Search

Rivaroxaban has been investigated in the EINSTEIN‐Jr program for the treatment of acute venous thromboembolism (VTE) in children aged 0 to 18 years and in the UNIVERSE program for thromboprophylaxis in children aged 2 to 8 years with congenital heart disease after Fontan‐procedure. Physiologically‐based pharmacokinetic (PBPK) and population pharmacokinetic (PopPK) modeling were used throughout the pediatric development of rivaroxaban according to the learn‐and‐confirm paradigm. The development strategy was to match pediatric drug exposures to adult exposure proven to be safe and efficacious. In this analysis, a refined pediatric PopPK model for rivaroxaban based on integrated EINSTEIN‐Jr data and interim PK data from part A of the UNIVERSE phase III study was developed and the influence of potential covariates and intrinsic factors on rivaroxaban exposure was assessed. The model adequately described the observed pediatric PK data. PK parameters and exposure metrics estimated by the PopPK model were compared to the predictions from a previously published pediatric PBPK model for rivaroxaban. Ninety‐one percent of the individual post hoc clearance estimates were found within the 5th to 95th percentile of the PBPK model predictions. In patients below 2 years of age, however, clearance was underpredicted by the PBPK model. The iterative and integrative use of PBPK and PopPK modeling and simulation played a major role in the establishment of the bodyweight‐adjusted rivaroxaban dosing regimen that was ultimately confirmed to be a safe and efficacious dosing regimen for children aged 0 to 18 years with acute VTE in the EINSTEIN‐Jr phase III study.

The combination of polymyxins, meropenem, and sulbactam demonstrated efficacy against multi-drug-resistant bacillus Acinetobacter baumannii. These three antibiotics are commonly used against major blood, skin, lung, and heart muscle infections.The objective of this study was to predict drug disposition and extrapolate the efficacy in these tissues using a physiologically based pharmacokinetic modeling approach that linked drug exposures to their target pharmacodynamic indices associated with antimicrobial activities against A. baumannii.An adult physiologically based pharmacokinetic model was developed for meropenem, colistin, and sulbactam and scaled to pediatrics accounting for both renal and non-renal clearances. The model reliability was evaluated by comparing simulated plasma and tissue drug exposures to observed data. Target pharmacodynamic indices were used to evaluate whether pediatric and adult dosing regimens provided sufficient coverage.The modeled plasma drug exposures in adults and pediatric patients were consistent with reported literature data. The mean fold errors for meropenem, colistin, and sulbactam were in the range of 0.710-1.37, 0.981-1.47, and 0.647-1.39, respectively. Simulated exposures in the blood, skin, lung, and heart were consistent with reported penetration rates. In a virtual pediatric population aged from 2 tolt; 18 years, the interpretive breakpoints were achieved in 85-90% of subjects for their targeted pharmacodynamic indices after administration of pediatric dosing regimens consisting of 30 mg/kg of meropenem, and 40 mg/kg of sulbactam three times daily as a 3-h or continuous infusion and 5 mg/kg/day of colistin base activity.The physiologically based pharmacokinetic modeling supports pediatric dosing regimens of meropenem/colistin/sulbactam in a co-administration setting against infections in the blood, lung, skin, and heart tissues due to A. baumannii.

Aims The objective of the current study was to evaluate paediatric dosing regimens for meropenem plus fosfomycin that generate sufficient coverage against multidrug-resistant bacteria. Methods The physiologically based pharmacokinetic (PBPK) models of meropenem and fosfomycin were developed from previously published pharmacokinetic studies in five populations: healthy subjects of Japanese origin, and healthy adults, geriatric, paediatric and renally impaired of primarily Caucasian origins. Pharmacodynamic (PD) analyses were carried out by evaluating dosing regimens that achieved a ≥90% joint probability of target attainment (PTA), which was defined as the minimum of the marginal probabilities to achieve the target PD index of each antibiotic. For meropenem, the percentage of time over a 24-hour period wherein the free drug concentration was above the minimum inhibitory concentration (fT > MIC) of at least 40% was its PD target. The fosfomycin PD index was described by fAUC/MIC of at least 40.8. Results For coadministration consisting of 20 mg/kg meropenem q8h as a 3-hour infusion and 35 mg/kg fosfomycin q8h also as a 3-hour infusion in a virtual paediatric population between 1 month and 12 years of age with normal renal function and a corresponding body weight between 3 and 50 kg, a joint PTA ≥ 90% is achieved at MICs of 16 and 64 mg/L for meropenem and fosfomycin coadministration, respectively, against Klebsiella pneumoniae and Pseudomonas aeruginosa. Conclusion The current study identified potentially effective paediatric dosing regimens for meropenem plus fosfomycin coadministration against multidrug-resistant bacteria.

This study applied territorial spatial planning control to a land use multi-scenario simulation in Changde, China, and measured the landscape ecological risk response. It embedded five planning control schemes, respectively, involving inertial development, urban expansion size quantity control, ecological spatial structure control, land use zoning control, and comprehensive control. Findings show that: (1) Woodland and arable land in Changde occupy 31.10% and 43.35% of land use, respectively, and constitute the main functional space of the research area. The scale of construction land in Changde has enlarged continuously, with ecological space represented by woodland and water constantly squeezed and occupied. (2) Comprehensive control has the most remarkable restraining effect on the disordered spread of construction land, while ecological space structure control is the most effective way to control ecological land shrinkage. (3) The overall landscape ecological risk index expanded over 2009–2018, presenting an S-type time evolution curve of “sharp increase–mitigation”. Landscape ecological risk presents a single-core, double-layer circle structure with the north and east regions as the core, attenuating to the periphery. (4) Landscape ecological risk under land use zoning control increased significantly more than in other scenarios. Comprehensive control best prevented landscape ecological risk and restrained the disorderly expansion of construction land.

Thrombosis remains an important complication for children with single ventricle physiology post-Fontan procedure and effective thromboprophylaxis is an important unmet medical need. To obviate conventional dose-finding studies and expedite clinical development, a rivaroxaban dose regimen for this indication was determined utilizing a model-informed drug development approach. A physiologically based pharmacokinetic (PBPK) rivaroxaban model was used to predict a pediatric dosing regimen that would produce drug exposures similar to that of 10 mg once daily in adults. This regimen was used in an open-label, multicenter Phase 3 study, which investigated the use of rivaroxaban for thromboprophylaxis in post-Fontan patients 2 to 8 years of age. The pharmacokinetics (PK) of rivaroxaban was assessed in Part A (n = 12) and in Part B (n = 64) of UNIVERSE. The safety and efficacy in the rivaroxaban group were compared to those in the acetylsalicylic acid group for 12 months. Pharmacodynamic endpoints were assessed in both parts of the study. Rivaroxaban exposures achieved in Part A and B were similar to the adult reference exposures. Prothrombin time also showed similarity to the adult reference. Exposure-response analysis did not identify a quantitative relationship between rivaroxaban exposures and efficacy/safety outcomes within the observed exposure ranges. A body-weight based dose regimen selected by PBPK modeling was shown in the UNIVERSE study to be appropriate for thromboprophylaxis in the post-Fontan pediatric population. Model-based dose selection can support pediatric drug development and bridge adult dose data to pediatrics, thereby obviating the need for dose-finding studies in pediatric programs. This article is protected by copyright. All rights reserved.

The emergence of polymyxin resistance in Gram-negative bacteria infections has motivated the use of combination therapy. This study determined the mutant selection window (MSW) of polymyxin B alone and in combination with meropenem and fosfomycin against A. baumannii strains belonging to clonal lineages I and III. To evaluate the inhibition of in vitro drug resistance, we investigate the MSW-derived pharmacodynamic indices associated with resistance to polymyxin B administrated regimens as monotherapy and combination therapy, such as the percentage of each dosage interval that free plasma concentration was within the MSW (%TMSW) and the percentage of each dosage interval that free plasma concentration exceeded the mutant prevention concentration (%T>MPC). The MSW of polymyxin B varied between 1 and 16 µg/mL for polymyxin B-susceptible strains. The triple combination of polymyxin B with meropenem and fosfomycin inhibited the polymyxin B-resistant subpopulation in meropenem-resistant isolates and polymyxin B plus meropenem as a double combination sufficiently inhibited meropenem-intermediate, and susceptible strains. T>MPC 90% was reached for polymyxin B in these combinations, while %TMSW was 0 against all strains. TMSW for meropenem and fosfomycin were also reduced. Effective antimicrobial combinations significantly reduced MSW. The MSW-derived pharmacodynamic indices can be used for the selection of effective combination regimen to combat the polymyxin B-resistant strain.

ObjectivesCeftazidime/avibactam is not active against MBL-producing bacteria. Combining ceftazidime/avibactam or avibactam with aztreonam can counter the resistance of MBL-producing Enterobacterales. The aim of this study was to evaluate whether the addition of avibactam could reduce or close the mutant selection window (MSW) of aztreonam in Escherichia coli and Klebsiella pneumoniae harbouring MBLs; MSW is a pharmacodynamic (PD) parameter for the selection of emergent resistant mutants.MethodsIn vitro susceptibility of 19 clinical isolates to ceftazidime/avibactam, aztreonam alone, and in co-administration (aztreonam/ceftazidime/avibactam and aztreonam/avibactam) was determined, as well as the mutant prevention concentration (MPC). The fraction of time within 24 h that the free drug concentration was within the MSW (fTMSW) and the fraction of time that the free drug concentration was above the MPC (fT>MPC) in both plasma and epithelial lining fluid (ELF) were determined from simulations of 10 000 profiles. The joint PTA was used to derive a joint cumulative fraction of response (CFR).ResultsAll isolates were resistant to ceftazidime/avibactam or aztreonam. Combining aztreonam and avibactam or ceftazidime/avibactam resulted in synergistic bactericidal activities against all isolates. Synergism was primarily due to the aztreonam/avibactam combination. For aztreonam/avibactam dosing regimens evaluated in clinical trials, fT>MPC values were >90% and >80%, whereas fTMSW measures were ConclusionsEffective antimicrobial combination optimized the PD parameters measuring selection for emergent mutants by increasing fT>MPC and reducing fTMSW.

The concentration-QTc (C-QTc) analysis is often applied in the first-in-human (FIH) study to demonstrate the absence of a QTc effect in support of a TQT waiver. However, a C-QTc analysis without properly designed sensitivity could fail to conclude the absence of a QTc effect at high concentrations, even though the compound is QTc negative. This is because the 90% confidence interval (CI) of the model-derived ∆∆QTc grows wider with increasing concentration, and the upper-bound could cross the 10-ms threshold, even though the slope is close to 0. So far, there is no simple math formula to calculate the sensitivity/specificity of a C-QTc analysis. A PK/QTc trial simulation scheme was applied to optimize the design features of a C-QTc trial in FIH studies by evaluating the study's sensitivity over a wide concentration range, circumventing the problem of not knowing the target concentration during FIH studies. It was also used to ensure that the specificity of the trial was well-controlled. Simulation showed that the study sensitivity can be quantitatively gauged by optimizing the dose range, the number of samples per subjects or subject number, and by sampling around Tmax, and at steady-state. The specificity of the trial can also be evaluated with this approach, and it is important to combine model-derived ∆∆QTc and slope estimate in the evaluation. The trial simulation approach helps maximize the probability of success of C-QTc analyses in FIH studies intended to support a TQT waiver.

ObjectivesThe aim of this report is to demonstrate pharmacokinetic (PK) and pharmacodynamic (PD) equivalence as well as similar efficacy, safety and immunogenicity between GP2013, a biosimilar rituximab, and innovator rituximab (RTX) in patients with rheumatoid arthritis (RA) with inadequate response or intolerance to tumour necrosis factor inhibitor (TNFi) treatment.MethodsIn this multinational, randomised, double-blind, parallel-group study, 312 patients with active disease despite prior TNFi therapy were randomised to receive GP2013 or either the EU (RTX-EU) or the US (RTX-US) reference product, along with methotrexate (MTX) and folic acid. The primary endpoint was the area under the serum concentration–time curve from study drug infusion to infinity (AUC0-inf). Additional PK and PD parameters, along with efficacy, immunogenicity and safety outcomes were also assessed up to week 24.ResultsThe 90% CI of the geometric mean ratio of the AUCs were within the bioequivalence limits of 80% to 125% for all three comparisons; GP2013 versus RTX-EU: 1.106 (90% CI 1.010 to 1.210); GP2013 versus RTX-US: 1.012 (90% CI 0.925 to 1.108); and RTX-EU versus RTX-US: 1.093 (90% CI 0.989 to 1.208). Three-way PD equivalence of B cell depletion was also demonstrated. Efficacy, safety and immunogenicity profiles were similar between GP2013 and RTX.ConclusionsThree-way PK/PD equivalence of GP2013, RTX-EU and RTX-US was demonstrated. Efficacy, safety and immunogenicity profiles were similar between GP2013 and RTX.Trial registration numberNCT01274182; Results.

Pharmacodynamics (PD) similarity is an important component to support the claim of similarity between two drugs or devices. This article investigates the trial design and statistical considerations in the equivalence test of PD endpoints. Using bone resorption marker CTX as a case study, the relationship between the PD readouts and drug potency was explored to evaluate the sensitivity of the PD endpoint and guide equivalence margin selection. For PD data that have high baseline variability, one conventional similarity assessment method was to apply baseline-normalization followed by the standard bioequivalence (BE) test (Lancet Haematol. 4:e350-61, 2017, Ann Rheum Dis. 2017). This study showcased the drawbacks of the conventional method for PD data that were close to inhibition saturation, as the baseline-normalization significantly skewed the distribution of the PD data toward non-log-normal. In such cases, the standard BE test can produce an inflated type I error. Alternatively, ANCOVA, when applied to the un-normalized PD data with the baseline as a covariate, produced a satisfactory type I error with sufficient power. Therefore, ANCOVA was recommended for equivalence test of PD markers that has a saturated inhibition profile and high variability at baseline. Moreover, the relationship between PD readouts and drug potency was used to explore the sensitivity of the PD endpoint and it could help justify the equivalence margins, since the standard 80% to 125% BE margin often does not apply to PD. Finally, a decision tree was proposed to help guide the design of the PD equivalence study in the choice of PD endpoints and statistical methods.

In order to facilitate inspection and maintenance for municipal public pipelines located below urban roads, more and more manholes were set up on the road surfaces. However, due to various inadequate design and construction etc., the manhole suffered most serious diseases such as collapse of wellhead, broken up of pavement around well, subsidence of manhole and damage of internal well structure. Which not only harms its life span, but also affects the driving safety and the city appearance. Firstly, this work investigated the manhole diseases of typical roads in cities of Changsha etc. and concluded that there are only two main types of road manhole diseases. The most prominent one is the pavement damages. The second one is the wellhead subsidence greater than 20mm. Secondly, the main causes of manhole disease was found out from the aspects of both design and construction. In design, the lack of details of wells and its surrounding backfill materials are main causes. In terms of construction, there are many problems such as insufficient attention to quality control, inadequate operation of key processes and lower level of construction refinement. Finally, systematic and comprehensive measures to deal with various kinds of diseases of manhole in not only design and construction but also prevention and restoration were presented.

This paper reported six rhodamine derived chemosensors for Hg(II) sensing, hoping to improve sensitivity of environmental Hg(II) monitoring. Up-conversion β-NaYF4:Yb3+/Er3+ nanocrystals were synthesized and used as an excitation host so that these chemosensors could be excited by near-IR laser radiation (980 nm). Emission of this excitation host overlapped well with absorption of these chemosensors, resulting in an efficient energy transfer which was confirmed by emission decay lifetime analysis. Sensing mechanism of these chemosensors was found as a complexing one with binding stoichiometry of 1:1. Host:chemosensor systems in buffer solution showed emission “turn-on” effect toward Hg(II) with good selectivity. Linear response was obtained. The correlation between sensing performance and chemosensor structure was discussed as well. It was found that S substituent in these chemosensors increased sensitivity and selectivity, but decreased photostability.

This article provides an overview of four case studies to demonstrate the utility of pharmacometric analysis in biosimilar development to help design sensitive clinical pharmacology studies for the demonstration of biosimilarity. The two major factors that determine the sensitivity of a clinical pharmacokinetic/pharmacodynamic (PK/PD) study to demonstrate biosimilarity are the size of the potential difference to be detected (signal) and the inter-subject variability (noise), both of which can be characterized and predicted using pharmacometric approaches. To maximize the chance to detect any potential difference between the proposed biosimilar and the reference drug, the dose selected for the clinical pharmacology study should fall on the steep part of the dose-response curve. Pharmacometric analysis can be used to characterize the dose-response relationship using PD- or PK/PD-linked models. The understanding of the PD endpoints in terms of dynamic range of the response and the location of the studied dose on the dose-response curve can provide strategic advantage in the trial design. To reduce the inter-subject variability (noise), pharmacometric analysis can help avoid high variability associated with low doses, and decrease variability by controlling certain covariates in the inclusion/exclusion criteria. Pharmacometric analysis also can help select or justify margins for the equivalence test of PD endpoints. Pharmacometric analysis will assume an ever-increasing role in the clinical development of biosimilar drugs, as it helps to ensure that sufficient sensitivity is built into the study design to detect potential PK and PD differences.

Detection and identification (ID) of all drug metabolites following liquid chromatography (LC)/mass spectrometry (MS) analysis of complex biological matrixes are not trivial. To facilitate detection of drug-derived materials that possess highly diagnostic isotopic patterns (e.g., chlorine- and bromine-containing compounds), we report an accurate-mass-based spectral-averaging isotope-pattern-filtering (AMSA-IPF) algorithm developed in the computational language R. The AMSA-IPF algorithm offers three significant improvements over the traditional isotope filtering method often provided by instrument vendors. First, spectral averaging is performed before the IPF to reduce scan-to-scan variability of ion intensities. Second, the IPF process is strictly based on accurate mass typically obtained on high resolution mass spectrometers. The designated isotopic ion-pairs (e.g., M + 2:M or M + 1:M, where M is the molecular ion and M + 1 and M + 2 are the isotopic ions) must fall into the predefined accurate mass tolerance window (e.g., 5 ppm) and at the same time satisfy the predefined relative abundance criteria. Third, both M + 1:M and M + 2:M ion pairs are inspected in the filtering process. The inclusion of M + 1:M ion pair enhanced the specificity of this algorithm by removing background ions that form M:M + 2 ion pairs within predefined isotope ratios by coincidence. The algorithm demonstrated excellent effectiveness in detecting drug-related ions in in vivo samples (plasma, bile, urine and feces) obtained from rats orally dosed with 14C-loratadine. The ion chromatograms of the filtered LC-MS data files showed near perfect qualitative correlation with the corresponding radioprofiles. AMSA-IPF will be another great tool to facilitate detection and ID of drug metabolites in complex LC-MS data without the help of radiolabels. The AMSA-IPF algorithm is applicable to not only compounds containing distinct natural isotopes (such as Cl and Br) but also compounds that contain synthetically incorporated isotopes (13C, 15N, etc) generating a distinct isotope pattern. The ability to detect and identify metabolites from nonradiolabeled studies will be extremely beneficial to achieve compliance with FDA's most recent guidance on metabolites in safety testing (MIST).

Objective— The purpose of this study was to determine the effect of 15-lipoxygenase-1 (15-LO-1) on cholesterol mobilization from macrophages. Methods and Results— Overexpression of human 15-LO-1 in RAW mouse macrophages led to enhanced cholesterol efflux, increased cholesteryl ester (CE) hydrolysis, and increased reverse cholesterol transport (RCT). Efflux studies comparing 15-LO-1 overexpressing cells to mock-transfected RAW macrophages resulted in a 3- to 7-fold increase in cholesterol efflux to apolipoprotein A-I and a modest increase in efflux to HDL. Additional experiments revealed an increase in mRNA and protein levels of ABCA1 and ABCG1 in the RAW expressing 15-LO-1 compared to controls. Efforts to examine whether the arachidonic acid metabolite of 15-LO-1, (15 S )-hydroxyeicosatetraenoic acid (HETE), was responsible for the enhanced efflux revealed this eicosanoid metabolite did not play a role. Enhanced steryl ester hydrolysis was observed in 15-LO-1 overexpressing cells suggesting that the CE produced in the 15-LO-1 expressing cells was readily mobilized. To measure RCT, RAW macrophages overexpressing 15-LO-1 or mock-transfected cells were cholesterol enriched by exposure to acetylated low-density lipoprotein and [ 3 H]-cholesterol. These macrophages were injected into wild-type animals and RCT was measured as a percent of injected dose of 3 H appearing in the feces at 48 hours. We found 7% of the injected 3 H in the feces of mice that received macrophages overexpressing 15-LO-1 and 4% in the feces of mice that received mock-transfected cells. Conclusions— These data are consistent with a model in which overexpression of human 15-LO-1 in RAW macrophages promotes RCT through increased CE hydrolysis and ABCA1-mediated cholesterol efflux.

A retention-time-shift-tolerant background subtraction and noise reduction algorithm (BgS-NoRA) is implemented using the statistical programming language R to remove non-drug-related ion signals from accurate mass liquid chromatography/mass spectrometry (LC/MS) data. The background-subtraction part of the algorithm is similar to a previously published procedure (Zhang H and Yang Y. J. Mass Spectrom. 2008, 43: 1181-1190). The noise reduction algorithm (NoRA) is an add-on feature to help further clean up the residual matrix ion noises after background subtraction. It functions by removing ion signals that are not consistent across many adjacent scans. The effectiveness of BgS-NoRA was examined in biological matrices by spiking blank plasma extract, bile and urine with diclofenac and ibuprofen that have been pre-metabolized by microsomal incubation. Efficient removal of background ions permitted the detection of drug-related ions in in vivo samples (plasma, bile, urine and feces) obtained from rats orally dosed with (14)C-loratadine with minimal interference. Results from these experiments demonstrate that BgS-NoRA is more effective in removing analyte-unrelated ions than background subtraction alone. NoRA is shown to be particularly effective in the early retention region for urine samples and middle retention region for bile samples, where the matrix ion signals still dominate the total ion chromatograms (TICs) after background subtraction. In most cases, the TICs after BgS-NoRA are in excellent qualitative correlation to the radiochromatograms. BgS-NoRA will be a very useful tool in metabolite detection and identification work, especially in first-in-human (FIH) studies and multiple dose toxicology studies where non-radio-labeled drugs are administered. Data from these types of studies are critical to meet the latest FDA guidance on Metabolite in Safety Testing (MIST).

Five major products (adducts A(1a), A(1b), A(2), A(3,) and B) from the reaction of guanosine (Guo) with 4-oxo-2(E)-nonenal (ONE) were detected by liquid chromatography-mass spectrometry (LC-MS). Tandem MS (MS/MS) analysis of these compounds suggested that modifications to the nucleoside had been introduced. Adducts A(1a), A(1b), A(2), and A(3) were heptanone-ethano-2'-Guo adducts that all decomposed to adduct B. Adducts A(1a) and A(1b) were isomeric hemi-ketal forms. Adducts A(2) and A(3) were diastereomers of the open chain ketone form. The structure of adduct B was shown by LC-MS/MS and NMR spectroscopy to be the heptanone-etheno-Guo (HepsilonGuo) adduct, 3-(D-erythropentafuranosyl)imidazo-7-(heptane-2' '-one)-9-hydroxyl[1,2-alpha]purine. The overall reaction of Guo with ONE was very similar to its reaction with 2'-deoxyguanosine. Reaction of ONE with yeast transfer RNA also resulted in the formation of HepsilonGuo. Finally, HepsilonGuo was detected and quantified in the RNA from rat intestinal epithelial cells that stably express cyclooxygenase-2. These data show that RNA is modified by the same bifunctional reactive electrophiles derived from lipid peroxidation that covalently modify DNA.

Background: GP2013 is being developed as a proposed biosimilar to EU approved originator Rituximab (RTX). The stepwise global development program for GP2013 is comprised of structural and functional characterization with highly sensitive analytical methods to ensure comparability to the originator at the structural and physicochemical level, followed by confirmatory nonclinical testing and clinical studies to prove similarity between these two compounds Methods: This is a prospective, multi-center, randomized, double blind, active-controlled, parallel group, confirmatory, Phase III trial comparing the efficacy, safety, pharmacokinetics (PK) and pharmacodynamics (PD) of GP2013 plus cyclophosphamide, vincristine, prednisone (GP2013-CVP) vs. RTX-CVP in 629 patients (159 centers; 26 countries) with previously untreated, advanced stage follicular lymphoma (FL). The study comprised of combination treatment phase (6 months), maintenance phase (2 years) and follow up until 3 years after randomization; combination phase is completed and maintenance phase is ongoing. Patients were randomized 1:1 to receive 8 cycles of either GP2013-CVP (n=314) or RTX-CVP (n=315), followed by a maintenance phase (double-blind treatment with GP2013 or RTX) in responders [(complete response (CR) & partial response (PR)]. Patients were stratified by Follicular Lymphoma International Prognostic Index (FLIPI) score risk group (low/intermediate risk [score 0 to 2] vs. high risk [score 3 to 5]) and geographic region. Percentage of patients with low risk , intermediate risk and high risk for GP2013-CVP vs RTX-CVP arms were 9.65% vs 11.1%, 34.0% vs 32.7% and 56.4% vs 56.2%, respectively. Comparison of overall response rate (ORR) between the two arms, based on blinded central review was the primary endpoint. Secondary endpoints included other efficacy parameters: CR, PR, progression-free survival (PFS) and overall survival (OS); safety; immunogenicity; PK and PD. Results: Baseline characteristics were well balanced between arms. The study met its primary objective showing equivalence of ORR (GP2013-CVP 87.1%, RTX-CVP 87.5%; difference GP2013-CVP - RTX-CVP was -0.40%, 95% confidence interval (CI)[-5.94%, 5.14%]). In combination phase, CR and PR rates were 14.8%, 72.3% and 13.4%, 74.1% for GP2013-CVP and RTX-CVP respectively. The median PFS and OS have not yet been reached, as data is still maturing. PK and PD of GP2013 were similar to RTX. The ratio of geometric mean for Cmax at cycle 4 day 1 was 1.00(90% CI [0.925; 1.09]). Similarity was also supported by the comparable results for AUC(0-21d), AUCall and Ctrough between GP2013 and RTX. Peripheral CD19+ B-cell counts were assessed as PD outcome; the ratio of geometric mean of area under effect-time curve (AUEC(0-21d)) was 0.939(90% CI [0.845; 1.04]), confirming the similarity between both products. Safety profile was similar in both arms with no new safety signals detected. In combination phase; adverse events (AE) were reported in 92.0% patients and incidence of serious AEs was similar between GP2013-CVP (22.8%) and RTX-CVP (20.0%) arms. The most common serious AE was febrile neutropenia (4.8% and 2.9% in GP2013-CVP and RTX-CVP arms respectively) (Table 1). A total of 11(1.8%) patients died during the combination phase, 4(1.3%) in GP2013-CVP and 7(2.2%) in RTX-CVP arm. Until data cut-off (10 Jul 2015), during the entire study, 35 patients died (18 in GP2013-CVP, 17 in the RTX-CVP arms); most common cause was Non-Hodgkin's lymphoma 8(2.6%) in GP2013-CVP and 6(1.9%) in RTX-CVP arms. Overall, 5(1.9%) patients in GP2013-CVP and 3(1.1%) in the RTX-CVP arm developed anti-drug antibodies. Conclusions: The proposed biosimilar GP2013 met the primary endpoint demonstrating equivalence in ORR to EU approved originator RTX. Similarity was demonstrated for efficacy, PK and PD parameters. Both arms demonstrated similar safety findings in patients with previously untreated advanced stage FL. Disclosures Pultar: Novartis Pharmaceuticals Corporation: Employment. Cherfi:Novartis Pharma AG: Employment. Zhu:Sandoz Inc (Parent organization: Novartis): Employment. Amersdorffer:HEXAL AG (Parent organization- Novartis): Employment.

10-Hydroxycamptothecin (HCPT) loaded PLA microbubbles, used as an ultrasound-triggered drug delivery system, were fabricated by a double emulsion-solvent evaporation method. The obtained microbubbles were characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and confocal laser scanning microscope (CLSM). In addition, the effect of diagnostic ultrasound exposure on BEL-7402 cells combined with HCPT-loaded PLA microbubbles was evaluated using cytotoxicity assay, CLSM and flow cytometry (FCM). It was found that the HCPT-loaded PLA microbubbles showed smooth surface and spherical shape, and the drug was amorphously dispersed within the shell and the drug loading content reached up to 1.69%. Nearly 20% of HCPT was released upon exposure to diagnostic ultrasound at frequency of 3.5MHz for 10min. Moreover, HCPT fluorescence in the cells treated only with the HCPT-loaded PLA microbubbles was discernible, but less intense, while those treated with the microbubbles in conjunction with ultrasound exposure was evident and intense, indicating an increased cellular uptake of HCPT by ultrasound exposure. Cytotoxicity test on BEL-7402 cells indicated that the HCPT-loaded PLA microbubbles combined with ultrasound exposure were more cytotoxic than the microbubbles alone. The results suggest that the combination of drug loaded PLA microbubbles and diagnostic ultrasound exposure exhibit an effective intracellular drug uptake by tumor cells, indicating their great potential for antitumor therapy.

Interleukin-12 (IL-12) is critical for resistance toToxoplasma gondiiduring both the acute and chronic stages of infection. However, the cellular and molecular pathways that regulate IL-12 production during chronic toxoplasmosis are incompletely defined. We recently discovered that 12/15-lipoxygenase (12/15-LOX), which oxidizes unsaturated lipids in macrophages, is a novel and selective regulator of IL-12 production. We now demonstrate the essential role of this enzyme in the chronic phase of toxoplasmosis. Although 12/15-LOX-deficient mice were resistant to acuteT. gondiiinfection, 80% of 12/15-LOX-deficient mice died during chronic toxoplasmosis, compared to no deaths in wild-type controls. The morbidity of chronically infected 12/15-LOX mice was associated with an increase in brain inflammation and parasite burden. These data suggest that the evolution of the immune response toT. gondiiis accompanied by an increasing requirement for 12/15-LOX-mediated signaling. Consistent with this conclusion, 12/15-LOX activity was enhanced during chronic, but not acute, toxoplasmosis. Furthermore, the enhanced susceptibility of 12/15-LOX-deficient mice to chronic toxoplasmosis was associated with reduced production of IL-12 and gamma interferon (IFN-γ) that was not evident during acute infection. Importantly, ex vivo IFN-γ production by 12/15-LOX-deficient splenocytes could be rescued by the addition of recombinant IL-12. These data establish that 12/15-LOX is a critical mediator of the chronic type 1 inflammatory response and that immune mediators can be subject to distinct cellular and/or molecular mechanisms of regulation at different stages of inflammation.

The rapid economic growth stimulates a surprising and uncontrolled urbanization wave in China, and necessarily brings about, in turn, poor environment quality and low life amenity. By what means the situation can be substantially transformed into a healthy track has been a puzzled and considerably difficult work challenging all Chinese. The article attempts to explore the conceptual model for establishing an ecological defense system in Yuelu District of Changsha, Hunan province, China, a rapid urbanized area, primarily focusing on its spatial pattern and adaptable scheme in order to control its continuing sprawl and improve local environment. For achieving the aim, authors first define the concept and its key qualities, then analyze and compare differing schemes with the principal purposes of defending urbanization and resolving prevailing urban ills, in terms of their principal goals, elements and spatial patterns. Finally, three elements are indentified, that is, the eco- base, eco-fortress and eco-corridor, and the skeleton and final scheme are set out, comprising 3 zones - enhancement zones, buffer zones and core conservation zones - and designing a web- like spatial pattern composing of urban green spaces, greenways, waterways, wetlands, forested hills and mountains, farmlands and villages. The system is designed as an eco-centric, forest- oriented, sustainable network of life support system. In order to accomplish its anticipated functions, it must make every efforts to obtain supports from legislation, administration, residents, enterprises and other institutions, to launch large-scale mass propagations and specific training, and ultimately to compete a high level priority over so called "gray" planning processes, at lest concurrently.

The formation of 15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE) as a product from rabbit lung 15-hydroxyprostaglandin dehydrogenase (PGDH)-mediated oxidation of 15(S)-hydroperoxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid was first reported more than 30 years ago. However, the pharmacological significance of 15-oxo-ETE formation has never been established. We have now evaluated 15-lipoxygenase (LO)-1-mediated arachidonic acid (AA) metabolism to 15-oxo-ETE in human monocytes and mouse RAW macrophages that stably express human 15-LO-1 (R15L cells). A targeted lipidomics approach was used to identify and quantify the oxidized lipids that were formed. 15-oxo-ETE was found to be a major AA-derived LO metabolite when AA was given exogenously or released from endogenous esterified lipid stores by calcium ionophore (CI) calcimycin (A-23187). This established the R15L cells as a useful in vitro model system. Pretreatment of the R15L cells with cinnamyl-3,4-dihydroxycyanocinnamate significantly inhibited AA- or CI-mediated production of 15(S)-hydroperoxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid [15(S)-HETE] and 15-oxo-ETE, confirming the role of 15-LO-1 in mediating AA metabolite formation. Furthermore, 15(S)-HETE was metabolized primarily to 15-oxo-ETE. Pretreatment of the R15L cells with the 15-hydroxyprostaglandin dehydrogenase (PGDH) inhibitor 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy-benzeneacetic acid (CAY10397) reduced AA- and 15(S)-HETE-mediated formation of 15-oxo-ETE in a dose-dependent manner. This confirmed that macrophage-derived 15-PGDH was responsible for catalyzing the conversion of 15(S)-HETE to 15-oxo-ETE. Finally, 15-oxo-ETE was shown to inhibit the proliferation of human vascular vein endothelial cells by suppressing DNA synthesis, implicating a potential antiangiogenic role. This is the first report describing the biosynthesis of 15-oxo-ETE by macrophage/monocytes and its ability to inhibit endothelial cell proliferation.

A retention-time-shift-tolerant background subtraction and noise reduction algorithm (BgS-NoRA) is implemented using the statistical programming language R to remove non-drug-related ion signals from accurate mass liquid chromatography/mass spectrometry (LC/MS) data. The background-subtraction part of the algorithm is similar to a previously published procedure (Zhang H and Yang Y. J. Mass Spectrom. 2008, 43: 1181-1190). The noise reduction algorithm (NoRA) is an add-on feature to help further clean up the residual matrix ion noises after background subtraction. It functions by removing ion signals that are not consistent across many adjacent scans. The effectiveness of BgS-NoRA was examined in biological matrices by spiking blank plasma extract, bile and urine with diclofenac and ibuprofen that have been pre-metabolized by microsomal incubation. Efficient removal of background ions permitted the detection of drug-related ions in in vivo samples (plasma, bile, urine and feces) obtained from rats orally dosed with (14)C-loratadine with minimal interference. Results from these experiments demonstrate that BgS-NoRA is more effective in removing analyte-unrelated ions than background subtraction alone. NoRA is shown to be particularly effective in the early retention region for urine samples and middle retention region for bile samples, where the matrix ion signals still dominate the total ion chromatograms (TICs) after background subtraction. In most cases, the TICs after BgS-NoRA are in excellent qualitative correlation to the radiochromatograms. BgS-NoRA will be a very useful tool in metabolite detection and identification work, especially in first-in-human (FIH) studies and multiple dose toxicology studies where non-radio-labeled drugs are administered. Data from these types of studies are critical to meet the latest FDA guidance on Metabolite in Safety Testing (MIST).

In this paper, ultrasound contrast agents based on a high molecular polymer-poly lactic acid (PLA) and loaded with hydroxycamptothecin (HCPT) were prepared by combining ultrasound method and a Shirasu porous glass (SPG) membrane emulsification technique. A special focus was on the optimization of production of HCPT-PLA microbubbles. Different factors, such as the power and the time of ultrasonic action, the ratio of inner aqueous phase against outer oil phase, and the concentration of PLA were evaluated, and the average size of HCPT-PLA microbubbles, the drug carrying efficiency, as well as the acoustically- triggered drug release at 3 kHz ultrasound were determined. The study showed that the HCPT-PLA microbubbles prepared using our optimized conditions, were sphere-like in shape with a mean diameter of 1-7 mum. The drug loading efficiency reached up to 56.48% In vitro, the drug release of HCPT-PLA microbubbles increased significantly at 3 kHz ultrasound for 30 s compared with that of ultrasound-free condition. In conclusion, the HCPT-PLA microbubbles has the characteristics desirable for an intravenously administered ultrasound contrast agent for further clinical use.

Oxidation of glutathione (GSH) to glutathione disulfide (GSSG) occurs during cellular oxidative stress. The redox potential of the 2GSH/GSSG couple, which is determined by the Nernst equation, provides a means to assess cellular redox status. It is difficult to accurately quantify GSH and GSSG due to the ease with which GSH is oxidized to GSSG during sample preparation. To overcome this problem, a stable isotope dilution liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) method has been developed using 4-fluoro-7-sulfamoylbenzofurazan (ABD-F) derivatization. ABD-F derivatization of the GSH thiol group was rapid, quantitative, and occurred at room temperature. The LC/MRM-MS method, which requires no sample clean-up, was validated within the calibration ranges of 5 to 400 nmol/mL in cell lysates for GSH and 0.5 to 40 nmol/mL in cell lysates for GSSG. Calibration curves prepared by adding known concentrations of GSH and GSSG to cell lysates were parallel to the standard curve prepared in buffers. GSH and GSSG concentrations were determined in two monocyte/macrophage RAW 267.4 cell lines with or without 15-LOX-1 expression (R15LO and RMock cells, respectively) after treatment with the bifunctional electrophile 4-oxo-2(E)-nonenal (ONE). R15LO cells synthesized much higher concentrations of the lipid hydroperoxide, 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), which undergoes homolytic decomposition to ONE. GSH was depleted by ONE treatment in both RMock and R15LO cells, leading to significant increases in their redox potentials. However, R15LO cells had higher GSH concentrations (most likely through increased GSH biosynthesis) and had increased resistance to ONE-mediated GSH depletion than RMock cells. Consequently, R15LO cells had lower reduction potentials at all concentrations of ONE. GSSG concentrations were higher in R15LO cells after ONE treatment when compared with the ONE-treated RMock cells. This suggests that increased expression of 15(S)-HPETE modulates the activity of cellular GSH reductases or the transporters involved in removal of GSSG.

3514 Background: In contrast to BRAFV600 mutated (BRAFm) advanced melanoma, BRAFm colorectal carcinoma (CRC) does not respond to BRAF inhibitors due to strong feedback activation of the epidermal g...

Though Abl inhibitors are often successful therapies for the initial stages of chronic myelogenous leukemia (CML), refractory cases highlight the need for novel molecular insights. We demonstrate that mice deficient in the enzyme 12/15-lipoxygenase (12/15-LO) develop a myeloproliferative disorder (MPD) that progresses to transplantable leukemia. Although not associated with dysregulation of Abl, cells isolated from chronic stage 12/15-LO–deficient (Alox15) mice exhibit increased activation of the phosphatidylinositol 3–kinase (PI3-K) pathway, as indicated by enhanced phosphorylation of Akt. Furthermore, the transcription factor interferon consensus sequence binding protein (ICSBP) is hyperphosphorylated and displays decreased nuclear accumulation, translating into increased levels of expression of the oncoprotein Bcl-2. The ICSBP defect, exaggerated levels of Bcl-2, and prolonged leukemic cell survival associated with chronic stage Alox15 MPD are all reversible upon treatment with a PI3-K inhibitor. Remarkably, the evolution of Alox15 MPD to leukemia is associated with additional regulation of ICSBP on an RNA level, highlighting the potential usefulness of the Alox15 model for understanding the transition of CML to crisis. Finally, 12/15-LO expression suppresses the growth of a human CML–derived cell line. These data identify 12/15-LO as an important suppressor of MPD via its role as a critical upstream effector in the regulation of PI3-K–dependent ICSBP phosphorylation.