Study On The Pharmacokinetics Of Liguzinediol And Its Benzoyl Derivative In Rats

Liguzinediol (LZDO),2,5-dihydroxymethyl-3,6-dimethylpyrazine, is a derivative synthesized from ligustrazine, which could be used to cure cardiovascular disease, like heart failure, with its good positive inotropic effect. Our group has studied the pharmacokinetics of absorption, distribution, excretion and metabolism of Liguzinediol and the research showed that Liguzinediol has a shot half-life and disparity was found between male and female rats.2,5-dibenzoyloxymethyl-3,6-dimethylpyrazinel is a derivative synthesized from Liguzinediol as a prodrug in order to defer the metabolize of Liguzinediol in vivo.Novel, simple, and sensitive methods for the determination of Liguzinediol and 2,5-dibenzoyloxymethyl-3,6-dimethylpyrazinel in rat plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) were developed. The pharmacokinetics were studied and used to assessed drug-likeness of 2,5-dibenzoyloxymethyl-3,6-dimethylpyrazinel.The main content reads as follows:1 Pharmacokinetics study of Liguzinediol in male and female rats1.1 Determination of Liguzinediol in rat plasma by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS)A novel, simple, and sensitive method for the determination of Liguzinediol in rats plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) were developed. Liguzinediol and the internal standard (caffeine) were extracted by a protein precipitation method. Separation of Liguzinediol and caffeine in plasma sample was carried out on Shim-pack XR-ODS column, eluting with a gradient mobile phase system, which consisted of methanol-0.1%(v/v) formic acid at a flow-rate of 0.4 mL/min.The mass spectrometer was operated in an ESI positive ion mode and quantification was carried out using the multiple reaction monitoring (MRM) mode.The fragmentation transitions for MRM were m/z 169.2�'122.2 for Liguzinediol and m/z 195.2�'110.2 for caffeine. The calibration curve was linear within the range of 10.26～20520ng·mL-1 and the lower limit of detection was of 0.02 ng·mL-1. The mean plasma extraction recovery of Liguzinediol was in the range of 75.60%-93.09%. The intra-day and inter-day precision (R.S.D) were 1.214%～4.675% and 2.195%～10.41%, respectively. The method has been successfully applied in pharmacokinetics study of Liguzinediol.1.2 Pharmacokinetics study of Liguzinediol in male and female ratsAfter oral administration of Liguzineidol (5,10、20 mg·kg-1) to Sprague-Dawley rats (n=36), the plasma drug concentration-curve analyzed by non compartment model. The half-life were 1.55±0.223h、1.76±0.319h、1.82±0.227h and 0.438±0.119h、0.464±0.182h、0.384± 0.185 h, respectively. The AUC(0-∞ were 13042±2033,29214±5666、60173±6228μg·L-1·h and 2313±452.7797±3167.10726±3960μg·L-1·h, respectively.After intravenous administration of Liguzineidol (5.10.20 mg·kg-1) to Sprague-Dawley rats (n=36), the plasma drug concentration-curve analyzed by non compartment model. The half-life were 1.43±0.467 h.2.122±0.282 h.1.98±0.369 h and 0.374±0.200 h.0.420±0.178 h.0.447±0.131 h, respectively. The AUC(0-o∞)Were 17484±2946.31434±3323.71644±8697 μg·L-1·h and 3436±964.6546±853.15946±2334μg·L-1·h, respectively.1.3 Determination of Liguzinediol in rat tissues by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and its application to tissues distribution studyAfter intravenous administration of Liguzinediol (10 mg·kg-1) to rats, the drug can be found in all determined tissues and mos of tissues have higher concentration, the tissues such as heart and muscle received more drug than other tissues. The parent drug can be detected in brain, it indicated that Liguzinediol can pass the blood-brain barrier. The concentration of Liguzinediol in tissues reduced obviously after 2h and Liguzinediol can hardly be found in rat tissues after 8 h.It shows that Liguzinediol can rapidly spread and eliminate.2 Synthesis of 2,5-dibenzoyloxymethyI-3,6-dimethylpyrazinel and its pharmacokinetics study in rats2.1 Synthesis of 2,5-dibenzoyloxymethyl-3,6-dimethylpyrazinel2,5-dibenzoyloxymethyl-3,6-dimethylpyrazinel was synthesized from Liguzinediol and benzoyl chloride with pyridine as catalyst. The structrues were comfimed by UV, IR, NMR and MS.Inotropic actions of ligustrazine derivatives in normal isolated rat hearts were investigated by Langendorff technique. The results showed that 2,5-dibenzoyloxymethyl-3,6-dimethylpyra-zine can induce positive inotropic effect on myocardium with no arrhythmia risk.2.2 Pharmacokinetics study of Liguzinediol benzoyl derivative in ratsA novel, simple, and sensitive methods for the determination of Liguzinediol and 2,5-dibenzoyloxymethyl-3,6-dimethylpyrazinel in rat plasma using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) were developed. Liguzinediol, and 2,5-dibenzoyloxymethyl-3,6-dimethylpyrazinel were extracted by a protein precipitation method. Separation of Liguzinediol,2,5-dibenzoyloxymethyl-3,6-dimethylpyrazinel, and caffeine(IS) in plasma sample was carried out on Shim-pack XR-ODS column, eluting with a gradient mobile phase system, which consisted of methanol-0.1%(v/v) formic acid at a flow-rate of 0.4 mL/min.The mass spectrometer was operated in an ESI positive ion mode and quantification was carried out using the multiple reaction monitoring (MRM) mode.The fragmentation transitions for MRM were m/z 169.2^122.2 for Liguzinediol, m/z 377.0�'255.0 for 2,5-dibenzoyloxymethyl-3,6-dimethylpyrazinel, and m/z 195.2�'110.2 for caffeine. The calibration curve were linear within the range of 5～500 ng·mL-1 and 2.5～100 ng·mL-1. The mean plasma extraction recovery of Liguzinediol and 2,5-dibenzoyloxymethyl -3,6-dimethylpyrazinel were in the range of 87.4%-95.0% and 83.4%-91.2%, respectively. The intra-day and inter-day precision (R.S.D) were less than 10%. The method has been successfully applied in pharmacokinetics study of Liguzinediol.After oral administration of 2,5-dibenzoyloxymethyl-3,6-dimethylpyrazinel (25、50 mg·kg-1) to Sprague-Dawley rats (n=12), the plasma drug concentration-curve analyzed by non compartment model. The Tmax were 2.333±1.443h and 2.500±0.500h with Cmax were 432.0±180.9 ng·mL-1 and 914.3±391.1 ng·mL-1. The half-life were 2.385±0.282h and 3.431± 0.775h. The CL/F were 0.140±0.0562L9h-19kg-1 and 0.119±0.0478L9h-1kg-1 and the AUC(0-∞) were 3443±1763、7761±3000μgL-1·h。2.3 Determination of Liguzinediol benzoyl derivative in rat tissues by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and its application to tissues distribution studyAfter oral administration of Liguzinediol benzoyl derivative (25 mg·kg-1) to rats, Liguzinediol can be found in most of determined tissues with low concentration. The concentration of Liguzinediol in tissues reduced obviously after 8h and Liguzinediol can hardly be found in rat tissues after 24 h. The tissues such as stomach and intestine received more drug than other tissues.Based on the pharmacokinetics study of Liguzinediol and its derivative, we compared the half-life、Tmax、Cl/F and AUC between them after oral administration and tissues distribution. The result showed that Liguzinediol benzoyl derivative, as a prodrug, could lengthen the half-life of Liguzinediol, but with a lower blood concentration and bioavailability.