Preclinical Pharmacological Study And Preliminary Exploration Of Pharmacological Mechanism For Dexmedetomidine Analogues

Objective:Dexmedetomidine（DEX）,as a new sedative hypnotic,has attracted much attention due to its high affinity to activateα₂-adrenergic receptor,resulting in sedative,anti-anxiety,anti-sympathetic activity and analgesic effect with minor depression of breath.However,patients are vulnerable to significant circulatory fluctuations and bradycardia even if the titration of DEX is gentle and continuous in perioperative period or ICU procedural sedation,not to mention the quick intravenous infusion.We aimed at developing new drugs that can retain the advantages of DEX such as sedative,anti-anxiety and analgesic effect without the circulation and respiration side effects.We obtained a series of analogues by chemical synthesis and modification on the structural basis of DEX.The analogues were comprehensively investigated in sedative efficacy,behavioral levels and changes of circulatory and respiratory state after a single intravenous injection in rats.Furthermore,we explored the pharmacokinetic characteristics of one analogue in rat plasma and identified metabolites after hepatocyte metabolism in vitro.Materials and Methods:The drugs were all administrated by a single rapid injection into tail vein of rats.Firstly,the target compounds with sedative effect were initially screened by dose climbing assay,and five compounds were screened out in total as follows:DA578,595,642,654,657.The ED₅₀ and LD₅₀ were determined by sequential method.Then,we evaluated the efficacy and adverse effects that induced by target compounds.The experiments of water maze and open field test were applied to evaluate the recovery of rats’behavioral state.Blood pressure,heart rate and respiratory parameters after drug administration in rats were also analyzed by the Taimeng BL-420N life monitoring system.Finally,an LC-MS/MS method was developed to determine plasma concentrations of drug DA657.Whole hepatocytes cell system experiments were performed in vitro to identify the metabolic processes of drug DA657 from different species（monkey,beagle Dog）.Results:Five target compounds that could produce potent sedation were selected out of the 92 analogues,named as:DA578,595,642,654,657,respectively.The ED₅₀ of the5 target compounds for inducing loss of righting reflex（LORR）are 13.7（0.013-0.015）mg/kg,11.4（10.82-12.12）mg/kg,4.1（3.84-4.46）mg/kg,5.4（5.25-5.65）mg/kg,3.7（3.44-3.92）mg/kg,and LD₅₀ are:33.5（8.25-9.56）mg/kg,48.85（43.07-55.42）mg/kg,50.89（47.09-54.99）mg/kg,25.91（23.13-29.02）mg/kg,35.35（31.45-39.96）mg/kg,respectively;The ED₅₀and LD₅₀ of DEX are 0.000139（0.0129-0.0150）mg/kg,8.88（8.25-9.56）mg/kg.The TI for the five new drugs were calculated to be:2.45,4.27,6.23,4.76,9.66,respectively.The onset time and recovery time of sedative effects from 595,642,654,657groups were significantly lower than that of DEX group（P<0.05）,except for drug DA578,which was similar to DEX.Mild tremor and respiratory inhibition occurred in all five groups.Only group 595 induced miosis,excitement and screaming phenomenon.No cardiac arrest,severe CNS symptoms were observed in all groups.In the water maze and open field tests,the group DA578 showed the worst memory not only due to its swimming escape latency time was the longest（58.91±12.22 s）and the percentage of dwelling time in target quadrant was minimum（19.18±8.14%）among all groups,but also its average speed of motion was slowest while the total resting time was kept the longest in open field test.Drug 595 impaired the memory moderately with shorter escape latency（54.21±9.43 s）than DA578 but significantly longer than blank solvent control group DMSO（32.2±6.37 s）.The drugs642,654 and 657 had the minimal inhibition on memory and autonomic activity of rats because of their similar levels of escape latency time and the percentage of dwelling time in target quadrant,although the motion speed and rest time at the moment of awakening(recovery of righting reflex,RORR_0min)were significantly worse than those in groups NS,DMSO and DEX which then returned to normal after recovery for 20 min.Drugs DEX,DA578,595,642,654,657 that dissolved in 75%DMSO all presented the consistent fluctuating trend on blood pressure of rats:a sharp decline within 1 min after administration then followed by a relatively slow recovery phase lasting for 5 to 10 min.Within 1 min after injection,SBP of group DEX,DA578,595,642,654,657 were maximally reduced to 62.47%,55.41%,71.22%,65.55%,69.97%,67.78%（all P<0.0001）,respectively.Along with SBP,DBP went down to 62.47%,57.14%,76.49%,77.91%,79.95%,80.64%（all P<0.0001）,and MBP dropped to65.42%,54.60%,74.94%,72.53%,75.48%,74.89%（all P<0.0001）,respectively.Subsequently,blood pressure in groups DA578,595,642,654,657 appeared a rapid transition to the rising phase and returned to normal with slightly reductions of no more than 9%compared to basal levels（all P>0.05）.Only in group DEX,its SBP,DBP,and MAP finally stabilized at approximately 33.92%,33.27%,and 33.97%,respectively（all P<0.0001）below basal levels without returning to normal until 30 min after dose.All the drugs DEX,DA578,595,642,654,657 in each group caused significant decrease in heart rate（HR）.HR in group DEX dropped off rapidly to 22%lower than base level within 1 min after dosing and then maintained at a level approximately 38.38%below the basal level（P<0.05）with no recovery until 30 min after dosing.However,although HR in groups DA578,595,642,654,and 657 touched the bottom quickly within 1 min after dosing,it began to climb back immediately and stabilized by 5 min after dosing at levels of approximately 23.78%,12.48%,2.11%,10.43%,4.28%（all P>0.05）lower to baseline,respectively.Obviously,DEX had the most pronounced inhibition of heart rate.Overall,the five new drugs all inhibited heart rate but to a lesser extent than DEX.Heart rate variability results:RMSSD and SD1 in DMSO,DEX,DA578,595,642,657 performed a transient elevation after dosing and fell back to normal within 5min after dosing,representing a transient enhanced parasympathetic tone and an increase of HRV.Similarly,SDNN and SD2 also rose at first and then decreased to normal,representing an increase in sympathetic activity,a transient decrease of HRV.TP describing the state of cardiac sympathetic parasympathetic balance rose transiently and rapidly returned to normal,indicating that the drug firstly strained the overall autonomic nervous tone,but did not last long.The ratio of LF/HF remained stable during the entire dosing period,indicating that the overall autonomic nervous system regulation of heart rate was not disturbed.The depression of respiratory:Groups DEX,DA578,595 significantly inhibited the respiration rate,while 642,654,657 showed milder respiratory depression after injection.The maximal percent of reduction on respiratory rate in groups DEX,DA578and 595 reached 64.47%,45.54%and 26.36%,respectively（all P<0.05）compared to baseline.However,drugs 642,654,657 only caused a respiratory rate reduction of no more than 10 breaths/min and shorter duration of abnormal breathing（all P>0.05）.Pharmacokinetic results:after injection of doses 3.64 mg/kg（low,L）,7.28 mg/kg（middle,M）,14.56 mg/kg（High,H）,the highest plasma C_max of the prototype DA657was 140834±73773μg/L,212086±113061μg/L,and 469208±325817μg/L,respectively.The elimination half-lives were at 3.51±1.55 h,2.14±0.89 h,1.96±1.06h,respectively.The V_Z was 235-439 L/kg and the CL was 124.70-132.78 L/h/kg.The C_max,AUC_0-t,and AUC_0-∞of prototype DA657 in plasma were evaluated to be linearly proportional to dosages in the dose range that we examined.The ED₅₀ of atipamezole hydrochloride antagonizing the effect of LORR from DEX and DA657 were 0.0105（0.0084-0.0132）mg/kg and 2.3473（1.9384-2.8425）mg/kg,respectively.The results of molecular docking simulation showed that the binding affinity of DA657 and DEX to the receptors was almost the same.The Libdock docking fraction of DEX to the three subtypes ofα_2A,α_2B andα_2C receptors was 87.32,101.83 and 82.88,respectively.The docking fraction of DA657 to the three subtypes ofα_2A,α_2B andα_2C receptors was 77.01,92.04 and 85.66,respectively.Metabolite identification in hepatocyte cell system:the drug DA657 was metabolized mainly through oxidation,glucuronide conjugation,cysteine conjugation,and glutathione conjugation pathways in hepatocytes of monkey and beagle dog in vitro.In all 12 metabolites were detected and named as follows:M1-1,M1-2;M2-1,M2-2;M3;M4-1,M4-2;M5-1,M5-2;M6;M7-1,M7-2.The percentage of peak area of the unmetabolized prototype DA657 was less than 1%after 1 h and 2 h in hepatocytes both from monkey and beagle dog.There were seven metabolites with peak area percentages greater than 5%in monkey hepatocytes when metabolized for1 h:M1-1,M1-2,M2-1,M2-2,M3,M5-1,M7-1 and seven metabolites with peak area percentages greater than 5%after 2 h of metabolism:M2-1,M2-2,M3,M4-1,M5-1,M6,M7-1,respectively.While only 5 metabolites with peak area percentages greater than 5%after 2 h of metabolism in hepatocytes of beagle dog were identified:M1-1,M2-1,M3,M5-1,M7-1.Conclusion:Drug DA578 had the similar sedative characteristics to DEX,the other four analogs 595,642,654 and 657 had the advantages of faster onset and moderately maintenance time over DEX.No severe central nervous system adverse reactions were observed after single intravenous injection.The recovery quality of drug DA578 was the worst.The recovery quality of 595 and 657 were better than DA578,but slightly worse than DEX.The recovery quality of new drugs 642 and 654 were the best and close to DEX.Above all,DA578,595,642,654 and 657 allowed the rats to recover well from LORR.The duration and degree of suppression on hemodynamics of DA578,595,642,654 and 657 were significantly less than those of DEX.The respiratory inhibition of the five new drugs was also lower than that of DEX.The analogs 642,654 and 657only slightly inhibited the respiratory rate,and the maximum inhibition was no more than 10 times/min.Although DA578 and 595 reduced respiratory rate,their respiratory rate were higher than DEX by 20%and 40%,respectively.According to the comprehensive evaluation of the pharmacodynamic properties of the five new compounds,DA657 with a moderate action time,better awakening quality,a less severe effect on circulation,respiration,and the highest TI,was proposed as the representative compound for subsequent studies.A specific,robust and precise LC-MS/MS method was validated.The standard linear range was 5-1500 ng/ml,and the lower limit of quantification（LLOQ）was 5ng/ml.The plasma concentrations of DA657 at different doses were determined accurately.The pharmacokinetic parameters of DA657 had a good linearity relationship between the dose range of 1-4ED₅₀.In addition,drug DA657 can be rapidly metabolized in hepatocytes from different species（monkey and beagle dog）in vitro.The types of metabolites were relatively stable with only one metabolite which named as M6,was exclusively found in monkey hepatocytes but not in dog hepatocytes.However,the insignificant species differences needed to be further explored.