Studies On Pharmacokinetics Of O-desmethylvenlafaxine Prodrugs And Bisoprolol

O-desmethylvenlafaxine, an active metabolite of venlafaxine, is an antidepressant with a pharmacological profile to that of venlafaxine acting via inhibition of serotonin and norepinephrine reuptake for the therapy of depression, obsession and anxiety disorders. Compared with venlafaxine, the administration of O-desmethylvenlafaxine was more easily to regulate the therapeutic doses and effect, reduce side effects or interaction with other drugs. Likewise, increased hydrophilicity of O-desmethylvenlafaxine caused by the formation of another hydroxyl group may reduce its oral absorption and increase pre-system side effect of unable absorption drugs. In order to improve its pharmacokinetic behavior, 7 ethers of O-desmethylvenlafaxine derivates were designed and synthesized at the first time. A series of studies were conducted on O-desmethylvenlafaxine and its derivates to offer some useful references and inspirations for further development of O-desmethylvenlafaxine prodrugs.In addition, a novel LC/MS/MS method was developed and validated for the determination of free bisoprolol in biological samples, which were successfully used in a pharmacokinetic study of domestic bisoprolol. In addition to an improvement in sensitivity, the volume of plasma required and the analytical time were dramatically reduced. This method can be extensively used in the pharmacokinetic study of bisoprolol.1. Syntheses of O-desmethylvenlafaxine prodrugs and pharmacokinetic screeningIn order to increase the lipophilicify of O-desmethylvenlafaxine and improve its oral bioavailability, structural modifications by chemical syntheses at hydroxyl group were carried out to obtain seven ethers of O-desmethylvenlafaxine derivatives at the first time. Studies on the in vivo metabolism showed four compounds including ODVP-1, ODVP-2, ODVP-3 and ODVP-5 had the characteristics of prodrugs.2. Studies on pharmacokinetics of O-desmethylvenlafaxine and its prodrugs in beagle dogsAfter oral administration of prodrugs, the prodrugs were quickly absorbed and metabolized into ODV in dogs. The pharmacokinetics of ODV and its prodrugs were investigated in dogs by the LC/MS/MS method. After oral administration of 0.013 mmo1/kg ODV or its prodrugs, Cmax of ODV (100 ng/mL) was achieved at 0.5 h, while Cmax of 91.5, 108 , 79.4 and 89.6 ng/mL were achieved at approximately 0.9, 1.2, 1.7 and 1.0 h for ODVP-1, ODVP-2, ODVP-3 and ODVP-5, respectively. The elimination half?life (t1/2) was calculated to be 2.07, 3.27, 2.45, 2.25 and 3.44 h for ODV, ODVP-1, ODVP-2, ODVP-3 and ODVP-5, respectively. The relative bioavailabilities of ODVP-1, ODVP-2, ODVP-3 and ODVP-5 to ODV at the same molar dose were calculated to be 127 %, 164 %, 118 % and 105 %, respectively. The concentration of released ODV after administration of same molar prodrugs ranked as follows: ODVP-2 > ODVP-1 > ODVP-3 > ODVP-5. Higher relative bioavailabilities of ODVP-1, ODVP-2 and ODVP-3 to ODV indicated great improvements of their pharmacokinetic behavior after structural modifications and ODVP-1, ODVP-2 and ODVP-3 were new drug candidates of ODV.The prodrugs of ODV have a relatively higher bioavailability. The main reason was that structural modifications by chemical syntheses at hydroxyl group increased lipophilicify of prodrugs.3. Metabolism Study of O-desmethylvenlafaxine and its prodrugs in rats The metabolic profiles of ODV and its prodrugs in rats were investigated after oral administration of 0.13 mmo1/kg ODV or its prodrugs. Two metabolites were measured in the tested animals, and were identified as O-desmethylvenlafaxine glucuronide (M1), N, O-didesmethylvenlafaxine glucuronide (M2), by comparisons of LC behavior and MSn data with reference substances. O-desmethylvenlafaxine glucuronide in rat fedes was found at the first time. It was demonstrated that the prodrugs underwent similar metabolic pathways to O-desmethylvenlafaxine in the tested animals after the prodrugs were rapidly reverted to parent drug in plasma.The results that there were no prodrugs in rat urine or feces showed that these prodrugs were ideal drug candidates of ODV, which further proved the better absorption and higher susceptibility to enzymatic hydrolysis of the prodrugs in plasma.4. Studies on degradation kinetics of prodrugs of desmethylvenlafaxineA sensitive, accurate and highly selective LC/MS/MS method was developed and validated at the first time for the determination of ODV and its prodrugs in rat plasma with diphenhydramine as the internal standard. The analytes of interest and internal standard (diphenhydramine) were extracted from plasma samples by diethylether–dichloromethane(3:2, v/v), and chromatographed on a Zorbax Eclipse XDB C8 column (150×4.6 mm, I.D., 5μm) with the mobile phase consisting of methanol: 10mM ammonium acetate(85:15, v/v) at a flow rate of 1.0 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer by mutiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The linear calibration curves of ODV and its three prodrugs were obtained in the concentration range of 1.25-750 ng/mL for ODV and 0.25-150 ng/ mL for three prodrugs with a lower limit of quantification (LLOQ) of 1.25 ng/mL for ODV and 0.25 ng/mL for three prodrugs using 100μL plasma. The intra- and inter-day precision in terms of RSD were both under 11.6 %, and the accuracy in terms of RE ranged from ?12.8 % to 14.0 %. The extraction recoveries of ODV and its prodrugs were all higher than 64.1 %.In addition, HPLC methods with diode array detector (DAD) method were developed and validated for the determination of ODV or its prodrugs in solutions, which was successfully used in the aqueous solubility determination and stability study of ODV and its prodrugs.Aqueous solubility of the prodrugs and parent drug in solutions of different pHs and temperatures were determined. At the same time,the appatent partition coefficient were investigated. As the substitute group changes from hydroxyl group to aromatic group, the solubility of ODVP-1 and ODVP-2 in the buffer decreased and the lipophilicity of all the prodrugs increased to a larger extent, compared with the parent drug. In comparison, the solubility of ODVP-3 increased in buffer. Partition coefficient values ranked as follows: ODVP-2 > ODVP-1≥ODVP-3 > ODV, which was mainly in accordance with the results of relative bioavailabilities.The stability of O-desmethylvenlafaxine prodrugs in several media, including buffered aqueous solutions at different pHs, and in biological fluids including 80 % plasma, 0.1 % liver and intestinal homogenates, were investigated in vitro by using HPLC or LC/MS/MS. The degradation of desmethylvenlafaxine prodrugs followed pseudo-first order kinetics, and the degradation was temperature-dependent. At pH 7.4 and 37°C the half-lives of approximately 5, 10, 24 days were calculated for ODVP-1, ODVP-2 and ODVP-3 in a phosphate solution respectively. The degradation half-life of ODVP-1, ODVP-2, ODVP-3 in 80% plasma were 1.36 ,2.17,5.78 min, respectively, while the degradation half-life of ODVP-1, ODVP-2, ODVP-3 in 0.1 % liver homogenates were 6.40, 8.98, 11.83 min, respectively.As esterase-mediated degradation of an ester prodrug may be a limiting factor for its trans-epithelial transport. The esterase activity in homogenates from various intestinal segments was investigated in order to identify a possible absorption window with low esterase activity and hence increased ester prodrug absorption. A significant difference was observed among the activity of various intestinal segments mucosa. The degradation rates of three prodrugs were all in the order duodenum > ileum > jejunum > colon.5. Studies on the effects of rat cytochrome p450 of desmethylvenlafaxine and its prodrugsIt was the first time to study the effects of O-desmethylvenlafaxine and its prodrugs on the P450 content and main P450 enzyme activity of rat liver microsomes, which were prepared by applying ultracentrifugation approach. Using phenacetin, debrisoquine, midazolam, tolbutamide and mephenytoin as specific substrate probes, the activities of CYP1A2, CYP2D6, CYP3A4, CYP2C9 and CYP2C19 were determined by calculating the formation rates of motabolites after the concentrations of the substrate metabolites—paracetamol, 4-hydroxydebrisoquine, 1-hydroxymedazolam, 4-tolbutamide and 4-hydroxy- mephenytion—in each sample were determined by LC/MS/MS. No induction was observed for hepatic phase I enzymes except that CYP2D6 activity was significantly decreased, by pretreatment with desmethylvenlafaxine and its prodrugs6. Pharmacokinetic study of bisoprolol in healthy volunteers An analytical method for the determination of bisoprolol in human plasma has been developed based on liquid chromatography–tandem mass spectrometry (LC/MS/MS). The analyte and internal standard (IS) diphenhydramine were cleaned-up by protein precipitation with acetonitrile, reconstituted in mobile phase, and separated by reversed phase high-performance liquid chromatography (HPLC) using methanol:10 mM ammonium acetate:formic acid (70:30:0.1 v/v/v) as mobile phase. Detection was carried out by multiple reaction monitoring (MRM) on a LC–MS/MS system and was completed within 2.5 min. The assay was linear over the range 0.5–100 ng/mL with a limit of quantitation (LLOQ) of 0.5 ng/mL. The intra- and inter-day precision levels were within 5.54 % and 9.95 %, respectively, while the accuracy was in the range of 89.4-113%. This method has been utilized in a pharmacokinetic study.After oral doses of bisoprolol 5.0 mg to 20 healthy volunteers, pharmacokinetics parameters of bisoprolol in human plasma were Cmax: 33.46±11.02 ng/mL , Tmax: 3.3±1.8 h , t 1/2 : 8.67±1.09 h, AUC0-t and AUC0-∞were 421.27±139.10 and 431.48±145.26 ng·h/mL, respectively.