| Amlodipine is a dihydropyridine calcium antagonists(calcium antagonist or slow channel blocker) , which directly acts on the vascular smooth muscle and decreased peripheral vascular resistance so that the blood pressure can be reduced. Telmisartan is a kind of oral administrated non-peptide angiotensin II type 1 (AT1)-receptor antagonist angiotensin that that lowers blood pressure through blockade of the renin–angiotensin–aldosterone system (RAAS), and widely used in treatment of hypertension. The work of this paper was designed to evaluate the pharmacokinetics features of the compound preparation in beagle dogs, so as to support the pharmacokinetics study in human and offer the reference for clinical researches.1. The establishment of analytical method of Amlodipine in biological matrixA HPLC/MS/MS method was developed to determine Amlodipine in biological matrix. The sample was extracted by ethyl acetate after the addition of 1.0mol/l sodium hydroxide solution, using clenbuterol hydrochloride as an internal standard. Separation was obtained on a Diamonsil C18 column (100mm×4.6mm i.d., 5μm; Dikma, Beijing, China). The isocratic mobile phase consisted of methanol and water (80:20, consisting 0.05% of formic acid ) was run at a flow rate of 0.8ml/min, with a diversion ratio of 3:2, which means the flow rate in the mass spectrometry is 0.32ml/min. oven temperature is 30℃, the injection volume is 20μl and the analysis time is 4.0min. The chromatographic system used provided good separation of the compound without interfering peaks from endogenous substances. The calibration curves were linear in the range of 2.03～507.5ng/ml in each sample with correlation coefficients above 0.99. The precision of intra-day and inter-day were evaluated by analysis of variance with the result of 2.71~4.78%and 3.71～5.99%, respectively. The method including accuracy, precision, specialization and linear range has been successfully used to support the pharmacokinetics study of Amlodipine.2. The establishment of analytical method of Telmisartan in biological matrixA reverse-phase high performance liquid chromatographic method was developed to determine telmisartan in biological matrix. The sample was pretreated by protein precipitation with acetonitrile, then directly injected into the system, using Naproxen as an internal standard. Separation was obtained on a Diamonsil C18 column (150mm×4.6mm i.d., 5μm; Dikma, Beijing, China) with fluorescence detection using an excitation wavelength of 305nm, and emission wavelength 365nm. The isocratic mobile phase consisted of acetonitrile-40mmol/L sodium dihydrogen phosphate- phosphoric acid=42:58:0.02 (v/v/v) was run at a flow rate of 1.0ml/min. oven temperature is 35℃, the injection volume is 20μl and the analysis time is 11.0min. The chromatographic system used provided good separation of the compound without interfering peaks from endogenous substances. The calibration curves were linear in the range of 5.025～1005 ng/ml in each sample with correlation coefficients above 0.99. The precision of intra-day and inter-day were evaluated by analysis of variance with the result of 2.19~4.87%and 4.25～5.64%, respectively. The dilutions of the high concentration samples do not effect the real plasma concentration, which demonstrates the method including accuracy, precision, specialization and linear range can been successfully used to support the pharmacokinetics study of Telmisartan.3. The pharmacokinetic studies of control drugs and different compositions of compound prescriptions in beagle dogsThis experiment contained three different compositions of compound prescriptions, 1:4, 1:6 and 1:8(amlodipine/ telmisartan). In one of the experiment(e.g. 1:4), six beagle dogs in a randomized crossover study, divided into three groups, were given a single oral dose of compound prescription (containing 10.8mg amlodipine and 43.2mg telmisartan), 10.8mg amlodipine and 43.2mg telmisartan, respectively. The blood samples were collected at different time after dosing. All collected blood samples were centrifuged to obtain plasma and the concentration of amlodipine and telmisartan were determined by LC-MS/MS and HPLC methods described as above. Pharmacokinetics parameter calculations were carried out using non-compartmental analysis method. The pharmacokinetics and bioequivalence of amlodipine and compound prescription (represented by amlodipine), telmisartan and compound prescription (represented by telmisartan) were compared. No significant difference appears in the pharmacokinetic parameters between the control drugs and compound prescription. The relative bioavailability of compound prescription (represented by amlodipine) was 82.7%～117.9%. The AUC and Cmax between amlodipine and compound prescription (represented by amlodipine) were bioequivalent. The relative bioavailability of compound prescription (represented by telmisartan) was 88.7%～120.8%. The AUC and Cmax between telmisartan and compound prescription (represented by telmisartan) were bioequivalent. The result showed that the combination of amlodipine and telmisartan has not influence each other in pharmacokinetics.4. The pharmacokinetic studies of different levels of compound prescription in beagle dogsIn the experiment, eighteen beagle dogs in a randomized crossover study, divided into three groups, were given three different levels of single oral doses of compound prescriptions ( low level contained 5.4mg amlodipine and 32.4mg telmisartan, medium level contained 10.8mg amlodipine and 64.8mg telmisartan and high level contained 21.6mg amlodipine and 129.6mg telmisartan), respectively. The blood samples were collected at different time after dosing. All collected blood samples were centrifuged to obtain plasma and the concentration of amlodipine and telmisartan were determined by LC-MS/MS and HPLC methods described as above. Pharmacokinetics parameter calculations were carried out using non-compartmental analysis method. Cmax and AUC0~72h of amlodipine at different levels were 63.08±12.37ng/ml and 790.17±57.35ng?h/ml, 86.83±14.40ng/ml and 1675.58±287.82ng?h/ml, 160.75±39.43ng/ml and 3501.24±859.69ng?h/ml, respectively. Cmax and AUC0~72h of telmisartan at different levels were 1221.58±241.64ng/ml and 6314.02±305.25ng?h/ml, 1949.52±243.30ng/ml and 12143.51±959.75ng?h/ml, 4472.15±485.71ng/ml and 24315.48±2039.78ng?h/ml, respectively. These date demonstrated that Cmax and AUC0~72h of the two drugs increased with the dose, showing apparent dose-dependent relationship.5. The pharmacokinetic studies of multiple oral doses of compound prescription in beagle dogsBeagle dogs who had been assigned to the medium level group (containing 10.8mg amlodipine and 64.8mg telmisartan) in the single-dose phase continued on to the multiple-dose phase and received compound prescription (containing 10.8mg amlodipine and 64.8mg telmisartan) one time daily for 4 days. This dose was chosen for the multiple-dose phase because it is likely to be the most effiective one in pharmacodynamic studies. Samples of venous blood (3ml each) were drawn before drug administration on days 3, 4 and 5 to determine the Cssmin. On day 5, the beagle dogs received compound prescription (containing 10.8mg amlodipine and 64.8mg telmisartan) one time and blood samples were drawn at different time after drug administration. All other experimental conditions were the same as those in the single-dose phase. Steady state was achieved after administration of compound prescription for 2 consecutive days. Cmax and AUC0~72h (steady state) of amlodipine and telmisartan were 101.45±24.40ng/ml and 1761.26±328.86ng?h/ml, 1975.80±73.38ng/ml and 13431.64±1222.22ng?h/ml, respectively. No significant differences in PK parameters were observed between the beagle dogs who received a single dose and those who received multiple doses of compound prescription (containing 10.8mg amlodipine and 64.8mg telmisartan). And no significant cumulation was observed, either. |
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