Demonstrating Assay Sensitivity Research Articles

Orexin 2 receptor-selective agonist danavorexton (TAK-925) promotes wakefulness in non-human primates and healthy individuals.

The orexin 2 receptor-selective agonist danavorexton (TAK-925) has been shown to produce wake-promoting effects in wild-type mice, narcolepsy-model mice, and individuals with narcolepsy type 1 and type 2. Here, we report wake-promoting effects of danavorexton in non-human primates and healthy men during their sleep phase. Electroencephalogram analyses revealed that subcutaneous administration of danavorexton significantly increased wakefulness in common marmosets (p < 0.05 at 0.1mg kg-1 , and p < 0.001 at 1mg kg-1 and 10mg kg-1 ) and cynomolgus monkeys (p ≤ 0.05 at 1mg kg-1 and 3mg kg-1 ). In a phase 1b crossover, randomized, double-blind, placebo-controlled and active-controlled study in sleep-deprived healthy participants (ClinicalTrials.gov identifier: NCT03522506), modafinil 300 mg (used to demonstrate assay sensitivity) and continuous infusion of danavorexton 44 mg and danavorexton 112 mg showed statistically superior wake-promoting effects to placebo (n=18). Measured using the Maintenance of Wakefulness Test, mean (standard deviation) sleep latencies during infusion of danavorexton 44 mg, danavorexton 112 mg and placebo were 21.4 (8.9), 31.8 (3.2) and 9.2 (6.4) min, respectively. Least-squares mean difference from placebo in average sleep latency was 16.8min with danavorexton 44 mg and 30.2min with danavorexton 112 mg (both p < 0.001). Karolinska Sleepiness Scale scores were statistically significantly lower (indicating decreased sleepiness) for participants receiving danavorexton than for those receiving placebo during infusion (danavorexton 44 mg, p=0.010; danavorexton 112 mg, p < 0.001). Together, these results indicate that an orexin 2 receptor agonist increases wakefulness in non-human primates and healthy individuals during their sleep phase.

A Localized CRISPR Assay that Detects Short Nucleic Acid Fragments in Unamplified Genetically Modified Samples.

Detecting short genetically modified (GM) nucleic acid fragments in GM crops and associated products is critically important for the global agriculture industry. Although nucleic acid amplification-based technologies have been widely used for genetically modified organism (GMO) detection, they still struggle to amplify and detect these ultra-short nucleic acid fragments in highly processed products. Here, we used a multiple-CRISPR-derived RNA (crRNA) strategy to detect ultra-short nucleic acid fragments. By combining confinement effects on local concentrations, an amplification-free CRISPR-based short nucleic acid (CRISPRsna) system was established to detect the cauliflower mosaic virus 35S promoter in GM samples. Moreover, we demonstrated assay sensitivity, specificity, and reliability by directly detecting nucleic acid samples from GM crops with a wide genomic range. The CRISPRsna assay avoided possible aerosol contamination from nucleic acid amplification and saved time due to an amplification-free approach. Given that our assay displayed distinct advantages over other technologies in detecting ultra-short nucleic acid fragments, it may have wide applications for detecting GM in highly processed products.

HERG block potencies for 5 positive control drugs obtained per ICH E14/S7B Q&As best practices: Impact of recording temperature and drug loss

According to the ICH S7B guideline, drug candidates are screened for hERG block prior to first-in-human testing to predict the likelihood of delayed repolarization associated with a rare, but life-threatening, ventricular tachyarrhythmia. The new ICH E14 Q&As guideline allows hERG results to be used in later clinical development for decision-making (Q&As 5.1 and 6.1). To pursue this path, the hERG assay should be conducted following the new ICH S7B Q&A 2.1 guideline, which calls for best practice considerations of the recording temperature, voltage protocol, stimulation frequency, recording/data quality, and concentration verification. This study investigated hERG block by cisapride, dofetilide, terfenadine, sotalol, and E-4031 – positive controls commonly used to demonstrate assay sensitivity – using the manual whole cell patch clamp method and an action potential-like voltage protocol presented at 0.2 Hz. Recordings were conducted at room and near physiological temperature. Drug concentrations were measured using samples collected during real patch clamp experiments and satellite experiments. Results showed temperature effects for E-4031, terfenadine, and sotalol, but not cisapride and dofetilide. Cisapride and terfenadine showed substantial concentration losses, largely due to nonspecific binding to the perfusion apparatus. Using concentrations measured from the real and satellite experiments to assess block potencies yielded comparable results, indicating that satellite sample collection may be viable for drugs with nonspecific binding concerns only. In summary, this study provides block potencies for 5 hERG positive controls, and serves as a case study for hERG assays conducted, and results illustrated in accordance with the new ICH E14/S7B Q&As.

The effects of intranasal esketamine on on-road driving performance in patients with major depressive disorder or persistent depressive disorder.

Background:Intranasal esketamine demonstrates rapid improvement of depressive symptoms. However, transient adverse effects (dissociation, sedation and dizziness) may occur, which could impact driving performance.Aims:To evaluate the effects of 84 mg intranasal esketamine on driving performance in unipolar major depressive disorder (MDD) or persistent depressive disorder (PDD) patients.Methods:The study consisted of two parts. Part A was a single-blind, double-dummy, randomized three-period, cross-over study to compare effects of esketamine versus placebo on next morning driving, 18 ± 2 h post-treatment. Alcohol was administered to demonstrate assay sensitivity. In Part B, same-day driving, 6 ± 0.5 hours post-treatment, was assessed during twice weekly esketamine administration for 3 weeks. Twenty-seven patients with mild-to-moderate MDD or PDD without psychotic features completed a 100 km on-the-road driving test on a public highway in normal traffic. The primary outcome was standard deviation of lateral position (SDLP; cm; weaving of car).Results:In Part A, alcohol impaired driving performance compared to placebo: Least-square means (95% CI), p-value for delta SDLP (cm) compared with placebo: (ΔSDLP = + 1.83 (1.03; 2.62), p < 0.001), whereas esketamine did not: (ΔSDLP = −0.23 (−1.04; 0.58), p = 0.572). In Part B, weekly driving tests showed no differences between placebo baseline SDLP and after esketamine administration over 3 weeks: Day 11: (ΔSDLP = −0.96 (−3.72; 1.81), p = 0.493), Day 18: (ΔSDLP = −0.56 (−3.33; 2.20), p = 0.686) and Day 25: (ΔSDLP = −1.05 (−3.82; 1.71), p = 0.451).Conclusions:In this study, esketamine did not impair on-road driving performance the next morning following a single dose, or on same day after repeated administration.

Abstract CT128: Effect of futibatinib on QT/QTc interval: a randomized, controlled, double-blind study

Abstract Background Futibatinib is an oral selective irreversible FGFR inhibitor with clinical activity in cholangiocarcinoma and other FGFR-deregulated tumors. A phase 1 thorough QT study was conducted to evaluate the effect of futibatinib on the heart rate (HR)-corrected QT (QTc) interval. Methods Healthy nonsmoking adults 18-55 years of age were randomized to receive single doses of each of the following treatments in different sequences: futibatinib (20 or 80 mg [therapeutic and supratherapeutic doses, respectively]), placebo, or moxifloxacin (positive QTc-prolongation control; 400 mg). At least 7 days were allowed between administration of each of the 4 treatments. Administration of futibatinib and placebo was double-blind; that of moxifloxacin was open-label. The primary endpoint was the difference in change from baseline in QTcF (QT interval corrected using Fridericia's formula) between futibatinib and placebo (ddQTcF) at the time points tested. Upper limits of the 2-sided 90% confidence intervals (CIs) of these differences of &amp;lt;10 ms indicated lack of QTc prolongation (per ICH E14 guidance). Secondary endpoints included change from baseline in other electrocardiogram (ECG) parameters, pharmacokinetics (PK), and safety. Results Enrolled participants (N=48) received ≥1 dose of futibatinib, were evaluable for futibatinib PK, and were included in the analysis population. The ddQTcF ranged from 0.13 to 2.47 ms with 20 mg futibatinib and from -0.04 to 2.46 ms with 80 mg futibatinib from 0.5 to 24 h after dosing. Upper limits of the 2-sided 90% CIs of these values remained well below 10 ms (&amp;lt;5 ms) throughout. The lower limits of 2-sided 97.5% CIs of the difference in change from baseline in QTcF between moxifloxacin and placebo all exceeded 5 ms (8.06-11.43 ms) at the time points analyzed (1.5, 2, 3, and 4 h), demonstrating assay sensitivity. No clinically significant effects of futibatinib on HR, other ECG parameters (eg, PR, QRS, or QT intervals), or ECG morphology were identified. Exposure-response modeling indicated no significant relationship between plasma futibatinib concentration and the QTcF interval. The futibatinib area under the concentration curve from time 0 to infinity and maximum plasma concentration (Cmax) increased by 4.5- and 3.3-fold, respectively, after administration of a single dose of 80 vs 20 mg. The Cmax values observed with 80 mg futibatinib (median, 553 ng/mL; range, 71-1160) exceeded any Cmax observed or expected with the 20-mg daily therapeutic dose at steady state. Seven participants (15%) experienced adverse events of any cause after receiving either futibatinib dose, all grade 1/2. Conclusions A single therapeutic or supratherapeutic dose of futibatinib did not prolong the QTc interval or adversely affect other measures of heart function. Futibatinib exposure increased dose-dependently, and administration was safe and well tolerated in healthy participants. Citation Format: Ikuo Yamamiya, John Laabs, Daryl Sonnichsen, Mark Mina, Yaohua He, Karim Benhadji. Effect of futibatinib on QT/QTc interval: a randomized, controlled, double-blind study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT128.

Association Between Vomiting and QT Hysteresis: Data from a TQT Study with the Endothelin A Receptor Antagonist Clazosentan.

This study investigated the potential QT liability of the selective endothelin-1 A receptor antagonist clazosentan at a therapeutic (20mg/h) and supratherapeutic (60mg/h) intravenous (i.v.) dose. A randomized, placebo- and moxifloxacin-controlled, double-blind, 3-period, crossover study was conducted in 36 healthy subjects receiving clazosentan (20mg/h followed by 60mg/h i.v. for 3h each), placebo (i.v. for 6h), and moxifloxacin (single oral dose of 400mg concomitantly with placebo i.v. for 6h). At least three replicate ECGs were extracted from Holter recordings at predefined time points from 1h pre-dose to 24h after end of infusion. Pharmacokinetic blood sampling was performed for concentration/QT analysis (primary endpoint). For moxifloxacin, the lower bound of the 90% confidence interval (CI) of baseline- and placebo-corrected QTcF (ΔΔQTcF) was > 5ms at its maximum plasma concentration together with a positive slope of the concentration/QT regression line demonstrating assay sensitivity. For clazosentan, time of peak exposure preceded maximum ΔΔQTcF by 4h indicating delayed QT-prolonging effects leading to invalidity of the concentration/QT analysis. The secondary by-time-point analysis revealed QT liability of clazosentan (i.e., upper bound of 90% CI ∆∆QTcF > 10ms). Delayed QT prolongation (i.e., hysteresis) was predominantly observed in subjects with nausea and vomiting, potentially caused by vagal reaction and/or decreases in potassium concentration. By contrast, there was no association with other adverse events, food intake, or concomitant medication. In conclusion, clazosentan at therapeutic and supratherapeutic doses has QT liability with hysteresis effects being associated with nausea and vomiting.

Randomized study of the effect of gadopiclenol, a new gadolinium-based contrast agent, on the QTc interval in healthy subjects.

We investigated the effect of gadopiclenol, a new gadolinium-based contrast agent, on the QTc interval at clinical and supraclinical dose, considering the relative hyperosmolarity of this product. This was a single centre, randomized, double-blind, placebo- and positive-controlled, 4-way crossover study. Forty-eight healthy male and female subjects were included to receive single intravenous (i.v.) administrations of gadopiclenol at the clinical dose of 0.1 mmol kg-1 , standard for current gadolinium-based contrast agents, the supraclinical dose of 0.3 mmol kg-1 , placebo and a single oral dose of 400 mg moxifloxacin. The largest time-matched placebo-corrected, mean change from-baseline in QTcF (ΔΔQTcF) was observed 3 hours after administration of 0.1 mmol kg-1 gadopiclenol (2.39 ms, 90% confidence interval [CI]: 0.35, 4.43 ms) and 5 minutes after administration of 0.3 mmol kg-1 (4.81 ms, 90%CI: 2.84, 6.78 ms). The upper limit of the 90% CI was under the threshold of 10 ms, demonstrating no significant effect of gadopiclenol on QTc interval. From 1.5 to 4 hours postdose moxifloxacin, the lower limit of the 90% CI of ΔΔQTcF exceeded 5 ms demonstrating assay sensitivity. Although there was a positive slope, the concentration-response analysis estimated that the values of ΔΔQTcF at the maximal concentration of gadopiclenol at 0.1 and 0.3 mmol kg-1 were 0.41 and 2.23 ms, respectively, with the upper limit of the 90% CI not exceeding 10 ms. No serious or severe adverse events or treatment discontinuations due to adverse events were reported. This thorough QT/QTc study demonstrated that gadopiclenol did not prolong the QT interval at clinical and supraclinical doses and was well tolerated in healthy volunteers. The positive slope of the QTc prolongation vs concentration relationship suggests that hyperosmolarity could be associated with QTc prolongation. However, the amplitude of this effects is unlikely to be associated with proarrhythmia.

Qualification and Clinical Validation of an Immunodiagnostic Assay for Detecting 11 Additional Streptococcus pneumoniae Serotype-specific Polysaccharides in Human Urine.

BackgroundIdentifying Streptococcus pneumoniae serotypes by urinary antigen detection (UAD) assay is the most sensitive way to evaluate the epidemiology of nonbacteremic community-acquired pneumonia (CAP). We first described a UAD assay to detect the S. pneumoniae serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, covered by the licensed 13-valent S. pneumoniae conjugate vaccine. To assess the substantial remaining pneumococcal disease burden after introduction of several pneumococcal vaccines, a UAD-2 assay was developed to detect 11 additional serotypes (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, and 33F) in individuals with radiographically confirmed CAP.MethodsThe specificity of the UAD-2 assay was achieved by capturing pneumococcal polysaccharides with serotype-specific monoclonal antibodies, using Luminex technology. Assay qualification was used to assess accuracy, precision, and sample linearity. Serotype positivity was based on cutoffs determined by nonparametric statistical evaluation of urine samples from individuals without pneumococcal disease. The sensitivity and specificity of the positivity cutoffs were assessed in a clinical validation, using urine samples obtained from a large study that measured the proportion of radiographically confirmed CAP caused by S. pneumoniae serotypes in hospitalized US adults.ResultsThe UAD-2 assay was shown to be specific and reproducible. Clinical validation demonstrated assay sensitivity and specificity of 92.2% and 95.9% against a reference standard of bacteremic pneumonia. In addition, the UAD-2 assay identified a S. pneumoniae serotype in 3.72% of nonbacteremic CAP cases obtained from hospitalized US adults. When combined with bacteremic CAP cases, the proportion of pneumonias with a UAD-2 serotype was 4.33%.ConclusionsThe qualified/clinically validated UAD-2 method has applicability in understanding the epidemiology of nonbacteremic S. pneumoniae CAP and for assessing the efficacy of future pneumococcal conjugate vaccines that are under development.

244. Development, Qualification, and Clinical Validation of an Immunodiagnostic assay for the Detection of 11 Additional S. pneumoniae Serotype-Specific Polysaccharides in Human Urine

BackgroundIdentifying Streptococcus pneumoniae (Sp) serotypes by urinary antigen detection assay (UAD) is the most sensitive and specific way to evaluate the changing epidemiology of non-bacteremic community-acquired pneumonia (CAP) and efficacy of pneumococcal vaccines. We first described an UAD to detect the Sp serotypes 1,-3,-4,-5,-6A,-6B,-7F,-9V,-14,-18C,-19A,-19F,-23F covered by the 13-valent Sp conjugate vaccine PCV13. To assess the pneumococcal disease burden of additional serotypes, a UAD-2 assay was developed to diagnose 11 additional Sp serotypes (-2,-8,-9N,-10A,-11A,-12F,-15B,-17F,-20,-22F,-33F).MethodsUAD-2 specificity was achieved by capturing highly purified pneumococcal polysaccharides with serotype-specific monoclonal antibodies using Luminex technology. Assay qualification assessed accuracy, precision, and sample linearity. Serotype positivity was based on cutoffs determined by non-parametric statistical evaluation of urine samples from individuals without pneumococcal disease. Clinical sensitivity and specificity of the positivity cutoffs were assessed in a clinical validation.ResultsThe UAD-2 was shown to be specific and reproducible. Clinical validation using urine samples from invasive disease patients demonstrated assay sensitivity and specificity of 92.2% and 95.9%, respectively compared with a gold standard of isolating and typing (by Quellung) Sp bacteria from patient samples. Analysis of 11,087 CAP patients showed a UAD-2 and UAD-1 serotype prevalence of 4.33% and 4.60%, respectively (bacteremic and non-bacteremic CAP combined).ConclusionThe qualified/clinically validated UAD-2 method has applicability in understanding the epidemiology of nonbacteremic Sp CAP as well as assessing vaccine efficacy of future pneumococcal conjugate vaccines.Disclosures All authors: No reported disclosures.

Effects of Dihydroartemisinin-Piperaquine Phosphate and Artemether-Lumefantrine on QTc Interval Prolongation

QT/QTc interval prolongation reflects delayed cardiac repolarization which can lead to Torsade de Pointes and sudden death. Many antimalarial drugs prolong QT/QTc interval. However, due to confounding factors in patients with malaria, the precise extent of this effect has been found to be highly variable among studies. We compared the effects of dihydroartemisinin-piperaquine phosphate (DHA-PQP) and artemether-lumefantrine (A-L) on QT interval duration in healthy volunteers. In this randomized, parallel groups, active moxifloxacin- and placebo-controlled study, prolongation of the QT/QTc interval following treatment with DHA-PQP in fasted and fed condition and A-L in fed state was investigated in healthy subjects (n = 287; Clinicaltrials.gov: NCT01103830). DHA-PQP resulted in significant mean (95% confidence interval (CI)) maximum increases in QTc Fridericia (QTcF) of 21.0 ms (15.7, 26.4) for DHA-PQP fasted, 35.9 ms (31.1, 40.6) for DHA-PQP high-fat/low-caloric and 46.0 ms (39.6, 52.3) for DHA-PQP high-fat/high-caloric breakfast. For A-L, the largest difference from baseline relative to placebo was 9.9 ms (95% CI: 6.8, 12.9). Increases in QTcF related to maximum plasma concentrations of piperaquine. Moxifloxacin demonstrated assay sensitivity. Increases in QTcF following DHA-PQP and A-L were clinically relevant. Food increased piperaquine exposure and QTcF interval prolongation emphasizing the need to administer DHA-PQP in the fasting state.

Thorough QT/QTc Evaluation of the Cardiac Safety of Secnidazole at Therapeutic and Supratherapeutic Doses in Healthy Individuals.

SYM‐1219, a novel oral granule formulation of secnidazole, is under development as single‐dose treatment for bacterial vaginosis. This 4‐way, randomized, crossover study evaluated the effects of SYM‐1219 on electrocardiographic (ECG) parameters in 52 healthy subjects. Subjects were administered single doses of SYM‐1219, 2 g (proposed therapeutic dose), 6 g (supratherapeutic dose), placebo, and moxifloxacin (positive control). Serial digital 12‐lead ECGs were recorded pre‐ and postdose; blood samples were taken to determine plasma secnidazole concentrations. A high‐precision QT technique measured ECGs. The primary end point was change from baseline QTcF (∆QTcF); data were analyzed with the objective of excluding QT effects >10 milliseconds at postdosing time points and with exposure‐response analysis. Safety and tolerability were assessed. Single doses of 2 g and 6 g SYM‐1219 did not have a clinically relevant effect on the QTcF interval; an effect >10 milliseconds could be excluded at all postdosing time points. A shallow slope of the exposure‐response relationship was seen (0.058 millisecond per μg/mL; 90%CI 0.042, 0.073); in this model, the effect on QTc can be predicted to be <10 milliseconds up to a secnidazole plasma concentration of ∼125 μg/mL, approximately 3.4‐fold higher than anticipated peak therapeutic plasma levels. The moxifloxacin QT response demonstrated assay sensitivity. The most frequently reported treatment‐emergent adverse events with SYM‐1219 were headache, dizziness, and nausea. This thorough QT study demonstrated that SYM‐1219 in doses and plasma concentrations up to 3‐fold above therapeutically relevant levels does not have a clinically concerning effect on ECG parameters, including the QT interval.

Quantification of 1,3-β-D-glucan from yeast added as a functional ingredient to bread

Due to their immunomodulatory effect, 1,3-β-G from yeast are used as functional ingredients, but reliable methods for their detection in foods are lacking. We have adapted a method based on fluorescence detection with aniline blue to quantify the amount of five commercial yeast β-glucan preparations added to crisp or yeast-leavened bread. This assay detected yeast β-glucan preparations added to different breads with an average recovery of 90, 96, 99 and 105%, while one of the preparations was overestimated, with an average recovery of 157%. The presence of cereal 1,3–1,4-β-D-glucans did not interfere with assay performance. The addition of 1,3-β-G at 0.2 and 0.5 g/100g is low compared to the recommended dose of 1,3-β-G per serving demonstrating assay sensitivity. However, more research is needed to fully understand the effect of 1,3-β-G conformation/structure on aniline blue interaction as well as the effect of baking on structure and dissolution properties of yeast β-glucans.

Tapentadol prolonged-release for moderate-to-severe chronic osteoarthritis knee pain: a double-blind, randomized, placebo- and oxycodone controlled release-controlled study

Objective: To assess efficacy and safety of tapentadol prolonged release (PR) for moderate-to-severe chronic osteoarthritis knee pain.Methods: Patients (n = 990) were randomized (1:1:1) to tapentadol PR, oxycodone controlled release (CR; reference compound for assay sensitivity), or placebo for a double-blind 3-week titration and 12-week maintenance period. Primary efficacy end-points were change from baseline in average pain intensity at week 12 of maintenance (US end-point) and over the entire maintenance period (non-US end-point) with “last observation carried forward” as imputation method for missing scores.Results: Both primary end-points were not significantly different for tapentadol PR nor for oxycodone CR vs placebo at week 12 (least squares [LS] mean difference = –0.3 [95% CI = –0.61–0.09]; p = 0.152 and 0.2 [95% CI = –0.16–0.54]; p = 0.279, respectively) and over the maintenance period (LS mean difference = –0.2 [95% CI = –0.55–0.07]; p = 0.135 and 0.1 [95% CI = –0.18–0.44]; p = 0.421, respectively). Considerably more patients receiving tapentadol PR than oxycodone CR completed the trial (58.3% vs 36.6%). This is consistent with better results with tapentadol PR on the overall health status (PGIC) compared to oxycodone CR. Indeed, respectively, 56% and 42.5% rated at least “much improved” at the end of treatment. Incidences of gastrointestinal adverse events were higher for both active treatments compared to placebo. Tapentadol PR was associated with a better gastrointestinal tolerability profile with incidences of constipation (17.9% vs 35%) and of the composite of nausea and/or vomiting (23.8% vs 46.8%) significantly lower vs oxycodone CR (p < 0.001).Conclusions: The study did not demonstrate assay sensitivity. The finding that both primary end-points for tapentadol PR were not met can, thus, not be interpreted. Tapentadol PR was better tolerated than oxycodone CR, largely due to fewer gastrointestinal side-effects.

Single Doses up to 800 mg of E-52862 Do Not Prolong the QTc Interval--A Retrospective Validation by Pharmacokinetic-Pharmacodynamic Modelling of Electrocardiography Data Utilising the Effects of a Meal on QTc to Demonstrate ECG Assay Sensitivity.

BackgroundE-52862 is a Sigma-1 receptor antagonist (S1RA) currently under investigation as a potential analgesic medicine. We successfully applied a concentration-effect model retrospectively to a four-way crossover Phase I single ascending dose study and utilized the QTc shortening effects of a meal to demonstrate assay sensitivity by establishing the time course effects from baseline in all four periods, independently from any potential drug effects.MethodsThirty two healthy male and female subjects were included in four treatment periods to receive single ascending doses of 500 mg, 600 mg or 800 mg of E-52862 or placebo. PK was linear over the dose range investigated and doses up to 600 mg were well tolerated. The baseline electrocardiography (ECG) measurements on Day-1 were time-matched with ECG and pharmacokinetic (PK) samples on Day 1 (dosing day).ResultsIn this conventional mean change to time-matched placebo analysis, the largest time-matched difference to placebo QTcI was 1.44 ms (90% CI: -4.04, 6.93 ms) for 500 mg; -0.39 ms (90% CI: -3.91, 3.13 ms) for 600 mg and 1.32 ms (90% CI: -1.89, 4.53 ms) for 800 mg of E-52862, thereby showing the absence of any QTc prolonging effect at the doses tested. In addition concentration-effect models, one based on the placebo corrected change from baseline and one for the change of QTcI from average baseline with time as fixed effect were fitted to the data confirming the results of the time course analysis.ConclusionThe sensitivity of this study to detect small changes in the QTc interval was confirmed by demonstrating a shortening of QTcF of -8.1 (90% CI: -10.4, -5.9) one hour and -7.2 (90% CI: -9.4, -5.0) three hours after a standardised meal.Trial RegistrationEU Clinical Trials Register EudraCT 2010 020343 13

Evaluation of opicapone on cardiac repolarization in a thorough QT/QTc study.

Opicapone, a novel third-generation catechol-O-methyltransferase inhibitor for use as adjunctive therapy in levodopa-treated Parkinson's disease patients, was investigated on cardiac repolarization in healthy adult volunteers. This was a single-center, randomized, double-blind, placebo-controlled, open-label active-controlled, 4-period crossover study conducted in 64 subjects. In each period, subjects received a single oral dose of 50 mg opicapone, 800 mg opicapone, placebo, or 400 mg moxifloxacin and 24-hour 12-lead Holter monitoring was performed on day -1 (baseline) and after each single dose. After a single oral administrations of 50 and 800 mg opicapone, opicapone was the major entity in the circulation, with a median tmax of 1.5-2.0 hours. Opicapone was rapidly eliminated, with an elimination half-life of 1-2 hours. There was no clinically relevant effect of 50 and 800 mg opicapone versus placebo on cardiac depolarization or repolarization. All upper bounds of the 1-sided 95% confidence interval (CI) were below 10 milliseconds, confirming that opicapone has no QT-prolonging effect. Moxifloxacin caused an increase in the QTcI, with a lower bound of the 2-sided 95% CI always higher than 5 milliseconds, around the tmax of peak concentration, demonstrating assay sensitivity. In conclusion, administration of opicapone at therapeutic (50 mg) and supratherapeutic (800 mg) doses did not induce a clinically significant prolongation of the QTc interval.

Using proximity biotinylation to detect herpesvirus entry glycoprotein interactions: Limitations for integral membrane glycoproteins

Herpesvirus entry into cells requires coordinated interactions among several viral transmembrane glycoproteins. Viral glycoproteins bind to receptors and interact with other glycoproteins to trigger virus–cell membrane fusion. Details of these glycoprotein interactions are not well understood because they are likely transient and/or low affinity. Proximity biotinylation is a promising protein–protein interaction assay that can capture transient interactions in live cells. One protein is linked to a biotin ligase and a second protein is linked to a short specific acceptor peptide (AP). If the two proteins interact, the ligase will biotinylate the AP, without requiring a sustained interaction. To examine herpesvirus glycoprotein interactions, the ligase and AP were linked to herpes simplex virus 1 (HSV1) gD and Epstein Barr virus (EBV) gB. Interactions between monomers of these oligomeric proteins (homotypic interactions) served as positive controls to demonstrate assay sensitivity. Heterotypic combinations served as negative controls to determine assay specificity, since HSV1 gD and EBV gB do not interact functionally. Positive controls showed strong biotinylation, indicating that viral glycoprotein proximity can be detected. Unexpectedly, the negative controls also showed biotinylation. These results demonstrate the special circumstances that must be considered when examining interactions among glycosylated proteins that are constrained within a membrane.

Tafenoquine at therapeutic concentrations does not prolong fridericia‐corrected QT interval in healthy subjects

Tafenoquine is being developed for relapse prevention in Plasmodium vivax malaria. This Phase I, single-blind, randomized, placebo- and active-controlled parallel group study investigated whether tafenoquine at supratherapeutic and therapeutic concentrations prolonged cardiac repolarization in healthy volunteers. Subjects aged 18–65 years were randomized to one of five treatment groups (n = 52 per group) to receive placebo, tafenoquine 300, 600, or 1200 mg, or moxifloxacin 400 mg (positive control). Lack of effect was demonstrated if the upper 90% CI of the change from baseline in QTcF following supratherapeutic tafenoquine 1200 mg versus placebo (ΔΔQTcF) was <10 milliseconds for all pre-defined time points. The maximum ΔΔQTcF with tafenoquine 1200 mg (n = 50) was 6.39 milliseconds (90% CI 2.85, 9.94) at 72 hours post-final dose; that is, lack of effect for prolongation of cardiac depolarization was demonstrated. Tafenoquine 300 mg (n = 48) or 600 mg (n = 52) had no effect on ΔΔQTcF. Pharmacokinetic/pharmacodynamic modeling of the tafenoquine–QTcF concentration–effect relationship demonstrated a shallow slope (0.5 ms/μg mL–1) over a wide concentration range. For moxifloxacin (n = 51), maximum ΔΔQTcF was 8.52 milliseconds (90% CI 5.00, 12.04), demonstrating assay sensitivity. In this thorough QT/QTc study, tafenoquine did not have a clinically meaningful effect on cardiac repolarization.

Cardiac safety of rasagiline, a selective monoamine oxidase type B inhibitor for the treatment of Parkinson’s disease: a thorough QT/QTc study

Rasagiline is a selective, irreversible monoamine oxidase type B inhibitor, developed for the treatment of Parkinson's disease. In compliance with current regulatory requirements, rasagiline underwent a thorough QT/QTc (TQT) study to assess its potential to prolong cardiac repolarization. The primary aim of this study was to evaluate the effects of clinical (1 mg/day) and supratherapeutic (2 mg/day and 6 mg/day) multiple oral doses of rasagiline on the baseline- and placebo-adjusted QTc interval (delta delta QTc (ddQTc)). Other electrocardiogram parameters, pharmacokinetic assessments, safety and tolerability as well as vital signs were investigated. This was a five-arm, randomized, double-blind, placebo- and active-controlled, and parallel study in healthy subjects. Moxifloxacin (400 mg) positive control was included to demonstrate assay sensitivity. 247 of 250 randomized subjects completed the study. Time-matched analysis of ddQTc yielded two-sided 90% confidence intervals for all rasagiline doses below the 10 ms regulatory threshold, showing no effect on cardiac repolarization. Concentration-effect analysis demonstrated no relationships between rasagiline (and its metabolite 1-aminoindan), plasma concentrations, and ddQTc. The pharmacokinetic profile of rasagiline was consistent with previous studies. Adverse events were mild to moderate in intensity and were similar across all treatment groups. There were no clinically significant changes in heart rate and systolic blood pressure. This TQT study demonstrated a favorable cardiac safety profile of rasagiline.

Albiglutide Does Not Prolong QTc Interval in Healthy Subjects: A Thorough ECG Study

IntroductionAlbiglutide, a selective once-weekly glucagon-like peptide-1 receptor agonist, is being developed for the treatment of type 2 diabetes mellitus. Albiglutide’s effect on cardiac repolarization (QTc interval) was assessed in a randomized, double-blind, placebo-controlled, parallel-group study in healthy subjects with a nested crossover comparison for moxifloxacin.MethodsSubjects were randomized to albiglutide (n = 85) or placebo (n = 89) and received injections of 30 mg albiglutide or placebo on Days 1 and 8 and 50 mg albiglutide or placebo on Days 15, 22, 29, and 36. In the placebo group, moxifloxacin was administered on Day −1 in half the subjects and on Day 40 in the other half. Blood samples for albiglutide plasma concentration were drawn on Days 4 and 39 and serial ECGs were extracted from continuous recordings on Days −2 (baseline), −1, 4, 39, and 40.ResultsDemographics were generally similar between albiglutide and placebo subjects: mean age was 29 years and BMI 25 kg/m2. Mean change-from-baseline QTcI (∆QTcI, which was corrected for individual heart rate) on Day 4 after a single dose of albiglutide 30 mg and on Day 39 after repeat dosing with albiglutide 50 mg once weekly was similar to the placebo response. The placebo-corrected ΔQTcI (ΔΔQTcI) on both albiglutide doses was small with the largest ΔΔQTcI of 1.1 ms (upper bound of 90% CI 3.8 ms) on Day 4 and −0.6 ms (upper bound of CI 1.8 ms) on Day 39. Moxifloxacin caused the largest mean effect on ΔΔQTcI of 10.9 ms and the lower bound of the CI was above 5 ms at all preselected timepoints, thereby demonstrating assay sensitivity. Albiglutide was well tolerated and there were no clinically relevant differences in safety data between albiglutide and placebo.ConclusionAlbiglutide at doses up to 50 mg in healthy subjects did not prolong the QTc interval.Electronic supplementary materialThe online version of this article (doi:10.1007/s13300-014-0055-1) contains supplementary material, which is available to authorized users.

Loxapine delivered as a thermally generated aerosol does not prolong QTc in a thorough QT/QTc study in healthy subjects

The objective of this study was to establish effects of inhaled loxapine on the QTc interval in this randomized, placebo-controlled, double-blind crossover study. Forty-eight healthy volunteers received a single inhaled placebo or 10 mg loxapine. Plasma concentrations of loxapine increased with a median Tmax of 1 minute and a mean Cmax of 312 ng/mL. After an initial rapid distribution phase, plasma concentrations of loxapine declined with a terminal half-life of 8 hours. Exposure to the active metabolite 7-OH-loxapine was 15% of the parent compound based on mean AUCinf and its terminal half-life was 12 hours. Inhaled loxapine did not increase QT intervals, as demonstrated by the upper bound of the 1-sided 95% CIs placed on the point estimate of the placebo-subtracted change of QTcI (ΔΔQTcI) being less than 10 milliseconds at all 11 post-dose times. The maximum ΔΔQTcI occurred at 1 hour post-dose (LSmean 5.42 milliseconds, upper confidence bound 7.75 milliseconds). The study outcome was validated by the demonstrated assay sensitivity using the positive control moxifloxacin maximum ΔΔQTcI occurred at 3 hour post-dose (LSmean 8.36 milliseconds, lower confidence bound 5.82 milliseconds). The analyses of QTc outliers, and the lack of emergent diagnostic findings for QTcI, QTcB, and QTcF; and simple mean placebo-subtracted changes of QTcI and QTcF supported the primary QT analysis conclusion that this is a negative finding and there is no apparent QT prolongation associated with the therapeutic dose of inhaled loxapine.