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R-/S-2-(2-hydroxypropanamido)-5-trifluoromethyl benzoic acid (R-/S-HFBA) is a novel COX inhibitor with remarkable anti-inflammatory and antiplatelet aggregation activities, but no gastrointestinal toxicity. In our previous study, the different pharmacokinetic profiles of the two enantiomers in rats were observed after administration of R-HFBA and S-HFBA. Stereoselective protein binding of the two enantiomers may be a reason for the different pharmacokinetic behaviors. In this study, we developed and validated an UPLC-MS/MS method for determining stereoselective binding of HFBA enantiomers to rat, dog, and human plasma in vitro. Chromatographic separation was achieved by gradient elution with a flow rate of 0.4 mL/min. MS/MS detection was operated in positive electrospray using multiple reaction monitoring (MRM) mode. The method was proved to be linear over the concentration range of 0.005 to 10 μg/mL with a lower limit of quantification of 0.005 μg/mL. The developed method was successfully employed to the plasma protein binding study of HFBA enantiomers. Equilibrium dialysis method was applied to assess drug-plasma protein interactions. The results showed that the enantiomers were both extensively bound to three species plasma and protein binding of R-/S-HFBA was concentration dependent. R-HFBA and S-HFBA showed significant species difference among rat, dog, and human plasma and stereoselective plasma protein binding., (© 2020 Wiley Periodicals, Inc.)

R-/S-2-(2-Hydroxypropanamido) benzoic acid (R-/S-HPABA), a marine-derived anti-inflammatory drug, however, the antiplatelet and antithrombotic effects have not been investigated. In this paper, the in vitro antiplatelet activities and in vivo antithrombotic effects of R-/S-HPABA were investigated, for the first time. The effects of R-/S-HPABA on platelet aggregation induced by adenosine diphosphate (ADP), collagen (COLL) and arachidonic acid (AA) were evaluated. In addition, the in vivo bleeding time, clotting time, collagen-epinephrine induced pulmonary thrombosis and common carotid artery thrombosis were also investigated in rats. R-/S-HPABA significantly inhibited ADP, COLL and AA induced platelet aggregation in rabbit platelet rich plasma in vitro compared with control group, to a degree similar to that of aspirin. Besides, R-/S-HPABA prolonged bleeding time and clotting time as well as increased the recovery rate obviously in pulmonary thrombosis. Moreover, the level of thromboxane B2 (TXB2) was decreased while the production of 6-keto-prostaglandin F1α (6-keto-PGF1α) was increased markedly by R-/S-HPABA. Furthermore, R-/S-HPABA reduced carotid artery thrombosis weight. These results illustrated that R-/S-HPABA could be a potent antiplatelet aggregation and antithrombotic agent. [ABSTRACT FROM AUTHOR]

The purpose of this study was to compare intestinal permeability between enantiomers of 2-(2-hydroxypropanamido) benzoic acid (( R) -/( S) -HPABA), a marine-derived antiinflammatory drug, using an in situ single-pass intestinal perfusion (SPIP) model in rats. Concentrations, isolated regions of small intestine, and p-glycoprotein (P-gp) inhibitor were performed to investigate their influences on the intestinal absorption of ( R) -/( S) -HPABA. In addition, a molecular docking method was performed to illustrate our prediction. The absorption rate coefficients ( K a) and permeability values ( P eff) of ( R) -/( S) -HPABA were calculated. The permeability of ( S)-HPABA was significantly ( P < 0.01) higher than that of ( R)-HPABA in jejunum, and ileum permeability of ( R) -/( S) -HPABA appeared best in ileum; the investigated concentrations ranged from 20 to 80 μg/mL, K a and P eff values of ( R) -/( S) -HPABA increased linearly; in the presence of P-gp inhibitor (verapamil), P eff values of two enantiomers were increased significantly; and the effect of P-gp on absorption of ( R)-HPABA is stronger than that of ( S)-HPABA in ileum segment. Based on these results, carrier-mediated transport or passive transport combined with carrier-mediated transport seems to be the mechanism for intestinal absorption of ( R) -/( S) -HPABA, and ( R) -/( S) -HPABA may be recognized as the P-gp substrate. In addition, the intestinal permeability of ( S)-HPABA is higher than that of ( R)-HPABA. [ABSTRACT FROM AUTHOR]

Highlights: [•] Development of a UHPLC–MS/MS method to determine HPABA in rat plasma. [•] The total analysis time was 2.0min. [•] The method was applied to pharmacokinetic study of HPABA in rats. [•] HPABA showed linear pharmacokinetic properties and high absolute bioavailability. [ABSTRACT FROM AUTHOR]

As the stereoselective plasma protein binding is an important factor for the differences in pharmacokinetic or pharmacodynamic properties of chiral drugs, we elucidated the binding of ( R ) -/ ( S ) - HPABA to plasma protein. A UHPLC–MS/MS method was developed and validated to the quantitation and equilibrium dialysis method was applied to the separation of bound and unbound drugs. Protein binding reached equilibrium within 12 h of incubation at 37 °C. Liquid-liquid extraction with ethyl acetate was used for sample preparation. The separation was achieved through a Thermo Syncronis C 18 column (50 mm × 2.1 mm, 1.7 μm; Thermo, USA) using gradient elution at a flow rate of 0.4 ml/min. All of the recovery and matrix effect of the method met the requirements of analytical method validation. The plasma protein binding of ( R ) -/ ( S )-HPABA show significant species dependence in enantioselectivity. In addition, the results of molecular docking showed that the combining capacity of ( R ) - HPABA with HSA was higher than that of ( S ) - HPABA. [ABSTRACT FROM AUTHOR]

A simple and sensitive UHPLC–MS/MS method was developed and validated to determine the pharmacokinetic profile of 2-(2-hydroxypropanamido) benzoic acid (HPABA) enantiomers and their prodrugs in rat plasma. Separation was performed on a Thermo Syncronis C 18 column (50 mm × 2.1 mm, 1.7 μm; Thermo, USA), which was protected by a high pressure column prefilter (2 μm) at a flow rate of 0.4 ml/min. Liquid-liquid extraction with ethyl acetate was used to process plasma samples. The separation of two enantiomers, prodrugs of ( R ) -/ ( S )-HPABA and internal standard was obtained within a cycle time of 4.5 min. The lower limit of quantification of ( R ) -/ ( S )-HPABA and prodrugs of ( R ) -/ ( S )-HPABA in plasma were 0.01 μg/ml and 0.2 μg/ml, respectively. ( S )-HPABA showed significantly higher AUC, C max and a longer t 1/2 than ( R )-HPABA, indicating higher bioavailability of the ( S )-HPABA. Additionally, inversion between HPABA enantiomers was not observed in rats. ( R ) -/ ( S )-HPABA showed higher C max and AUC than those of their prodrugs. However, the values of t 1/2 of prodrugs were higher than those of ( R ) -/ ( S )-HPABA. Furthermore, the higher V z values of prodrugs might improve the targeting of ( R ) -/ ( S )-HPABA in rat tissues. [ABSTRACT FROM AUTHOR]

A RP-HPLC method was developed and validated for the degradation kinetic study of 2-(2-hydroxypropanamido) benzoic acid (HPABA), a promising anti-inflammatory drug, which would provide a basis for further studies on HPABA. The effects of pH, temperature, buffer concentration and ionic strength on the degradation kinetics of HPABA were discussed. Experimental parameters such as degradation rate constants ( k ), activation energy ( E a ), acid and alkali catalytic constants ( k ac , k al ), shelf life ( t 1/2 ) and temperature coefficient ( Q 10 ) were calculated. The results indicated that degradation kinetics of HPABA followed zero-order reaction kinetics; degradation rate constants ( k ) of HPABA at different pH values demonstrated that HPABA was more stable in neutral and near-neutral conditions; the function of temperature on k obeyed the Arrhenius equation ( r = 0.9933) and HPABA was more stable at lower temperature; with the increase of ionic strength and buffer concentration, the stability of HPABA was decreased. The major unknown degradation product of HPABA was identified by UHPLC/TOF–MS/MS with positive electrospray ionization. Results demonstrated that the hydrolysis product was the primary degradation product of HPABA and it was deduced as anthranilic acid. [ABSTRACT FROM AUTHOR]

An ultra high performance liquid chromatography combined with Fourier transform ion cyclotron resonance mass spectrometry (UHPLC-FT-ICR-MS) method was developed to investigate the in vivo metabolism of 2-(2-hydroxypropanamido) benzoic acid (HPABA), a marine-derived anti-inflammatory drug, for the first time. Plasma, urine, feces and bile samples were collected from male and female rats after a single intragastric administration of HPABA at a dose of 100 mg/kg. Besides the parent drug, a total of 13 metabolites (3 phase I and 10 phase II metabolites) were detected and tentatively identified through comparing their mass spectrometry profiles with those of HPABA. Results demonstrated that metabolic pathways of HPABA in rats included decarboxylation, hydroxylation, dehydrogenation, glucuronidation, glycine conjugation and N-acetyl conjugation. In summary, this work provided valuable information regarding the metabolism of HPABA in rats, which would contribute to better understanding of its safety and mechanism of action. [ABSTRACT FROM AUTHOR]