Liver-targeting Molecular Mechanism And Preliminary Study On Pharmacokinetic Of Mn(Ⅱ) MRI Contrast Agent

Objective:To investigate the molecular mechanism of cell uptake of Mn-BnO-TyrEDTA by hepatocytes.To evaluate the safety of Mn-BnO-TyrEDTA by a preliminary study on pharmacokinetics.Methods:1.The cytotoxicity of Mn-BnO-TyrEDTA on RAW264.7 cells was evaluated by Cell Counting Kit-8 assay.2.Over-expressed OATP1B1 in HEK-OATP1B1 cells was transfected and the cell uptake of Mn-BnO-TyrEDTA in HEK-293 and HEK-OATP1B1 cells was performed by using BSP as a competitive inhibitor.3.Carotid artery cannula and common bile duct cannula in SD rats were used to collect blood and bile at different time points(periods)before and after administration,respectively.Mn2+concentration of the sample was detected by ICP-MS.The in vivo biodistribution in nine tissues(heart,brain,Liver,spleen,lungs,kidney,bone,muscle,small intestine)of Mn content was quantitatively measured by ICP-MS.The urine and feces of SD rats were collected,and LC-MS qualitatively analyzed whether the complex is intact Mn-BnO-TyrEDTA in vivo.Results:1.At low drug loading concentration(<125 μM),it was found that RAW264.7 cells were significantly viable with viability over 80%.At high concentration(>250 μM),the survival rate of RAW264.7 cells dropped to 75%,indicating that Mn-BnO-TyrEDTA did not show any appreciable cytotoxicity.2.The uptake rate of Mn-BnO-TyrEDTA by HEK-OATP1B1 cells was approximately 2-fold higher than that of HEK-293 cells when incubating with 0.1 mM drug loading for 30 min(HEK-OATP1B1:Vmax=73.73±10.11 pmol/mg,HEK-293:Vmax=34.68±2.3 pmol/mg).BSP can significantly inhibited the uptake of Mn-BnO-TyrEDTA.3.Mn-BnO-TyrEDTA exhibits a rapid biexponential plasma clearance with a half-life of distribution(tl/2α)of 0.83±0.24 minutes and a half-life of elimination(t1/2β)of 9.33±1.92 minutes.4.The rapid systemic distribution of Mn-BnO-TyrEDTA was observed after administration.The excretion of Mn-BnO-TyrEDTA was via both renal and hepatobiliary routes with Mn levels returning to baseline within 24 h.The excreted of Mn-BnO-TyrEDTA detected by LC-MS was intact,implying that no transmetalation and metabolism occurred in vivo.Conclusion:The mechanism of hepatic targeting of Mn-BnO-TyrEDTA is the hepatic uptake of the amphiphilic anion contrast agent mediated by OATPs expressed by functional hepatocytes.Mn-BnO-TyrEDTA is rapidly and intactly excreted by hepatobiliary and renal clearance pathways and Mn levels returned to baseline within 24 h.The hepatic targeting and dual excretion pattern of complex are very similar to the liver-specific Gd-EOB-DTPA.