Study On The Pharmacodynamics Andpharmacokinetics Interaction Of Harmine Combined With Memantine

1.Background and objectivesHarmine?HAR?is a-carboline alkaloid widely distributed in plants and animals in nature.The previous study of our team found that HAR has the function of inhibiting acetylcholinesterase and improving learning and memory disorders,which is expected to be developed into a new drug for the treatment of Alzheimer disease?AD?and other diseases.However,a high dose of HAR will cause central tremor toxicity of Parkinson's disease?PD?symptoms,which limits the development and clinical use of new drugs for HAR.Memantine?MEM?is a mild non-competitive N-methyl-D-aspartate receptor antagonist.Studies have found that MEM can not only reduce excitatory toxicity,relieve PD symptoms of central tremor,but also improve the symptoms of AD.In summary,this subject using Chinese medicine pharmacology,analytical chemistry,molecular biology and pharmacokinetic and other disciplines theory and research methods,thorough the effects of MEM on the memory damage model of HAR combined with MEM,the effects of MEM on the tremor induced by HAR,and the pharmacokinetic drug interactions between HAR and MEM were studied in terms of pharmacodynamics and pharmacokinetics.The pharmacodynamics and pharmacokinetic interactions between HAR and MEM were clarified,which provided an important research idea and theoretical basis for the development of HAR as a new effective drug for the treatment of AD and the clinical medication and drug interaction between HAR.2.Methods?1?The study on drug-drug interaction between drug efficacy and toxicity of harmine and memantineFirst,the effect of HAR combined with MEM on the memory damage model.Male C57BL/6 mice were randomly divided into 10 groups:control group,model group?Scopolamine group?,HAR-L group?5 mg/kg HAR?,HAR-M group?10 mg/kg HAR?,HAR-H group?20 mg/kg HAR?,HAR-L combined MEM group?5 mg/kg HAR and 4 mg/kg MEM?,HAR-M combined MEM group?10 mg/kg HAR and 4 mg/kg MEM?,HAR-H combined MEM group?20 mg/kg HAR and 4 mg/kg MEM?,positive drug group?Donepezil group,5 mg/kg?and MEM group?4 mg/kg MEM?,respectively.The control group and model group were given normal saline.After continuous administration of the corresponding drugs for 7 days in each group,except the control group,normal saline was given,and the other 9groups received intraperitoneal injection of 1 mg/kg of scopolamine every day starting on day8,followed by another 7 days,followed by a water maze space search experiment on day 15.After intraperitoneal injection of scopolamine for 30 minutes the next day,hippocampal and cortical brain tissues were taken.Using Morris water maze behavior tester,the improvement effect of HAR and HAR combined MEM on memory damage model was investigated.Then,the performance of HAR and MEM in improving learning and memory was explored through analytical chemistry,molecular biology,pharmacology and other technologies in terms of relevant neurotransmitters,protein expression and neuron staining.Secondly,the effect of MEM on HAR induced tremor.SD male rats were randomly divided into five groups:control group?given normal saline?,HAR group?10 mg/kg HAR?,MEM-L combined HAR group?2 mg/kg MEM and 10 mg/kg HAR?,MEM-M combined HAR group?4 mg/kg MEM and 10 mg/kg HAR?,and MEM-H combined HAR group?8 mg/kg MEM and10 mg/kg HAR?,respectively.Rats were given the corresponding dose of MEM by intraperitoneal injection,followed by 10 mg/kg HAR 10min later.The control group was successively given the same amount of normal saline,and the follow-up experiment was conducted immediately.Tremor detector was used to evaluate the tremor behavior,and then through the analysis of chemical methods,using ultra performance liquid-mass spectrometry technology related neurotransmitters.The mechanism of HAR inducing central tremor and the effect of HAR and MEM on the level of neurotransmitter were preliminarily explored,and then the neurons were stained to observe their state.?2?The study on pharmacokinetic drug interaction between tetrandrine and memantine Firstly,the Agilent 1290-Agilent 6410 triple quadrupole mass spectrometer was used to establish the biological sample analysis method for simultaneous detection of MEM,HAR and harmol?HOL,the major metabolites of HAR?.Biological sample analysis methods including selectivity,residue effect,standard curve,Lower limit of detection?LLOD?,Lower limit of quantitation?LLOQ?,precision,matrix effect,recovery rate and stability were investigated.Then the pharmacokinetic drug interactions between HAR and MEM in rats were investigated.SD male rats were randomly divided into seven groups.One group was given MEM?5 mg/kg?dissolved in ultra-pure water by gavage,namely the MEM group.The rats in the three groups were given HAR at low?20 mg/kg?,medium?40 mg/kg?and high?80mg/kg?doses.The other three groups were treated with low?20 mg/kg?,medium?40 mg/kg?and high?80 mg/kg?HAR combined with 5 mg/kg MEM.Blood samples were collected from the orbital venous plexus at 0?before administration?,0.08,0.17,0.25,0.33,0.5,0.75,1.0,2.0,4.0,8.0,12.0 and 24.0 h after administration,and quantitative analysis was performed on the plasma MEM,HAR and HOL by UPLC-MS/MS method.3.Results?1?The study on drug-drug interaction between drug efficacy and toxicity of harmine and memantineIn the effect of HAR combined with MEM on the memory impairment model,Morris water maze behavioral experiments showed that three doses of HAR?5,10,20 mg/kg?or three doses of HAR combined with MEM?4 mg/kg?can shorten the swimming distance and the incubation period of the mice?P<0.05,P<0.01 or P<0.001?,increase the number of passages?P<0.05,P<0.01 or P<0.001?.Moreover,the combined administration groups had a greater effect on them than the HAR groups alone.Neurotransmitter detection and Western Blot?WB?results showed that ACh/Ch was significantly increased in hippocampus and cortex of high-dose HAR group and three-dose HAR combination groups?P<0.01 and P<0.001?.The protein expression of ACh E in hippocampus and cortex of HAR-H group and three doses of HAR combined group was significantly lower?P<0.05,P<0.01 or P<0.001?.The expression of Ch AT protein in the middle and high dose HAR group and the medium and high dose HAR combined with MEM in the hippocampus was increased?P<0.05,P<0.01?.Both alone and in combination,the Glu content in the hippocampus and cortex was reduced?P<0.001?,the GABA content was increased?P<0.01 and P<0.001?,and the Glu/GABA was significantly decreased?P<0.001?,and the combined dose was more significant than the HA alone?P<0.05,P<0.01 or P<0.001?.In addition,the 5-HT/5-HIAA was significantly increased of the hippocampus and cortex in the medium-high-dose HAR group and the medium-high-dose HAR combined with the MEM group?P<0.001?.Neuron staining showed that both HAR and HAR combined with MEM could improve hippocampal neuronal damage induced by scopolamine.In the effect of MEM on tremor induced by HAR,the experimental results showed that moderate tremor was stably obtained when 10 mg/kg of HAR was administered intraperitoneally.Three doses of MEM in the low,medium and high?2,4,8 mg/kg?intraperitoneal doses delayed the latency of HAR?10 mg/kg?induced tremor in a dose-dependent manner?P<0.001?.In addition,low-and medium-dose MEM can shorten the duration of tremor?P<0.001?,reduce the degree of tremor,and the low dose is better?P<0.01?.High doses of MEM increased the duration of MEM and the intensity of tremor?P<0.05 or P<0.001?.Neurotransmitter results showed a significant increase in Glu/GABA,ACh/Ch,and 5-HT/5-HIAA in hippocampus and cortex after administration of HAR?P<0.001?.After combined with MEM,Glu/GABA and ACh/Ch were significantly decreased?P<0.05,P<0.01 or P<0.001?,whereas 5-HT/5-HIAA did not decrease after combined administration.Neuronal staining showed that HAR could damage hippocampal neurons.After combined administration of low and medium doses,neurons had no damage,while high dose of MEM combined with HAR had slight damage.?2?The study on pharmacokinetic drug interaction between tetrandrine and memantine A biological sample analysis method for simultaneous detection of MEM,HAR and HOL by ultra-high performance liquid chromatography-mass spectrometry was established,and the method was systematically verified.The results of the methodological investigation showed that the analytes and IS to be tested were not endogenously interfered in the MRM mode.The peak retention times of MEM,HAR,HOL,and IS were 3.58,2.78,1.95,and 2.30 min,respectively.No residual effect,and the analyte to be tested had a LLOD of 0.1 ng/m L and an LLOQ of 0.4 ng/m L.In five concentrations of QC samples?n=6?of 0.40,1.00,40.00,300.00and 400.00 ng/m L,the within-run precision CV%was between 1.84%and 8.86%.The accuracy of RE%is not higher than 17.62%in LLOQ,and the other four concentrations are not higher than 9.90%.Among the five concentrations,the CV%of the inter-assay precision is not higher than 6.02%,and the RE%of the between-run precision is not higher than10.12%.In three concentrations of QC samples?n=6?,the matrix effect of the analyte to be tested was 96.39-102.47%,and the SD was below 7.50%.In addition,the matrix effect of the internal standard was 99.04±1.68%?n=6?.The recoveries of the analytes MEM,HAR and HOL at the three concentrations were 97.23%to 104.86%,and the SD was not higher than6.34%?n=6?.The IS recovery was 102.88±0.66%?n=6?.The QC samples were placed at room temperature for 4 h,4°C refrigerator for 24 h,-20°C for one month and repeated freeze-thaw three times,and the SD was determined to be between 0.01%and 16.50%,and the CV%was not more than 6.68%.The analyte and IS stock solutions were also examined for stability at room temperature for 24 h,4°C for three days,and-20°C for one month.The quantitative analysis method was sensitive and rapid,and the selectivity,residual effect,inter-batch precision,matrix effect,recovery and stability of the method were in line with the relevant requirements of the bioanalytical method validation guide.The pharmacokinetic drug interaction between HAR and MEM was studied using a newly established quantitative assay.The results showed that compared with the MEM group,the AUC_0-t and MRT of MEM were significantly increased after combined with HAR?P<0.01 or P<0.001?,and the dose-dependent decrease of CL in MEM was combined with three doses of HAR?P<0.01 or P<0.001?.The C_max of MEM in the combination group was lower than that in the MEM group.The C_maxof MEM was higher than that of the low-dose HAR combined MEM group in the middle and high doses of HAR combined with MEM.The C_maxof HAR in the co-administered groups were higher than that in the HAR group?P<0.05 or P<0.001?.The AUC_0-t in the co-administered groups were higher than that in the HAR group?P<0.05?.The CL of HAR was decreased in the co-administered groups compared with the single-administered group?P<0.05?.In the combination of the single and low dose groups,the concentration of HOL in some plasma samples was lower than LLOQ and the complete drug time curve could not be drawn.In the HAR-M combined MEM group,the C_max of HOL was higher than that of the HAR-M group,and the HAR-H combined with MEM was higher in the MRT than in the HAR-H group?P<0.05?.The AUC_0-t of HOL in the HAR-H combined MEM group was higher than that in the HAR-H group?P<0.01?.The CL in the HAR-H combined MEM group was significantly lower than that in the HAR-H group?P<0.001?.4.ConclusionsIn pharmacodynamic studies,both HAR and HAR combined with MEM showed significant improvement in scopolamine-induced memory impairment model and learning and memory ability in mice,and the combined drug-administered group improved the memory impairment model better than HAR group.Low-dose MEM has a greater effect on the attenuation of tremor caused by HAR than the medium dose,and high doses of MEM increase the tremor caused by HAR.In pharmacokinetic research,we have established a quantitative analysis method for biological samples that are sensitive,rapid,and stable for simultaneous detection of MEM,HAR,and HOL.Pharmacokinetic studies found that HAR can slow the metabolism of MEM,increase the residence time of MEM,and then expand the safety window of MEM.MEM increases the exposure of HAR and its metabolite HOL in rat plasma.In summary,MEM can enhance the effect of HAR on learning and memory,on the other hand,MEM has the effect of attenuating the toxicity of HAR central tremor,and the pharmacokinetic characteristics of the two combined drugs can guide the combination of the two.This study provides important research ideas and theoretical basis for the development of HAR as an effective drug for the treatment of AD,clinical application of HAR and drug interaction.