Effects Of Acute Hypoxia On Pharmacokinetics And Its Drug Transporter Regulatory Mechanism

【Background】 There are more than 2 millions people exposed to high altitude every year in china, but the dosage and dosing interval of drugs were not changed in high altitude when they fall ill, the safty of drug can not be guaranteed under hypoxia, therefore, we should pay more attation on the research of drug safety at high altitude. Pharmacokinetics is the key role in rational use of medicines, but there are few researchs for us to reference. How did the pharmacokinetics changed under hypoxia? It is not clear. Many factors can affect the drug’s pharmacokinetics, such as gastric emptying, blood rheology, heart and lung function, liver and kidney function, P450 enzymes, drug transporters, etc., most previous studies have focused on the impact of hypoxia on hepatic drug metabolizing enzyme, few researchs have been published on the drug transporters. Actually, drug transporters were more important than the enzymes, because the enzymes were almost exist in the liver, and have few effects on absorption, distribution and excretion of the drugs except metabolism. On the contrast, drug transporters is a key factor in activing the drugs transport across the cell membrane into the body, such as MDR1, BCRP, MRP2, MRP3, OCT, OATP, OAT, PEPT, etc., which are widely present in the small intestine villus epithelial cells, renal tubular epithelial cells, hepatocytes and biliary epithelial cells, they play an important role in drug absorption, distribution, metabolism and excretion(ADME), especially P-gp, which widely distributed in many tisuses and has a number of substrate, it is very important in drug absorbing and transporting. Therefore, it is very important to study the drug transporters changes under hypoxia and its impact on ADME of drugs, especially the regulatory mechanisms under hypoxia, which will play an important role in improving the rational use of drugs at high altitude.【Aims】 To investigate the effect on pharmacokinetic changes of levofloxacin and other drugs after acute altitude hypoxia; discover the different expression of drug transporters(P-gp) in small intestine, liver and kindey, and its effects on the absorption and transport of levofloxacin in the small intestine under hypoxia; discuss the the regulatory mechanisms of P-gp under hypoxia, and provide data support and theoretical basis for rational use of drugs at high altitude.【Methods】 1. To compare the differences of physiological parameters, biochemical parameters, drug pharmacokinetics between high altitude and plain area, by oral administrate drugs at plain area and administered again when acute exposured to high altitude after a 7-10 day washout period, examin the effects of hypoxia on the pharmacokinetic parameters of levofloxacinand other drugs. 2. High throughput PCRarray are used to screen the expression profile difference in rats’ small intestine, liver and kidney between high altitude and plain area, q RT-PCR and Western Blot are used to verify the changes of drug transporters which were common and significant changed in those tissues, especially P-gp. 3. To evaluate the influence of hypoxia on absorption and transport of P-gp’s specific substrate levofloxacin in the small intestine through single-pass intestinal perfusion model in vitro, everted intestinal sacs model, and Caco-2 cell monolayer model. 4. Based on the high-throughput microarray expression which had show that lnc RNA-H19 is a key role in regulating the expression of MDR1 under hypoxia, the H19 expression was depleted through lentivirus stably transfected in Caco-2 cells,q RT-PCR, Western Blot were used to examine the P-gp and associated transcription factor(HIF-1a, NF-κB, PXR, CAR) expression while H19 was slienced, all these researchs are in order topredict which is the upstream or downstream target genes of H19.【Results】 1. Flight stress has no significant effects on the physiological parameters, biochemical parameters and the pharmacokinetics of levofloxacinin in rats, on the other hand, hypoxia had significantly alter the oxygen saturation, the function of hepatic and renal at high altitude. The pharmacokinetic parameters of levofloxacin were significantly changed, the mainly performance is hypoxia will increase the AUC, t1 / 2, and Cmax, and decrease CL in rats at high altitude. We had evaluated the pharmacokinetics of 16 drugs at high altitude, there are 10 drugs for bioavailability increased, metabolism and excretion slowed down; in contrast, 4 drugs for bioavailability decreased, metabolism and excretion enhanced; the remaining 2 drugs have nosignificant changes. 2. 5, 18 and 8 drug transporters’ expression had decreased more than two times in small intestine, liver and kidney respectively through high throughput PCRarray, on the other hand, 12,3 and 4 drug transporters’ expression had increased more than two times in small intestine, liver and kidney respectively; the common and significant changed in those tissues is P-gp, q RT-PCR found that the relative levels of MDR1 were significantly reduced about 50.80% in small intestine, and incerased about 1.15 times and 49.00% in liver and kidney respectively.Western blot results showed that the relative levels of P-gp were significantly reduced about 71.30% in small intestine, and incerased about 1.33 and 1.83 times in liver and kidney respectively. All the results showed that the drug transporters which are the main factors affecting the pharmacokinetic changes had changed significantly and exhibit an apparent tissue specificity during hypoxia. 3. In the single-pass intestinal perfusion model, decreased expression of P-gp caused the intestinal wall permeability increased by 56.16%, 226.00%, 77.74% and 141.00% in the time intervals at 30-60 min, 60-90 min, 90-120 min and 120-150 min, everted intestinal sac model experiments showed that hypoxia can significantly improve the absorption of levofloxacin in the duodenum, jejunum and ileum; Caco-2 cell monolayer model experiments showed that hypoxia can slowing the rate of transport of levofloxacin from side A to side B, on the contrary, the transport rate from side B to side A was increased, hypoxia can enhances the efflux efficiency of P-gp in Caco-2 cells. 4. The lnc RNA-H19 expression and P-gp expression under hypoxia were positive correlated, silence H19 could significantly inhibit the expression of P-gp, and reduce the expression of PXR and CAR, which is the related transcription factors of P-gp, but had no significant effect on the expression of HIF-1a and NF-κB, the results indicated that lnc RNA-H19 is a key molecules in the regulatory networks of MDR, PXR and CAR are the downstream gene of the H19, and HIF-1a and NF-κB are not the downstream gene of the H19.【Conclusion】 Hpoxia can significantly affect the pharmacokinetic parameters of levofloxacin after acut exposed to high altitude, and the changes of drug transporter expression is an important factor in pharmacokinetic changes, especially the changes of P-gp, it can significantly affect the absorption and transport of levofloxacin in the small intestine; P-gp and its related transcription factors were significantly reduced while silencing the lnc RNA-H19 which is the upstream gene of P-gp. The preliminary results indicated that lnc RNA-H19 is a key gene in the regulatory networks of MDR1, which might regulating the P-gp expression by affecting DNA binds proteins expression( PXR CAR) to prevent transcription factor combining with the gene promoter of MDR1. This study provided data support and theoretical basis for rational use of drugs at high altitude.