (fCmax/MIC >8-fold) was achieved, while the post-dialysis dosing resulted in a significantly lower trough concentration. The result of simulated dose fractionation demonstrated that both fCmax/MIC and fAUC(0-24)/MIC are strong predictors of drug effectiveness, but the PK/PD model would provide a more precise prediction of antibacterial activity as well as valuable information on dose selection in ESRD patients receiving haemodialysis., Conclusions: Our study supports the original FDA label with regard to the dosing regimen of gentamicin in ESRD patients, which offers adequate clinical benefit as well as an acceptable safety profile., (© The Author 2015. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Introduction: Identifying the optimized dosing regimen and algorithm is critical in the development of antibiotics. Suboptimal regimens and inappropriate choice of drug give rise to drug-resistant bacteria which have limited the therapeutic utility of many commercially available antimicrobial agents. Strategies to optimize therapy of antimicrobial candidates to speed up the development process are urgently needed., Areas Covered: We examined pharmacokinetics and pharmacodynamics of antimicrobial agents with modeling and simulation approaches. The approach that is based on minimum inhibitory concentration to evaluate antimicrobial dosing strategy is widely utilized in drug development. The modeling approach utilizing information from time-kill kinetic studies is a tool that can provide more information on the time-course of bacterial response to a particular dosing regimen. Animal studies of dosing regimens that mimic human pharmacokinetics are another option to evaluate antimicrobial efficacy. Empirical, semi-mechanistic and mechanistic models of bacterial dynamics and development of drug resistance in response to drug therapy are discussed., Expert Opinion: Both theories and applications of these approaches provide an overall understanding of how the tools can streamline drug development process and help make crucial decisions. Many opportunities and potentials are presented to incorporate more rigorous integration of PK-PD modeling approaches even at preclinical stage to extrapolate to clinical settings, thus enabling successful trials and optimizing dosing strategies in relevant populations where the drug is mostly used.

The aim of this study was to evaluate whether genetic polymorphisms in CYP3A5 and ABCB1 are responsible for the interindividual variability observed in quetiapine pharmacokinetics. Pharmacokinetic data from a randomized crossover study evaluating 2 quetiapine 25 mg immediate-release tablets after single oral dose were used to develop a population pharmacokinetic model. The single nucleotide polymorphisms (SNPs) evaluated for the genotype effects of quetiapine pharmacokinetics were CYP3A5 A6986G and ABCB1 C3435T, along with other demographic variables and formulations. A one-compartment distribution model with linear elimination plus four transit compartments for the delayed absorption adequately described quetiapine disposition. CYP3A5 *1/*1 individuals (n = 3) had 29% increased clearance compared to *1/*3 and *3/*3 individuals. The impact of an increased clearance was evaluated by simulations. By computing the probability of target attainment (PTA) of steady-state therapeutic goal at 1-hour and 12-hour time points after 50-400 mg twice-daily regimens, the results indicated that CYP3A5 genotype has minimal impact on the PTA of the 1-hour concentrations but a significant impact on the 12-hour concentrations. The interpretation based on the simulations does not call for a genotype-based dosing scheme and is consistent with consensus guidelines for quetiapine that therapeutic drug monitoring is considered useful. Clinical Pharmacology in Drug Development., (© 2015, The American College of Clinical Pharmacology.)

The pharmacokinetic properties of a new molecular entity are important aspects in evaluating the viability of the compound as a pharmacological agent. The sesquiterpene lactone lychnopholide exhibits important biological activities. The objective of this study was to characterize the pharmacokinetics of lychnopholide after intravenous administration of 1.65 mg/kg (n = 5) and oral administration of 3.3 mg/kg (n = 3) lychnopholide in rats (0.2 ± 0.02 kg in weight) through nonlinear mixed effects modeling and non-compartmental pharmacokinetic analysis. A highly sensitive analytical method was used to quantify the plasma lychnopholide concentrations in rats. Plasma protein binding of this compound was over 99 % as determined by a filtration method. A two-compartment body model plus three transit compartments to characterize the absorption process best described the disposition of lychnopholide after both routes of administration. The oral bioavailability was approximately 68 %. The clearance was 0.131 l/min and intercompartmental clearance was 0.171 l/min; steady-state volume of distribution was 4.83 l. The mean transit time for the absorption process was 9.15 minutes. No flip-flop phenomenon was observed after oral administration. The pharmacokinetic properties are favorable for further development of lychnopholide as a potential oral pharmacological agent., (Georg Thieme Verlag KG Stuttgart · New York.)

The aim of this study was to examine the relationships between N-acetyltransferase genotypes, pharmacokinetics, and tolerability of granular slow-release para-aminosalicylic acid (GSR-PAS) in tuberculosis patients. The study was a randomized, two-period, open-label, crossover design wherein each patient received 4 g GSR-PAS twice daily or 8 g once daily alternately. The PAS concentration-time profiles were modeled by a one-compartment disposition model with three transit compartments in series to describe its absorption. Patients' NAT1 and NAT2 genotypes were determined by sequencing and restriction enzyme analysis, respectively. The number of daily vomits was modeled by a Poisson probability mass function. Comparisons of other tolerability measures by regimens, gender, and genotypes were evaluated by a linear mixed-effects model. The covariate effects associated with efavirenz, gender, and NAT1*3, NAT1*14, and NAT2*5 alleles corresponded to 25, 37, -17, -48, and -27% changes, respectively, in oral clearance of PAS. The NAT1*10 allele did not influence drug clearance. The time above the MIC of 1 mg/liter was significantly different between the two regimens but not influenced by the NAT1 or NAT2 genotypes. The occurrence and intensity of intolerance differed little between regimens. Four grams of GSR-PAS twice daily but not 8 g once daily ensured concentrations exceeding the MIC (1 mg/liter) throughout the dosing interval; PAS intolerance was not related to maximum PAS concentrations over the doses studied and was not more frequent after once-daily dosing. We confirm that the slow phenotype conferred by the NAT1*14 and NAT1*3 alleles resulted in higher PAS exposure but found no evidence of increased activity of the NAT1*10 allele., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Owing to significant dose-related toxicity, the adult stavudine dose was reduced in 2007. The paediatric dose, however, has not been reduced. Although the intended paediatric dose is 1 mg/kg twice daily (b.i.d.), the current weight-band dosing approach results in a mean actual dose of 1.23±0.47 mg/kg. Both efficacy and mitochondrial toxicity depend on the concentration of the intracellular metabolite stavudine triphosphate (d4T-TP). We simulated the effect of reducing the paediatric dose to 0.5 mg/kg. A physiologically-based pharmacokinetic model consisting of 13 tissue compartments plus a full ADAM model was used to describe the elimination of stavudine. The volume of distribution at steady-state and apparent oral clearance were simulated and the resulting AUC profile was compared with literature data in adult and paediatric populations. A biochemical reaction model was utilised to simulate intracellular d4T-TP levels for both the standard and proposed reduced paediatric doses. Simulated and observed exposure after oral dosing showed adequate agreement. Mean steady-state d4T-TP for 1.23 mg/kg b.i.d. was 27.9 (90% CI 27.0-28.9) fmol/10(6) cells, 25% higher than that achieved by the 40 mg adult dose. The 0.5 mg/kg dose resulted in d4T-TP of 13.2 (12.7-13.7) fmol/10(6) cells, slightly higher than the adult dose of 20 mg b.i.d. [11.5 (11.2-11.9) fmol/10(6) cells], which has excellent antiviral efficacy and substantially less toxicity. Current paediatric dosing may result in even higher d4T-TP than the original 40 mg adult dose. Halving the paediatric dose would significantly reduce the risk of mitochondrial toxicity without compromising antiviral efficacy., (Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

The aim of this study was to use the pharmacokinetic information of avicularin in rats to project a dose for humans using allometric scaling. A highly sensitive and specific bioanalytical assay to determine avicularin concentrations in the plasma was developed and validated for UPLC-MS/MS. The plasma protein binding of avicularin in rat plasma determined by the ultrafiltration method was 64%. The pharmacokinetics of avicularin in nine rats was studied following an intravenous bolus administration of 1 mg/kg and was found to be best described by a two-compartment model using a nonlinear mixed effects modeling approach. The pharmacokinetic parameters were allometrically scaled by body weight and centered to the median rat weight of 0.23 kg, with the power coefficient fixed at 0.75 for clearance and 1 for volume parameters. Avicularin was rapidly eliminated from the systemic circulation within 1 h post-dose, and the avicularin pharmacokinetic was linear up to 5 mg/kg based on exposure comparison to literature data for a 5-mg/kg single dose in rats. Using allometric scaling and Monte Carlo simulation approaches, the rat doses of 1 and 5 mg/kg correspond to the human equivalent doses of 30 and 150 mg, respectively, to achieve comparable plasma avicularin concentrations in humans., (Georg Thieme Verlag KG Stuttgart · New York.)

The aim of this study was to develop a semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) model to evaluate the in vitro synergy between vertilmicin and ceftazidime against Pseudomonas aeruginosa. The in vitro antimicrobial activity of vertilmicin alone was initially assessed by static and dynamic time-kill experiments against three bacterial strains, including MSSA, MRSA and P. aeruginosa. The combined killing effect with ceftazidime was then evaluated in a static time-kill study against P. aeruginosa. Vertilmicin displayed a concentration-dependent killing effect against the three bacterial strains, and its short half-life may possibly have a dramatic impact on antimicrobial activities. A two-compartment pharmacodynamic model consisting of drug-susceptible and -resistant compartments was developed to characterise the relationship between drug exposure and bacterial response for the time-kill curves from both monotherapy and combination therapy. Loewe additivity was incorporated into the pharmacodynamic model to describe the drug-drug interactive effect in the combination therapy. For monotherapy, the estimated EC50 of the dynamic time-kill study against each strain was close to its MIC but was higher than that of the static time-kill study. The EC50 of combination therapy was estimated at 2.67 mg/L compared with 4.54 mg/L in monotherapy, indicating an enhanced bactericidal capacity. The drug-drug interactive effect was not significantly synergistic but highly varied at each specific combination. Potential synergistic combinations could be screened using PK/PD modelling and simulation. These results demonstrated that PK/PD modelling provides an innovative approach to assist dose selection of combination vertilmicin and ceftazidime for future clinical study design., (Copyright © 2014 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.)

The empirical scaling from adult to pediatric using allometric size adjustments based on body weight continued to be the mainstream method for pediatric dose selection. Due to the flexibility of a polynomial function to conform to the data trend, an empirical function for simulating age-matched weight and body mass index by gender in the pediatric population is developed by using a polynomial function and a constant coefficient to describe the interindividual variability in weight. A polynomial of up to fifth order sufficiently described the pediatric data from the Center for Disease Control (CDC) and the World Health Organization (WHO). The coefficients of variation to describe the variability were within 17%. The percentages of the CDC simulated weights for pediatrics between 0 and 5 years that fell outside the WHO 90% and 95% confidence boundaries were well within the expected percentage values, indicating that the CDC dataset can be used to substitute for the WHO dataset for the purpose of pediatric drug development. To illustrate the utility of this empirical function, the CDC-based age-matched weights were simulated and were used in the prediction of the concentration-time profiles of tenofovir in children based on a population pharmacokinetic model whose parameters were allometrically scaled. We have shown that the resulting 95% prediction interval of tenofovir in newborn to 5 years of age was almost identical whether the weights were simulated based on WHO or CDC dataset. The approach is simple and is broadly applicable in adjusting for pediatric dosages using allometry.

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis prompted the reintroduction of para-aminosalicylic acid (PAS) to protect companion anti-tuberculosis drugs from additional acquired resistance. In sub-Saharan Africa, MDR/XDR tuberculosis with HIV coinfection is common, and concurrent treatment of HIV infection and MDR/XDR tuberculosis is required. Out of necessity, patients receive multiple drugs, and PAS therapy is frequent; however, neither potential drug interactions nor the effects of HIV infection are known. Potential drug-drug interaction with PAS and the effect of HIV infection was examined in 73 pulmonary tuberculosis patients; 22 (30.1%) were HIV coinfected. Forty-one pulmonary MDR or XDR tuberculosis patients received 4 g PAS twice daily, and in a second crossover study, another 32 patients were randomized, receiving 4 g PAS twice daily or 8 g PAS once daily. A PAS population pharmacokinetic model in two dosing regimens was developed; potential covariates affecting its pharmacokinetics were examined, and Monte Carlo simulations were conducted evaluating the pharmacokinetic-pharmacodynamic index. The probability of target attainment (PTA) to maintain PAS levels above MIC during the dosing interval was estimated by simulation of once-, twice-, and thrice-daily dosing regimens not exceeding 12 g daily. Concurrent efavirenz (EFV) medication resulted in a 52% increase in PAS clearance and a corresponding >30% reduction in mean PAS area under the concentration curve in 19 of 22 HIV-M. tuberculosis-coinfected patients. Current practice recommends maintenance of PAS concentrations at ≥1 μg/ml (the MIC of M. tuberculosis), but the model predicts that at only a minimum dose of 4 g twice daily can this PTA be achieved in at least 90% of the population, whether or not EFV is concomitantly administered. Once-daily dosing of 12 g PAS will not provide PAS concentrations exceeding the MIC over the entire dosing interval if coadministered with EFV, while 4 g twice daily ensures concentrations exceeding MIC over the entire dosing interval, even in HIV-infected patients who received EFV., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

A highly sensitive and specific LC-MS/MS method for the quantitation of largazole thiol, the active species of the marine-derived preclinical histone deacetylase inhibitor, largazole (prodrug), was developed and validated. Largazole thiol was extracted with ethyl acetate from human or rat plasma along with the internal standard, harmine. Samples were separated on an Onyx Monolithic C18 column by a stepwise gradient elution with 0.1% formic acid in methanol and 0.1% aqueous formic acid employing multiple reaction monitoring (MRM) detection. Linear calibration curves were obtained in the range of 12.5-400 ng/mL with 200 µL of human plasma. The overall intra-day precision was from 3.87% to 12.6%, and the inter-day precision was from 7.12% to 9.8%. The accuracy at low, medium and high concentrations ranged from 101.55% to 105.84%. Plasma protein bindings of largazole thiol in human and rat plasma as determined by an ultrafiltration method were 90.13% and 77.14%, respectively. Plasma drug concentrations were measured by this LC-MS/MS method. The pharmacokinetics of largazole thiol in rats was studied following i.v. administration at 10 mg/kg and found to follow a two-compartment model. Largazole thiol was rapidly eliminated from systemic circulation within 2 h. The established LC-MS/MS method is suitable for the analysis of largazole thiol in human plasma, as well.

The antiviral efficacy of stavudine depends on the trough concentration of its intracellular metabolite, stavudine-triphosphate (d4T-TP), while the degree of stavudine's mitochondrial toxicity depends on its peak concentration. Rates of mitochondrial toxicity are high when stavudine is used at the current standard pediatric dose (1 mg/kg twice daily [BID]). Evidence from adult work suggests that half of the original standard adult dose (i.e., 20 mg BID) may be equally effective, with markedly less mitochondrial toxicity. We present a population pharmacokinetic model to predict intracellular d4T-TP concentrations in pediatric HIV-infected patients administered a dose of 0.5 mg/kg BID. Our model predicted that the reduced pediatric dose would result in a trough intracellular d4T-TP concentration above that of the reduced 20-mg adult dose and a peak concentration below that of the 20-mg adult dose. The simulated pediatric intracellular d4T-TP at 0.5 mg/kg BID resulted in median peak and trough values of approximately 23.9 fmol/10(6) cells (95% prediction interval [PI], 14.2 to 41 fmol/10(6) cells) and 14.8 fmol/10(6) cells (95% PI, 7.2 to 31 fmol/10(6) cells), respectively. The peak and trough concentrations resulting from a 20-mg BID adult dose were 28.4 fmol/10(6) cells (95% PI, 17.3 to 45.5 fmol/10(6) cells) and 13 fmol/10(6) cells (95% PI, 6.8 to 28.6 fmol/10(6) cells), respectively. Halving the current standard pediatric dose should therefore not compromise antiviral efficacy, while markedly reducing mitochondrial toxicity.

Development of central nervous system-acting drugs would be enhanced by suitable biomarkers that reflect the targeted pathophysiologic brain state. The electroencephalogram (EEG) has several characteristics of an ideal biomarker and can be promptly adapted to pre-clinical and clinical testing. The aim of this study was to evaluate EEG as a measure of the wakefulness-promoting effect of armodafinil in sleep deprived healthy subjects. Armodafinil pharmacodynamics were simultaneously assessed by EEG- and behavioral-based measures including a well-established measure of alertness. Using two quantitative EEG-based measures-power spectral and event-related brain activity analyses-we observed that armodafinil mitigated the slowing of brain activity and the decrease of the event-related brain activity caused by sleep deprivation. Armodafinil-induced changes in EEG are in agreement and explain up to 73.1% of the armodafinil-induced changes in alertness. Our findings suggest that EEG can serve as a marker of the wakefulness-promoting drug effect., (© 2013, The American College of Clinical Pharmacology.)

The SNP A6986G of the CYP3A5 gene (*3) results in a non-functional protein due to a splicing defect whereas the C3435T was associated with variable expression of the ABCB1 gene, due to protein instability. Part of the large interindividual variability in tacrolimus efficacy and toxicity can be accounted for by these genetic factors. Seventy-two individuals were examined for A6986G and C3435T polymorphism using a PCR-RFLP-based technique to estimate genotype and allele frequencies in the Jordanian population. The association of age, hematocrit, platelet count, CYP3A5, and ABCB1 polymorphisms with tacrolimus dose- and body-weight-normalized levels in the subset of 38 pediatric renal transplant patients was evaluated. A Markov model was used to evaluate the time-dependent probability of an adverse event occurrence by CYP3A5 phenotypes and ABCB1 genotypes. The time-dependent probability of adverse event was about double in CYP3A5 non-expressors compared to the expressors for the first 12 months of therapy. The CYP3A5 non-expressors had higher corresponding normalized tacrolimus levels compared to the expressors in the first 3 months. The correlation trend between probability of adverse events and normalized tacrolimus concentrations for the two CYP3A5 phenotypes persisted for the first 9 months of therapy. The differences among ABCB1 genotypes in terms of adverse events and normalized tacrolimus levels were only observed in the first 3 months of therapy. The information on CYP3A5 genotypes and tacrolimus dose requirement is important in designing effective programs toward management of tacrolimus side effects particularly for the initial dose when tacrolimus blood levels are not available for therapeutic drug monitoring.

The aim of the study is to investigate the influence of ABCB1(3435) and CYP3A5(6986) polymorphisms, tacrolimus troughs and clinical factors on the time of adverse events associated with tacrolimus in pediatric kidney transplant patients. Clinical data, adverse events, tacrolimus troughs, corresponding doses, ABCB1 3435C > T and CYP3A5 6986A > G genotypes were collected from 38 pediatric kidney transplant patients in a retrospective study for over 2 years post-transplant. We used a marginal Cox proportional hazard model to evaluate the influence of clinical factors and single nucleotide polymorphisms (SNPs) on tacrolimus-associated adverse events. CYP3A5 genotype, the Bayesian predicted tacrolimus concentrations, hematocrit and mean corpuscular volume are significant risk factors of adverse events over a 2-year-period. CYP3A5*1 genotype was associated with 36% relative risk of CYP3A5*3/*3 genotype. In the 9-month period, the additional factor, ABCB1 3435TT genotype, was shown to be associated with 38% relative risk of the CC and CT genotypes. For graft loss, acute and chronic rejection, only tacrolimus concentration and hematocrit, but not CYP3A5 or ABCB1 polymorphisms, are important factors influencing their occurrences., (© The Author(s) 2013.)

Background: Mathematical models of dynamical systems facilitate the computation of characteristic properties that are not accessible experimentally. In cell biology, two main properties of interest are (1) the time-period a protein is accessible to other molecules in a certain state - its half-life - and (2) the time it spends when passing through a subsystem - its transit-time. We discuss two approaches to quantify the half-life, present the novel method of in silico labeling, and introduce the label half-life and label transit-time. The developed method has been motivated by laboratory tracer experiments. To investigate the kinetic properties and behavior of a substance of interest, we computationally label this species in order to track it throughout its life cycle. The corresponding mathematical model is extended by an additional set of reactions for the labeled species, avoiding any double-counting within closed circuits, correcting for the influences of upstream fluxes, and taking into account combinatorial multiplicity for complexes or reactions with several reactants or products. A profile likelihood approach is used to estimate confidence intervals on the label half-life and transit-time., Results: Application to the JAK-STAT signaling pathway in Epo-stimulated BaF3-EpoR cells enabled the calculation of the time-dependent label half-life and transit-time of STAT species. The results were robust against parameter uncertainties., Conclusions: Our approach renders possible the estimation of species and label half-lives and transit-times. It is applicable to large non-linear systems and an implementation is provided within the PottersWheel modeling framework (http://www.potterswheel.de).

Purpose: The aim of this investigation was to demonstrate that nonlinear mixed-effects population pharmacokinetic (PK) modeling can be used to evaluate data from studies of drug transport/excretion into human milk and hence to estimate infant exposure., Methods: A sparse dataset from a previously published study of the use of oral tramadol for post-cesarean pain management in 75 lactating women was used. Milk and plasma samples were collected during days 2-4 of lactation, and tramadol and O-desmethyltramadol (ODT) concentration measurements in these samples were available. Absolute infant dose was obtained from the concentration measurements and estimated milk volume ingested, and expressed in micrograms per kilogram per day. Relative infant dose was calculated as a percentage of the absolute infant dose divided by the maternal dose (μg/kg/day). Nonlinear mixed-effects modeling was used to fit a population PK model to the data., Results: The disposition of tramadol and ODT in plasma and the transition of these substances into milk were characterized by a five-compartment population PK mixture model with first-order absorption. The polymorphic ODT formation clearance in the plasma compartment was able to be characterized in both CYP2D6-poor and -extensive metabolizers. Milk creamatocrit was a significant covariate in ODT transfer between the plasma and milk compartments. The estimated relative infant doses in extensive and poor metabolizers, respectively, were 2.16 ± 0.57 and 2.60 ± 0.57% for tramadol, and 0.93 ± .20 and 0.47 ± 0.10% for ODT., Conclusions: This study demonstrates that a population PK approach with sparse sampling of analytes in milk and plasma can yield quality information about the transfer process and that it also can be used to estimate the extent of infant exposure to maternal drugs via milk.

What Is Already Known About This Subject: There are presently no published data on tramadol transfer into breast milk or on its effects in the breastfed infant., What This Study Adds: We have provided quantitative data on the absolute and relative infant doses of rac-tramadol and it rac-O-desmethyl metabolite for the breastfed infant. We have also demonstrated a novel sparse sampling data collection method for investigating infant exposure via milk., Aims: To investigate the transfer of rac-tramadol and its rac-O-desmethyl metabolite into transitional milk, and assess unwanted effects in the breastfed infant., Methods: Tramadol HCl (100 mg six hourly) was administered to 75 breastfeeding mothers for postoperative analgesia on days 2-4 after Caesarian section. Milk and plasma samples were collected after administration of four or more doses. Rac-tramadol and rac-O-desmethyltramadol were measured by high performance liquid chromatography. Milk : plasma ratio (M : P) and infant doses were calculated by standard methods. The behavioural characteristics of the exposed breastfed infants and a matched control group of infants not exposed to tramadol were also studied. RESULTS At steady-state, mean (95% CI) M : P was 2.2 (2.0, 2.4) for rac-tramadol and 2.8 (2.5, 3.1) for rac-O-desmethyltramadol. The estimated absolute and relative infant doses were 112 (102, 122) microg kg(-1) day(-1) and 30 (28, 32) microg kg(-1) day(-1), and 2.24% (2.04, 2.44)% and 0.64% (0.59, 0.69)% for rac-tramadol and rac-O-desmethyltramadol, respectively. The exposed infants and control breastfed infants had similar characteristics, including Apgar scores at birth and Neurologic and Adaptive Capacity Scores., Conclusions: The combined relative infant dose of 2.88% at steady-state was low. The similarity of NACS in exposed infants and controls suggests that there were no significant behavioural adverse effects. We conclude that short-term maternal use of tramadol during establishment of lactation is compatible with breastfeeding.

In clinical trials in patients with acute or unstable coronary syndromes and/or undergoing percutaneous coronary intervention, oral glycoprotein (GP) IIb/IIIa antagonists did not show therapeutic benefit over aspirin during long-term administration. Moreover, high-dose oral administration of these agents was associated with greater fatality risk compared with that of lower doses. This article postulates that continuous exposure of the GP IIb/IIIa receptor (integrin alpha(IIb)beta(3)) to these agents may result in some form of resistance or activation of other biological systems. These toxicological mechanisms may help explain some factors that could potentially contribute to the failure of these agents in clinical trials. Several hypotheses are presented: (i) modulation of platelet response because of long-term exposure to GP IIb/IIIa antagonists; (ii) role of related integrins and associated proteins to compensate for the loss of platelet activity because of dysfunctional GP IIb/IIIa receptors occupied by inhibitors; (iii) effects of the GP IIb/IIIa antagonists on other cellular systems such as the caspase and procaspase enzymes in apoptosis and possibly the ryanodine receptor involved in sarcoplasmic reticulum calcium release. These toxicological mechanisms could potentially limit the utility of these oral agents in long-term administration.

Roxifiban (DMP 754) is a glycoprotein (GP) IIb/IIIa antagonist. Following oral administration to humans, roxifiban is metabolized to its primary active zwitterionic form, XV459, and several minor, active, hydrolyzed and hydroxylated metabolites, namely, M1a (DPC-AD3508), M1b (DPC-AD6128), M2 (SW156), M3 (DPC-AG2185), M8a (DPC-AF5814), and M8b (DPC-AF5818). Quantification of these metabolites in humans was not workable with a previous analytical method due to ion suppression of at least four of the analytes by a competitive displacer, DMP 728. This compound, which is another GP IIb/IIIa antagonist with very high affinity for the platelet receptor, was added to harvested blood samples in millimolar quantity to liberate XV459 from the GP IIb/IIIa receptor. An automated ion exchange solid phase extraction (IX-SPE) procedure was developed to selectively extract the seven metabolites of roxifiban and its deuterated internal standard while specifically excluding DMP 728. Among the six hydroxylation metabolites, there were two pairs of epimeric diastereomers (M1a/M1b and M8a/M8b) and one pair of geometric isomers (M2/M3), corresponding to three critical chromatographic pairs that needed to be base-line resolved because of the lack of specificity of MS/MS detection for these isomers. A new LC/MS/MS assay was developed to simultaneously quantify the seven metabolites in human plasma. The assay method was validated under GLP conditions over the concentration range of 0.5 to 80 nM for each of the analytes and successfully applied to assaying approximately 500 plasma samples from clinical trials., (Copyright 2003 Elsevier Science B.V.)

Introduction: Intravenous platelet glycoprotein (GP) IIb/IIIa receptor inhibitors have a significant beneficial impact on the outcomes of patients undergoing high-risk coronary interventions and in the stabilization of patients with unstable angina pectoris refractory to conventional medical treatment. The role of long-term treatment with oral platelet GP IIb/IIIa receptor inhibitors in patients with coronary artery disease is unproven. This study examined the dose-response effect on inhibition of platelet aggregation by roxifiban (DMP754), a novel oral platelet GP IIb/IIIa receptor inhibitor, and its safety and tolerability in patients with a history of chronic stable angina pectoris., Methods: Ninety-eight patients were randomized to receive either a placebo or 1 of 8 oral dosages of roxifiban. Twenty-two patients were enrolled in multiple-dose regimens, bringing the total study population to 120. The oral dosages were 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, or 2.5 mg/day for up to 30 days., Results: Pharmacodynamic response of roxifiban was clearly dose-dependent. Platelet aggregation inhibition in response to 10 micromol/L slope adenosine diphosphate was sustained throughout the study period (up to 1 month). No serious adverse events, including significant major bleeding events, were associated with roxifiban treatment. Minor bleeding was reported in 5% of participants in the placebo group (1 of 21 cases) versus 26% in the study group (26 of 99 cases). Incidence of minor bleeding associated with roxifiban 2 and 2.5 mg/day was significantly (p < or = 0.05) greater than that with placebo. Adverse events, including gastrointestinal disorders, platelet and clotting disorders, and urinary tract disorders, were observed in 1 of 21 cases (5%) in the placebo group and in 12 of 99 cases (12%) in the study group. Reversible thrombocytopenia without other complications developed in two patients., Conclusions: Roxifiban-induced inhibition of platelet aggregation was dose-dependent and sustained throughout the study period: higher drug dosages correlated with higher levels of platelet inhibition and higher incidence of minor bleeding events. No serious adverse events were observed at any dosage. Thus, roxifiban appears to be a potent oral platelet GP IIb/IIIa receptor inhibitor that is clinically well-tolerated and deserves further study as a new treatment strategy in patients with chronic stable angina pectoris.

Aims: To determine whether the variability in cytochrome P(450) (CYP)3A4 metabolic function is exhibited at both transcription and translation levels and to examine the population distribution of CYP3A4 enzyme kinetics, protein, and mRNA., Methods: Enzyme kinetics of testosterone 6beta-hydroxylation, immunoblot CYP3A4 protein, and CYP3A4 mRNA were determined in a microsomal bank of human livers. The distribution of these determinations was analyzed using cumulative distribution (probit) plots and normality test variable (NTV) to detect deviation from normality., Results: Mean hepatic CYP3A4 protein and relative CYP3A4 mRNA were 35+/-23 pmol/mg and 79+/-59 (CYP3A4/beta-actin), respectively. Kinetic parameter estimates of testosterone 6beta-hydroxylation were 611+/-684 pmol/mg/min for maximum rate of the reaction (V(max)) and 206+/-48 micro M for the Michaelis constant (K(m)). The CYP3A4 gene expression and its activity exhibited a relatively high degree of interindividual variability. Furthermore, significant correlation between CYP3A4 protein and V(max) of testosterone 6beta-hydroxylation (r=0.82, P<0.001) as well as CYP3A4 protein and its mRNA (r=0.52, P<0.01) was observed. Cumulative distribution plots and histograms for the CYP3A4 protein, its mRNA, and maximum testosterone 6beta-hydroxylation exhibited evidences of deviation from log-normal distribution. The minimum NTV value for the distribution of CYP3A4 protein corresponding to the inflection point in the probit occurred at approximately 10% cumulative frequency. The percentage of low CYP3A4 protein phenotype was consistent for CYP3A4 activity and its mRNA. In contrast, the distribution of K(m) of testosterone 6beta-hydroxylation does not show evidence of bimodality., Conclusions: The distribution of CYP3A4 metabolic function, protein, and mRNA is non-normal and may represent a regulatory polymorphism in hepatic CYP3A4 gene expression. In contrast, assessment of the possibility of a structural variant of CYP3A4 through evaluation of the standard deviation relative to the mean kinetic constant value suggests that structural mutation of CYP3A4 may not be a major factor affecting interindividual variation in CYP3A4 metabolic function.

Objective: To characterize the distribution of mathematically derived human hepatic CYP1A1 activity using differential inhibition of ethoxyresorufin O-deethylation (EROD) by fluvoxamine., Methods: Quantitative CYP1A1- and CYP1A2-mediated EROD activities were determined in 42 human livers using differential inhibition of EROD by fluvoxamine. CYP1A2-specific activity was also measured by phenacetin O-deethylation and caffeine 3-demethylation. Distributions of CYP1A1-mediated EROD and CYP1-A2 probe activities were analyzed using cumulative distribution (probit) plots and the Kolgomorov-Smirnov test. Age effect on CYP1A1- and CYP1A2-mediated EROD activities was evaluated using descriptive statistics and analysis of variance., Results: The derived CYP1A1 protein concentration of 0.58 +/- 1.04 pmol/mg was only 4% of the derived CYP1A2. Since CYP1A1 is intrinsically far more active than CYP1A2 in mediating EROD, contribution of CYP1A1 to EROD represented approximately 25-40% of CYP1A2 contribution. Three of the 42 livers exhibited no CYP1A1-mediated EROD. Approximately 8% of the individuals showed high CYP1A1 activity phenotype based on cumulative distribution curve analysis. Hepatic CYP1A1 activity was more variable than that of CYP1A2. The variance of CYP1A1-mediated EROD was significantly different from that of CYP1A2, using the Kolgomorov-Smirnov statistical test. Even though not statistically significant, an age-related pattern in CYP1A1-mediated activity was identified: activity was high in the pre-puberty group, then decreased in the young/mature adult group and, finally, a slight increase was observed in old age., Conclusions: Distribution pattern in CYP1A1-mediated EROD suggests that the low derived CYP1A1 expression is most likely induced rather than constitutive. CYP1A1 activity deviates from log-normal distribution; the variations in hepatic CYP1A1 activity may affect the conversion of procarcinogens to carcinogens. The age-related trend in CYP1A1-mediated EROD activity hints that CYP1A1 responsiveness to inducers may change with age as well as with exposure to environmental inducers. These findings prompt (1) future genotyping studies to determine whether increased CYP1A1 inducibility is a result of genetic factors and (2) studies to address whether CYP1A1 inducibility changes with age.