Quantitative HPLC-MS/MS determination of Nuc, the active metabolite of remdesivir, and its pharmacokinetics in rat.

Remdesivir is a nucleotide analog prodrug that has received much attention since the outbreak of the COVID-19 pandemic in December 2019. GS-441524 (Nuc) is the active metabolite of remdesivir and plays a pivotal role in the clinical treatment of COVID-19. Here, a robust HPLC-MS/MS method was developed to determine Nuc concentrations in rat plasma samples after a one-step protein precipitation process. Chromatographic separation was accomplished on Waters XBrige C18 column (50 × 2.1 mm, 3.5 µm) under gradient elution conditions. Multiple reaction monitoring transitions in electrospray positive ion mode were m/z 292.2 → 163.2 for Nuc and 237.1 → 194.1 for the internal standard (carbamazepine). The quantitative analysis method was fully validated in line with the United States Food and Drug Administration guidelines. The linearity, accuracy and precision, matrix effect, recovery, and stability results met the requirements of the guidelines. Uncertainty of measurement and incurred sample reanalysis were analyzed to further ensure the robustness and reproducibility of the method. This optimized method was successfully applied in a rat pharmacokinetics study of remdesivir (intravenously administration, 5 mg kg-1). The method can act as a basis for further pharmacokinetic and clinical efficacy investigations in patients with COVID-19. Graphical abstract.

Discovery and optimization of 2-((1H-indol-3-yl)thio)-N-benzyl-acetamides as novel SARS-CoV-2 RdRp inhibitors.

The emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the global pandemic coronavirus disease (COVID-19), but no specific antiviral drug has been proven effective for controlling this pandemic to date. In this study, several 2-((indol-3-yl)thio)-N-benzyl-acetamides were identified as SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) inhibitors. After a two-round optimization, a new series of 2-((indol-3-yl)thio)-N-benzyl-acetamides was designed, synthesized, and evaluated for SARS-CoV-2 RdRp inhibitory effect. Compounds 6b2, 6b5, 6c9, 6d2, and 6d5 were identified as potent inhibitors with IC50 values of 3.35 ± 0.21 µM, 4.55 ± 0.2 µM, 1.65 ± 0.05 µM, 3.76 ± 0.79 µM, and 1.11 ± 0.05 µM, respectively; the IC50 of remdesivir (control) was measured as 1.19 ± 0.36 µM. All of the compounds inhibited RNA synthesis by SARS-CoV-2 RdRp. The most potent compound 6d5, which showed a stronger inhibitory activity against the human coronavirus HCoV-OC43 than remdesivir, is a promising candidate for further investigation.

Application of graphene-ionic liquid-chitosan composite-modified carbon molecular wire electrode for the sensitive determination of adenosine-5'-monophosphate.

In this paper, a graphene (GR) ionic liquid (IL) 1-octyl-3-methylimidazolium hexafluorophosphate and chitosan composite-modified carbon molecular wire electrode (CMWE) was fabricated by a drop-casting method and further applied to the sensitive electrochemical detection of adenosine-5'-monophosphate (AMP). CMWE was prepared with diphenylacetylene (DPA) as the modifier and the binder. The properties of modified electrode were examined by scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy. Electrochemical behaviors of AMP was carefully investigated with enhanced responses appeared, which was due to the presence of GR-IL composite on the electrode surface with excellent electrocatalytic ability. A well-defined oxidation peak of AMP appeared at 1.314 V and the electrochemical parameters were calculated by electrochemical methods. Under the selected conditions, the oxidation peak current of AMP was proportional to its concentration in the range from 0.01 µM to 80.0 µM with the detection limit as 3.42 nM (3σ) by differential pulse voltammetry. The proposed method exhibited good selectivity and was applied to the detection of vidarabine monophosphate injection samples with satisfactory results.

Measurement of adenine nucleotides in plasma.

The goal of this study was to identify the most important variables affecting bioluminescent ATP, ADP and AMP measurements in plasma and to develop an assay that takes these variables into account. Blood samples were drawn from conscious dogs. A 'stop solution' containing EDTA was prepared, which greatly retarded plasma ATP degradation by chelating Mg(+2) and Ca(+2) that are co-factors for many ATPases. Stop solution and blood were mixed using a two-syringe withdrawal system. Samples were centrifuged twice in order to remove red blood cells, and ATP was measured in the supernatant using the firefly luciferase assay. Sample pH was adjusted to the optimal range (7.75-7.95) and Mg(2+) (necessary for the luciferase reaction) was added back to the sample within the luminometer 2 s prior to luciferase addition. Four assay tubes were prepared for each plasma sample, containing standard additions of 0-15 pmol added ATP, in order to quantify native plasma ATP content. In separate plasma/stop solution samples ADP + ATP was measured after converting ADP to ATP via the pyruvate kinase reaction, and AMP + ADP + ATP was measured after addition of both myokinase and pyruvate kinase. Addition of forskolin and isobutylmethylxanthine (IBMX) to the stop solution to inhibit platelets resulted in lower ATP concentrations. Measurement of ATP and haemoglobin from lysed erythrocytes revealed that haemolysis exerts a strong influence on plasma ATP concentration that must be taken into account.

Open multicentre study of the P2T receptor antagonist AR-C69931MX assessing safety, tolerability and activity in patients with acute coronary syndromes.

Platelet aggregation is the central process in the pathophysiology of acute coronary syndromes. ADP contributes to thrombosis by activating platelets, and AR-C69931MX is a specific antagonist of this process acting at the P2T receptor. At 5 hospitals, 39 patients with unstable angina or non-Q wave myocardial infarction, who were receiving aspirin and heparin, were administered intravenous AR-C69931MX with stepped dose increments over 3 h to a plateau of either 2 microg/kg/min for 21 h (Part 1; n = 12) or up to 69 h (Part 2; n = 13) or 4 microg/kg/min for up to 69 h (Part 3: n = 14). Safety parameters, platelet aggregation (PA) induced by ADP 3 micromol/L (impedance aggregometry), bleeding time (BT) and plasma concentrations of AR-C69931XX were assessed. AR-C69931MX was well tolerated. 33 patients completed the study. There were no deaths at 30 days and no serious adverse events attributed to AR-C69931MX. Trivial bleeding (56%) was common. At 24 h, mean inhibition of PA was 96.0 +/- 8.6, 94.9 +/- 14.4 and 98.7 +/- 2.1% and BT was 9.5 +/- 8.4, 14.0 +/- 9.7 and 16.0 +/- 11.1 min for Parts 1, 2 and 3 respectively. At 1 h post-infusion, mean inhibition of PA was 36.2 +/- 39.2, 20.7 +/- 25.9 and 40.7 +/- 36.7% respectively. 90% patients had a plasma half-life for AR-C69931XX of <9 min. In conclusion, AR-C69931MX is a potent, short-acting platelet ADP receptor antagonist suitable for further studies as an antithrombotic agent.