Investigating the endothelin receptor antagonist aprocitentan in resistant hypertension: design and baseline characteristics of the PRECISION study

The endothelin (ET) system plays an important role in hypertension, especially in volume and salt-dependent forms, which are common in patients with resistant hypertension (RHT). Therapies targeting the ET system may provide a new treatment option. A Phase 2 dose-finding study demonstrated an effect of the dual ET receptor antagonist aprocitentan monotherapy on blood pressure (BP) [1]. PRECISION is a randomized, parallel-group, Phase 3 study assessing the short-term effect of 2 doses of aprocitentan (12.5 mg and 25 mg) and its long-term sustained effect on BP. Following randomization, patients entered a 4-week, double-blind (DB), placebo-controlled part, followed by aprocitentan 25 mg for 32 weeks, and a 12-week, placebo-controlled, randomized withdrawal part (Figure 1). The primary endpoint is the change in systolic BP (SBP) from randomization to week 4 and the key secondary endpoint is the change in SBP from re-randomization to week 40. The study enrolled 1971 patients with RHT diagnosed according to the site's medical practice, from 193 centres worldwide. Entry criteria included sitting SBP [SiSBP] ≥140 mmHg, measured by unattended automated office BP (uAOBP, reducing the white coat effect), despite the use of ≥3 antihypertensive medications. During the screening period of 4 to 12 weeks, secondary causes of hypertension were excluded by the investigator and patients still having SiSBP ≥140 mmHg were switched from their individual antihypertensive drugs to a single-tablet, triple combination of valsartan 160 mg /amlodipine 5 or 10 mg /hydrochlorothiazide 25 mg o.d. (minimizing medical inertia and improving drug adherence) for at least 4 weeks before entering the placebo run-in (RI) period. The screening failure rate of 53% is indicative of the high incidence of apparent RHT within the hypertensive population. Patients continuing having SiSBP ≥140 mmHg on triple therapy (true RHT) then entered the 4-week, single-blind placebo RI period. Of these patients, 20% failed to be randomized, most frequently because of SiSBP <140 mmHg, possibly due to a placebo effect. As of 12 March 2021, 860 patients were in the placebo RI period and 664 patients were randomized, 30% from North America, 62% from Europe, and 8% from Asia/Australia. Mean age was 61.8 years (standard deviation [SD]=10.8). 40% of patients were women, 11% were Black, 5% Asian, and 83% White. Mean body mass index (BMI) was 33.6 kg/m2 (SD=6.4) and mean estimated glomerular filtration rate (eGFR) was 76.8 mL/min/1.73 m2 (including 21% chronic kidney disease [CKD] stage 3–4 patients). Medical history included diabetes (54%), myocardial infarction (30%), stroke (23%), congestive heart failure (19%), and sleep apnoea (15%). In addition to the standardized antihypertensive medication, 59% of patients used beta-blockers. Mean baseline SiSBP/SiDBP was 153/88 mmHg. Results of the trial will be available in 2022. FUNDunding Acknowledgement Type of funding sources: Private company. Main funding source(s): The PRECISION study is sponsored by Idorsia Pharmaceuticals Ltd.

Validation and Implementation of molecular RT-PCR COVID-19 Testing Platforms

Introduction/Objective COVID-19 is caused by the SARS-CoV-2 coronavirus that has led to a worldwide pandemic with an unprecedented need for fast and accurate testing under FDA Emergency Use Authorizations (EUA). Despite the promise of the Alinity-m as an upgrade from the Abbott m2000 for optimization of high throughput testing and random access for laboratory workflow, validation studies comparing Alinity-m to already used Abbott m2000 are sparse in the literature, particularly for the veteran population. Methods/Case Report The validation study for the Alinity m (Chicago IL) was performed in three parts 1) Method to method sample/patient correlation study, 2) precision study (at 250 virus copies/mL, 500 virus copies/mL and 1000 virus copies/mL versus 4 negatives), and 3) verification and confirmation of the accuracy of the assay at the lower limit of detection (LOD). For the validation study. The patient specimens were used side by side for both assays. The results from the Abbott m2000 (Chicago IL), which was already established in our lab, were used as a reference for validation. Results (if a Case Study enter NA) The validation with a method to method correlation included 157 valid specimens from which concordant results were obtained for all 157 specimens on both the Alinity-m and Abbott m2000. The precision or reproducibility of the Alinity-m was verified at all concentrations. The limit of detection verification on diluted samples determined the limit of detection to be 20 virus copies/mL (>95% of dilution samples agreed with positive results at this level), which confirmed even below the manufacturer provided LOD of 100 virus copies/mL. Conclusion Alinity m was validated for clinical use with study demonstrating that it is 1) equivalent in the method to method correlation, precision, and LOD determination. 2)The validation of Alinity m, holds great promise with its optimized throughput for testing of large numbers of specimens with less technician handling and random access, is a valuable addition to the literature of test validations under the FDA EUA. 3)The validation of tests under the FDA EUA is unprecedented and provides an important way to improve patient care during this extraordinary pandemic.

Validation of a commercial human ELISA to measure hyaluronic acid concentration in feline plasma

Our goal was to validate a human hyaluronic acid (HA) ELISA (Hyaluronic acid plus ELISA; TECOmedical Group) for use in feline plasma. Plasma from 5 healthy cats and 5 critically ill cats was used for validation of the assay. Validation methods performed included intra- and inter-assay variability, spike-and-recovery, and dilutional linearity. All measurements were performed in duplicate. The precision study revealed good intra-assay CV of 7.4–8.9%; inter-assay CV was 3.4–4.2%. Extraction efficiency via spiking tests yielded mean recovery of 89.6%. The assay met criteria for acceptable linearity using 3 serial dilutions. Our results demonstrate that this commercial HA ELISA had acceptable analytical performance using feline plasma and could be a useful tool in the veterinary clinical research setting.

Clinical Evaluation of the Torq Zero Delay Centrifuge System for Decentralized Blood Collection and Stabilization

Blood sample collection and rapid separation—critical preanalytical steps in clinical chemistry—can be challenging in decentralized collection settings. To address this gap, the Torq™ zero delay centrifuge system includes a lightweight, hand-portable centrifuge (ZDrive™) and a disc-shaped blood collection device (ZDisc™) enabling immediate sample centrifugation at the point of collection. Here, we report results from clinical validation studies comparing performance of the Torq System with a conventional plasma separation tube (PST). Blood specimens from 134 subjects were collected and processed across three independent sites to compare ZDisc and PST performance in the assessment of 14 analytes (K, Na, Cl, Ca, BUN, creatinine, AST, ALT, ALP, total bilirubin, albumin, total protein, cholesterol, and triglycerides). A 31-subject precision study was performed to evaluate reproducibility of plasma test results from ZDiscs, and plasma quality was assessed by measuring hemolysis and blood cells from 10 subject specimens. The ZDisc successfully collected and processed samples from 134 subjects. ZDisc results agreed with reference PSTs for all 14 analytes with mean % biases well below clinically significant levels. Results were reproducible across different operators and ZDisc production lots, and plasma blood cell counts and hemolysis levels fell well below clinical acceptance thresholds. ZDiscs produce plasma samples equivalent to reference PSTs. Results support the suitability of the Torq System for remotely collecting and processing blood samples in decentralized settings.

Optimization of an Ultrasound-Assisted Extraction Method for the Analysis of Major Anthocyanin Content in Erica australis Flowers

Erica australis plants have been used in infusions and folk medicine for years for its diuretic and antiseptic properties and even for the treatment of infections. In addition, a recently published thorough study on this species has demonstrated its antioxidant, antibiotic, anti-inflammatory, anticarcinogenic and even antitumoral activities. These properties have been associated with the high content of anthocyanins in E. australis leaves and flowers. The aim of the present research is to optimize an ultrasound-assisted extraction methodology for the recovery of the anthocyanins present in E. australis flowers. For that purpose, a Box Behnken design with response surface methodology was employed, and the influence of four variables at different values was determined: namely, the composition of the extraction solvents (0–50% MeOH in water), the pH level of those solvents (3–7), the extraction temperature (10–70 °C), and the sample:solvent ratio (0.5 g:10 mL–0.5 g:20 mL). UHPLC-UV-vis has been employed to quantify the two major anthocyanins detected in the samples. The extraction optimum conditions for 0.5 g samples were: 20 mL of solvent (50% MeOH:H2O) at 5 pH, with a 15 min extraction time at 70 °C. A precision study was performed and the intra-day and inter-day relative standard deviations (RSDs) obtained were 3.31% and 3.52%, respectively. The developed methodology has been successfully applied to other Erica species to validate the suitability of the method for anthocyanin extraction.

Simultaneous determination of notoginsenoside R1 and ginsenosides Rg1, Re, Rb1 in dietary supplements by HPLC-DAD

A method using solid phase extraction (SPE) and high performance liquid chromatography with diode array detector (HPLC-DAD) has been optimized for the simultaneous determination of notoginsenoside R1 and three ginsenosides Rg1, Re, Rb1 in solid, oil, and liquid dietary supplements. The substances were separated by an InertSustain C18 column (250 mm × 4.6 mm i.d.; particle size 5 μm) with a gradient program composed of acetonitrile and water. Linearities were in the range of 4.0 - 400 μg/mL with the coefficients of determination were more than 0.999. The limits of detection and limits of quantitation were 2.13 - 6.89 μg/g and 7.11 - 22.98 μg/g, respectively. The recovery values of the compounds were in the range of 87.2 - 103.5%. The precision study showed the intra-day relative standard deviation (RSD) of 1.41 - 2.91%, and inter-day RSD of 1.87 - 4.85%, which meet the AOAC International requirements. The method was applied to determine the content of notoginsenoside R1, ginsenoside Rg1, ginsenoside Re, and ginsenoside Rb1 in 20 dietary supplement samples containing Ginseng and Pseudoginseng.

A Nanodrop Spectrophotometric Method and Stability Indicating for Determination of Amlodipine Besylate in Pharmaceutical Formulations of Kurdistan of Iraq

A nanodrop spectrophotometric method was developed and validated for determination of amlodipine besylate (AB) in bulk and tablet dosage form. The maximum absorption of amlodipine was shown at 357 nm using acetonitrile as a solvent. The developed method was found to be linear (R2 = 0.9990) within the concentration range of 2-10 µg/mL. The precision study showed acceptable values of RSD% (less than 1%). LOD and LOQ values were found to be 0.34 and 1.14 µg/mL, respectively. Accuracy study showed good recovery 99% Awalodipine and 98.88% Amloneer, in locally commercial tablets. The present method was applied successfully for stability indicating study of AB in Awalodipine and Amloneer products manufactured in (Erbil and sulaymaniyah, respectively)/ Kurdistan of Iraq. The stability-indicating study was investigated under acidic, basic, oxidative, photolytic, and thermal conditions. The results of both products showed that AB is unstable in acidic, alkaline, and oxidative conditions under heating at 60℃ up to 5 hrs. While under photolytic and thermal conditions, the degradation percentage was less than 15% indicating to the stability of AB in both Awalodipine and Amloneer tablets according to International Conference on Harmonization (ICH) guideline of drugs. It can be concluded that the main factor that affects the degradation of AB is the passage of time.