| With the development of modern medical science and technology,human life expectancy continues to extend.The world population has already entered an aging state,and the safety of medication for the elderly is becoming more and more prominent.This safety problem is mainly due to the changes in drug disposal in the body caused by the aging of the physiological functions of cells and organs in the elderly,and the drug interactions caused by the combined use of multiple drugs in the elderly.However,the pharmacokinetic effects of aging are not always well predicted due to differences in drug properties.In addition,the pharmacodynamic effect or toxicity of a drug is more related to the concentration at its target site,suggesting that we should pay more attention to the change of drug concentration at the therapeutic target site.The study on the changes and mechanism of the pharmacokinetics of specific drugs in the elderly will guide the clinical personalized use of drugs more scientifically.Alzheimer’s disease is an age-related neurodegenerative disease,and currently no drug could cure it but only improve cognitive function.Acetylcholinesterase inhibitors,which are the clinical first choice for medication,often cause adverse reactions in gastrointestinal tract,cardiac system,nervous system and so on in the elderly.More than 70%cases are serious,which hindered continuous medication use to obtain improvements in elderly patients.Fluoropezil is a new acetylcholinesterase inhibitor developed by Shanghai Institute of Medica Material.Compared with the first-line drug Donepezil,it has higher selectivity and inhibition potential to acetylcholinesterase.It is expected to have better clinical efficacy and safety.In the present study,the anti-Alzheimer’s disease drug,fluoropezil,was the main research subject.Using a variety of in vivo and in vitro models,the disposition process of fluoropezil in human was summarized and the aging effect on the pharmacokinetics of fluoropezil and its potential mechanism were systematically researched and clarified.According to the research conclusion,guidance and suggestions for the clinical medication of fluoropezil in the elderly was proposed.The main results are summarized as follows:1.The metabolic characteristics and enzyme phenotype of fluoropezil in humanAfter oral absorption,fluoropezil was extensively metabolized in human.A total of 14 metabolites were detected and identified from plasma,urine and fecal samples by ultra performance liquid chromatography-quadrupole-time of flight(UPLC-UV/QTOF)mass spectrometry.The metabolic pathways of fluoropezil in human included Noxidation(M11),N-defluorobenzyl(M1),O-demethylation(M2),O-demethylation and monooxidation(M3),di-demethylation(M5),O-demethylation and glucuronidation(M9),monooxidation and glucuronidation(M10),O-demethylation and Ndefluorobenzyl(M12),N-defluorobenzyl and hydrogenation(M13),hydrogenation(M14),monooxidation and hydrogenation(M15),glucuronidation(M16).The structures of the main metabolites M1 and M11,which were also the main metabolites of fluoropezil in human liver microsomes(accounting for 79.3%),were confirmed by chemical synthesized standard reference.The metabolic phenotype study in human liver microsomes and recombinant enzymes proved that CYP3 A4 and CYP3 A5 are the main subtypes of enzymes that mediate the oxidative metabolism of fluoropezil,accounting for 64%of the metabolic contribution,followed by CYP2C8,CYP2C9 and CYP2D6.In the structural characterization process of metabolite M11,we found that opposite stereoselective nitrogen oxidation preference of fluoropezil was observed in vivo and in vitro.The in vivo biotransformation of fluoropezil preferred cis nitrogen oxidation,while under the in vitro chemical synthesis condition,fluoropezil tended to produce trans nitrogen oxidation configuration.In order to explain this phenomenon,fluoropezil as a ligand was docked to the human CYP3A4 protein(PDB:6MA7).The results exhibited that the heme iron in the catalytic center of CYP3A4 enzyme was closer to the N atom of fluoropezil when it was in cis oxidation orientation than in trans oxidation orientation.However,under chemical conditions with hydrogen peroxide,the steric hindrance of forming trans N-oxidation conformation of fluoropezil was smaller,so it was easy to add oxygen in trans state.In addition,we found that fluoropezil N-oxides metabolites M11 in fresh human whole blood tended to be reduced to the parent drug and could serve as a drug reservoir.Thus,it is necessary to evaluate the in vivo disposition of the metabolite.2.Effect of aging on the pharmacokinetics of fluoropezil and its metabolites:in exploration of potential mechanismIn order to evaluate the effect of aging on the pharmacokinetics of fluoropezil in healthy volunteers,we established a rapid,sensitive and reliable liquid chromatography-tandem mass spectrometry(LC-MS/MS)method for simultaneous determination of fluoropezil,N-oxidized fluoropezil(M11)and N-deflurobenzyl metabolite(M1)in elderly(62-73 years old)and young(24-39 years old)subjects’plasma after an oral administration of 4 mg fluoropezil.After oral administration of fluoropezil,the plasma peak time of fluoropezil was about 1 hour in both aged and young subjects,and there was no significant difference,suggesting that the intestinal absorption of fluoropezil was rapid and the absorption rate was not significantly affected.The systemic exposure(AUC0-t)of fluoropezil in elderly subjects was 1.49 times of that in young subjects(91.0±27.0 h*ng/mL vs 59.2±7.95 h*ng/mL,p<0.01),and the half-life was prolonged by 141%in the elderly(74.2±34.1 h vs 30.8 ± 5.18 h,p<0.001).Moreover,the apparent distribution volume increased by 49%(3600±961 L vs 2570±239 L,p<0.01),and the clearance rate decreased significantly by 44.7%(38.8±15.6 L/h vs 59.4±11.8 L/h,p<0.05),suggesting that fluoropezil eliminates more slowly and has a risk of accumulation in the elderly.The main metabolites M1 and M11 in plasma accounted for 27.2%and 15.4%of the parent drug exposure in young subjects,respectively.This ratio decreased significantly in elderly subjects,to 18.6%and 10.2%,respectively,indicating the metabolism formation was influenced.In addition,there was no significant difference in the protein binding rate of fluoropezil,Ml and M11 between the aged and the young subjects,indicating that the change of plasma free drug concentration in the elderly subjects was consistent with the change of the total concentration.In another word,the free fluoropezil concentration in the elderly’s plasma was higher than that in the young,suggesting that the therapeutic dose of fluoropezil in the elderly population was potentially lower than in young people.In order to elucidate the mechanism of age-related pharmacokinetic changes,natural aged rats(23-26 months old)and young rats(2-5 months old)were used for further study.The reasons for choosing rats as the research species included the following considerations.The metabolic pathways of fluoropezil in rats were similar to human,and the enzymes mediating the metabolism of fluoropezil in rats and humans were both mainly CYP3A enzymes.After oral administration,the rats showed similar pharmacokinetic characteristics to those of humans.The plasma exposure of fluoropezil in aged rats was 2.23 times higher than that in young rats(214±72.7 h*ng/mL vs 96.0±44.1 h*ng/mL,p<0.05),and the half-life was 1.5 times longer(9.23±1.40 h vs 6.01±1.08 h,p<0.05).The Cmax showed an increasing trend without significant difference.There was no difference in plasma peak time of fluoropezil between the two groups.The proportion of plasma exposure of metabolites M1 and M11 to the parent drug exposure both decreased in aged rats.Firstly,the influence from aging effect on absorption process was evaluated.After an intravenous administration of 0.5 mg/kg fluoropezil to rats,the plasma exposure of fluoropezil in aged rats was 1.81 times of that in young rats,and the half-life was prolonged by 117%.The proportion of M1 and M11 exposure to the parent drug decreased in aged rats.These pharmacokinetic changes were similar to those after intragastric administration,suggesting that the absorption process of fluoropezil was not significantly affected by aging.In addition,after intravenous administration,the plasma clearance rate of fluoropezil in aged rats was 46.8%of that in young rats,indicating that its clearance slowed down in aged rats.The mean renal clearance rates of fluoropezil in aged and young rats were 18.8 mL/h/kg and 29.2 mL/h/kg,respectively,accounting for less than 2%of plasma clearance.The further results of excretion study illustrated that fluoropezil was mainly eliminated by metabolism,and the recovery rate of the parent drug in urine and feces was no more than 3%dose.The results of fluoropezil in vitro metabolism exhibited that the internal clearance rate of fluoropezil in rat liver was more than 50 times higher than that in intestine,and the difference in human liver clearance and intestinal clearance was more than 8 times as well.It was suggested that fluoropezil was mainly eliminated by liver metabolism,and the decrease of liver clearance rate in the elderly was the main reason for the decrease of plasma clearance rate.In order to further clarify the effect of aging factor on metabolic clearance,excretion tests were carried out.In the excretion study,fluoropezil was cleared from the body mainly in the form of metabolites in both human and rats.Metabolites M1 and M11 were the main forms of excretion,accounting for more than 44%of the dose in young rats,and their recovery dose percent decreased significantly in aged rats(28.5%),indicating that aging factors reduced the bioformation of M1 and M11 in vivo.However,there was no significant difference in the parent drug recovery percent between the two groups.In order to explain this phenomenon,the semi-quantitative analysis of other metabolites in urine and feces was performed.It was found that the recovery of metabolites related to monooxidation metabolic pathway decreased in aged rats.On the other hand,the recovery of some metabolites related to O-demethylation in urine and feces of aged rats increased slightly,suggesting that aging factors mainly influenced metabolism through N-dealkylation and monooxidation pathway,and partial compensatory metabolism occurred through other metabolism paths.This also suggested that the activity of some metabolic enzymes(internal clearance)in the liver of aged rats changed.The results of enzymatic kinetics studies using liver microsomes incubation from aged and young rats further proved that the intrinsic clearance rate of fluoropezil in liver microsomes of aged rats was 47.4%of that in young rats.The formation velocity of M1 and M11 also decreased significantly.In order to further determine which metabolic enzymes were affected by aging,specific CYP450 enzyme probes were used to characterize the changes of enzyme activities,which contributed to fluoropezil’s metabolism in aged and young rats.The results indicated that there was no significant difference in other enzyme activity except that the activity of CYP3 A decreased significantly.Further kinetic studies showed that the metabolic velocity of CYP3 A substrate midazolam in liver microsomes of aged rats was about 1/6 of that in young rats.The results of this study suggested that the decrease of liver clearance rate caused by the decrease of CYP3A enzyme activity was the main factor leading to the changes of plasma pharmacokinetics of fluoropezil.3.Brain distribution and uptake mechanism of fluoropezilBrain is a heterogeneous organ,which contains a variety of structural regions,and the distribution of drugs in different regions is closely related to its efficacy.Fluoropezil,as a brain-targeted drug,the study on the mechanism of its entry into the brain and the regional distribution of the drug in the brain will provide kinetic and spatial information for elucidating its efficacy.The Kp,brain of fluoropezil in rats were 2.28,2.50 and 2.21 after intragastric administration of 0.5 mg/kg,5 mg/kg and 25 mg/kg fluoropezil,suggesting that the exposure in brain was higher than that in plasma and fluoropezil was easy to distribute into brain.Desorption electrospray ionization coupling to time of flight mass spectrometry with deuterated internal standard normalization work flow for imaging fluoropezil was established.The imaging results indicated that fluoropezil could rapidly(within 1 hour)and widely distributed in various brain regions of rats,including acetylcholinesterase receptor-enriched striatum,cortex,hippocampus and other structural regions.The results of transporter study suggested that fluoropezil was not the substrate of multiple organic cation transporters(OCT1,OCT2),carnitine transporter(OCTN2),organic anion transporters(OAT1,OAT3),organic anion transporting polypeptide(OATP2B1)uptake transporter on the blood-brain barrier,nor was it effluxed by P-glycoprotein(Pgp)or breast cancer resistant protein transporter(BCRP).The free rate of fluoropezil in plasma and brain tissue was determined by LC-MS/MS method.It was found that the free rate of fluoropezil in plasma was 2.09 times higher than that in brain tissue,which means the affinity of fluoropezil to brain tissue was higher than that in plasma.It was the main driving force of passive diffusion of fluoropezil with high permeability from plasma to brain,resulting in higher exposure in brain than in plasma.4.The effect of aging on the brain distribution of fluoropezil and the clinical dose modification suggestionWhen studying the effect of aging on pharmacokinetics,the attention was mainly paid to the changes of plasma pharmacokinetics.While the changes of drug concentration in tissues and organs,especially in the therapeutic target organs,can better reflect the possible drug efficacy changes caused by age,and are more related to the adjustment of drug dose.From the previous research results,fluoropezil mainly diffused into the brain through passive diffusion,and the difference in affinity between brain tissue and plasma protein created the potential energy difference that drove it into the brain.Would aging influence the potential energy difference between brain and plasma and the spatial distribution in the brain?After intragastric administration,the free rate of fluoropezil in brain tissue and plasma was determined.It was found that there was no significant difference in the ratio of free rate in brain tissue and plasma between aged and young rats,indicating that the change of drug concentration of fluoropezil in rat brain was consistent with that in plasma.In addition,the results of DESI-MSI imaging showed that the brain distribution regions of aged and young rats were the same,and there was no significant difference between the two groups.These results show that the aging factor does not change the blood-brain distribution ratio and spatial distribution,and there is a good correlation between brain drug concentration and plasma drug concentration.In clinic,the appropriate dose reduction of fluoropezil in the elderly population can be considered,which can decrease the adverse reactions while ensuring the therapeutic effect.5.ConclusionThe present research systematically studied the metabolism,pharmacokinetics,brain distribution process of fluoropezil,and further explored the aging effect and its potential mechanism.The relationship between the age-related liver clearance rate changes and the pharmacokinetics and brain distribution of fluoropezil in vivo was established.The innovation and significance of the research are as follows:(1)The present study for the first time clarified the metabolic fate and phenotype of fluoropezil in human.A total of 14 metabolites were detected,and metabolic pathway included monooxidation,N-defluorobenzyl,O-demethylation,N-oxidation and so on.Fluoropezil was mainly metabolized by CYP3A enzyme,and the contribution rate was 64%.The preferred stereoselective configuration of N-oxidation metabolites M11in vivo catalyzed by CYP3A enzymes was proved to be cis rather than the dominant configuration of chemical synthesis.Moreover,M11 can be reduced to the parent drug in the fresh whole blood.(2)The influence of aging factors on the pharmacokinetics of fluoropezil and its two main metabolites in healthy volunteers were studied for the first time.It was found that the plasma exposure of fluoropezil in elderly subjects increased by 49%,the halflife prolonged by 1.41 times,and clearance slowed down by 44.7%.Besides,the proportion of plasma exposure of the main metabolites M1(N-defluorobenzyl fluoropezil)and M11 to the parent drug decreased significantly.(3)It was clarified that the main factor of the effect of aging on the pharmacokinetics of fluoropezil is that aging leads to a significant decrease in CYP3A enzyme activity and caused CYP3Amediated fluoropezil liver clearance reduction.(4)The method of DESI-MSI combined with deuterated internal standard normalization was successfully established for the first time to image the distribution characteristics of fluoropezil in striatum,cortex and hippocampus,and the strategy workflow of DESI-MSI imaging method optimization process was summarized,which will provide reference for other researchers.(5)The uptake mechanism of fluoropezil into the brain was firstly clarified.Fluoropezil was uptaken into brain mainly through passive diffusion driven by high affinity potential energy to the brain.It was further found that the increase of fluoropezil plasma exposure caused by age would lead to a consistent increase in distribution in the target organ brain.Thus,it is suggested that when using fluoropezil in clinic,the liver clearance rate changes in the elderly should be paid attention to,and the dose should be properly modified to maximize therapeutic efficacy and minimize adverse reactions. |
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