Study On The Pharmacokinetics Of Vitamin B6 And Acetaminophen In Simulated Weightlessness SD Rats

With the development of the space station in China,the space flight cycle will be longer and longer,and the astronauts will have to stay in space for a long time.The physiological condition of astronauts will be significantly affected by the adverse factors such as space radiation,weightlessness,narrow space of working and living.Studies have shown that astronauts in weightlessness can easily induce diseases related to the blood circulation,urinary system,immune system,motor system and nervous system.Vitamin B6 is one of the most important coenzymes in the body.It is involved in more than 80 biochemical reactions in the body.Hundreds of enzymes that are related to biosynthesis or metabolism need the participation of vitamin B6 in order to function properly.Vitamin B6 is a water-soluble vitamin,which can not be synthesized by human body.It can only be supplemented by exogenous intake.The lacking of vitamin B6 in human body can lead to biochemical dysfunction,which can induce many diseases,including most of the common diseases of astronauts,so it is necessary to supply the right amount of vitamin B6 to astronauts.Acetaminophen?APAP?is an essential antipyretic and analgesic drug for astronauts,who are inevitably suffering from pain in their bones and muscles,as well as fever caused by trauma and infection during the mission in space.Since excess vitamin B6 and APAP will cause damage to the body,especially the latter can cause severe liver dysfunction and even life-threatening effects.While the effect of weightlessness on the pharmacokinetics of the two drugs have not been reported in detail.Objective:To investigate the changes of body weight gain,organ coefficient,liver function,renal function,myocardial enzyme,vitamin B6,and APAP-related metabolic enzymes in simulated weightlessness 1W,2W,3W,4W and control group;study the pharmacokinetics of the two drugs in SD rats under simulated weightlessness,provides a theoretical basis for aerospace drug use.Methods:1.Determination of relevant physiological parameters of simulated weightlessness rats with hanging tail64 SD rats were randomly divided into 8 groups?n=8?,of which 4 groups were separately established simulated weightlessness models of 1W,2W,3W and 4W,using the method of unloading the hind limbs of rats.Their body weight growth rate,viscera coefficient,liver function,kidney function and myocardial enzyme spectrum were measured and compared with the normal fed rats in the same period..The expression of pyridoxal kinase?PDXK?,pyridoxine oxidase?PNPO?,UGT1A1,UGT1A6,UGT1A9,SUT1A1 and SULT1A3,CYP1A2,CYP2E1,CYP3A1 protein and mRNA in liver,kidney,lung,duodenum,jejunum,ileum were measured and statistically analyzed.2.Pharmacokinetics of simulated weightlessness SD rats with oral vitamin B6Shimadzu LC-30 UPLC,API 4000 mass spectrometer,Inertsil ODS-3 C18column?4.6×150mm,5?m?coupled with a C18 guard cartridge?4.6×10mm,5?m?.The mobile phase consisted of methanol?A?and water(containing 0.1%formic acid and 1mmol·L^-1ammonium acetate)?B?with gradient elution:0¹.0min,5%A;1.0¹.1min,5%A?70%A;1.1⁴.5min,70%A;4.5⁴.6min,70%A?5%A;4.6⁶.5min,5%A.Flow rate at 0.6mL·min^-1,injection volume was 10?L.The elution time was 6.5min.Mass spectrometry was detected by electrospray ionization source?ESI?,positive ion multireaction monitoring?MRM?scanning analysis.Vitamin B6 m/z 170.1?151,Internal standard caffeine m/z 195.2?137.9,The plasma samples were pretreated by methanol precipitation protein,The supernatant was evaporated and reconstituted with 30%methanol?containing 0.1%formic acid?solution.After control and simulated weightlessness SD rats were given vitamin B6(1.8mg·kg^-1)by oral administration,blood samples were taken at various time intervals,the C-T curve was measured according to the above blood sample treatment method and LC-MS/MS test conditions,and the relevant pharmacokinetics parameters were calculated by DAS 2.0 software.3.Pharmacokinetics of simulated weightlessness SD rats with oral APAPShimadzu LC-2030C 3D HPLC,LC-2030/2040 DAD detector,Inertsil ODS-3 C18column?4.6×150mm,5?m?coupled with a C18 guard artridge?4.6×10mm,5?m?.The mobile phase consisted of methanol?A?and water?B?with gradient elution:0².5min,25%A;2.5².6min,25%A?55%A;2.6⁵.0min,55%A;5.0min⁵.1min,55%A?25%A;5.1⁸.0min,25%A.Flow rate at 1.0 mL·min^-1,injection volume was20?L.The elution time was 8.0min.The content of acetaminophen in plasma samples of SD rats was determined by methanol precipitation protein method.After control and simulated weightlessness SD rats were given APAP(90.0mg·kg^-1)by oral administration,blood samples were taken at various time intervals,the C-T curve was measured according to the above blood sample treatment method and HPLC test conditions,and the relevant pharmacokinetics parameters were calculated by DAS 2.0 software.Results:1.Determination of relevant physiological parameters of simulated weightlessness rats with hanging tail.The results showed that the body weight growth rate of simulated weightlessness rats decreased gradually,the organs coefficient of heart and kidney increased,and the changes of liver,kidney and myocardial enzymes were different compared to the control group.The expression levels of PDXK protein and mRNA was the control group>1W>2W>3W>4W;The expression levels of PNPO was 2W>1W>3W>4W>control group.The expression levels of UGT1A1,SULT1A1 and SULT1A3 was2W>1W>3W>4W>control group.The expression level of UGT1A6 was the control group>2W>3W>4W>1W.The expression levels of CYP1A2,CYP2E1 and CYP3A1 in simulated weightlessness SD rats were all increased.All the above changes were significant difference?P<0.05?or obvious difference?P<0.01?.2.Pharmacokinetics of simulated weightlessness SD rats with oral vitamin B6The method was with good specificity.It appears a good linear relationship within the range of 2²000ng·mL^-1,the linear equation is Y=0.0335X+0.0552,r=0.9972.Inter-Day precision was 10.51%,intra-day precision was 7.788%.The accuracy was±13.14%;The stability was 91.77%¹02.0%;The recovery rate was 88.61%⁹5.83%;The matrix effect was 96.50%¹07.7%,that is,each item of the determination method meets the requirements.Compared to the control group,the T_max of vitamin B6 in the simulated weightlessness groups was advanced 35%⁴0%;Cmax,AUC increased 63%and 40%in1W,and increased about 91%and 66%in 2W⁴W.The CLz/F decreased 27%in 1W and decreased 41%in 2W⁴W.3.Pharmacokinetics of simulated weightlessness SD rats with oral APAPThe separation of target APAP and internal subject acetyl aniline was good,and there was no interference with the endogenous material in plasma,that is,the method was good.In the range of 0.1⁴0?g·mL^-1,the linear equation is y=0.5539x+0.0574,R2=0.9994.Inter-Day precision 8.329%,intra-day precision 7.416%;The accuracy±12.18%;The stability 89.51%¹04.6%;The recovery rate 90.05%⁹3.57%,The matrix effect99.27%¹04.7%,that is,the determination method all meet the requirements.The different pharmacokinetic parameters of the simulated redenomination also varied in degree and trend compared with the control group.Compared to the control group,the AUC of vitamin B6 in the simulated weightlessness groups was decreased 39%and 26%in 1W?2W respectively,and increased 57%in 3W⁴W;MRT decreased 32%and 21%in1W?2W respectively,and increased 17%in 3W⁴W;Cmax increased about 52%in3W⁴W;CLz/F increased 75%and 35%in 1W?2W respectively,and decreased 36%in3W⁴W;Vz/F increased 63%and 30%in 1W?2W respectively,and decreased 17%in3W⁴W.Conclusion:This study confirmed that simulated weightlessness had a number of effects on the physical performance of rats.Body weight gain was inhibited,and liver,kidney,and myocardium functions were severely impaired.This damage was exacerbated with the time of simulated weightlessness increased from 1W to 4W.From 1W to 4W of simulated weightlessness,the variation trend and degree of vitamin B6 and APAP pharmacokinetic parameters were also different,but all were basically consistent with the changes of related metabolic enzymes.It is suggested that we should formulate a safe and reasonable dosing plan according to the flight time of astronauts to ensure that the astronauts have the best physiological functions and complete the mission smoothly.