Effect Of Tianma Gouteng Decoction On Metabolism Of Amlodipine In Rats And The Effect Of Bupivacaine On Proliferation Of Phenylephrine In Rats

Amlodipine?AML?,third generation of dihydropyridine Ca²⁺ channel blocker,is the first-line drug for clinical treatment of hypertension.The main mechanism of action is that AML selectively blocks Ca²⁺ influx across the cell membrane via Ca²⁺ channels,thereby relaxing vascular smooth muscle to decrease blood pressure.AML is recommended for the treatment of the hypertension or coronary heart disease accompanied with chronic heart failure.It has been reported that AML can improve the symptoms of patients with heart failure and reduce mortality.As for those with severe chronic heart failure using digitalis,diuretics and ACEI,no cardiovascular complications or increase in mortality was observed after their combination with AML.With respect to patients with impaired renal function,no adjustment is needed in the dosage of AML.Tianma Gouteng Decoction?TGD?,a classical traditional Chinese medicine formula,always helps to counter hypertension in clinical treatment.Foreign researchers have reported that 80% of patients with hypertension need two or more antihypertensive drugs in combination to stabilize blood pressure,therefore,combined antihypertensive drugs has become a routine treatment of hypertension in clinical practice.It is common to treat hypertension with Chinese medicine preparation combined with Western medicine.In terms of complex traditional Chinese medicine's composition,the pharmacokinetics is more complex in the treatment of hypertension with Chinese herbs combined with modern drugs,the changes of which are of great significance.In the present study,we analyzed the effect of compound preparation TGD on drug metabolism of AML and its metabolites in rats by LC-MS/MS.Bupivacaine?BUP?is one of the most commonly used local anesthetics in clinical practice.In the previous study,we have found that Bupivacaine exerts dual effects on ?1 adrenoceptor-mediated vasoconstriction of isolated rat aortic rings: short-term treatment enhances the response,whereas long-term treatment inhibits it.However,the mechanism is not clearly demonstrated.Therefore,in our study,we used isolated rat aortic rings to observe changes in enhancement of ?1 adrenoceptor-mediated vasoconstriction by BUP.Meanwhile,pharmacological tool drugs were used to analyze the role endothelium-derived relaxing factor?EDRF?played in enhancement of ?1 adrenoceptor-mediated vasoconstriction by BUP.Part one The effect of compound preparation TGD on the pharmaco-kinetics of AML and its metabolite in ratsObjective: To develop a LC-MS/MS method for the determination of AML and its metabolites,and to analyze pharmacokinetic effects of TGD on AML and its metabolites.Methods: Healthy SD rats were randomly divided into control group and the experimental group.Rats in the experimental group were given TGD?0.5 g/kg,1 time/day?intragastrically,and those in control group were given equal volume of distilled water for 15 consecutive days.On the last day,the rats in the two groups were given AML of 1mg/kg.After intragastric administration of AML,blood was collected.The plasma concentration of AML and its metabolites at different time point was determined by LC-MS/MS method and the pharmacokinetic parameters were calculated.Results: The standard curve equation of AML was Y= 1.89305X-0.0321939?r²=0.996789?,and the linear range was 0.15⁶0 ng/m L for AML.The recoveries of quality control samples at three concentrations?low,medium and high?for AML were 80.16%,69.98% and 66.67%,respectively.Matrix effects of quality control samples were 100.35%,100.04% and 109.81%,respectively.The RSD values were 7.11%,4.27% and 14.90%,respectively;The RSD values of intra-day and inter-day concentration for AML were 13.40%,10.00%,9.80%,and 13.00%,6.80%,14.20%,respectively;precision values were all less than 8%.The accuracy values were 2.71%,2.69% and 2.64%,respectively.Pharmacokinetic parameters of AML in rats treated with distilled water were as follows: AUC_0-t,64.40±25.54?g/L·h;AUC_0-?,74.58±29.81 ?g/L·h;C_max,5.73±3.16 ?g/L;T_max,1.37±0.44 h;t_1/2,6.91±1.64 h;CL,0.02±0.006 L/h/kg;V: 222.73±97.80 L/kg;t_1/2Ka : 0.31 ± 1.88 h.Pharmacokinetic parameters of AML in rats treated with TGD were as follows: AUC_0-t,49.54±29.66?g/L·h;AUC_0-?,48.45±29.66 ?g/L·h;C_max,4.28±3.05 ?g/L;T_max,1.12±0.35 h;t_1/2,5.38±1.16 h;Cl,0.42±0.028 L/h/kg;V,3623.3±1952.07 L/kg;t_1/2Ka: 0.65±0.72 h.Compared with control group,there was significant decrease in t_1/2 accompanied with an remarkable increase in Cl,V and t_1/2Ka?P<0.05?.Pharmacokinetic parameters of metabolites M1?m/z=363.0869?in control group were as follows: AUC_0-t,43.16±11.99 ?g/L·h;AUC_0-?,45.09±12.48 ?g/L·h;C_max,5.55±1.59 ?g/L.Pharmacokinetic parameters of M1?m/z= 363.0869?in rats treated with TGD were as follows: AUC_0-t,32.56±10.42 ?g/L·h;AUC_0-?,33.52±11.27 ?g/L·h;C_max,5.37±2.00 ?g/L.Significant decrease was found in AUC_0-?,as compared with control group?P<0.05?.Pharmacokinetic parameters of metabolites M3?m/z=365.1025?in control group were as follows: AUC_0-t,14.85±3.65 ?g/L·h;AUC_0-?,15.38±3.81 ?g/L·h;C_max,1.92±0.54 ?g/L,Pharmacokinetic parameters of M3?m/z= 365.1025?in rats treated with TGD were as follows: AUC_0-t,10.92±3.68?g/L·h;AUC_0-?,11.27±3.72 ?g/L·h;C_max,1.73±0.58 ?g/L.Compared with control group,AUC_0-t and AUC_0-? were remarkably decreased?P<0.05?.Pharmacokinetic parameters of metabolites M4?m/z =377.1026?in control group were as follows: AUC_0-t,22.23±7.04 ?g/L·h;AUC_0-?,23.29±7.67 ?g/L·h;C_max,2.84±0.91 ?g/L,Pharmacokinetic parameters of M4?m/z=377.1026?in rats treated with TGD were as follows: AUC_0-t,16.30±5.39 ?g/L·h;AUC_0-?,16.55±5.50 ?g/L·h;C_max,2.70±1.01 ?g/L.Compared with control group,significant decrease was noted in AUC_0-??P<0.05?.Pharmacokinetic parameters of metabolites M6?m/z=406.1290?in control group were as follows: AUC_0-t,36.46±17.91 ?g/L·h;AUC_0-?,84.14±63.44 ?g/L·h;C_max,3.27±1.90 ?g/L.By contrast,pharmacokinetic parameters of metabolites M6?m/z=406.1290?in rats treated with TGD were as follows:AUC_0-t,10.92±3.68 ?g/L·h;AUC_0-?,11.27±3.72 ?g/L·h;C_max,1.73±0.58 ?g/L.Compared with control group,significant decreases were found in AUC_0-t,AUC_0-? and C_max?P<0.05?.Pharmacokinetic parameters of other metabolites did not differ significantly.Conclusion: TGD may inhibit the absorption of AML in the gastrointestinal tract and may promote the excretion of AML and its seven major metabolites,resulting in a shortening of the AML t_1/2 and speeding up of CL.Part two The effect of bupivacaine on proliferation of phenylephrine in ratsObjectives: To investigate the role of endothelium in the enhancement of phenylephrine-mediated vasoconstriction by bupivacaine in the isolated rat aorta.Methods: The isolated rat aortic rings were prepared,and the vascular endothelium was removed chemically or physically.Phenylephrine-mediated vasoconstriction was recorded.Results: A pretreatment with bupivacaine at 30 ?mol·L^-1 for 20 min significantly increased the Emax value of vasoconstrictive responses to phenylephrine from 2.22±0.07 g of solvent-controlled group to 2.50±0.05 g?P<0.01?in the isolated endothelium-intact rat aorta.However,the Emax value was not significantly changed by a pretreatment with bupivacaine at 30 ?mol·L^-1 for 5,10 or 15 min?P>0.05?.A pretreatment with bupivacaine at 30 ?mol·L^-1 for 20 min slightly but significantly inhibited the vasoconstrictive responses to low concentration of phenylephrine in the isolated endothelium-denuded rat aorta?P<0.05?.In the isolated endothelium-intact rat aorta under a combined treatment with indometacin,Ch TX,apamin and L-NAME,the vasodilator responses to acetylcholine were completely suppressed,and a pretreatment with bupivacaine at 30 ?mol·L^-1 for 20 min did not significantly affect the vasoconstrictive responses to phenylephrine?P>0.05?.Conclusion: Bupivacaine enhances ?1-adrenoceptor-mediated vasoconstriction by its inhibition on vascular endothelium in the isolated endothelium-intact rat aorta,and this kind of inhibition by bupivacaine on vascular endothelium potentiates indirectly the vasoconstrictive responses to phenylephrine.