Fluconazole’s Transport Mechanisms Across The Blood-Brain Barrier And Blood -CSF Barrier In Rats

Background:The CNS is a sanctuary organ which is protected by two major barriers namely the blood-brain barrier (BBB),the blood cerebrospinal fluid barrier (BCSFB) and the blood cerebrospinal fluid brain barrier.BBB is considered as the most important barrier.The BBB is consist of tight junctions(TJ) that interconnect adjacent endothelial cells and occlude the paracellular spaces, endothelial cells basement membrane with nucleotidase and nonspecific cholinesterase and a layer of astrocytic end feet and pericytes under the basement membrane.According to the molecular structure of blood-brain barrier,the permeability of BBB should be the smallest depends on molecules’s size and lipophilicity. However, the investigator found that vincristine and epipodophylotoxin displayed poor BBB penetration, despite their high lipophilicity. P-GP, a member of ATP-binding cassette (ABC) transporter superfamily was found connected with the observed poor BBB penetration of lipophilic drugs by Cordon-Cardo et al. in the late 1980s. These investigators clearly demonstrated that P-GP was highly expressed on the apical surface of the endothelial cells of the brain capillaries facing the blood circulation. P-GP identifies a variety of different drugs and pumps them out of BBB,which strengthens the blood-brain barrier and blood-brain barrier’s function. Recent studies have shown that P-GP is predominately expressed on the surface of capillary endothelial cells facing the blood circulation and acted as efflux transporter, while it localizes subapically, conferring an apical-to-basolateral drug-transport barrier on the choriod plexus. This research finding may be related with that fluconazole concentration in CSF disproportionate with its clinical efficacy,but it remains to be proved.Objective:Compare SD rats fluconazole concentration in plasma, CSF and bECF with or without administration of P-GP inhibitor verapamil. Study the pharmacokinetics of fluconazole after its absorption into plasma, CSF and bECF. Thus demonstrate if fluconazole is P-GP’s substrate and the differences between blood brain barrier and blood cerebrospinal fluid barrier in vivo.Method:Observe unbound fluconazole concentration in plasma, CSF and bECF with or without administration of P-GP inhibitor verapamil by microdialysis combined with high performance liquid chromatography(HPLC) after single intravenous injection of fluconazole(40mg/Kg). Calculate area under concentration-time curve of fluconazole,the permeation rate of blood brain barrier and blood cerebrospinal fluid barrier and its correlation with P-GP’s function.Results:Successfully established brain two-site synchronously microdialysis S.D. Rat models. After a single I.V. administration of fluconazole, the Single Fluconazole Group: the distribution of fluconazole in plasma, bECF and CSF had significant difference(P<0.05); The concentration of fluconazole in bECF(3.13～4.15μg/ml) had reached the MIC of cryptococcus neoformans(3.12～6.25μg/ml) wtihin 180 minutes; the penetration rate of BCSFB (AUCCSF-05h/AUCplasma-0-5h) was significantly higher than BBB (AUCbECF-0～5h/AUCplasma-0～5h) within 5 hours. Fluconazole+Verapamil Group: the distribution of fluconazole in plasma, bECF and CSF had obvious difference(P<0.05); the penetration rate of BBB was significantly higher than BCSFB within 5 hours; the concentration of fluconazole in plasma and CSF had no significant change compared with the Single Fluconazole Group, while the bECF concentration elevated significantly and reached the MIC of cryptococcus neoformans within the whole 5 hours.Conclusion:Brain two-site synchronously microdialysis has been successfully used to acquire dialysate samples of CSF and bECF. The distribution of fluconazole in CSF and bECF and the penetration rate through BBB and BCSFB had significant difference, thus it’s rigorous to use CSF concentration to predict brain target concentration. The distribution of fluconazole in CSF decreased while that increased in bECF when used in combination with varapamil.So we can draw a conclusion that P-GP plays an important role in BBB and BCSFB transport and P-GP inhibitor offers a new research perspective in improving drug penetration through BBB.