Animals
Adult male Wistar rats (weight, 250±20 g) were purchased from the Experimental Animal Center of Jilin University (Jilin, China). Adult male beagle dogs (weight, 12±1 kg) were provided by Tianyao Pharmaceutical Co., Ltd. (Tianjin, China) All animals were housed individually at 22±2°C and a relative humidity of 50±10% with a 12-h light/dark cycle, with free access to food and water. The present study was approved by the Ethical Committee of Jilin University. Rats were sacrificed via an overdose of sodium pentobarbital (150 mg/kg) by intraperitoneal injection.
Reagents and instruments
ODV succinate monohydrate, ODVP-1 hydrochloride, ODVP-2 hydrochloride, ODVP-3 succinate, ODVP-4 hydrochloride, ODVP-5 hydrochloride, ODVP-6 hydrochloride and ODVP-7 hydrochloride (derivative content, >98%) were provided by Jilin Institute of Pharmaceutical Research (Jilin, China). The chemical structures of these compounds are shown in Fig. 1. Ethyl ether and dichloromethane were purchased from the Jiayu Chemical Co., Ltd., (Tianjin, China) and Beijing Chemical Factory (Beijing, China), respectively. An Agilent 1100 high performance liquid chromatography (HPLC) system (Agilent Technologies, Inc., Santa Clara, CA, USA) and QTRAP type Triple Quadrupole mass spectrometer (Applied Biosystems; Thermo Fisher Scientific, Inc., Foster City, CA, USA) were used for analysis. LD5-2A centrifuge (Beijing Medical Centrifuge Factory, Beijing, China) and test tubes (Nantong Haizhixing Experimental Co., Ltd., Nantong, China) were used for sample collection.
Administration regimen for the pharmacokinetic screening of the ODV ester derivatives in rats
The seven ODV ester derivatives were prepared in normal saline for oral administration. A total of 21 healthy male Wistar rats were randomly allocated into seven groups (n=3 per group). After 12 h of fasting, with free access to water, a dose of 0.043 mmol/kg ODVP-1, ODVP-2, ODVP-3, ODVP-4, ODVP-5, ODVP-6 or ODVP-7 in 0.5 ml normal saline was intragastrically administered to each of the seven groups. Blood samples (1.0 ml) were extracted from the retrobulbar venous plexus at 5, 10, 30 and 1 h after administration. The samples were centrifuged in heparinized and dichlorvos-treated test tubes (terminal concentration of dichlorvos, 200 µg/ml) for 10 min at 4°C (1,570 × g). The plasma samples were then preserved at −80°C for further analysis.
Chromatography and mass spectrometry for the pharmacokinetic screening of the ODV ester derivatives in rats
The chromatographic column was a ZORBAX Eclipse XDB C8 column (I.D., 4.6×150 mm; particle diameter, 5 µm; Agilent Technologies, Inc.) and the mobile phase was methanol: 10 mM ammonium acetate (85:15, v/v). The flow rate was 1.0 ml/min, the injection volume was 10 µl and the column temperature was set at 20°C. The samples were determined with positive ion mode, the scanned mode was multiple reaction monitoring and the ion reaction used for ODV qualitative analysis was m/z 264.3→m/z 58.0.
Pretreatment of plasma samples and HPLC-MS/MS determination for the pharmacokinetic screening of the ODV ester derivatives in rats
Plasma samples (0.5 ml) were added into a Sep-Pak C18 solid phase extraction column (Waters Corporation, Milford, MA, USA) and activated by methanol and water at a pass speed of 30 drops/min. The samples were washed with 2 ml water and eluted with 2 ml methanol. The collected eluate was concentrated to ~200 µl using a gentle stream of N2, and 10 µl was collected for HPLC-MS/MS analysis.
Administration regimen for the pharmacokinetic study of prodrugs in beagle dogs
Pharmacokinetic evaluation was performed in beagle dogs. The test animals were randomly divided into five groups (n=4). Food and water were freely available during the entire experimental period. After a 12 h fast, each group was administered a single dose of 0.013 mmol/kg ODV, ODVP-1, ODVP-2, ODVP-3 or ODVP-5.
Sample collection for the pharmacokinetic study of prodrugs in beagle dogs
Blood samples (1.0 ml) were extracted from the small saphenous vein of the legs of beagle dogs prior to and at and 0.083, 0.167, 0.25, 0.5, 0.75, 1, 2, 3, 4, 8, 12 and 24 h after administration. Blood samples were centrifuged in heparinized and dichlorvos-treated test tubes (terminal concentration of dichlorvos, 200 µg/ml) for 10 min at 4°C (1,570 × g). Plasma samples were stored in a refrigerator protected from light at −80°C for future analysis.
Plasma sample treatment for the pharmacokinetic study of prodrugs in beagle dogs
Plasma samples (100 µl) were placed in a test tube with a stopper, and 100 µl internal standard solution and 100 µl sodium carbonate (0.1 M) were added to the test tube and allowed to mix. A total of 3 ml ethyl ether-dichloromethane (60:40, v/v) was then added prior to 10 min of eddy-mixing, followed by shaking for 10 min (240 times/min). The samples were then centrifuged for 5 min at 2,136 × g. The upper organic phase was placed in a new tube, and after blow-drying with N2 stream at 25°C, 200 µl mobile phase was added to the residue for dissolution. After a second round of eddy-mixing, 10 µl of the samples were collected for LC/MS/MS analysis.
Chromatography and mass spectrometry for the pharmacokinetic study of prodrugs in beagle dogs
The chromatographic conditions and mass spectrometer conditions are the same as described above in the ‘Chromatography and mass spectrometry for the pharmacokinetic screening of the ODV ester derivatives in rats’ section. The ion reaction used for qualitative analysis was m/z 264.1→m/z 107.0 (ODV) and m/z 256.3→m/z 167.1 (internal standard, benzhydramine).
Determination of the concentration of ODVP-1, ODVP-2, ODVP-3 and ODV in plasma, brain, and hypothalamus in rats
ODVP-1, ODVP-2, ODVP-3 and ODV were prepared in normal saline for oral administration. Eighty rats were dosed orally with ODVP-1, ODVP-2, ODVP-3 or ODV (0.043 mmol/kg in 0.5 ml). Food was restricted from 12 h predosing to 60 min postdosing. At 0.25, 0.5, 1, 2 and 4 h postdosing, blood samples were drawn from the sinus and collected in heparinized and dichlorvos-treated test tubes (terminal concentration of dichlorvos, 200 µg/ml) for plasma isolation. At 0.25, 0.5, 1, 2 and 4 h postdosing, rats were perfused with 40 ml phosphate-buffered saline (4°C) via the left ventricle, and the brain was removed and the hypothalamus was dissected. The brain and hypothalamus were placed in ice-cold methanol: water (50:50, v/v) in either 2.0 or 1.0 ml, respectively, and maintained on ice until tissue homogenization. Plasma and brain tissues were stored at −80°C for further processing. For determination of the concentration of ODVP-1, ODVP-2, ODVP-3 or ODV in tissues, three standard curves (determination of ODVP-1 and ODV, ODVP-2 and ODV, and ODVP-3 and ODV) of plasma or brain tissue (1.0–1,000 ng/ml) were generated from untreated animals. Samples and standards (100 µl) were spiked with 100 µl diazepam solution (1.0 µg/ml) in acetonitrile to serve as an internal standard. A total of 3.5 ml N-hexane-dichloromethane-dimethyl carbinol (300:150:15, v/v/v) was then added, and 10 min of eddy-mixing was performed followed by shaking for 10 min (240 times/min). The samples were then centrifuged for 5 min at 2,136 × g. The upper organic phase was placed in a new tube, and after blow-drying with N2 stream at 25°C, 200 µl methanol: 0.05% formic acid (50:50, v/v) was added to the residue for dissolution. After a second round of eddy-mixing, 20 µl sample was collected for LC/MS/MS analysis.
Statistical analysis
BAPP2.2 software (China Pharmaceutical University, Nanjing, China) was used to calculate the pharmacokinetic parameters. The trapezoidal method was selected for calculation of the AUC0-t value. Based on the semi-logarithmic map method, the t1/2 was calculated in accordance with the endmost four concentration points of the elimination phase. The Cmax and Tmax were calculated with the measured values, the AUC0-t after ODV administration was set as 100%, and the relative bioavailability was calculated according to the AUC0-t of ODV after the administration of each ODV prodrug.