title: Penetration of verapamil across blood brain barrier following cerebral ischemia depending on both paracellular pathway and P-glycoprotein transportation.
abstract: BACKGROUND
  Blood brain barrier (BBB) dysfunction is a common facet of cerebral ischemia, and the alteration of drug transporter, P-glycoprotein (P-gp), has been documented.
  
  
  OBJECTIVE
  This study explores influence of damaged BBB and elevated P-gp on cerebral verapamil penetration after ischemia both in vivo and in vitro.
  
  
  METHODS
  Middle cerebral artery occlusion (MCAO) induced ischemia/reperfusion (I/R) of rats, and Na(2)S(2)O(4) induced hypoxia/reoxygenation (H/R) damage of rat brain mirovessel endothelial cells (RBMECs) respectively, served as BBB breakdown model in vivo and in vitro. Evans-Blue (EB) extravagation and (125)I-albumin were used to quantify BBB dysfunction; UPLC-MS/MS analytical method was performed to determine accurately the concentration of verapamil in brain tissue and cell. Flow cytometry, immunohistochemistry and western blotting were applied to evaluate transport function and protein expression of P-gp.
  
  
  RESULTS
  Overexpressed ICAM-1 and MMP-9 mediated BBB dysfunction after ischemia, which induced EB leakage and (125)I-albumin uptake increase. Enhanced accumulation of verapamil in brain tissue, but intracellular concentration reduced evidently after H/R injury. Transcellular transportation of verapamil elevated when P-gp function or expression was inhibited after H/R injury.
  
  
  CONCLUSIONS
  These data indicated that BBB penetration of verapamil under ischemia condition was not only depending on BBB breakdown, but also regulated by P-gp.
authors.affiliation: Department of Physiology, School of Pharmacy, China Pharmaceutical University, Nanjing 210009, PR China.
authors.forename: Weirong
authors.initials: W
authors.lastname: Fang
authors.forename: Peng
authors.initials: P
authors.lastname: Lv
authors.forename: Xiaohan
authors.initials: X
authors.lastname: Geng
authors.forename: Erxin
authors.initials: E
authors.lastname: Shang
authors.forename: Qichuan
authors.initials: Q
authors.lastname: Yang
authors.forename: Lan
authors.initials: L
authors.lastname: Sha
authors.forename: Yunman
authors.initials: Y
authors.lastname: Li
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active; D001812:Blood-Brain Barrier / Q000378:metabolism*; D015153:Blotting, Western; D002545:Brain Ischemia / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000378:metabolism*; D002196:Capillaries / Q000187:drug effects / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D002534:Hypoxia, Brain / Q000503:physiopathology; D007150:Immunohistochemistry; D007202:Indicators and Reagents; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis; D008297:Male; D013058:Mass Spectrometry; D020780:Matrix Metalloproteinase 9 / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000378:metabolism; D020112:Rhodamine 123; D014700:Verapamil / Q000378:metabolism*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002121:Calcium Channel Blockers; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D018799:Intercellular Adhesion Molecule-1; D020112:Rhodamine 123; D014700:Verapamil; D020780:Matrix Metalloproteinase 9