| 2,5-dihydroxymethy1-3,6-dimethylpyrazine(Liguzinediol),is a compound with lower toxicity,heart safety,which could be used for the treatment of acute heart failure.It has good positive inotropic effect to normal rat and in vitro heart by means of affecting sarcoplasmic reticulum calcium release,increasing in rats in vivo and in vitro cardiac contractile force.Our group has studied the pharmacokinetics,sexua difference and metabolism of Liguzinediol in rats.According to the requirements of the first type of new drug development,Novel,simple,and sensitive methods for the determination of Liguzinediol in beagle dog plasma using liquid chromatography coupled to tandem mass spectrometry(LC-MS/MS)were developed.We studied the pharmacokinetics of beagle dog after intravenous and oral administration of Liguzineidol.We also have studied the pharmacokinetics of absorption,distribution,excretion and metabolism of Liguzinediol,provided necessary experimental basis for the further develop and clinical research of Liguzineidol.The main content reads as follows:1 Determination of Liguzinediol in beagle dog plasma and pharmacokinetics study of Liguzinediol in beagle dogTo establish a simple and sensitive method for determination of Liguzinediol by LC-MS/MS and study the pharmacokinetics of Liguzinediol in Beagle dogs,respectively.Methods:Separation of Liguzinediol and caffeine in plasma sample was eluting with a gradient mobile phase system,at a flow-rate of 0.4 mL·min-1,carried out on Shim-pack XR-ODS column(50 mm×2.0 mm,2.2 ?m).Measurement of Liguzinediol was performed by positive ion electro spray ionization in multiple reaction monitoring mode,monitoring the transitions m/z 169.2?122.2 and m/z 195.2?110.2 for Liguzinediol and caffeine,respectively.Each group contained 6 beagle dogs(three males and three females).They were administrated an intravenous dose of 1.6,3.2 and 6.4 mg/kg respectively.The pharmacokinetic parameters were obtained with the DAS 2.1 software.Results:Under the doses of 1.6,3.2,6.4 mg·kg-1,tl 2 was 0.37,0.58 and 0.57 h;CL was 1.354,0.686 and 1.202 L·min-1·kg-1;AUC0?t was 1195.43,2598.18 and 5448.652 ?g·L-1·h;AUC0?? was 1202.76,2602.37 and 5453.751 ?g·L-1·h.A good linearity was observed over the concentration range of 5?10 000 ng·ml-1(r = 0.999 6)with the lower limit of quantification at 5 ng·mL-1.They met the requirements of the analysis of biological samples with RSD of the intra-and inter-day precisions were less than 10%.Conclusion:Liguzinediol can be metabolized rapidly in beagle dogs.The developed method was successfully applied to the pharmacokinetic study in beagle dogs.After intravenous administration of Liguzineidol(1.6,3.2,6.4 mg·kg-1)to beagle dogs,the plasma drug concentration-curve analyzed by non compartment model.The half-life were 0.369±0.021 h,0.584±0.246 h,0.567±0.087 h,respectively.The AUC(o-?)were 1195.41±172.46,2598.2± 1147.1,5448.7±943.7 ng·h·mL-1.After oral administration of Liguzineidol(3.2 mg kg-1)to beagle dogs,the plasma drug concentration-curve analyzed by non compartment model.The half-life were0.431 ±0.106 h.The Tmax were 0.333±0.129 h.The Cmax were 2703.2±719.3 ?g L-1.The AUC(0-?)were 2066.1±358.0 ng h mL-1.2 Plasma protein binding of Liguzineidol in ratsEquilibrium dialysis was employed to determine protein binding.The serum proteins of rat were used in this studies.The mean protein binding was 3.88%,in which the range of concentration of Liguzineidol was 5?25?g· ml-1.Low protein binding rate shows that only a small amount of Liguzinediol combined with plasma protein,a large number of Liguzinediol in free form exists in the blood.3 Determination of Liguzinediol in rat tissues by liquid chromatography coupled to tandem mass spectrometry(LC-MS/MS)and its application to tissues distribution studyA methods for the determination of Liguzinediol in rat tissues using liquid chromatography coupled to tandem mass spectrometry(LC-MS/MS)were developed.After intravenous administration of Liguzinediol(10 mg·kg-1)to rats,the drug can be found in all determined tissues.But sexual difference is obvious.Compared with the serum concentration at the same time,the drug content in the organization is relatively less.After intravenous administration of Liguzinediol(10 mg·kg-1)to rats,the drug can be found in all determined tissues and most 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 1h and Liguzinediol can hardly be found in rat tissues after 8 h.It shows that Liguzinediol can rapidly spread and eliminate.Distribution of Liguzinediol in male rats in the heart,muscle,kidney and pancreas is more than the others,in the stomach,intestine and liver is less,and which in other viscera is more uniform.The drug can be measured in brain,explain that Liguzinediol can through the blood brain barrier.After dosing for 10 min,the concentration of Liguzinediol is highest in each group.But 1h later after dosing,drug concentration in each group significantly decreased,Liguzinediol can not be found in most organizations after 8h,and its distribution in the body is relatively quickly,and without accumulation.Compared with male rats,after intravenous administration of Liguzinediol(10 mg·kg-1)to female rats,it shows that the drug can also be distributed to most of the organization in a short period of time,but at the same time point in the organization content is significantly higher than the male rats,and pharmacokinetic study of Liguzinediol shows the metabolism of female rats are slow than the male rats.The distribution in muscles,heart,kidneys and ovaries is more,the stomach,the fat distribution is less,and the other viscera distribution is more uniform.After dosing for 10 min,the concentration of Liguzinediol is highest in each group.2h later after dosing,drug concentration in each group significantly decreased.After dosing for 8h,drug concentration in each group decreased obviously.4 Excretion of Liguzinediol in ratsAfter intravenous administration of Liguzinediol 10 mg kg-1 to rats,the parent drug can be found in bile,urine and feces.A simple and sensitive method for determination of Liguzinediol in bile,urine and feces by LC-MS/MS has been established.After intravenous administration of Liguzinediol 10 mg kg-1 to rats,the parent drug can be found in bile,urine and feces.The bile excretion of Liguzinediol amounted to 0.49%in the male rats and 0.99%in the female rats after the dosage within 24h.The urine excretion of Liguzinediol amounted to 1.22%in the male rats and 5.31%in the female rats after the dosage within 48h.The bile excretion of Liguzinediol amounted to 3.71%in the male rats and 7.42%in the female rats after the dosage within 48hr.About 80%or more of the drug remains to be proven.The metabolites of Liguzinediol in rats in urine,feces and bile have been studied,and a LC-MS/MS method for detecting the metabolites in rat bile,urine and feces has been established.After scanning the bile,urine and feces samples with the TOF MS mode,it has detected multiple metabolic product.We aim at the mass spectrum response of the top 5 kinds of metabolites using MDF-IDA real-time acquisition method to scan again.At last we find a Phase ? products and four types of Phase ? products. |
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