Effects Of Polysorbate 80 On Activity And MRNA Expression Of Cytochrome P450 Enzymes In Rats

Objective: Pharmaceutical excipients are the material basis for the production and development of pharmaceutical preparations which play a key role in the formulation and production of pharmaceutical preparations.Medicinal excipients are often regarded as inert substances.At present,the attention paid to excipients mainly focuses on the quality and physical and chemical properties of excipients,but the biological effects of excipients have not attracted enough attention.However,there is increasing evidence that pharmaceutical excipients indirectly or directly affect the metabolism,efficacy,and safety of drugs in the body.Polyoxyethylene sorbitan monooleate(polysorbate 80,PS80)is a pharmaceutical excipient that is widely used in liquid,semi-solid,and solid formulations.Meanwhile,Cytochrome P450 enzymes are phase I metabolic enzymes that play a key role in drug metabolism.At present,relevant literature has reported potential effects of PS80,but the effect of PS80 on the expression of drug metabolizing enzymes and m RNA is still unclear.The purpose of this study was to investigate the effect of PS80 on the expression of Cyp1a2,Cyp2b1,Cyp2c6,Cyp2c11,Cyp2c7,Cyp2d2,and Cyp3a1 enzymes in rats from the level of enzyme activity and m RNA expression in order to provide guidance for clinical drug use.Methods: Firstly,a method for the simultaneous determination of the corresponding probe substrates(phenacetin,amfedone,diclofenac acid sodium,omeprazole,amodiaquine,dextromethorphan,midazolam)was established for the simultaneous determination of seven major metabolic enzymes,Cyp1a2b1,Cyp2c6,Cyp2c11C11,Cyp2c7Cyp2d2 and Cyp3a1,in rats plasma at the same time,for the determination of the corresponding probe substrates(phenacetin,amfedone,diclofenac sodium,omeprazole,amodiaquine,dextromethorphan,midazolam).Secondly,48 SD male rats were randomly divided into 8 groups: four of them were PS80 tail vein administration group: normal saline Group,PS80 low,middle and high dose groups(37.5,75,150mg/kg)were injected tail vein intravenously for PS80 for 14 consecutive days;the other four groups were administered with PS80 intragastric administration group:ultrapure water group,PS80 low,middle The high-dose group(250,500,1000 mg/kg)was administered intragastrically to PS80 for 20 consecutive days.After each group was given on the last day of administration,each group of rats was injected with a mixed probe solution via the tail vein,and blood was collected from the rat's fundus venous plexus at the set time point,and LC-MS/MS method was used to measure different groups.The plasma concentrations of 7 kinds of probe substrates in rat plasma were calculated,and the main pharmacokinetic parameters of each group of probe substrates were calculated using DAS2.0 software,and statistical analysis was conducted to investigate the effect of PS80 on the activity of Cyp450 enzyme in rats.At the same time,another 48 male SD rats were randomly divided into 8 groups randomly and administered in the same group as the above.Rats were sacrificed by cervical dislocation on the last day.Liver tissues were taken and the major cytochromes in each group were determined by fluorescent quantitative PCR.The m RNA expression level of P450 isoforms was analyzed to determine whether PS80 had an effect on the gene expression levels of the seven Cyp450 enzymes in rats.Results: This study established a simultaneous determination of seven probe substrates in rat's plasma,phenacetin,bupropion,diclofenac sodium,omeprazole,amodiaquine,dextromethorphan and midazolam.The LC-MS/MS analysis method was validated by the methodological validation;the results of the PS80 enzyme activity and m RNA expression of the cytochrome P450 in rats were basically consistent: 1.PS80 tail vein administration group: 1PS80 medium,high the activity of Cyp1a2 and Cyp2c11 was significantly inhibited in the dose group,and the m RNA expression was down-regulated.2The high and middle doses of PS80 significantly inhibited the activity of Cyp2b1 and the low,medium,and high doses of PS80 significantly down-regulated m RNA expression.3PS80 high dose group significantly inhibited the activity of Cyp3a1 enzyme and down-regulated the m RNA expression;4PS80 dose group had no significant effect on the enzyme activity and m RNA expression levels of Cyp2c7,Cyp2c6,and Cyp2d2.PS80 intragastric administration group: 1PS80 low,medium and high dose group significantly inhibited the activity of Cyp2b1 enzyme and down-regulated the m RNA expression;2PS80 low,medium and high dose groups significantly inhibited the activity of Cyp1a2 enzyme and PS80 Both high-dose and high-dose groups significantly down-regulated the m RNA expression;3PS80 medium and high-dose groups significantly inhibited the activity of Cyp2c11 and down-regulated the m RNA expression;4PS80 high-dose group significantly inhibited the activity of Cyp3a1 and down-regulated its m RNA The expression of 5PS80 in each dose group had no significant effect on the enzyme activity and m RNA expression levels of Cyp2c7,Cyp2c6,and Cyp2d2.Conclusion: PS80 has a certain effect on Cyp450 enzyme activity and m RNA expression.Except for Cyp2c7,Cyp2c6,and Cyp2d2,PS80 shows different inhibitory effects on Cyp1a2,Cyp2b1,Cyp2c11,and Cyp3a1 enzymes.In conclusion,this study shows that when PS80 is used in combination with substrates metabolized by Cyp1a2,Cyp2b1,Cyp2c11,and Cyp3a1 enzymes(CYP1A2,CYP2B6,CYP2C19,and CYP3A4 in humans),the possible interaction between PS80 and these substrates should be fully considered to avoid adverse reactions.