| The weightless environment of aerospace causes changes in the physiological functions of the body,which in turn affects the pharmacokinetics and pharmacodynamics of the drug,it may brings significant risks to aerospace medicine.Under the weightlessness environment,the levels of vitamins are significantly reduced and the body is oxidized.,bone metabolism disorders and other injuries are aggravated.Therefore,astronauts must take vitamin supplements to maintain normal physiological functions.Folic acid is an important water-soluble B vitamin,which is involved in the transfer of a carbon unit in the body in the form of a coenzyme and plays an important role in amino acid and nucleotide metabolism.The lack of FA or excessive accumulation of FA in the body under microgravity will inevitably cause damage to related tissues in the body.After the astronauts enter the space,frequent changes in the day and night will cause the biological clock to be disordered,which will easily cause astronaut insomnia.Zolpidem binds selectively to the benzodiazepineω1 receptor.ZPD rarely produces tolerance and physical dependence during the normal treatment period,and it is the first-line drug for astronauts in weightless environment.1.PurposeInvestigate the pharmacokinetic parameters of FA and ZPD in normal and different time simulated weightlessness SD rats models;Determine the expression of related metabolic enzymes in normal and different time simulated weightlessness SD rats and the absorption parameters of FA and ZPD.In order to provide reference for aerospace medication under weightless conditions.2.Method2.1 Determination and validation the SD rats of FA and ZPD by LC-MS/MS method in plasma samples.The mass spectrometer was used as API 4000 and it was supplied by AB.The liquid chromatograph was Shimadzu LC-30.The column was selected from AgilentC18（4.6×100 mm,3.5μm）.FA:The mobile phase was methanol:water（0.1%formic acid）（55:45）.The mobile phase flow rate was set to 0.6 mL·min-1,the sample injection volume was 5μL,and the sample elution time was 6min.Mass spectrometry conditions were analyzed using negative ion multiple reaction monitoring（MRM）scans and electrospray ionization（ESI）was used.FA m/z 442.0→m/z 295.1,internal standard caffeine:m/z 195.2→m/z 137.9.The method for determining the content of FA in plasma samples of SD rats under weightlessness was established.The validation of the methods mainly included recovery,specificity,linearity,sensitivity,stability,precision,matrix effect,and accuracy.ZPD:The mobile phase was methanol:water（10mM ammonium acetate）（85:15）.The mobile phase flow rate was set to 0.6 mL·min-1,the sample injection volume was 5μL,and the sample elution time was 7min.Mass spectrometry conditions were analyzed using negative ion multiple reaction monitoring（MRM）scans and electrospray ionization（ESI）was used.ZPD m/z 308.0→m/z 235.2,internal standard caffeine:m/z 195.2→m/z 137.9.The method for determining the content of ZPD in plasma samples of SD rats under weightlessness was established.The validation of the methods mainly included recovery,specificity,linearity,sensitivity,stability,precision,matrix effect,and accuracy.2.2 Study on the pharmacokinetics of oral FA and ZPD in normal and weightlessness SD rats.FA:30 male SD rats（220±20g）were divided into normal,weightless state groups 1,2,3,and 4W（n=6）.Orally administered by gavage,the dose was 0.5208 mg·kg-1.At the time of oral administration of each group 10min、20min、40min、1h、1.5h、2h、3h、4h、6h、8h、12h,0.5-1.0mL of blood was taken from the eyelids.Then 12,000 rpm·min-1centrifuged at low temperature for 10 min and rat plasma was aspirated.ZPD:30 male SD rats（220±20g）were divided into normal,weightless state groups 1,2,3,and 4W（n=6）.Orally administered by gavage,the dose was 1.0416 mg·kg-1.At the time of oral administration of each group 5min、10min、15min、20min、30min、45min、1h、1.5h、2h、3h、6h,0.5-1.0mL of blood was taken from the eyelids.Then 12,000rpm·min-1 centrifuged at low temperature for 10 min and rat plasma was aspirated.Add 10μL of the internal standard solution,vortex and mix.Pipette 360μL of methanol for extraction.Shake well for 5 min after extraction.Centrifuge at 12,000 rpm for 10 min.Pipette the supernatant and filter.After the sample was completely dried,100μL of the mobile phase was added to reconstitute the solution,shake well,and then the supernatant was centrifuged.Then determined the samples according to established assays.2.3 Determination of folic acid metabolizing enzymes in related tissues of normal and weightlessness SD rats’biological samples.The weightless SD rats of 1,2,3,4 weeks and blank group took the liver,lung,kidney,duodenum,jejunum and ileum tissues were treated with 0.9%NaCl.After pretreatment,the expression of MTHFR,CBS and MS were determined by RT-PCR and Western-Blot.2.4 Intestinal absorption experiment of FA and ZPDAgelient 1200 was applied as HPLC system.We used ODS-3 as column of Intertsil,The particle size was 5μm,and the column was 4.6×150mm.The FA mobile phase was composed of phosphoric acid（pH5.0）（Potassium dihydrogen phosphate 2.0g,water650mL,1moL?L-11 phosphoric acid 7mL,270mL methanol,use 1moL?L-11 phosphoric acidor ammonia to adjust pH 5.0,dilute with water to 1000mL）.The flow rate of the HPLC was set to 1.2 mL·min-1,isocratic elution,sample injection volume was 10μL,and the UV detection wavelength was 280 nm.The ZPD mobile phase was composed of acetonirtile（20%）-methanol（17%）-phosphate buffer（63%）,0.15%phosphoric acid and triethylamine solution were added to the buffer salt.The flow rate of the HPLC was set to 1.0 mL·min-1,isocratic elution,the sample injection volume was 10μL,and the UV detection wavelength was 254 nm.3.Result3.1 Determination the SD rats of FA and ZPD by LC-MS/MS method in plasma samples.In the range of 202000 ng·mL-1 the FA plasma standard curve y=2.22e-4x+0.00357（r=0.9998）showed a good linear relationship.In the range of 1200 ng·mL-1the ZPD plasma standard curve y=2.18e-2x+0.0932（r=0.9999）showed a good linear relationship.Methodological verification results met the requirements for biological sample determination.3.2 Study on the pharmacokinetics of oral FA and ZPD in normal and weightlessness SD rats.FA:After orally administrated FA(0.5208 mg·kg-1),the C-T curves of SD rats at 1,2,3,4 weeks who under the weightlessness were significantly different from normal rats.The results showed there was a significant difference in Cmax、AUC（0-t）、Tmaxax and t1/2between normal group and weightless group（P<0.05）.As the time of weightlessness increases,the plasma FA Cmax of rats gradually decreased,the rats of weightlessness in 4weeks decreased to 50%of the normal group.The AUC（0-t）was the highest in the second week,then gradually decreased,the rats of weightlessness in 3 weeks and 4 weeks decreased 20%.Tmax was showed an increasing state in the front of three weeks,the rats of weightlessness in 3 weeks increased 80%,but the fourth week was similar to the normal group,and the t1/2/2 increased significantly in fourth week,increased 101%.ZPD:After orally administrated ZPD(1.0416 mg·kg-1),the C-T curves of SD rats at1,4 weeks who under the weightlessness were no significantly different,there was a significant difference in Cmax、Tmax and AUC（0-t）between the normal group and the weightlessness SD rats of 2 weeks（P<0.05）.The weightlessness SD rats of 2 weeks Cmaxax decreased 25%,Tmax increased 98%,AUC（0-t）decreased 16%.There was a significant difference in AUC（0-t）between the normal group and the weightless 3 weeks SD rats（P<0.05）,decreased about 14%.There was no significant difference Cmax、Tmaxax and t1/2between the normal group and the weightless group of 3 weeks（P>0.05）.There was no significant difference in Cmax、Tmax、AUC（0-t）and t1/2/2 between the normal group and weightless groups of 1 week and 4 weeks（P>0.05）.3.3 Determination of folic acid metabolizing enzymes in related tissues of normal and weightlessness SD rats’biological samples.The results showed that mRNA and protein expression of MTHFR in the kidney were higher in the blank group,mRNA and protein expression in the three weeks of weightless rats in lung were higher than those in the other four groups.The expression of mRNA and protein in the 2 weeks of weightless rats in the duodenum was higher than that in the other four groups.The expression of mRNA and protein in the jejunum was the highest in 4weeks of weightless rats,and the expression of mRNA and protein in the 1 week of weightless rats in the ileum was highest.CBS expressed the highest mRNA and protein content around the 4 weeks of weightless rats in the liver and lung.The expression of mRNA and protein in the weightless rats of the kidney was lower than normal group.The mRNA and protein expression in 2 weeks of weightless rats in the duodenum and jejunum were higher than other four groups.1 week of weightless rats in the ileum was higher than the other four groups.MS in 1 week of weightless rats had the highest expression of mRNA and protein in the livers and ileum.The expression of mRNA and protein in the weightless rats of the kidney were lower than that in the normal group.The 3 weeks of weightless rats had the expression of protein was high in lung.The expression of mRNA and protein in the 2weeks of weightless rats in the duodenum were higher than that in the other four groups.The expression of mRNA and protein in the jejunum around the 4 weeks of weightless rats were the highest.3.4 Intestinal absorption experiment of FA and ZPDFA and ZPD have good linearity in the range of 5-200μg·mL-1.The FA regression equation was Y=32.5053X-75.9772,R2=0.99902.The average spiked recovery was99.87%.The precision of the method was 0.36%,the stability was good.The ZPD regression equation was Y=27.2334X-10.5722,R2=0.9999,the average spiked recovery was 99.63%.The precision RSD was 0.33%,the stability was good,and the FA in duodenum intestinal absorption parameter values were Ka=1.46±0.28(×10-4)(s-1),Peff=1.96±0.52(×10-4)(cm·s-1).The ZPD in duodenum intestinal absorption parameter values were Ka=1.95±0.37(×10-4)(s-1),Peff=3.24±1.38(×10-4)(cm·s-1).4.ConclusionBased on the above research results,it is necessary to adjust the amount of FA and ZPD in the state of weightlessness.However,there are many difficulties in the study of pharmacokinetics of human under weightless conditions.Therefore,it is necessary to combine normal and simulated weightlessness physiological parameters,FA and ZPD physicochemical parameters,intestinal absorption parameters,pharmacokinetic parameters to establish normal and simulated weightless rat PBPK model and normal human PBPK model.Then in order to calculate the body’s drug dosage in weightless state. |
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