Simulation Of Human Pharmacokinetics Across Different Species Using Atipamezole As A Model Drug

Atipamezole?ATI?is commonly used to recover animals from sedation-anesthesiainducedby?₂-adrenoceptoragonists?especially dexmedetomidine?.It can also effectively alleviate the adverse reactions induced by the combination of metopidine-midazolam-ketamine.Recent experimental animal studies suggest atipamezole can enhance sexual activity and increase pain-related responses.Bsaed on its potential clinical applications,it was picked up as an IND to be developed by our institute.Combined with systemic pre-clinical pharmacokinetics of atipamezole and in vitro and in vivo inevestigation,our study illustrated the pharmacokineticcharacteristicsofatipamezoleandestablishedPBPK?physiologically based pharmacokinetic?model of animal and human.The aim of this study is to overcome the different species barrier,predict human pharmacokinetics based on pre-clinical data,and provide more human relevant data for clinical research.The research of includes the following contents:1.The quantification method of atipamezole in dog and rat plasma was established by using liquid chromatography-tandem mass spectrometry?LC-MS/MS?.The methodology was systematicly verified including specificity,precision,accuracy,matrix effect,recovery,stability and and dilution reliability.2.Systematically study the pharmacokinetics of rats and beagle dogs after intramuscular and intravenous adminstration of different doses of atipamezole.The pharmacokinetic characteristics were obtained and compared.3.The distribution of free drug in the main tissues including brain,liver and kidney after intramuscular admininstration of atipamezole was obtained after corrected by specific free fraction.The dose denpendent brain exposure was also investigated.4.The total recovery of atipamezole through urine,feces and bile was investigated after intramuscular administration of atipamezole in rats.5.In vitro metabolic stability and metabolites mapping of atipamezole in liver microsomes of different species were compared.The metabolic pathway mediated by CYP was conducted.6.Physicochemical and ADME related parameters used for PBPK model construction were acquired which include the LogD,solubility,the blood:plasma partitioning ratio(R_bp),the tissue:plasma partition coefficient?K_p?,the phenotypeing,the enzyme kinetics and CYP inhibition etc.7.PBPK models of the atipamezole in rats and human were established and validated.Pharamcokinetics of atipamezole in Chinese was predited ultimately.The main results are as follows:1.The quantification method of atipamezole was successfully established and validated according to the latest guidelines of CFDA.The specificity,precision,accuracy,matrix effect,recovery,stability and and dilution reliability all met the standards of the quantification in biological samples.After intramuscular administration of four doses?0.1,0.3,1,3mg/kg?in rats,atipamezole was absorbed rapidly with the peak time(T_max)of 15-30min.The blood concentration was reduced to less than 1/20 of the peak concentration 14h after the dosing.AUC and C_max increased with the increase of dose.In the dosage range of0.1-1mg/kg,both the exposure and the peak concentration increased porportionally with the dose.However,non-linear exposure occurred at the dosage of 3mg/kg.After intramuscular administration of three doses?0.2,0.5,1.25mg/kg?in beagle dogs,atipamezole was absorbed rapidly with the peak time(T_max)of 8-30min.The blood concentration was reduced to less than 1/20 of the peak concentration 14h after the dosing.AUC and C_max increased with the increase of dose.In the dose range of0.2-0.5mg/kg,both the exposure and the peak concentration increased proportionally.However,non-linear exposure occurred at the dosage of 1.25mg/kg.The absolute bioavailability of atipamezole in rats and beagle dogs was 61%and82%,respectively.There was no statistically significant difference in the main pharmacokinetic paramters between male and female beagle dogs after intramuscular of atipamezole.2.Atipamezole could distribute to the tissues or organs rapidly after intramuscular administration at 1mg/kg in rats.In most tissues the concentration reached the peak level at 15min post dosing.Concentration of atipamezole in the tissues decreased below the quantitative lower limit?1ng/g?after 24h and no accumulation could be observed in tissues.The exposure of brain is higher than that of plasma,indicating that atipamezole is prone to across the blood-brain barrier.It is also easily exposed in tissues with abundant blood flow such as liver,kidney and lung,but it is low in fat and muscle,indicating that the tissue permeability mechanism of atipamezole is mainly driven by blood perfusion.3.After intramuscular administration of 1mg/kg in rats,the total recovery of atipamezole through urine,feces and bile were?0.28±0.48?%suggesting that the kidney clearance of atipamezole is negligible and it is mainly eliminated by biotransformation.4.Using UPLC/LTQ-Orbitrap-MS to identify and compare the in vito metabolites of atipamezole in liver microsomes of different species,three oxidation metabolites were all found.Although the amount of the three metabolites is different,but no species diffidence occurred in the in vitro bio-transformation,suggesting that rats and beagle dogs can be used for preclinical drug safety evaluation.Using the recombinant human CYP isoforms,we identified that atipamezole is bio-transformated to different metabolites in CYP2A6,2B6,2D6,2C19 and 3A4.5.The blood:plasma partitioning ratio(R_bp)for atipamezole in rat and dog were 1.07 and 0.9,respectively.CYP2B6,CYP2C19,CYP2D6 and CYP2A6 were involved in the phase I metabolism of atipamezole,with contribution of 64.4%,22.8%,12.7%and 0.1%,respectively.Atipamezole showed strong inhibition against seven CYP enzymes in liver microsomes of different species,with IC₅₀ between0.005-9.68?M,but no time-dependent inhibition and self-inhibition was involved.6.The PBPK model of rat and human was established using Gastroplus^TMM software.The PBPK model could successfully simulate the dose-dependent pharmacokinetics and brain tissue distribution characteristics in rats.The PBPK model of human was established according to the human pharmacokinetics reported in the literature.However,atipamezole did not exhibit significant nonlinear pharmacokinetics similar in rats after increasing the dose.After intramuscular administration of atipamezole,the exposure both plasma and brain were increased linearly with the dose less than 500mg but non-linear exposure occurred at the dose more than 1000mg.The predicted results will provide useful information for subsequent clinical trials and are expected to be confirmed through clinical studies.In summary,we attempt to explore a pathway by PBPK apparoach to overcome the cross-species barriers to simulate the pharmacokinetic of human using atipamzole as a model drug.The reasons led to nonlinear pharmacokinetics of atipamezole were delinated and the prospective human pharmacokinetics different from animals was predicted.