A high-throughput mass spectrometry assay to measure the catalytic activity of phosphatidylserine decarboxylase (PISD) is described. PISD converts phosphatidylserine to phosphatidylethanolamine during lipid synthesis. Traditional methods of measuring PISD activity are low throughput and unsuitable for the high-throughput screening of large compound libraries. The high-throughput mass spectrometry assay directly measures phosphatidylserine and phosphatidylethanolamine using the RapidFiretrade mark platform at a rate of 1 sample every 7.5 s. The assay is robust, with an average Z' value of 0.79 from a screen of 9920 compounds. Of 60 compounds selected for confirmation, 54 are active in dose-response studies. The application of high-throughput mass spectrometry permitted a high-quality screen to be performed for an otherwise intractable target.
An IgG mouse monoclonal antibody (10F05) against polyethylene glycol has been generated. The antibody reacts with PEG regardless of the linker used for PEG attachment, and is able to recognize a PEGylated peptide in plasma at concentrations as low as 3 pg/mL. The antibody is readily purified in substantial quantities. The PEG IgG will find significant utility in the sensitive detection of PEG derivitives during the pharmacokinetic characterization of PEGylated compounds.
Ang1 is a soluble ligand to receptor Tie2, and increasing the circulating Ang1 level may improve vascular stabilization under certain disease conditions. Here, we found that the circulating Ang1 level was significantly increased in cynomolgus monkeys treated with non-neutralizing anti-Ang1 antibodies. Improving the antibodies’ pharmacokinetic properties by IgG Fc mutations further increased the circulating Ang1 level. However, the mutations decreased the thermal stability of the molecules, which may limit their use as therapeutic antibodies. Nevertheless, we showed that non-neutralizing antibodies may have therapeutic potential by increasing the level of a target molecule in the circulation.
22 Background: The objective of this study is to determine the pharmacokinetics (PK) and pharmacodynamics (PD) of dabigatran (a small molecule thrombin inhibitor) and its antidote (a humanized Fab against dabigatran) in the monkey and to develop a combined mechanistic mathematical model to describe the data. Methods: There were three groups: control, antidote alone and dabigatran etexilate (DE) + antidote. Rhesus monkeys (n = 2/group) received either 12 mg/kg/day of DE or vehicle orally on Days 1–4, 15–18 and 29–32 with a single IV dose of the antidote administered 90 minutes after DE on Days 4, 18 and 32. Doses of the antidote were 30, 90 or 175 mg/kg, respectively. PK parameters of the antidote and sum dabigatran (dabigatran plus its glucuronides) were determined after measurements of plasma concentrations. Coagulation activity was measured using a diluted thrombin time assay to determine the activity of the unbound sum dabigatran. Results: The PK of the antidote were not affected by dabigatran. Clearance of the antidote was low (0.87 mL/min/kg) and steady-state volume of distribution was small (0.06 L/kg), indicating that the antidote was mostly restricted to plasma. The plasma profile of the antidote was bi-phasic with a short initial phase t1/2 of 0.4 hour (h) and a terminal phase t1/2 of 4.3 h. Immediately after antidote dosing, plasma concentrations of sum dabigatran increased, a consequence of the rapid redistribution of dabigatran and its glucuronides from tissue to plasma due to binding to the antidote. Complete reversal of dabigatran's anticoagulant activity was observed immediately after antidote dosing at all three dose levels, as measured by the diluted thrombin time assay, which indicates that all dabigatran was bound to the antidote. The degree to which this reversal effect was maintained over an extended period (24 h) was dose-dependent. A mechanistic ordinary differential equation model, based on the mass action kinetics for describing the distribution, binding and elimination of dabigatran and its antidote, was developed by combining the PK models for dabigatran and the antidote and adding the binding interaction (1:1 stoichiometry) between the two compounds. The distribution and elimination parameters of the dabigatran-antidote complex were assumed to be the same as those of the antidote, based on similar measured PK parameters of the antidote with and without dabigatran in the monkey. The combined PK/PD model of dabigatran and antidote was able to describe the in vivo PK/PD data observed in monkeys. Conclusion: The dabigatran-specific antidote successfully reversed the anticoagulant activity of dabigatran in the monkey in a dose-dependent manner, and our combined mathematical model accurately describes monkey PK/PD data of sum dabigatran and its antidote. Insights gained from this model will be used to guide model development for clinical trials. Disclosures: Toth: Boehringer Ingelheim: Employment. Gan:Boehringer Ingelheim: Employment. van Ryn:Boehringer Ingelheim: Employment. Dursema:Boehringer Ingelheim: Employment. Isler:Boehringer Ingelheim: Employment. Coble:Boehringer Ingelheim: Employment. Burke:Boehringer Ingelheim: Employment. Lalovic:Boehringer Ingelheim: Employment. Olson:Boehringer Ingelheim: Employment.
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