Purification And Preparation Of Two Types Of Isomers Of The Gadoxetate For Rabbit MR Imaging And Pharmacokinetic Study

Gadoxetate disodium（trade name:Primovist）,is a liver-specific contrast agent,obtained by adding lipophilic moiety（an ethoxybenzyl group,EOB）to the Gd-DTPA,thus the molecular structure has both hydrophilic and lipophilic properties.It can be excreted by both liver and kidney concurently.Gadoxetate can be specifically uptake into hepatocytes by Organic Anion Transporting Peptides（OATPs）in the basolateral membrane of hepatocytes and excreted through the biliary system as prototype.Gadoxetate has two types of isomers,Gd-A and Gd-B,which exist in a relatively constant proportion（65%:35%）under the condition of 25～120℃and p H value of 5～9（65%:35%）;Animal studies have found after it’s intravenous administration,the ratio of the two in rat bile was much higher than 65%:35%,suggesting that the uptake and clearance of Gd-A by the liver is much higher than that of Gd-B.The two types of isomers have different pharmacokinetics.In theory,purified Gd-A has higher liver affinity and MRI imaging efficiency than Gd-B.Using pure Gd-A instead of Gadoxetate for liver MR imaging may be shortening the imaging time and improving the efficiency of diagnosis and may also reduce the amount of drug administration and the time course in body,thereby reducing the metabolic burden of gadolinium and the risk of deposition in the body.However,there is no animal or human pharmacokinetic study of pure Gd-A.In addition,the equilibrium ratio of isomerization of Gd-A and Gd-B at room temperature is 65%:35%,and the purified Gd-A will transform into Gd-B slowly.How to keep the purified Gd-A at a high purity for a long time?There is no relevant report on this question.This study tried to verify the followings:1)the feasibility of Gd-A purification and maintain purity in long-term storage,2)the difference between the Gd-A and Gd-B on MR imaging and pharmacokinetics,and whether pure Gd-A has potential clinical value?Using reversed phase high-performance liquid chromatography to separate Gadoxetate to obtain purified Gd-A and Gd-B（in aqueous solutions）,which are further subjected to nitrogen（gas）blowing and freeze-drying to obtain the Gd-A and Gd-B solid powders,and test whether the two isomers（Gd-A and Gd-B solid powders）can maintain purity for a long time under different temperature conditions after vacuum packaging.Twelve 6-month-old male New Zealand white rabbits were randomly divided into two groups A and B.MR imaging and pharmacokinetic studies after intravenous administration of pure Gd-A and pure Gd-B were carried out with the same doses,during which the following were measured and compared:the time to peak of the signal intensity of the live(Time to peak;t_{peak A},t_{peak B}),the signal intensity of the liver at peak(Signal intensity;SI_{peak A},SI_{peak B}),and plasma clearance（Plasma clearance;PCL-A,PCL-B）and the half-life(Half-life;t_1/2A,t_1/2B).For statistical comparison,independent sample t-test（Student’s t-test）was used,and p<0.05 was considered as statistically significant.We found that the purity of the Gd-A and Gd-B separated by reversed-phase HPLC technology was above 99%.The solid powders of Gd-A and Gd-B obtained by nitrogen blowing and freeze-drying and packaged in vacuum glass bottles for two months（60 days）remain the purity of 99%,99%,98%,95%for Gd-A and 99%,9%,97%,94%for Gd-B when stored at room temperature（25℃）,low temperature（-20℃）,higher temperature（50℃）and high temperature（80℃）,respectively.After intravenous bolus injection of Gd-A and Gd-B,the time to peak of the liver parenchyma on MRI T1WI dynamic imaging,t_{peak A}and t_{peak B},were 3.49±0.99 min and 11.5±2.03 min,respectively.The difference between the two was statistically significant（p<0.001）.The peak signal intensities,SI_{peak A}and SI_{peak B},of the liver parenchyma were 825.7±92.9 and 714.5±78.8,respectively,p=0.049.The plasma clearance of Gd-A and Gd-B,PCL-A and PCL-B,were 25.41±5.174 ml/min and 6.734±1.834 ml/min,p<0.001,respectively;The half-lives(t_1/2A and t_1/2B)were 22.91±3.42 min and35.27±4.31 min,respectively,p<0.001.In short,the solid powders of Gd-A and Gd-B can be stored at room temperature or low temperature for a long time and maintain high purity after vacuum packaging,which provides feasibility for animal and clinical experiments.The imaging efficiency of Gd-A rabbit liver is better than that of Gd-B.The peak time of liver parenchyma of Gd-A is significantly shorter than Gd-B.The signal intensity of the liver peak Gd-A is also higher than that of Gd-B;at the same time,due to Gd-A The plasma clearance rate of Gd-A is high,and its half-life in rabbits is shorter than that of Gd-B,which further reduces the risk of gadolinium deposition in the body.Therefore,pure Gd-A has potential clinical application value.