Elimination Rate Constant Research Articles

Pharmacokinetics of Two Forms of Recombinant Insulin-Like Growth Factor 1 in the Mouse Blood

The pharmacokinetics of two substances containing recombinant insulin-like growth factor 1 (IGF1) as an active substance was considered. The first substance (IGF1) contains the actual recombinant IGF1, and the second (IGF2) contains IGF1, which is translated from plasmid DNA encoding the IGF-1 gene for this protein. It was established that, with intramuscular administration of IGF1, the delay time for its entry into the bloodstream is 1.5–2 h, while that with IGF2 is 24–25 h. This indicates the presence of various mechanisms of accumulation of these substances in the systemic circulation. The maximum concentration of IGF1 in the blood was determined 5 h after administration and that of IGF2 was determined 125 h after administration. The maximum concentrations of these substances are comparable to each other. The concentration of IGF1 in the blood decreases to its initial value 12 h after its administration, and the concentration of IGF2 does so after 216 h. The clearance parameters (Cl) and elimination constants (Kel) of the considered substances also had significant differences, which confirms the presence of fast and slow dynamics of the decrease of their maximum concentrations after intramuscular injection. The different dynamics of the accumulation of substances in the blood and their elimination from the bloodstream after administration, as well as the different values ​​of the area parameters under the pharmacokinetic curve (AUCt, AUC∞) demonstrate that IGF2 has been in the systemic circulation for a longer time than IGF1. This is essential for the appearance and severity of pharmacodynamic effects.

The capability of Several Population-based Approach Software to Analyze Sparse Drug Plasma Concentration Data after Intra-Venous Bolus Injection

Monolix, NONMEM, and WinBUGS-PKBUGS are among available software package for population-based modeling. The sparse condition of drug plasma concentration versus time ( Cp-time ) data is prevalent in clinically based studies involving patients. It is not ethical in this case, to collect a many and large volumes of blood samples. This study was aimed to simulate the capability of Monolix, NONMEM, and WinBUGS-PKBUGS to analyze very sparse Cp-time data after an intravenous bolus drug administration and to estimate the minimum number of Cp-time data required for an adequate analysis. Data of Cp-time were obtained based on simulation using the pharmacokinetic one-compartment open model following an intravenous bolus administration of 50 mg of a hypothetical drug. In this respect, six random values of k (rate constant of elimination) and Vd (volume of distribution) with mean and standard deviation values of 0.3 ±0.1 per hour and 30 ± 10 L, respectively, were used to create simulated Cp-time data of 6 subjects. Simulated Cp-time data in each subject were randomly ranked to choose data based on the intended number of samples in each subject. Several sparse Cp-time data scenarios, starting from a very limited state, i.e., with a total of 6 Cp-time data (1 datum per subject) to a rich situation with 48 Cp data (8 data per subject), were examined. The goodness of fit evaluations, as well as the similarity of individual values of k and Vd to the respective real values (p>0.05), indicate that nonlinear-mixed-effect-model using Monolix, NONMEM and WinBUGS-PKBUGS can appropriately describe sparse Cp-time data even with only 2 data per subject. This fact is an important finding to support the demand of analytical tool for a limited number of Cp-time data such as obtained in therapeutic drug monitoring event.

Endocytosis mechanism in physiologically-based pharmacokinetic modeling of nanoparticles

BackgroundThe physiologically based pharmacokinetic (PBPK) model is a useful tool to predict the pharmacokinetics of various types of nanoparticles (NPs). The endocytosis mechanism plays a key role in pharmacokinetics of NPs. However, the effect of endocytosis mechanism both in the blood and tissue are seldom considered in PBPK model. ObjectivesTo investigate the biodistribution of intravenously injected pegylated AuNPs in mice and human using PBPK model considering the endocytosis mechanism both in the blood and tissue. MethodsTaking polyethylene glycol-coated gold nanoparticles (AuNPs) as an example, we developed a PBPK model to explore biodistribution of different size AuNPs. In the model, we considered the role of endocytosis mechanism both in the blood and tissue. In addition, the size-dependent permeability coefficient, excretion rate constant, phagocytic capacity, uptake rate, and release rate were derived from literatures. The mice PBPK model was extrapolated to the human by changing physiology parameters and the number of phagocytic cell (PCs). ResultsAuNPs were primarily distributed in the blood, liver, and spleen regardless of particle size, and almost all captured by the PCs in the liver and spleen, while few was captured in the blood. There are more organ distribution and longer circulation for smaller NPs. The 24-h accumulation of AuNPs decreased with increasing size in the most organ, while the accumulation of AuNPs showed an inverted U-shaped curve in the liver and slight U-shaped curve in the blood. The human results of model-predicted displayed a similar tendency with those in mice. Size, partition coefficients, and body weight were the key factors influencing the organ distribution of AuNPs. ConclusionsThe size played an important role on the distribution and accumulation of AuNPs in various tissues. Our PBPK model was well predicted the NPs distribution in mice and human. A better understanding of these mechanisms could provide effective guides for nanomedine delivery.

Development and validation of a liquid chromatography-tandem mass spectrometry method for determination of ibuprofen in human plasma

Ibuprofen is widely used to reduce fever, pain, and inflammation and in the treatment of patent ductus arteriosus (PDA) in preterm infants. However, to study the impact of pharmacokinetics of ibuprofen on the rate of ductus arteriosus closure in humans, a quantitative method is required. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a highly sensitive, accurate, reliable, fast, and simple bioanalytical method. In this study, we developed and utilized an LC-MS/MS method to determine ibuprofen bioavailability. One of the novel aspects of this work is the small specimen volume required for the analysis (i.e., 50 µL). The calibration curve was linear across a concentration range of 0.15–50 μg/mL. The coefficient of variation for intra-day and inter-day precision was within 0.78%–7.21% and accuracy was within 97.52%–107.21% of the nominal values for low, medium, and high quality control (QC) concentration levels—QCL, QCM, and QCH (0.45, 9.0, and 40.0 μg/mL, respectively). For ibuprofen concentrations at the lower limit of quantitation (LLOQ), intra-day and inter-day accuracy were 98.11% and 99.81%, respectively; however, the intra-day and inter-day precision were 1.89% and 5.37%, respectively. The pharmacokinetic study involved 18 neonatal subjects who were grouped into low- or high-dose ibuprofen groups. The median maximum concentration for low- and high-dose regimens were 16.05 (14.21–19.32) and 24.10 (20.63–32.03) µg/mL, respectively, and the median elimination rate constant was 0.041 (0.026–0.047) and 0.071 (0.050–0.073) h−1, respectively. Thus, the fully validated LC-MS/MS method was determined to be suitable for analyzing unknown plasma samples for ibuprofen pharmacokinetic, bioavailability, and bioequivalence studies.

1573. Population Pharmacokinetic Analyses for Cefepime in Adult and Pediatric Patients

BackgroundCefepime (CEF) is commonly used for adult and pediatric infections. Several studies have examined CEF’s pharmacokinetics (PK) in various populations; however, a unifying PK model for adult and pediatric subjects does not yet exist. We developed a combined population model for adult and pediatric patients and validated the model.MethodsThe initial model includes adult and pediatric patients with a rich cefepime sampling design. All adults received 2 g CEF while pediatric subjects received a mean of 49 (SD 5) mg/kg. One- and two-compartment models were considered as base models and were fit using a non-parametric adaptive grid algorithm within the Pmetrics package 1.5.2 (Los Angeles, CA) for R 3.5.1. Compartmental model selection was based on Akaike information criteria (AIC). Covariate relationships with PK parameters were visually inspected and mathematically assessed. Predictive performance was evaluated using bias and imprecision of the population and individual prediction models. External validation was conducted using a separate adult cohort.ResultsA total of 45 subjects (n = 9 adults; n = 36 pediatrics) were included in the initial PK model build and 12 subjects in the external validation cohort. Overall, the data were best described using a two-compartment model with volume of distribution (V) normalized to total body weight (TBW/70 kg) and an allometric scaled elimination rate constant (Ke) for pediatric subjects (AIC = 4,138.36). Final model observed vs. predicted plots demonstrated good fit (population R2 = 0.87, individual R2 = 0.97, Figure 1a and b). For the final model, the population median parameter values (95% credibility interval) were V0 (total volume of distribution), 11.7 L (10.2–14.6); Ke for adult, 0.66 hour−1 (0.38–0.78), Ke for pediatrics, 0.82 hour−1 (0.64–0.85), KCP (rate constant from central to peripheral compartment), 1.4 hour−1 (1.3–1.8), KPC (rate constant from peripheral to central compartment), 1.6 hour−1 (1.2–1.8). The validation cohort has 12 subjects, and the final model fit the data well (individual R2 = 0.75).ConclusionIn this diverse group of adult and pediatrics, a two-compartment model described CEF PK well and was externally validated with a unique cohort. This model can serve as a population prior for real-time PK software algorithms. Disclosures All authors: No reported disclosures.

Different factors determined the toxicokinetics of organic chemicals and nanomaterials exposure to zebrafish (Danio Rerio)

Little is known about how the chemical properties (molecular structure, such as the hydrophobic and hydrophilic end group for organic chemical, and particle size for nanomaterials (NMs)) quantitatively affect the toxicokinetics (TK) in organisms especially in short-term, single-species studies. A novel method based on a first-order one compartment TK model which described the monophasic uptake pattern and two-compartment TK model which adequately described the biphasic metabolism pattern was used to determine the bioconcentration and TK rate constants of organic compounds (n = 17) and nanomaterials (NMs, n = 7) in zebrafish. For both one and two compartment model, the uptake (kin) and elimination (kout) rate constants were fitted using a one- and two-compartment first-order kinetic model, and bioconcentration factors (BCF) and 95% depuration times (t95) for all tested chemicals were calculated, respectively. The results showed that there was significant difference in TK parameters kin, kout, and BCF between organic chemicals and nano metal oxides. For organic compounds, significant correlations were found between the kin and BCF and the octanol-water partition coefficient (Kow) and molecular mass. For nano metal oxides, there was a significant negative correlation between the kin or BCF and particle size, but a positive correlation between kin and Zeta potential of nanoparticles and also a significant positive correlation between kout and particle size or specific surface area. Those findings indicated that NMs particle size does matter in biological influx and efflux processes. Our results suggest that the TK process for organic compound and NMs are correlated by different chemical properties and highlight that the Kow, the absorption kin, metabolism k12 and k21, elimination rate kout, and all the parameters that enable the prediction and partitioning of chemicals need to be precisely determined in order to allow an effective TK modeling. It would therefore appear that the TK process of untested chemicals by a fish may be extrapolated from known chemical properties.

Non-Linear Rituximab Pharmacokinetics and Complex Relationship between Rituximab Concentrations and Anti-Neutrophil Cytoplasmic Antibodies (ANCA) in ANCA-Associated Vasculitis: The RAVE Trial Revisited.

Rituximab is approved in patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) and leads to a decrease of ANCA levels. The objectives of this study were to investigate the non-linear pharmacokinetics of rituximab and the relationship between its concentrations and ANCA levels in AAV patients. Ninety-two AAV patients from the RAVE (Rituximab in ANCA-Associated Vasculitis) trial were assessed. Both ANCA anti-myeloperoxidase (MPO-ANCA) and anti-proteinase3 (PR3-ANCA) levels were used as biomarkers. The pharmacokinetics of rituximab were described using a semi-mechanistic two-compartment model that included a latent target antigen turnover and allowed the estimation of specific target-mediated elimination in addition to its non-specific elimination of rituximab. The effect of rituximab on the ANCA level was described using a semi-mechanistic compartment model with a negative feedback (Friberg) model with no transit compartment. A population modeling approach was used. Our pharmacokinetic and pharmacokinetic-pharmacodynamic (PK-PD) models satisfactorily described both concentration-time and concentration-effect relationship data. The mean (inter-individual standard deviation) estimated non-specific clearance was 0.15L/day (0.30%) and the target-mediated elimination rate constant was 2.4 × 10-5 nmol/day. The elimination half-lives for MPO-ANCA and PR3-ANCA were 24 and 18days, respectively. A non-linear target-mediated elimination of rituximab was detected in AAV patients. Our PK-PD model allowed quantification of the association between rituximab concentrations and ANCA levels. This decrease was deep but delayed, and more sustained in patients with MPO-ANCA than in those with PR3-ANCA. Our results suggest that repeating courses of rituximab might improve the clinical response to rituximab.

P743The efficacy and the safety of the action of the complex drug of fibrin-specific streptokinase in arterial thrombosis in vivo

Abstract Introduction The use of streptokinase (SK) in the clinic is limited by the absence of fibrin-specificity, low degree of recovery of the vessel lumen and hemorrhagic manifestations. We have developed a system of local delivery of SK based on fibrin-specific liposomes, which is a complex drug of streptokinase (SKf), containing “free” SK and SK “associated” with liposomes conjugated with monoclonal antibodies FnI-3C (IgG2 class) to fibrin in a ratio of 40%/60% respectively. The purpose of this study was to evaluate the efficacy and the safety of the action of the SKf in different methods of its administration in arterial thrombosis in vivo. Methods Experiments were performed with 60 outbred male dogs weighing 15–18 kg on a 2-hour model thrombus of the femoral arteries. Experimental group (n=30) received SKf, control group (n=30) - native SK. The drugs were administered intravenously (IV) and intraarterially (IA) in the dose of SK 75000 ME. The observation time was 180 minutes. The pharmacokinetic parameters of the SKf and native SK were studied. Ultrasound, morphological and morphometrical methods of the arteries investigations were used. P<0.05 was taken as statistically significant. Results The SKf had better pharmacokinetic parameters than native SK. The duration of the half-life of SKf was significantly longer than that of native SK 24,1 [21,76; 27,53] min vs. 1,83 [1,64; 1,93] min, (p<0.001). The elimination constant of SKf was lower than that of native SK 0,0291 [0,0175; 0,0407] 1/min vs. 0,3867 [0,1679; 0,5135] 1/min, (p<0.001). Differences between pharmacokinetic parameters of SKf and the native SK, and the presence of fibrin-specificity of SKf contributed to increase of its thrombolytic effect. According to the data of the morphological analysis, the lysis of the central part of the thrombus was noted during the action of native SK. At the same time, the release of SKf initially caused the lysis of the central part of the thrombus with the further dissolution of the thrombus at the periphery. The morphometric evaluation of the artery samples showed that SKf leads to a significant increase in the degree of free vascular lumen compared to the native SK in different methods of its administration: IV - 65,25 [62,67; 68,53]% vs. 52,82 [50,60; 55,71]% respectively, p<0.001; IA - (74,27 [70,39; 77,39]% vs. 68,56 [67,36; 70,87]% respectively, p<0.001. The hemorrhagic complications were not revealed for different methods of SKf administration during the observation period. Conclusions The use of SKf with different methods of administration leads to highly efficient and safe fibrinolyc action in arterial thrombosis in vivo.

Copper, Nickel and Manganese Residues in Tissues and Organs of Rabbits Dosed with Edible Clay

The study examined the kinetics of Copper (Cu), Nickel (Ni) and Manganese (Mn) in tissues and organs of rabbits treated with edible clay from Enyigba, Ebonyi state Nigeria. Clay samples were obtained from Enyigba mine. Fifty four of the sampled rabbits were grouped into 3: group 1 and group 2 were administered with 2000 mg/kg and 4000 mg/kg of edible clay respectively while group 3 served as control. Concentrations of these metals were detected in the brain, heart, kidney, liver, lungs, skeletal muscle and blood of each of the rabbits using atomic absorption spectroscopy (AAS). One - way ANOVA was used in analyzing the concentrations with significant results (p < 0.05) recorded. The study results showed that while Cu concentrations were predominant in the liver (15.33±1.18 µg/g), kidney (8.25±1.18 µg/g) and heart (9.43±2.36 µg/g) of 2000 mg/kg treated rabbits on day 1 post-treatment, Ni concentrations were the highest among the three elements especially in the blood (221.59±17.05 µg/g) and skeletal muscle (153.41±19.68 µg/g) of 2000 mg/kg treated rabbits on day 1 post-treatment. Concentrations of Mn were mostly detected in the liver (13.99±4.85 µg/g) of 2000 mg/kg treated rabbits on day 1 post-treatment. Most of the concentration values increased in 4000 mg/kg treated rabbits on day 1 post-treatment. Elimination constants as well as half-life eliminations of the metals studied showed that absorbed elements were still present in most organs and tissues of treated rabbits. The study recommends research for possible microbial toxins that may be found in the edible clay studied.

Feed-to-Fillet Transfer of Selenite and Selenomethionine Additives to Plant-Based Feeds to Farmed Atlantic Salmon Fillet

This study investigated the transfer kinetics of dietary selenite and selenomethionine (SeMet) to the fillet of farmed Atlantic salmon (Salmo salar). The uptake and elimination rate constants of the two selenium (Se) forms were determined in Atlantic salmon fed either selenite- or SeMet-supplemented diets followed by a depuration period. The fillet half-life of selenite and SeMet was 779 ± 188 and 339 ± 103 days, respectively. The elimination and uptake rates were used in a simple one-compartmental kinetic model to predict levels in fillet based on long-term (whole production cycle) feeding with given dietary Se levels. Model predictions for Atlantic salmon fed plant-based feeds low in natural Se and supplemented with either 0.2 mg of selenite or SeMet kg-1 gave a predicted fillet level of 0.042 and 0.058 mg Se kg-1 wet weight, respectively. Based on these predictions and the European Food Safety Authority risk assessment of Se feed supplementation for food-producing terrestrial farm animals, the supplementation with 0.2 mg of selenite kg-1 would likely be safe for the most sensitive group of consumers (toddlers). However, supplementing feed to farm animals, including salmon, with 0.2 mg of SeMet kg-1 would give a higher (114%) Se intake than the safe upper intake limit for toddlers.

Interest of adjusting urine cannabinoids to creatinine level to monitor cannabis cessation therapy in heavy smokers with psychiatric disorders

Up to 25% of hospitalized patients in a psychiatric department exhibit troubles linked to cannabis use. Weaning patients with psychiatric disorders off drugs of abuse requires specific care to improve their clinical outcome. The present study aims to develop a predictive model of urinary excretion of creatinine-normalized cannabinoids (UCNC ) and to determine UCNC thresholds corresponding to the widely used cut-offs of 20ng/mL and 50ng/mL for cannabinoids. One hundred thirty-two patients with 452 urine samples were included between 2013 and 2017. Urinary cannabinoids and UCNC elimination curves were computed for each patient. Using a mono-exponential mixed effect model with 88 samples from 26 subjects exhibiting at least 3 decreasing UCNC in a row, the average calculated elimination rate constant was 0.0108±0.0026h-1 , corresponding to a mean elimination half-life of 64±12hours. The use of UCNC is of particular interest because of a high inter- and intra-individual variability of urinary creatinine concentration (from 0.06 to 3.81mg/mL). Moreover, UCNC allows for the detection of diluted or concentrated urine specimens that may lead to false positive (FP) or false negative (FN) results. Receiver operator characteristic (ROC) curves were used to assess UCNC thresholds of 32.4 and 124.7ng/mg that provide a strong discrimination between positive and negative samples for cannabinoids cut-offs of 20 and 50ng/mL respectively. The developed model and the defined UCNC thresholds allowed for the accurate prediction of the time needed to reach a negative UCNC result that could be used by clinicians to optimize clinical care.

Evaluation of a non-parametric modelling for meropenem in critically ill patients using Monte Carlo simulation.

In critically ill patients treated with meropenem, the proposed pharmacokinetics/pharmacodynamics (PK/PD) efficacy index is to keep the free drug concentration 4-5 times above the minimum inhibitory concentration (MIC) of the germ isolated, for 100% of the interval regimen. The objectives were to design a population pharmacokinetics model for meropenem in critically ill patients and to evaluate different dosage schemes that achieve the optimal PK/PD objectives. This retrospective, observational, single-centre study included 80 critically ill patients (154 samples) treated with meropenem between May 2011 and December 2017. Patient data, concentrations, treatment and bacteriological variables were collected from electronic medical records. Total and free concentrations of meropenem were modelled in Pmetrics. Monte Carlo simulations were performed to assess the probability of achieving the PK/PD target for different dosage regimens. For patients with available data, the number of patients with a free concentration 4 times higher or lower than the observed MIC for the P. aeruginosa and E. coli was investigated. A one-compartment model with first-order elimination adequately described serum total and free meropenem concentrations. The only variable that significantly influenced the elimination constant of meropenem was the creatinine clearance (CLcr) calculated using the CKD-EPI formula. The highest probability of achieving the pharmacodynamic objective was with 3-h infusion dosage regimens. Sixty percent and 89% of patients attained a free drug concentration 4 times above the MIC for P. aeruginosa and E. coli respectively. This study proposed different dosing regimens depending on renal clearance strata and the MIC of the germ targeted.

In vivo effect of Kaempferia parviflora extract on pharmacokinetics of acetaminophen

Kaempferia parviflora is widely used as a food supplement and a herbal medicine for vitalization. Previous study has shown that K. parviflora had CYP2E1 inducer activity. It is likely to affect the metabolism of CYP2E1 substrates such as acetaminophen which is a common household pain relief medicine. This study investigated the possible pharmacokinetic interaction between K. parviflora and acetaminophen in rats. Acetaminophen (100 mg/kg, p.o) was administered to rats for nine consecutive days. On days 4–9, K. parviflora extract (250 mg/kg, p.o) was given to the acetaminophen-treated rats. After co-administration with K. parviflora, the concentrations of acetaminophen during day 5–8 markedly decreased compared with acetaminophen-only group. At day 9, the pharmacokinetic parameters of acetaminophen in the presence of K. parviflora extract also decreased, including area under the concentration-time curve (from 1.68 ± 0.16 to 0.34 ± 0.04 mg.min/mL), the maximum concentration (from 19.10 ± 1.90 to 4.48 ± 0.56 µg/mL), and half-life (from 21.29 ± 1.36 to 10.81 ± 1.24 min). In addition, clearance and the elimination rate constant of acetaminophen were significantly increased (from 0.003 ± 0.000 to 0.006 ± 0.001 L/min and 0.03 ± 0.00 to 0.07 ± 0.01 min−1, respectively) in the presence of K. parviflora extract. These findings provide the data for in vivo herb-drug interaction between K. parviflora extract and acetaminophen. Therefore, the concomitant use of K. parviflora as a food supplement and acetaminophen should occasion therapeutic and safety concerns.

Population pharmacokinetics of total and unbound concentrations of intravenous posaconazole in adult critically ill patients

BackgroundThe population pharmacokinetics of total and unbound posaconazole following intravenous administration has not yet been described for the critically ill patient population. The aim of this work was, therefore, to describe the total and unbound population pharmacokinetics of intravenous posaconazole in critically ill patients and identify optimal dosing regimens.MethodsThis was a prospective observational population pharmacokinetic study in critically ill adult patients with presumed/confirmed invasive fungal infection. A single dose of 300 mg posaconazole was administered intravenously as an add-on to standard antifungal therapy, and serial plasma samples were collected over 48 h. Total and unbound posaconazole concentrations, measured by chromatographic method, were used to develop a population pharmacokinetic model and perform dosing simulations in R using Pmetrics.ResultsFrom eight patients, 93 pairs of total and unbound concentrations were measured. A two-compartment linear model with capacity-limited plasma protein binding best described the concentration-time data. Albumin and body mass index (BMI) were included as covariates in the final model. Mean (SD) parameter estimates for the volume of the central compartment (V) and the elimination rate constant were 72 (43) L and 42.1 (23.7) h−1, respectively. Dosing simulations showed that high BMI was associated with a reduced probability of achieving target total and unbound posaconazole concentrations. Low serum albumin concentration was associated with a reduced probability of attaining target total but not unbound posaconazole concentrations.ConclusionsAn important clinical message of this study is that critically ill patients with increased BMI may require larger than approved loading doses of intravenous posaconazole when considering currently recommended dosing targets. Variability in plasma albumin concentration appears unlikely to affect dosing requirements when the assessment is based on unbound concentrations. Where available, therapeutic drug monitoring of unbound concentrations may be useful.

Accumulation, biodegradation and toxicological effects of N-ethyl perfluorooctane sulfonamidoethanol on the earthworms Eisenia fetida exposed to quartz sands

While N-ethyl perfluorooctane sulfonamidoethanol (EtFOSE) is a precursor of perfluorooctane sulfonate (PFOS), its bioaccumulation, transformation and toxicological effects in earthworms (Eisenia fetida) exposed to quartz sands are poorly understood. The present study showed that except for parent EtFOSE, N-ethylperfluorooctane sulfonamide acetate (EtFOSAA), N-ethyl perfluorooctane sulfonamide (EtFOSA), perfluorooctane sulfonamide acetate (FOSAA), perfluorooctane sulfonamide (FOSA) and PFOS were detected in earthworms, with EtFOSAA as the primary biotransformation product. The biota-to-sand accumulation factor (BSAF) and uptake rate coefficient (ku) of EtFOSE were 5.7 and 0.542/d, respectively. The elimination rate constants (ke) decreased in the order EtFOSA (0.167/d) ∼ FOSAA (0.147/d) > FOSA (0.119/d) ∼ EtFOSAA (0.117/d) > EtFOSE (0.095/d) > PFOS (0.069/d). No significant effects were observed in malondialdehyde (MDA) contents and acetylcholinesterase (AChE) activities between EtFOSE treatments and controls. EtFOSE could cause significant accumulation of reactive oxygen species (ROS) in earthworms. Peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) were significantly activated by 41.4–74.3%, 37.2–44.4% and 32.4–52.3% from day 4–10, respectively, while 8-Hydroxy-2-deoxyguanosine (8-OHdG) levels were elevated by 47.7–70.3% from day 8–10, demonstrating that EtFOSE induced oxidative stress and oxidative DNA damage in earthworms. Significant increase of glutathione-S-transferase (GST) with 41.6–62.8% activation (8–10 d) gave indirect evidence on the conjugation of EtFOSE or its corresponding metabolites during phase II of detoxication. This study provides important information on the fate and potential risks of EtFOSE to terrestrial invertebrates.

PS926 IN VITRO AND IN VIVO STUDIES OF NOVEL ASPARAGINASE MUTANTS FOR THE TREATMENT OF ACUTE LYMPHATIC LEUKEMIA

Background:E. coli derived L‐asparaginase (EcA) is an essential medicine in the combination chemotherapy for Acute Lymphoblastic Leukemia (ALL). Since it is exogenously derived, silent inactivation and hypersensitivity due to anti‐drug antibody have often necessitated discontinuation of the drug.Aims:Using a rational protein engineering approach, we created four EcA mutants with improved thermal stability, better serum stability, less antigenicity and less glutaminase activity. The mutant with most favourable pharmacological profile will be developed as a novel asparaginase for therapy of ALL.Methods:We identified and mutated several B‐cell epitopes and amino acid residues at the EcA interface that are responsible for activity, stability and antigenicity. Steady state enzyme kinetics was measured at 37oC (optimum temperature for EcA) and 62oC (EcA degrades at 65oC). The kinetic parameters, such as Km and Vmax, were calculated from linear fits of Michaelis‐Menten equation. Glutaminase activity of the mutants was measured and compared to the wild type EcA. The cytotoxicity of the EcA mutants was verified by performing MTT assay in ALL sensitive REH cell lines. Further indirect ELISA was performed to check the antigenicity of the mutants. The binding of the mutants to the commercially available l‐asparaginase antibody was analysed. Moreover, to evaluate the in vivo immunogenicity, mice were immunized with primary and booster doses of EcA mutants over 66 days followed by the measurement of IgG and IgM titers. In addition, the binding of wild‐type EcA and mutants to pre‐existing anti‐asparaginase antibodies present in ALL patient serum receiving asparaginase therapy was studied by indirect ELISA. Finally the activity‐time profile of the EcA mutants was plotted and was used to calculate the pharmacokinetic parameters, which were further compared with the commercially available l‐asparaginase drug.Results:At 37oC catalytic activity of the mutants was comparable to wild type. At 62oC, the catalytic activity of Mutant A, B and C was 155%, 96.5% and 98.2% respectively, compared to 56.9% for the wild type. All four mutants showed significantly lower glutaminase activity as compared to wild type EcA. In MTT assay, the wild type EcA showed 25.31% of cell kill, whereas mutant B showed highest cytotoxicity with 60% cell kill in Reh ALL cell lines. In terms of antigenicity, Mutant D was found to be 48% less antigenic compared to wild EcA, followed by Mutant B (26.4% less) and Mutant C (23.7% less). Pharmacokinetic profiling showed that all mutants had higher AUC and lower elimination rate constant, with Mutant B having highest AUC (1127.7 ± 363.6 vs. 72.12 ± 28.03 min∗ug/ml) and lowest elimination rate constant (0.048 ± 0.017 vs. 0.79 ± 0.42 ml/min) compared to commercially available L‐asparaginase. Mice immunized with Mutant B variant showed approximately 10‐fold lower IgG and IgM titers as compared to antibody responses induced by wild‐type EcA. Further, the binding of mutant B to serum antibody from the ALL patients was approximately 2‐fold less compared to wild‐type EcA.Summary/Conclusion:Considering all parameters, Mutant B was found to have the most favorable characteristics and emerged as the lead candidate for future development. This formulation is expected to overcome the existing deficiencies and clinical challenges encountered with the EcA formulations. Clinical development of this drug candidate is envisaged.

Predicting mercury bioavailability in soil for earthworm Eisenia fetida using the diffusive gradients in thin films technique.

In general, the diffusive gradients in thin films (DGT) technique is an effective tool for evaluating metal bioavailability; however, its applicability is subject to the type of metal and organism involved. In this study, the accumulated masses of Hg in DGT probes and in the earthworm species Eisenia fetida were monitored for 10days, to test if the DGT technique can be used as a predicting method for the bioavailability of soil Hg to earthworms. In the Hg exposure tests using soils prepared with different peat moss concentrations of 5, 10, 15, and 20% and varying pH values of 4.6, 5.6, and 6.2, the experimentally determined DGT-soil accumulation factor (DSAF) and biota-soil accumulation factor (BSAF) both increased as the peat moss content decreased and the pH increased. According to a one compartment model, this was a result of the increased Hg uptake rate constant (k1) and the relatively stable Hg elimination constant (k2) under lower peat moss and higher pH conditions. It is interesting to note that the Hg uptake rates by DGT and earthworms were considerably higher for fresh soils than for aged soils, while porewater (and acid-extractable) Hg concentrations were rather similar between the two types of soils. Across diverse soil properties, steady-state Hg in earthworm tissue showed a strong positive correlation with DGT-measured Hg flux ([earthworm Hg] = 354(DGT-Hg flux)-34, r2 = 0.88), while meager correlations were found between Hg concentration in earthworms and that in porewater (and acid-extractable). The overall results indicate that DGT-measured Hg flux is a better tool than conventional methods for predicting Hg bioavailability for earthworms inhabiting diverse types of soil.

PO-01-018 Comparative pharmacokinetic and bioavailability studies of monotropein, kaempferol 3-O-glucoside and quercetin 4'-O-glucoside after oral and intraveneous administration of MOTILIPERM in rats

Background and ObjectiveThis study has evaluated the pharmacokinetic parameters and bioavailabilities of monotropein, kae-mpferol-3-O-glucoside, and quercetin-4’-O-glucoside after administration of MOTILIPERM in rats.Material and MethodsFollowing the administration of MOTILIPERM, the plasma concentrations of each compound in rats were simultaneously determined by using liquid chromatography tandem mass spectrometry (LC-MS/MS).ResultsThe pharmacokinetic parameters of monotropein in rats were AUCinf 20,020.44±3944.67 and 11,915.53±1190.91 min·ng/mL and Cmax 286.99±38.37 and 56.23±9.02 ng/mL for intravenous and oral administration, respectively. The pharmacokinetic parameters of kaempferol-3-O-glucoside in rats were AUCinf 287.86±126.17 min·ng/mL and not estimated; Cmax 5.80±1.87 and 1.24±0.41 ng/mL for intravenous and oral administration, respectively. The pharmacokinetic parameters of querce-tin-4’-O-glucoside in rats were AUCinf 511.38±248.11 and 481.44±65.72 min·ng/mL; Cmax 10.72±2.70 and 2.83±0.34 ng/mL for intravenous and oral administration, respectively. ConclusionThe absolute bioavailabilities of monotropein and quercetin-4’-O-glucoside for oral administration were evaluated and calculated as 3.0 and 4.7%, respectively. The absolute bioavailability of kaemp-ferol-3-O-glucoside was not calculated because the elimination rate constant could not be estimated. These results may be applied to the basic data in a further study in order to develop functional foods or herbal medicinal products.

ELEMENTAL COMPOSITION OF TISSUES AND TOXICOKINETICS OF ARSENIC AND ANTIMONY ON INTAKE IN MALE WHITE RATS OF THE WISTAR LINE WITH DRINKING WATER

The biogeochemical province of antimony and arsenic created in the area of massive sulphide deposits forms a level of their exchange in the biosphere that is different from the usual conditions. These elements along with a number of common properties of the semi-metals have significant features of biological action. The aim of the study was to assess the variability of the elemental composition of the liver tissues of white rats with oral intake of wastewater from the tailings of Komsomolsk gold mine (Kemerovo region) with a high content of antimony and arsenic. The objects of the study were wastewater from the slurry pits of Komsomolsk gold mine (Kemerovo region); liver tissue of white rats of the Wistar line, whole blood, urine, excrements. The contents of sulfur, chlorine, potassium, calcium, titanium, manganese, chromium, iron, nickel, copper, zinc, selenium, bromine, rubidium, strontium, molybdenum, arsenic, mercury, lead, antimony in the tissues of the liver, blood, urine, excrements before priming, after 3 weeks of priming and after 1, 2 and 4 weeks during the recovery period after priming have been determined. It has been established that combined oral 3 weeks intake of arsenic in a dose of 10,0-15,1 µg / kg / day and antimony in a dose of 4,2-6,1 µg / kg / day in male white rats of the Wistar line is accompanied by their accumulation in the liver and blood and the excretion with urine and excrements, the parameters of the toxicokinetics of arsenic indicate its absorption and elimination from the blood about the same rate, whereas in liver, the rate of elimination of arsenic is somewhat higher than the rate of absorption (t 1/2 - 38 and 49 days and constants of elimination and absorption - 0.018 and 0.014, respectively). The predominance of elimination processes over absorption for antimony in the blood (t 1/2 - 12 and 22 days, constants of elimination and absorption – 0.058 and 0.032, respectively) has been established.

Effects of Silymarin on the In Vivo Pharmacokinetics of Simvastatin and Its Active Metabolite in Rats.

Herein, the effect of silymarin pretreatment on the pharmacokinetics of simvastatin in rats was evaluated. To ensure the accuracy of the results, a rapid and sensitive UPLC–MS/MS method was established for simultaneous quantification of simvastatin (SV) and its active metabolite simvastatin acid (SVA). This method was applied for studying the pharmacokinetic interactions in rats after oral co-administration of silymarin (45 mg/kg) and different concentrations of SV. The major pharmacokinetic parameters, including Cmax, tmax, t1/2, mean residence time (MRT), elimination rate constant (λz) and area under the concentration-time curve (AUC0–12h), were calculated using the non-compartmental model. The results showed that the co-administration of silymarin and SV significantly increased the Cmax and AUC0–12h of SVA compared with SV alone, while there was no significant difference with regards to Tmax and t1/2. However, SV pharmacokinetic parameters were not significantly affected by silymarin pretreatment. Therefore, these changes indicated that drug-drug interactions may occur after co-administration of silymarin and SV.