Concentration-effect Curves Research Articles

A validated LC-MS/MS method for the determination of hederasaponin C: Application to pharmacokinetics-pharmacodynamics studies in the therapeutic area of acetic acid-induced ulcerative colitis in rats.

Hederasaponin C (HSC), one of the main components of Pulsatilla chinensis, is considered as a potential drug for the treatment of inflammatory bowel disease. In the present research, we developed a pharmacokinetics-pharmacodynamics model to describe the concentration-effect course of drug action following the intraperitoneal injection of HSC in colitis rats. A sensitive UPLC-MS/MS method was established for the the determination of HSC in rat plasma to explore the pharmacokinetics properties. The separation was performed on an Accucore C18 column (2.1 × 100 mm, 2.6 μm) with a flow phase consisting of acetonitrile and 0.1% formic acid water. The assay method was validated and demonstrated good adaptability for application in the pharmacokinetics study. Then the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) in colon tissues were measured using an ELISA assay. The levels of TNF-α, IL-1β and IL-6 were decreased after HSC administration, suggesting that HSC can significantly improve the level of inflammatory syndrome factor. The pharmacokinetics study showed that the time to peak concentration of HSC was 1 h. The concentration-effect curves showed a hysteresis loop. There was also a hysteresis between the peaked concentration and the maximum effect of HSC. The present study established in vivo pharmacokinetics-pharmacodynamics models and the results showed a great potential of HSC for treating ulcerative colitis.

Reproductive toxicity of enrofloxacin in Caenorhabditis elegans involves oxidative stress-induced cell apoptosis

Fluoroquinolone antibiotics (FQs) that persist and bioaccumulate in the environment have aroused people's great concern. Here, we studied the adverse effects of FQs in soil animals of Caenorhabditis elegans via food-chronically exposure. The result shows C. elegans exposed to FQs exhibited reproductive toxicity with small-brood size and low-egg hatchability. To study the underlying mechanism, we conduct a deep investigation of enrofloxacin (ENR), one of the most frequently detected FQs, on nematodes which is one of commonly used animal indicator of soil sustainability. The concentration-effect curves simulated by the Hill model showed that the half effect concentrations (EC50) of ENR were (494.3 ± 272.9) µmol/kg and (107.4 ± 30.9) µmol/kg for the brood size and the hatchability, respectively. Differential gene expression between the control and the ENR-exposure group enriched with the oxidative stress and cell apoptosis pathways. The results together with the enzyme activity in oxidative stress and the cell corpses suggested that ENR-induced reproductive toxicity was related to germ cell apoptosis under oxidative stress. The risk quotients of some soil and livestock samples were calculated based on the threshold value of EC10 for the egg hatchability (2.65 µmol/kg). The results indicated that there was possible reproductive toxicity on the nematodes in certain agricultural soils for the FQs. This study suggested that chronic exposure to FQs at certain levels in environment would induce reproductive toxicity to the nematodes and might reduce the soil sustainability, alarming the environment risks of antibiotics abuse.

OA16 Therapeutic certolizumab pegol drug levels to achieve good EULAR response in patients with rheumatoid arthritis: results from the Biologics in Rheumatoid Arthritis Genetics and Genomics Study Syndicate (BRAGGSS) cohort

Abstract Background/Aims Rheumatoid arthritis (RA) is a systemic inflammatory disease which is often treated with biologic disease-modifying anti-rheumatic drugs (bDMARDs) like tumour-necrosis factor inhibitors (TNFi) including certolizumab pegol (CTZ). A proportion of patients (approximately 30%) do not respond to treatment, and previous studies have shown that low drug levels and poor adherence are associated with poor response; however, an optimal drug level has not been defined or applied in clinical practice. The aim of this analysis is to determine an optimal CTZ drug level associated with a “good” European Alliance of Associations for Rheumatology (EULAR) response in fully adherent and non-adherent patients with RA. Methods In a prospective observational cohort study, patients with RA were treated with CTZ. At baseline, and at 6 months, 4-component DAS28 scores, as well as self-reported treatment adherence data were collected. Drug levels were measured using ELISA during follow-up at 6 months from treatment initiation. Patients were considered fully adherent if they self-reported never having altered, forgotten or omitted any dose of their biologic drug at follow-up. Patients without drug levels were excluded. In fully adherent patients, the relationship between random CTZ levels and clinical efficacy after 6 months was determined using a concentration-effect curve, and a receiver-operator characteristics (ROC) curve established a therapeutic cut-off concentration using the EULAR response classification. Median drug levels in “good” EULAR responders were compared between adherent and non-adherent patients to provide an estimated therapeutic range. Results Of 347 RA patients taking CTZ recruited, 111 (32.0%) had drug levels and EULAR response measured at 6 months, of which 42 (37.8%) also provided self-reported treatment adherence data. Of those, 37 (88.1%) reported being fully adherent. In 37 fully adherent RA patients taking CTZ, clinical efficacy was positively correlated with increasing CTZ concentration reaching a maximum median DAS28CRP improvement of 2.99 (IQR 2.67-3.40) with levels above 30mg/L. Levels exceeding 30mg/L showed no significant additional benefit. The ROC curve showed an area under the curve (AUC) of 0.714 (95% CI 0.526-0.901) for random CTZ levels with EULAR good response versus non/moderate responders. A cut-off of 30mg/L had a sensitivity and specificity of 86.4% and 60.0%, respectively, to classify good responders in fully adherent patients. Fully adherent good responders (n = 37) had a median drug level of 34.5mg/L (IQR 31-38), whereas good responders in the remaining cohort (n = 76) had a median drug level of 23.5 (IQR 15-30). Conclusion In RA patients taking CTZ, random drug levels were higher in EULAR good responders, and in fully adherent patients. A target lower threshold drug level cut-off of 30mg/L or a target range from 23.5mg/L to 34.5mg/L may be useful in clinical practice. Disclosure R.M. Hum: None. P. Ho: None. N. Nair: None. D. Plant: None. A.W. Morgan: None. J.D. Isaacs: None. A.G. Wilson: None. K.L. Hyrich: None. A. Barton: None.

Muscarinic Receptor Activation Reduces Force and Arrhythmias in Human Atria Independent of IK,ACh.

In human hearts, muscarinic receptors (M-R) are expressed in ventricular and atrial tissue, but the acetylcholine-activated potassium current (IK,ACh) is expressed mainly in the atrium. M-R activation decreases force and increases electrical stability in human atrium, but the impact of IK,ACh to both effects remains unclear. We used a new selective blocker of IK,ACh to elaborate the contribution of IK,ACh to M-R activation-mediated effects in human atrium. Force and action potentials were measured in rat atria and in human right atrial trabeculae. Cumulative concentration-effect curves for norepinephrine-induced force and arrhythmias were measured in the presence of carbachol (CCh; 1 µM) or CCh together with the IK,ACh -blocker XAF-1407 (1 µM) or in time-matched controls. To investigate the vulnerability to arrhythmias, we performed some experiments also in the presence of cilostamide (0.3 µM) and rolipram (1 µM), inhibiting PDE3 and PDE4. In rat atria and human right atrial trabeculae, CCh shortened the action potential duration persistently. However, the direct negative inotropy of CCh was only transient in human, but stable in rat atria. In rat and human atria, the negative inotropic effect was insensitive to blockage of IK,ACh by XAF-1407. In the presence of cilostamide and rolipram about 40% of trabeculae developed arrhythmias when exposed to norepinephrine. CCh prevented these concentration-dependent norepinephrine-induced arrhythmias, again insensitive to XAF-1407. Maximum catecholamine-induced force was not depressed by CCh. In human atrium, the direct and the indirect negative inotropic effect of CCh are independent of IK,ACh. The same applies to the CCh-mediated suppression of norepinephrine/PDE-inhibition-induced arrhythmias.

Protective Effects of Grape Juice on Vascular Damage Induced by Chlorine Free Radical in Rats.

Grapes and their derivatives have antioxidant and cardioprotective properties. Therefore, we hypothesized that grape juice (GJ) could improve vascular oxidative damage caused by chlorine radicals (OCl−), which are excessively produced in vascular tissue during cardiovascular diseases (mainly diabetes and hypertension). The antioxidant capacity of GJ was analyzed by an electrochemical method, followed by administration in rats (100 or 300 mg/kg/d, via the oral) for seven days. Then, rats were sacrificed, and their aortas were isolated and subjected to isometric recordings or immuno-histochemical analyses with or without exposure to OCl− (5, 20, or 100 μM, 60 min). Concentration-effect curves for acetylcholine (ACh) and sodium nitroprusside (SNP) were derived to analyze endothelium-dependent or independent vasore-laxation. The GJ presented with high antioxidant capacity, and treatment with GJ did not alter vascular relaxation induced by ACh or SNP. After exposure to OCl−, endothelium-denuded arteries showed preserved relaxation with SNP, whereas endothelium-intact arteries showed reduced relaxation with ACh. OCl− at various concentrations induced significantly decreased relaxation of arteries (80.6±4.2%, 55.4±4.7%, and 28.1±5.9%, respectively) vs. control arteries (96.8±2.4%). However, treatment with GJ prevented loss in relaxation caused by 5 and 20 μM OCl− and improved relaxation after exposure to 100 μM OCl−. Exposure to OCl− induced increased nitrotyrosine immunostaining of endothelial cell layers, which was improved by GJ treatment. Altogether, vascular damage caused by OCl− was prevented by treatment with GJ, and GJ prevented nitrosative stress in these vessels.

Extra Virgin Oil Polyphenols Improve the Protective Effects of Hydroxytyrosol in an In Vitro Model of Hypoxia-Reoxygenation of Rat Brain.

Hydroxytyrosol (HT) is the component primarily responsible for the neuroprotective effect of extra virgin olive oil (EVOO). However, it is less effective on its own than the demonstrated neuroprotective effect of EVOO, and for this reason, it can be postulated that there is an interaction between several of the polyphenols of EVOO. The objective of the study was to assess the possible interaction of four EVOO polyphenols (HT, tyrosol, dihydroxyphenylglycol, and oleocanthal) in an experimental model of hypoxia-reoxygenation in rat brain slices. The lactate dehydrogenase (LDH) efflux, lipid peroxidation, and peroxynitrite production were determined as measures of cell death, oxidative stress, and nitrosative stress, respectively. First, the polyphenols were incubated with the brain slices in the same proportions that exist in EVOO, comparing their effects with those of HT. In all cases, the cytoprotective and antioxidant effects of the combination were greater than those of HT alone. Second, we calculated the concentration–effect curves for HT in the absence or presence of each polyphenol. Tyrosol did not significantly modify any of the variables inhibited by HT. Dihydroxyphenylglycol only increased the cytoprotective effect of HT at 10 µM, while it increased its antioxidant effect at 50 and 100 µM and its inhibitory effect on peroxynitrite formation at all the concentrations tested. Oleocanthal increased the cytoprotective and antioxidant effects of HT but did not modify its inhibitory effect on nitrosative stress. The results of this study show that the EVOO polyphenols DHPG and OLC increase the cytoprotective effect of HT in an experimental model of hypoxia-reoxygenation in rat brain slices, mainly due to a possibly synergistic effect on HT’s antioxidant action. These results could explain the greater neuroprotective effect of EVOO than of the polyphenols alone.

Redefining the age-specific therapeutic ranges of lamotrigine for patients with epilepsy: A step towards optimizing treatment and increasing cost-effectiveness

ObjectiveThe pharmacokinetics of lamotrigine exhibits age-related characteristics. Nevertheless, current evidence regarding the therapeutic range of lamotrigine has been derived almost exclusively from studies in adult patients, and the applicability of this therapeutic range to the pediatric population remains unclear. The purpose of this study was to establish the appropriate age-specific therapeutic ranges of lamotrigine corresponding to adequate clinical responses for patients with epilepsy. MethodsThis prospective cohort study of therapeutic drug monitoring included 582 Chinese epilepsy patients receiving lamotrigine monotherapy. Patients were divided into three age-related subgroups: (1) toddler and school-age group (2–12 years old, n = 168), (2) adolescent group (12–18 years old, n = 171), and (3) adult group (>18 years old, n = 243). Patients with a reduction in seizure frequency of 50 % or greater than baseline were defined as responders, and the remaining patients were non-responders. The relationship between lamotrigine serum concentrations and clinical response was assessed using multivariate logistic regression analysis. A receiver operating characteristic curve was generated to determine the representative cut-off values of lamotrigine trough levels, to distinguish responders from non-responders. The upper margin of the therapeutic range of lamotrigine was determined by developing concentration-effect curves for the three age-related subgroups. ResultsThe median trough levels of lamotrigine were significantly higher in responders than in non-responders from all three age-related groups (P < 0.0001). Results of logistic regression analysis revealed that higher serum concentrations of lamotrigine predicted a higher probability that seizure frequency would be reduced by more than 50 % compared to baseline (adjusted odds ratio: 1.228, 95 % CI: 1.137–1.327; P < 0.0001), and younger children were less likely to be responders (adjusted odds ratio: 1.027, 95 % CI: 1.012–1.043; P = 0.001). Based on a trade-off between sensitivity and specificity, the optimal cut-off values for lamotrigine trough concentrations corresponding to clinical response were 3.29 mg/L, 2.06 mg/L, and 1.61 mg/L in the toddler and school-age group, adolescent group, and adult group, respectively. By reducing interpatient variability, the results of the concentration-effect curves suggested no additional clinical benefit from a continued increase of doses for lamotrigine concentrations exceeding 9.08 mg/L, 8.43 mg/L, and 10.38 mg/L in the toddler and school-age group, adolescent group, and adult group, respectively. In conclusion, the therapeutic ranges of lamotrigine trough concentrations corresponding to adequate clinical response were 3.29–9.08 mg/L in the toddler and school-age group, 2.06–8.43 mg/L in the adolescent group, and 1.61–10.38 mg/L in the adult group. ConclusionsThe study determined age-specific therapeutic ranges corresponding to optimal clinical efficacy for lamotrigine. Our findings lay the foundation for catalyzing novel opportunities to optimize treatment and reduce therapeutic costs. Based on the age-specific therapeutic ranges identified in this study, individualized and cost-effective algorithms for lamotrigine treatment of epilepsy patients may be developed and validated in larger cohort studies of therapeutic drug monitoring.

The Rho kinase (ROCK) inhibitor Y-27632 reduces the β2-adrenoceptor density but enhance cAMP formation in primary equine bronchial epithelial cells

The present study addresses the effect of the Rho-kinase (ROCK) inhibitor Y-27632 on the β2-adrenoceptor density and β-agonist-stimulated intracellular second messenger cAMP formation in primary equine bronchial epithelial cells (EBEC). Y-27632 significantly decreased the β2-adrenoceptor number (Bmax) without markedly affecting the receptor affinity (dissociation constant, KD) to the radioligand [125I]-iodocyanopindolol (ICYP). In contrast, Y-27632 augmented the β-agonist-stimulated intracellular cAMP production. Herein, Y-27632 markedly increased the maximal cAMP responses (Emax) (isoproterenol > epinephrine > norepinephrine) but did not shift the β-agonist concentration-effect curves to the left. The β2-selective antagonist ICI 118.551 and the β1/β2-antagonsit propranolol but not the β1-selctive antagonist CGP 20712A reversed the isoproterenol-induced cAMP formation equally in Y-27632-treated and control EBEC, suggesting the effect was merely related to the β2-subtype. These results show that Y-27632 differentially regulates the receptor density and function. Thus, these findings provide the first evidence that the functional interaction of the β2-adrenoceptor and Rho-kinase (ROCK) signaling pathways decreases the receptor expression but enhances receptor downstream cAMP formation. This differential regulation of the receptor density and function by Y-27632 should be further reconsidered with regard to the beneficial effect of the drug in asthma therapy.

Exploring group size for statistical analysis of real-time signalling experiments.

Classical pharmacological bioassays generally use observed effects from a concentration series, at a single equilibrium time point to construct a concentration-effect curve, representing one experiment. However, if the full kinetic profile of the effect data for each concentration was evaluated simultaneously, then the analysis would be more powerful. In this work, we explore if more precise parameters can be achieved by using the full kinetic method. We used a simulation estimation study to explore the influence of kinetic analysis on the precision of the Emax model parameter estimates (Emax and C50 ). We compared a full kinetic approach in which all effect versus time data from a theoretical real-time signalling experiment were analysed simultaneously with a 'reference' approach. The theoretical real-time signalling experiment was based on a previously published CB2 receptor-binding experiment. The reference method with a group size (n) of 5 provided highly precise parameter estimates (coefficient of variation [CV] 3.4% for Emax and 0.72% for C50 ). A full kinetic method provided more precise estimates than the reference with equal or smaller group sizes. Note that group size 'n' here refers to the number of technical replicates rather than the number of biological replicates. A full kinetic method can yield more precise parameter estimates than the equilibrium method. Such an approach may be more useful for researchers.

Effects of omecamtiv mecarbil on failing human ventricular trabeculae and interaction with (-)-noradrenaline.

Omecamtiv mecarbil (OM) is a novel medicine for systolic heart failure, targeting myosin to enhance cardiomyocyte performance. To assist translation to clinical practice we investigated OMs effect on explanted human failing hearts, specifically; contractile dynamics, interaction with the β1–adrenoceptor (AR) agonist (−)‐noradrenaline and spontaneous contractions. Left and right ventricular trabeculae from 13 explanted failing hearts, and trabeculae from 58 right atrial appendages of non‐failing hearts, were incubated with or without a single concentration of OM for 120 min. Time to peak force (TPF) and 50% relaxation (t 50%) were recorded. In other experiments, trabeculae were observed for spontaneous contractions and cumulative concentration‐effect curves were established to (−)‐noradrenaline at β1‐ARs in the absence or presence of OM. OM prolonged TPF and t 50% in ventricular trabeculae (600 nM, 2 µM, p < .001). OM had no significant inotropic effect but reduced time dependent deterioration in contractile strength compared to control (p < .001). OM did not affect the generation of spontaneous contractions. The potency of (−)‐noradrenaline (pEC50 6.05 ± 0.10), for inotropic effect, was unchanged in the presence of OM 600 nM or 2 µM. Co‐incubation with (−)‐noradrenaline reduced TPF and t 50%, reversing the negative diastolic effects of OM. OM, at both 600 nM and 2 µM, preserved contractile force in left ventricular trabeculae, but imparted negative diastolic effects in trabeculae from human failing heart. (−)‐Noradrenaline reversed the negative diastolic effects, co‐administration may limit the titration of inotropes by reducing the threshold for ischemic side effects.

Compromised right ventricular contractility in an ovine model of heart transplantation following 24 h donor brain stem death

BackgroundHeart failure is an inexorably progressive disease with a high mortality, for which heart transplantation (HTx) remains the gold standard treatment. Currently, donor hearts are primarily derived from patients following brain stem death (BSD). BSD causes activation of the sympathetic nervous system, increases endothelin levels, and triggers significant inflammation that together with potential myocardial injury associated with the transplant procedure, may affect contractility of the donor heart. We examined peri-transplant myocardial catecholamine sensitivity and cardiac contractility post-BSD and transplantation in a clinically relevant ovine model. MethodsDonor sheep underwent BSD (BSD, n = 5) or sham (no BSD) procedures (SHAM, n = 4) and were monitored for 24h prior to heart procurement. Orthotopic HTx was performed on a separate group of donor animals following 24h of BSD (BSD-Tx, n = 6) or SHAM injury (SH-Tx, n = 5). The healthy recipient heart was used as a control (HC, n = 11). A cumulative concentration-effect curve to (-)-noradrenaline (NA) was established using left (LV) and right ventricular (RV) trabeculae to determine β1-adrenoceptor mediated potency (-logEC50 [(-)-noradrenaline] M) and maximal contractility (Emax). ResultsOur data showed reduced basal and maximal (-)-noradrenaline induced contractility of the RV (but not LV) following BSD as well as HTx, regardless of whether the donor heart was exposed to BSD or SHAM. The potency of (-)-noradrenaline was lower in left and right ventricles for BSD-Tx and SH-Tx compared to HC. ConclusionThese studies show that the combination of BSD and transplantation are likely to impair contractility of the donor heart, particularly for the RV. For the donor heart, this contractile dysfunction appears to be independent of changes to β1-adrenoceptor sensitivity. However, altered β1-adrenoceptor signalling is likely to be involved in post-HTx contractile dysfunction.

Oxycodone Effect on Pupil Constriction in Recreational Opioid Users: A Pharmacokinetic/Pharmacodynamic Meta-Analysis Approach.

Understanding the effect of oxycodone pharmacokinetics (PK) on µ-opioid receptor binding benefitsfrom an integrated approach to compiling the results of multiple studies. The current pharmacokinetic/pharmacodynamic (PK/PD)model analysis brings together various studies to support the interpretation of newly collected PK/PD data, putting the new results into the perspective of the full concentration-effect curve. A two-step modeling approach was applied to characterize the PK of oxycodone and its PK/PD relationshipfor the pupil diameter as a biomarker forµ-opioid receptor binding inrecreational opioid users. First, a model-based meta-analysis (MBMA) was used to quantify the state-of-the-art knowledge from seven published studies, each of which contained part of the data needed for full characterization. Subsequently, the estimated parameters with uncertainty from the MBMA were used as prior information for a model developed on newly collected clinical data after intranasal administration in a clinical abuse potential trial. The inclusion of intravenous data in the MBMAshowed thatthe PK of oxycodone can be described by a two-compartmental model, andallowed for the estimation of absolute bioavailability after intranasal and oral administration.A hysteresis loop was observed when plotting plasma concentrations and pupil constriction, which was approximated using an effect compartment. The totality of literature data enabled the identification of a Hill equation for the drug effect.The model with prior information fitted successfully to the newly collected data, where most parameter estimates had their confidence intervals overlapping with the prior distribution. The new data led to a slightly lower intranasal absorption rate constant, explaining the longer apparent half-life of oxycodone in the newly collected data. The PK/PD model parameters were confirmed by the new data, leading to the following estimates: half maximal inhibitory concentration (IC50) of26.5ng/mL, maximum pupil restriction of 66.0% from baseline, and a Hill factor of 1.05. The new data confirmed the PK profile and the PK/PD relationship identified using the MBMA, resulting in similar parameter estimates except for the intranasal absorption rate constant. The latter was lower than in the MBMA and explained the slightly longer apparent half-life of oxycodone in the newly collected data.

γ-Hydroxybutyric Acid: Pharmacokinetics, Pharmacodynamics, and Toxicology.

Gamma-hydroxybutyrate (GHB) is a short-chain fatty acid present endogenously in the brain and used therapeutically for the treatment of narcolepsy, as sodium oxybate, and for alcohol abuse/withdrawal. GHB is better known however as a drug of abuse and is commonly referred to as the "date-rape drug"; current use in popular culture includes recreational "chemsex," due to its properties of euphoria, loss of inhibition, amnesia, and drowsiness. Due to the steep concentration-effect curve for GHB, overdoses occur commonly and symptoms include sedation, respiratory depression, coma, and death. GHB binds to both GHB and GABAB receptors in the brain, with pharmacological/toxicological effects mainly due to GABAB agonist effects. The pharmacokinetics of GHB are complex and include nonlinear absorption, metabolism, tissue uptake, and renal elimination processes. GHB is a substrate for monocarboxylate transporters, including both sodium-dependent transporters (SMCT1, 2; SLC5A8; SLC5A12) and proton-dependent transporters (MCT1-4; SLC16A1, 7, 8, and 3), which represent significant determinants of absorption, renal reabsorption, and brain and tissue uptake. This review will provide current information of the pharmacology, therapeutic effects, and pharmacokinetics/pharmacodynamics of GHB, as well as therapeutic strategies for the treatment of overdoses. Graphical abstract.

Methadone-mediated sensitization of glioblastoma cells is drug and cell line dependent

Purposed,l-methadone (MET), an analgesic drug used for pain treatment and opiate addiction, has achieved attention from oncologists and social media as possible chemoensitizing agent in cancer therapy, notably brain cancer (glioblastoma multiforme, GBM). MET has been reported to enhance doxorubicin-induced cytotoxicity in GBM cells via activation of the µ-opioid receptor (MOR). Here, we extended this work and quantified the toxic effect of MET in comparison to other opioids alone and in combination with doxorubicin and the clinically more relevant alkylating drug temozolomide (TMZ), using a set of GBM cell lines and primary GBM cells.MethodsMOR expression in GBM cells was investigated by immunofluorescence and immunoblotting. Resistance to drugs alone and in combination with anticancer drugs was assessed by MTT assays. Concentration effect curves were fitted by nonlinear regression analysis and IC50 values were calculated. Apoptosis and necrosis rates were determined by annexin V/propidium iodide (PI)-flow cytometry.ResultsMET alone was cytotoxic in all GBM cell lines and primary GBM cells at high micromolar concentrations (IC50 ~ 60–130 µM), observed both in the metabolic MTT assay and by quantifying apoptosis and necrosis, while morphine and oxycodone were not cytotoxic in this concentration range. Naloxone was not able to block MET-induced cytotoxicity, indicating that cell death-inducing effects of MET are not MOR-dependent. We recorded doxorubicin and TMZ concentration- response curves in combination with fixed MET concentrations. MET enhanced doxorubicin-induced cytotoxicity in only one cell line, and in primary cells it was observed only in a particular MET concentration range. In all assays, MET was not effective in sensitizing cells to TMZ. In two cell lines, MET even decreased the cell's sensitivity to TMZ.ConclusionMET was found to be cytotoxic in GBM cells in vitro only at high, clinically not relevant concentrations, where it was effective in inducing apoptosis and necrosis. Sensitizing effects were only observed in combination with doxorubicin, but not with TMZ, and are dependent on cell line and the applied drug concentration. Therefore, our findings do not support the use of MET in the treatment of GBM in combination with TMZ, as no sensitizing effect of MET was observed.

Von Willebrand Factor Predicts Mortality in ACS Patients Treated with Potent P2Y12 Antagonists and is Inhibited by Aptamer BT200 Ex Vivo.

von Willebrand factor (VWF) is crucial for arterial thrombosis and its plasma levels are increased in acute coronary syndromes (ACSs). The effects of conventional platelet inhibitors are compromised by elevated VWF under high shear rates. BT200 is a third-generation aptamer that binds and inhibits the A1 domain of human VWF. This article aims to study whether VWF is a predictor of mortality in ACS patients under potent P2Y12 blocker therapy and to examine the effects of a VWF inhibiting aptamer BT200 and its concentrations required to inhibit VWF in plasma samples of patients with ACS. VWF activity was measured in 320 patients with ACS, and concentration effect curves of BT200 were established in plasma pools containing different VWF concentrations. Median VWF activity in patients was 170% (interquartile range % confidence interval [CI]: 85-255) and 44% of patients had elevated (> 180%) VWF activity. Plasma levels of VWF activity predicted 1-year (hazard ratio [HR]: 2.68; 95% CI: 1.14-6.31; p < 0.024) and long-term (HR: 2.59; 95% CI: 1.10-6.09) mortality despite treatment with potent platelet inhibitors (dual-antiplatelet therapy with aspirin and prasugrel or ticagrelor). Although half-maximal concentrations were 0.1 to 0.2 µg/mL irrespective of baseline VWF levels, increasing concentrations (0.42-2.13 µg/mL) of BT200 were needed to lower VWF activity to < 20% of normal in plasma pools containing increasing VWF activity (p < 0.001). VWF is a predictor of all-cause mortality in ACS patients under contemporary potent P2Y12 inhibitor therapy. BT200 effectively inhibited VWF activity in a target concentration-dependent manner.

The aptamer BT200 effectively inhibits von Willebrand factor (VWF) dependent platelet function after stimulated VWF release by desmopressin or endotoxin

Von Willebrand factor (VWF) plays a major role in arterial thrombosis. Antiplatelet drugs induce only a moderate relative risk reduction after atherothrombosis, and their inhibitory effects are compromised under high shear rates when VWF levels are increased. Therefore, we investigated the ex vivo effects of a third-generation anti-VWF aptamer (BT200) before/after stimulated VWF release. We studied the concentration-effect curves BT200 had on VWF activity, platelet plug formation under high shear rates (PFA), and ristocetin-induced platelet aggregation (Multiplate) before and after desmopressin or endotoxin infusions in healthy volunteers. VWF levels increased > 2.5-fold after desmopressin or endotoxin infusion (p < 0.001) and both agents elevated circulating VWF activity. At baseline, 0.51 µg/ml BT200 reduced VWF activity to 20% of normal, but 2.5-fold higher BT200 levels were required after desmopressin administration (p < 0.001). Similarly, twofold higher BT200 concentrations were needed after endotoxin infusion compared to baseline (p < 0.011). BT200 levels of 0.49 µg/ml prolonged collagen-ADP closure times to > 300 s at baseline, whereas 1.35 µg/ml BT200 were needed 2 h after desmopressin infusion. Similarly, twofold higher BT200 concentrations were necessary to inhibit ristocetin induced aggregation after desmopressin infusion compared to baseline (p < 0.001). Both stimuli elevated plasma VWF levels in a manner representative of thrombotic or pro-inflammatory conditions such as arterial thrombosis. Even under these conditions, BT200 potently inhibited VWF activity and VWF-dependent platelet function, but higher BT200 concentrations were required for comparable effects relative to the unstimulated state.

Determining the Properties of Omecamtiv Mecarbil on Human Explanted Cardiac Trabeculae and Its Interaction with Current Inotropes via the Beta 1-Adrenoceptor

Omecamtiv mecarbil (OM) is a first in class selective myosin activator, developed to increase contractile force without altering intracellular calcium concentrations in patients with advanced heart failure (AHF). We sought to investigate the effect of OM on contractility, arrhythmia genesis and interaction with Beta 1-adrenoceptor (B1AR) inotropes directly on explanted human failing heart. Trabeculae from 7 explanted hearts with AHF were dissected and set up to contract at 1 Hz. Trabeculae were incubated with or without OM (600 nM or 2 µM) for 120 mins. Cumulative concentration-effect curves were established to (-)-noradrenaline at B1ARs in the presence of the B2AR blocker (50 nM ICI 118,551) and phenoxybenzamine to block α1ARs and neuronal uptake. Contractile force and duration were recorded and observed for spontaneous contractions. OM prevented time dependent reduction (fade) in contractile force (Fig A, P=0.013 at 120mins). Trabeculae exhibited a concentration dependent increase in systolic ejection time (SET), associated with increased stroke volume, but this reduces the time for diastole. (Fig B). The concentration of (-)-noradrenaline required for 50% of maximal contractile force, the LogEC50, remained unchanged in the presence of OM (Mean LogEC50: -6.1 (control), -6.2 (600 nM), -6.1 (2 µM); P=0.7). (-)-Noradrenaline reversed OM mediated increases in SET. There was no significant increase in spontaneous contractions with OM concentrations up to 2 µM. OM maintains contractile force, independent of a B1AR mediated mechanism, without an increase in spontaneous contraction. In isolation OM increases SET at the cost of diastolic performance. Concomitant B1AR activation by (-)-noradrenaline normalises SET, improving ventricular filling. This finding may prove clinically relevant, especially when titrating traditional inotropes which increase metabolic demand.

A semi-compartmental model describing the pharmacokinetic-pharmacodynamic relationship.

Frequently, we encounter the phenomenon of hysteresis in kinetic-dynamic modeling. The hysteresis loop in the concentration-effect curve suggests a time discrepancy caused by various pharmacokinetic and pharmacodynamic factors. To collapse the hysteresis loop and to simplify the concentration-effect relationship, several kinetic-dynamic modeling approaches including the effect compartment link model, turnover model (indirect response model), and tolerance/rebound model, have been used. The semicompartmental model is one method to describe the hysteresis of the pharmacokinetic-pharmacodynamic relationship. Furthermore, this semi-compartmental model differs from other models (full parametric approaches) as it does not require pharmacokinetic parameters to estimate pharmacodynamic parameters and ke0. Therefore, we could employ a semi-compartmental approach in case it is difficult to apply the compartment model to pharmacokinetic data, as required for the pharmacodynamic analysis of inhalational anesthetics.

025 Determining the Effects of Omecamtiv Mecarbil on Human Failing Ventricular Trabeculae and Interaction With (-)-Noradrenaline Via the β1-adrenoceptor

Omecamtiv mecarbil (OM) is a novel medicine for systolic heart failure, targeting myosin to enhance cardiomyocyte performance. However, OM had negative diastolic effects and evidence of ischaemia at supratherapeutic doses. To assist translation to clinical practice we investigated OMs effect on ventricular trabeculae from explanted human failing hearts, specifically; contractile dynamics, interaction with the β1–adrenoceptor (AR) agonist (-)-noradrenaline and arrhythmia genesis. Ventricular trabeculae from explanted failing hearts were paced under tension and incubated with or without a single concentration of OM for 120 mins. Time to peak force (TPF) and 50% relaxation (t50%) were recorded. Cumulative concentration-effect curves were established to (-)-noradrenaline at β1-ARs in the absence or presence of OM, whilst β2-AR, α1-ARs and neuronal uptake were blocked. Stimulators were switched off and trabeculae observed for arrhythmic contractions. OM prolonged TPF and t50% in ventricle (600 nM, 2 mM, p<0.001) but had no effect on inotropic force. OM did not affect the generation of arrhythmic contractions. The potency of (-)-noradrenaline (pEC50 6.05±0.10), for inotropic effect, was unchanged in the presence of OM 600 nM or 2 μM. (-)-Noradrenaline reversed the negative diastolic effects of OM at maximal β1-AR effect. In ventricular trabeculae from explanted human failing hearts, OM prolonged TPF and t50%, but had no effect on contractile force, generation of arrhythmic contractions or potency of (-)-noradrenaline at β1-ARs. The competing diastolic effects of OM and (-)-noradrenaline may prove clinically important. If used together in patients with cardiogenic shock.

Особливості спазмолітичної дії препарату «Беластезин»

Мета дослідження – порівняльне вивчення спазмолітичної активності препарату «Беластезин», таблетки та атропіну на моделі ацетилхолін-залежної скоротливої активності (міогенної та нейрогенної) ізольованих смужок гладеньких м’язів кишківника (ГМК) мурчаків. Дослідження було проведене на 10 мурчаках-самцях (масою 300–350 г). Для запобігання можливості інтерферуючого впливу бензокаїну на скоротливі реакції гладеньких м’язів вилучення алкалоїдів з таблеткової маси проводили у водному розчині, а на смужках ГМК тестували лише фракції водорозчинних сполук. Зважаючи на загальну подібність механізму дії та фізіологічних ефектів, специфічну активність Беластезину порівнювали з атропіну сульфатом. Введення досліджуваного препарату та атропіну в діапазоні концентрацій (10–9–10–6 моль/л) у камеру зі смужками одночасно з ацетилхоліном (АХ) (10–6 моль/л) викликало зниження амплітуди та інтегральної інтенсивності АХ-стимульованої скоротливої активності ГМК. Беластезин відрізнявся від атропіну за характером антагоністичної взаємодії з М-холінорецепторами, про що свідчили зміни в нахилі кривої «концентрація-ефект» (рівняння Больцмана). Беластезин у діапазоні низьких концентрацій (10–10–10–7 моль/л) підвищував інтенсивність скоротливої діяльності смужок у разі електростимуляції, тоді як атропін знижував скоротливу активність ГМК. Відмінності в дії препарату, які були відмічені за умов функціонального тесту, дозволяють висловити припущення, що Беластезин виявляв активність більш характерну для скополаміну (гіосцину), ніж для атропіну (гіосціаміну). В умовах дозозалежної АХ-стимуляції скоротливої активності ГМК Беластезин викликав суттєве зрушення кривих «концентрація-ефект» порівняно з помірним ефектом атропіну. Досліджуваний препарат також зменшував амплітуду максимальної відповіді на відміну від атропіну. Отримані результати свідчать про те, що природна суміш алкалоїдів у складі Беластезину більш ефективна, ніж атропін щодо усунення спастичної діяльності гладеньких м’язів, яка спричинена стимуляцією М-холінорецепторів.