| BackgroundCalcium sensitizer is positive myocardial drugs by increasing calcium channel sensitivity, which can both enhance cardiac function and expand blood vessel. Calcium sensitizers mainly promote the myocardial contraction protein to Ca2+, but not increasing the concentration, which decreases arrhythmia and overload of Ca2+. The discovery of calcium sensitive agent provides a new path for the treatment for heart failure, as well as new field for anti-heart failure drug study.At present, the calcium sensitizer what we only know is just levosimendan provided by Orion company from Finland, used in acute and chronic heart failure when the conventional therapy effect stays poor. As far as we know, Calcium sensitizer pre-clinical research still maintain less, and study of calcium sensitizers could be helping researchers further understand the mechanism in application for therapy of heart failure or other ischemic diseases.Piperphentonamine hydrochloride, PPTA, is first synthesized creatively by china, which has already authorized several domestic/oversea invention patents. Experiment has been confirmed PPTA can enhance affinity between myocardial protein and calcium iron, prevent myocardial ischemia/reperfusion injury and Ca2+overload, open Ca2+ sensitive K+ channel, and promote Na+ channel inactivation.Recent study shows that PPTA can protect cerebral ischemia/reperfusion injury in rat, and PC-12 cells after OGD damage. The result indicates PPTA reduces LDH, iNOS, bax/bcl-2 and caspase-3 expression of PC-12 after OGD damage, exhibiting anti-apoptosis and pre-oxidation.Pre-experiments have found that PPTA can be metabolized in the body more active substances, which as one of the major metabolite M6, however, its molecular structure has not been verified, as well as specific preclinical study, including M6 synthetic route, pharmacokinetic and pharmacodynamic study. The further research on M6 may provide a new consideration for deeper development and clinical application to PPTA. This paper is based on M6 synthetic route design, structural verification, pharmacokinetics and preliminary pharmacodynamic study, in order to develop a new target effect of M6, explore new direction to protect organs in vivo.Five parts has been divided for this study. The first part aimed to design, synthesize M6, and confirm the structure of the M6 by mass spectrometry and NMR analysis, for better understanding better understanding of the structure differences between M6 and PPTA. In the second experiment we used HPLC-UV method to detect determination of M6 in biological samples, and elected appropriately internal standard, flow phase proportion, wavelength and so on, in order to established successfully stable, convenient and fast method of determination detection for further study on plasma and tissue detection. The third part was based on pharmacokinetic studies on high, medium, low dose in rats. In the meantime, we detect M6 determination in organs or tissues distribution after tail vein injection in high dose to explore the characteristic distribution of M6 in rats. The fourth part aimed to study the plasma protein binding rate of M6 in human, rat and pig plasma. We evaluated plasma protein binding rate of M6 in different species through detecting liberation drug determination in plasma of M6. The fifth part accessed condition of PC-12 cell, primary rat myocardial cell and neurons by CCK8 method growth after OGD damage (oxygen glucose deprivation) simulating ischemia and hypoxia biological injury and high glucose, oxygen recovery, providing reference to further research whether M6 could be applied to myocardial ischemia-reperfusion or brain ischemia-reperfusion injury, expecting to develop new effective and protective drugs for anti-ischemia-reperfusion injury.Part 1 The study on synthesis and confirmation of Piperphentonamine hydrochloride metabolites M6ObjectiveTo establish reasonable, scientific M6 synthesis route, and confirm the structure of the M6 by mass spectrometry and NMR analysis, for better understanding better understanding of the structure differences between M6 and PPTAMethodsPPTA is dissolved in methanol and mixed with certain Pd-C, Ph2S. After about 7 hours reaction under normal pressure at room temperature 25℃, the solution was under heating for concentrating to 200 mL and placed until crystal came out. Residual solution was added orderly sodium borohydride, saturated sodium bicarbonate solution and 400mL ethyl acetate under ice cooling condition, stirred for thirty minutes, and extracted once with 400mL ethyl acetate. We get yellow oil after dried with anhydrous sodium sulfate and added ethanol solution of hydrochloric acid for scratching for solid.95% ethanol solution was added and filtered for solid, which was washed with methanol and dissolved with heating. After five hours in the refrigerator we filtered to give wet sample and repeat the operation to obtain the taget product.All target products were collected and detected by mass spectrometry and NMR analysis.ResultsThe data of 1H NMR of M1:1H NMR δ:2.66-2.78(m,7H),2.94 (t, J=8.4 Hz, 2H),3.04 (t, J=7.2Hz,2H),3.19-3.22(m,3H),3.32-3.35(m,1H),5.98(s,2H),6.67(d, J=8.8 Hz,2H),6.74(dd, J=1.6,8.0Hz,1H),6.89(d, J=8.0 Hz,1H),6.90(d, J=1.6 Hz,1H),6.98(d, J=8.8 Hz,2H),9.23(s,1 H),10.80(s,1H).The data of 13C NMR of M1:13C NMR 8:28.03,29.07,36.10,43.88,49.60, 55.89,100.86,108.34,109.13,115.10,121.79,129.00,130.61,130.81,146.01, 147.38,155.52,206.73.The data of mass spectrometry of M1:ESI-MS:m/z 356.2[M+H]+; HRESI-MS: C21H25NO4+H ([M+H]+),measured value was 356.1858.The data of 1HNMR of M6:1HNMR δ:1.57-1.63(m,1H),1.73(m,1H),1.85(m, 1H),2.44-2.60(m,3H),2.75(s,3H),2.95 (t, J=8.4 Hz,2H),3.16-3.23(m,4H),3.48(s, 1H),4.86(s,1H),5.98 (s,2H),6.67(d, J=8.4 Hz,2H),6.74(dd, J=1.6,8.0 Hz,1H), 6.86(d, J=7.6 Hz,1H),6.90(d, J=1.6 Hz,1H),6.98(d, J=8.4 Hz,2H),9.18(s,1 H), 10.56(s,1 H).The data of 13C NMR of Mδ:13C NMR 8:29.08,30.40,30.65,52.69,100.85, 108.33,109.12,115.04,121.78,129.03,130.68,132.05,145.99,147.38,155.28.The data of mass spectrometry of M6:ESI-MS:m/z 358.2[M+H]+; HRESI-MS: C21H27NO4+H ([M+H]+), measured value was 358.2019。ConclusionThe structure of M6 was confirmed that 5-{(3,4-methylenedioxy phenyl ethyl) methylamino}-1-p-hydroxyphenyl-3-hydroxy-pentane hydrochloride. The chemical structural formula has shown structural difference between M6 and PPTA was position where alkenyl group dehydrogenated. Pre-tests showed, M1 and M6 performed better dissolving ability than PPTA, as well as chemical stability, soluble in water.Part 2 Establishment of determination detection of M6 in biological sample by HPLC-UVObjectiveTo establish effective, stable, fast and convenient HPLC-UV determination detection of M6 in biological sampleMethodsInternal standard method was used on plasma and tissue homogenate determination detection. amlodipine besyla was elected as internal standard. Chromatographic condition:ODSC18 column (waters,4.6x150 mm,5μm), mobile phase was 1% acetic water:acetonitrile=86:14 at flow rate of 1.0 mL·min-1;column temperature at 40℃;sample side was 20μL; UV detection wavelength of M6 was 265nm; pretreatment of biological samples was used 5% acetic-acetidin for fluid-fluid extraction; 40℃ constant speed nitrogen blowing for drying, and 20μL supernatant for HPLC detection after mobile phase dissolving and high speed centrifugation.ResultsMethod validation has confirmed amlodipine besyla did not interfere with the determination detection of M6, showing well specificity. Linear logical results illustrated standard curve of M6 was Y=0.0023X-0.0007(R2=0.9999) having a good linear relationship range from 3.125-200μg·mL-1; within-run precision for rat plasma of M6 were 3.22%,3.75%,1.40%(n=5),between-run precision were 5.08%, 5.29%,4.46%, showing good instrument precision; RSD of Repeatability experiment of M6 were 6.53%,1.84%,1.61%, showing good Repeatability. Recovery of M6 were 92.18±2.04(%),89.50±0.35(%),89.92±1.49(%). Total recovery>80%,fitting to detection requirement.RSD of stability, repeated freezing and thawing 3 times,-4℃ storage for 12h and room temperature 25℃ placed after 12h, were 1.75%, 2.64%,3.40% for 200μg·mL-1,2.52%,2.83%,7.50% for 25μg·mL-1,and 6.36%, 6.48%,7.87% for 3.125μg·mL-1 (n=5),fitting to Methodology requirement.ConclusionIt was convenient, accurate, less interference, high sensitivity for M6 fitting to determination detection in vivo.Part 3 Pharmacokinetics study on M6 in SD ratsObjectiveTo observe characteristic of pharmacokinetic parameters and tissue distribution of M6 after tail vein injection in high, medium and low doseMethodsM6 dose plans for study on plasma pharmacokinetics:18 SD rats were divided into 3 groups randomly, high, medium and low dose,16 mg·kg-1、12 mg·kg-1、8 mg·kg-1, each group had 3 male and 3 female rats. M6 was injected by tail vein injection and each rat plasma was collected at pre-dose time,1 min,3 min,5 min, 10 min,15 min,30 min,1 h,2 h,4 h,6 h,8 h. Determination of plasma was calculated. Plasma determination-time curve was drawn by origin 8.0. Pharmacokinetic parameters were calculated by PKsolver data processing software. M6 dose plans for tissue distribution:36 SD rats were divided into 6 groups randomly, 1min group, 10min group,30min group, 1h group,2h group,4h group. Each group had 3 male and 3 female rats. Tail vein injection was processed in 16 mg·kg-1 dose, and samples were collected at 1min,10min,30min, 1h,2h,4h. Plasma was collected quickly and sufficiently, as well as heart, liver, spleen, lung, kidney, stomach, Testis/ ovary, muscle, intestine and brain. The samples in each group must maintain consistency, rationality and were handled by removing organ surface fat, blotted up excess normal saline after washing surface residual blood, and stored in --70℃. Each tissue samples were scaled according to actual weight and mix with normal saline at the ratio of weight:volume to 1:1.5. homogenate were made and centrifugal at 4℃、 4000rpm/min,7min. HPLC was analysis. Results were processed by Microsoft Excel 2007.ResultsThe pharmacokinetic procedure of M6 after tail vein injection in SD rats corresponded with non-compartmental model in high, medium and low dose. Results of plasma pharmacokinetic of M6 showed double-peak concentration-time curve. The parameters of high, medium and low dose group were followed, AUC0-t were 133.4±135.39、100.15±32.67、77.49±70.43 mg·L-1·min (p<0.05), Cmax分别 2.52±0.86、2.63±1.07、1.94±0.99mg·L-1;CL were 0.12±0.06、0.08±0.01、 0.08±0.03L·min-1;tmax were 3.43±3.26、2.6±0.89、7.67±5.75min;MRT were 85.99±44.24、81.13±26.57、67.81±20.19min;t1/2 were 54.09±22.21、57.04±21.09、 44.94±15.43min.High selection of M6 was founded in rat tissues or organs, most of which concentrated extremely in liver and spleen. Results of sequence were kidney, heart, lung, spleen, intestine, brain, plasma, liver, stomach, ovary or testis, muscle. Concentration of brain was 7.23±0.19μg/ml, suggesting certain permeability of M6 to blood brain barrier. The concentration sequence were in accordance ovary or testis, lung, intestine, spleen, kidney, liver, brain, plasma, stomach, heart, muscle after 10min. however, detection difference in ovary or testis was large, as concentration in ovary was higher than the one in testis, which may suggest difference to organs selection exist. The concentration of brain was the highest,8.3±2.91μg/ml. After 30min, high aggregation was found in liver, spleen, stomach, more than 100μg/ml, higher than other organs, most in spleen. Results showed selection of M6 to spleen. Concentration in all organs became less after 30min.ConclusionCL in rats of M6 would be high as the high dose injection. MRT of group in high dose was 85.99±44.24min, higher than those in medium and low dose, positive correlation with dose. As the structure differences between M6 and PPTA exist, the characteristics of pharmacokinetic and tissues distribution of M6 have distinguished from PPTA.Results also indicated the high selection to M6, in the meantime, M6 was detected in brain indicating M6 could get through blood brain barrier.Part 4 The study of plasma protein binding rate of M6 in human, pig and rat plasmaObjectiveTo observe the plasma protein binding rate of M6 in human, pig and rat plasma.MethodsExternal standard method was used to detect protein binding rate of M6 in human, pig and rat plasma by ultra-filtration method.To calculate the plasma protein binding rate through detection of ultra filtrate of the sample concentration. Through plasma protein binding rate (%)= (the actual concentration-ultra filtrate concentration)/actual concentration ×100%, we evaluated plasma protein binding rate in M6 in various genera.ResultsThe experiment showed that M6 standard curve was Y=4.5268X+3.8703(R2=0.9999). Curve fitting was better, and satisfied to detection requirements.The recovery of M6 in high, medium and low concentration were 97.42±0.19%、 97.41±3.36%、99.81±11.49%, more than 95%; Ultra-filtration membrane did not interfere to filtration of M6, satisfying the detection of free concentration.High plasma protein binding of M6 has been tested, which were more than >99% in human plasma,70.59±0.161% in rat plasma,74.59±0.0.084% in pig plasma. The rates in other genera were higher than 93%, showing M6 was high intensity of binding protein rate.ConclusionPlasma protein binding rate of M6 was 74.59±0.0.084%, but more than 99% of medium and low concentration. It suggest insufficient binding exist when M6 of high concentration mixed with pig plasma. This phenomenon was corresponded to the rate in rat plasma, which was considered the reliability between binding and concentration.Part 5 Effect of M6 in PC-12 cell, primary rat myocardial cell and neuron after OGD damageObjectiveTo observe the effect of M6 from OGD damage in PC-12 cell, primary rat myocardial cell and neuron.MethodsPC-12 cell, primary rat myocardial cell and neuron each were divided into control group, model group, dose group (20μmol/L、12μmol/L、4μmol/L).Simulate OGD damage after 2h and recover oxygen and glucose 24h to test each group growth condition by CCK8.The results showed that OD values of the model and control group had significant difference (p< 0.05), artificial simulation of OGD damage was confirmed, and statistically significant inhibition or injury.M6 reduced the OGD damage in PC-12 cell on the whole. The high dose 20μmol/L was the best and statistically significant different with model group (p<0.0.5), correlated with the dose, which were as the same as the model of OGD damage in primary rat myocardial cell. The results showed anti-OGD damage protection of M6 was better in primary rat myocardial cell in different dose and had statistically significant difference with the model (p<0.0.5). Besides, the anti-OGD damage in high, medium and low dose group of M6 were better than the model groups (p<0.05).ConclusionOn the whole, M6 demonstrated anti-OGD damage in PC-12 cell, primary rat myocardial cell and neuron. M6 were expected to be new target drugs for further pharmacodynamic research. |
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