Analysis Method And Preclinical Pharmacokinetic Study Of PDE4 Inhibitor Chlorbipram

As a potential target for depression and Alzheimer’s disease, PDE4 has been studied for more than 20 years. The development of PDE4 inhibitors may provide some possibilities for the treatment of these diseases. However, the further study of PDE4 inhibitors has been hampered by the severe gastrointestinal side effects such as nausea and vomiting. How to reduce the adverse effects of PDE4 inhibitors and to keep their pharmacological activities at the same time are the difficulties in the development of the class of drugs. According to the enzyme activity test respectively, we screened a representative compound from these compounds which named chlorbipram by our group. Preliminary studies confirmed that:1. chlorbipram has good inhabitation on PDE4 in vitro and did not perform emesis activity in beagle dogs during the 120 min observation period; 2. chlorbipram significantly improved the symptoms of anxiety and depression in different models of depression, such as acute depression model and chronic unpredictable mild stress model; 3. chlorbipram can reverse the cognitive dysfunction in the aged mice and the Aβ25-35 induced AD rat model in a certain extent. Therefore, chlorbipram has a good development prospect. However, whether a candidate compound can emerge from preclinical and clinical studies not only depends on the pharmacological and toxicological effects, but also depends on the pharmacokinetics profiles and tissue distribution. These properties will directly affect the reliability of new compounds. In the preliminary stages of designing and screening candidate drugs, pharmacokinetic studies are beneficial for optimization of the structure of compounds by providing information regarding the drug half-life and feasibility of transport to the site of action.Our study was chiefly to make an evaluation of preclinical pharmacokinetics for chlorbipram in Kunming mice and SD rats, estimate the preclinical pharmacokinetics, investigate the relationship between dosage and parameters of pharmacokinetics, calculate the absolute bioavailability of chlorbipram after oral administration and examine the the distribution of chlorbipram in brain tissue. If chlorbipram can pass through the blood-brain-barrier, then we will consider the distribution of chlorbipram in the sub-region of brain tissue.1 Method validation for chlorbipram in plasma and brain tissue samplesAt first, we established and validated a novel, simple and sensitive UFLC-MS/MS method for the quantitative determination of the PDE4 inhibitor chlorbipram and studied the pharmacokinetics of chlorbipram in biological samples (plasma and brain tissue of Kunming mouse and SD rat). ZXI14 was selected to be the internal standard which has a similar chemical structure to chlorbipram. The liquid-liquid extraction (LLE) method with ethyl acetate was used for both pretreatment of plasma and brain homogenates. The Chromatographic separation was accomplished on a Acquity UPLC BEH C18 column (50mm×2.1mm, particle size 1.7μm) with a gradient mobile phase consisting of water and methanol at a flow rate of 0.25 ml/min. Detection was performed in the multiple reaction monitoring (MRM) mode with the precursor-to-product ion transitions m/z 492.6→231.3 (chlorbipram) and m/z 452.8→191.2 (IS, ZXI14) used for quantitation using electrospray ionization (ESI) in the positive ion mode.In our study, Calibration curves were constructed in the range of 0.5-200 ng/ml for mouse plasma and 0.25-100 ng/g for mouse brain, the intra-and inter-day precision values of these two samples were less than 13.0%. Mean extraction recoveries of IS and QC samples assayed at three concentrations were more than 78.3% in these two samples. The matrix effects in mouse plasma and brain were ranged from 91.0% to 101.5% for IS and all QC sample concentrations, which indicated that the matrix exerted minimal effects on the method response to analytes. After three freeze-thaw cycles, the concentrations of analytes in mouse and rat plasma deviated by less than ±15% from their nominal concentrations. In stability studies (short-term, long-term and post-preparative), the relative errors for three QC sample concentrations were within ±15.0%.As before, the linear range for SD rat plasma was 0.5-500.0 ng/ml and 0.25-100.0 ng/g for rat brain tissue samples. The intra-and inter-day precision values were less than 11.7% for brain tissue QC samples. Mean extraction recoveries of IS and QC samples assayed at three concentrations were 77.7%-86.0% in these two samples. The matrix effects in rat plasma and brain ranged from 93.8% to 101.8% for IS and all QC sample concentrations as well, which indicated that the matrix exerted minimal effects on the method response to analytes. In stability studies, the relative errors for three QC sample concentrations were within ±15.0% as well. These data indicated that the developed method was suitable for application to the determination of clorbipram in these biological samples and the pharmacokinetic studies of chlorbipram in Kunming mouse and SD rat.2 The pharmacokinetics and brain distribution study of chlorbipram after administration in Kunming mice.In the experiment,180 healthy male Kunming mice were divided into 4 groups. They were oral administrated of low-dose, medium-dose and high-dose (2.4 mg/kg, 3.6 mg/kg,5.4 mg/kg) and intravenous administrated 0.6mg/kg, respectively. Their blood and brain tissue samples were collected at different time points after administration (n=5). The concentrations of chlorbipram in plasma and brain samples were measured by the analysis methods established in this research before. The plasma concentration-time curves were based on the measured concentration data, and the pharmacokinetic profiles and the absolute bioavailability were analyzed by a non-compartmental model. According to the brain concentration-time curve, we can also get the information if chlorbipram could pass though the blood-brain-barrier.The results of determination showed that pharmacokinetic parameters of plasma in Kunming mice such as Cmax±S.D. were 11.3±2.0 ng/ml,28.5±3.5 ng/ml and 30.5±7.3 ng/ml ng/ml; tmax±S.D. were 12.0±4.5 min,15.0 min and 15.0 min; t1/2 ±S.D. were 58.8±29.4 min,55.9±18.3 min and 96.9±45.3 min; AUC0-t±S.D. were 635.1±85.5 ng min/ml,948.7±179.8 ng min/ml and 1628.0±270.5 ng min/ml after single oral dose of chlorbipram at three grades (2.4,3.6 and 5.4 mg/kg). According to the data proceding, we can conclude that it is basic linear dynamics characteristics of the absorption of the drug in Kunming mice. In the range of 2.4 mg/kg to 5.4mg/kg, AUC0-t were linearly related to the three different dosages (R2=0.991) and Cmax were also showed a certain correlation (R2=0.729).And then, the Cmax、t1/2、AUC0-t were 159.6±26.5 ng/ml,45.9±5.7min and 4172.9±349.0 ng min/ml after intravenous administration of chlorbipram(0.6 mg/kg) respectively. By calculation, the absolute bioavailability of chlorbipram are 3.8±0.4%,3.8±0.8% and 4.4±1.0% respectively after oral dose of chlorbipram at three dosages.The pharmacokinetic parameters of brain in Kunming mice such as tmax were 17±7.6、14±2.2、27±6.7 min; T1/2 were 147.3±39.3、69.2±47.2、81.1±14.0 min; Cmax were 4.73±0.53、4.60±0.78、9.4±0.6 ng/g after single oral dose of chlorbipram at three grades (2.4,3.6 and 5.4 mg/kg). The tmax、t1/2、Cmax were 10 min,47.2±8.5 min and 83.5±11.0 ng/g respectively after intravenous administration. Interesting, after intravenous administration of chlorbipram (0.6 mg/kg), the concentration of chlorbipram in mouse brain reached Cmax at about 10 min, which is delayed comparing to the one in plasma, suggested that chlorbipram could pass through the blood-brain-barrier. Moreover, the trends of mean brain concentration-time curve of chlorbipram at three dosages were consistent with the one in plasma, which also pointed out that chlorbipram could cross over the blood-brain-barrier further.3 The pharmacokinetics and brain sub-region distribution study of chlorbipram after administration in SD rat.At first,18 male SD rats (200-250 g) were divided into 3 groups. They were orally administrated 2 mg/kg,4 mg/kg and intravenous administrated 1 mg/kg chlorbipram, respectively. The concentration of chlorbipram in SD rat plasma was determinated by rat plasma sample analysis methods established in this research before. The plasma concentration-time curves were based on the measured cocentration data, and the pharmacokinetic parameters and the absolute bioavailability were calculated by a non-compartmental model. Additional 18 male SD rats (200-250 g) were divided into 3 groups, which were all oral administrated 4 mg/kg chlorbipram. Brain was collected at 3 time-points, which are in the phase of absorption, elimination and at tmax, respectively. And then, Hippocampus, prefrontal cortex, striatum and remaining brain tissue were separated and collected. The concentration of the brain tissues were detected by the established method before.The result of determination showed that pharmacokinetic parameters in SD rats such as Cmax±S.D. were 14.1±1.3 ng/ml and 28.5±5.4 ng/ml; tmax±S.D. were 32.5±6.1 min and 30 min; t1/2±S.D. were 175.2±45.6 min and 105.6±38.3 min; AUC0-t±S.D. were 1973.6±170.1 ng min/ml and 2866.0±534.9 ng min/ml after single oral dose of chlorbipram of 2 and 4 mg/kg; the Cmax、t1/2、AUC0-t were 394.18±52.47 ng/ml, 37.38±10.0 min and 13885.1±1031.3 ng min/ml respectively after intravenous administration.The absolute bioavailabilities of chlorbipram are 7.1±0.8% and 5.0±0.8% after single oral dose of chlorbipram in SD rats. The results of sub-region distribution in brain tissue showed that the distribution of chlorbipram in the prefrontal cortex and hippocampus was more compared with that in striatum.