| Yonkenafil,(2-(2-ethoxy-5-(4-ethylpiperazin-1-ylsulfonyl)phenyl)-5-methyl-7-propyl-3H-pyrrolo [2,3-d]pyrimidin-4(7H)-one), a pyrrolopyrimidinone analogue of cyclic guanosine monophosphate (cGMP), was developed for the treatment of male erectile dysfunction (ED). It is a potent and selective inhibitor of cGMP-specific phosphodiesterase type V which is the predominant isozyme metabolizing cGMP in the corpus cavernosum. Currently, it is being evaluated in preclinical trials. The pharmacodynamics trials by comparing yonkenafil with placebo and sildenafil have demonstrated that yonkenafil has an IC50 of 2.0 nM (cf. sildenafil 4.5 nM), better acceptability and only few gastrointestinal side effects. Thus, yonkenafil is more suitable for a low-dose, short-term administration. In this thesis, a novel LC/MS/MS method for the quantification of yonkenafil in bio-samples has been developed and fully validated. The thesis aimed to study the preclinical pharmacokinetics of yonkenafil in rats and beagle dogs by LC/MS/MS, to exactly predict the transformation of yonkenafil in human, to rationally evaluate the efficiency and security of yonkenafil, and to offer some references and inspirations for the clinical studies. 1. Absorption study of youkenafil in rats and beagle dogs. A sensitive, selective and rapid method for the determination of yonkenafil in rat and beagle dog plasma was fully validated and successful applied for the absorption study of yonkenafil in rats and beagle dogs. The results were shown below:The dynamics process of yonkenafil in both rats and beagle dogs fitted one-compartment model after yonkenafil was given to rats and beagle dogs at a single dose (8.0, 16.0 and 32.0 mg/kg for rat; 3.0, 6.0, 12.0 mg/kg for beagle dog) by intragastric or oral administration. Linear pharmacokinetic characteristics in the dose range were found because the pharmacokinetics parameters (AUC0-t,AUC0-∞and Cmax) were linearly increased with the increase of dose. While the other pharmacokinetic parameters (t1/2, CL, MRT and Vd) were not varied with the increase of dose. Sex differences were not found in the absorption pharmacokietics in rats and beagle dogs. The absolute bioavailability of yonkenafil in rats after intragastric administration was 29.5 %. The absolute bioavailability of yonkenafil in dogs after oral administration was 23.7 %.2. Tissue distribution study of yonkenafil in rats. A sensitive and selective LC/MS/MS method for the quantification of yonkenafil in rat tissues was presented and partly validated in fat and muscle. The results were shown below:Yonkenafil distributed fast after intragastric administration. Tmax was between 20 min and 1h. The concentrations of yonkenafil at 6 h postdose in all tissues except intestinal contents and wash were smaller than 10% of Cmax, which illustrated that yonkenafil was not cumulated in tissues. The concentrations of yonkenafil in intestinal contents and wash, stomach contents and wash, intestine, stomach, lungs and liver were relative high at 1 h postdose.The plasma protein binding ratio was tested using equilibrium dialysis. A relative high plasma binding ratio (>97%) was found after equilibrium, which was similar to other synthetic phosphodiesterase V inhibitors.3. Metabolism and excretion studies of yonkenafil in rats. The metabolism study of yonkenafil was performed on an ion trap mass spectrometry using rat bile and urine samples collected before and after administration (0-12 h). The results showed that yonkenafil had extensive metabolism. Six metabolites were found in bile samples, and the structures of three of them were identified. We haven't found any metabolite in urine.The LC/MS/MS methods for the determination of yonkenafil and its three main metabolites in bile, urine and feces were developed to study the excretion of yonkenafil in rats. After an intragastric administration of 16.0 mg/kg yonkenafil, yonkenafil was little excreted in urine and the cumulative amount within 120 h reached to 0.05 % of the dose, the unchanged cumulative amount in feces within 120 h reached to 2.13 % of the dose, and the unchanged cumulative amount in bile within 36 h reached to 0.20 % of the dose.The results above further indicated that yonkenafil changed to a lot of metabolites in rats after intragastric administration. Only a little ratio of yonkenafil excreted as unchanged yonkenafil. The percents of administered dose excreted as M3, M4 and M5 in rats were 52.8 %.4. The influence of yonkenafil on CYP450 enzymes in rat liver microsomes. The influence of yonkenafil on the microsome proteins, CYP450 enzymes and the activity of the main CYP450 subfamilies (CYP1A2, CYP 2D6, CYP 3A4, CYP 2C9 and CYP 2C19 ) in rat liver microsomes was investigated after Wistar rats were continuously intragastric administrated at a dose of 32 mg/kg/day in a week. And the LC/MS/MS methods for the determination of the main CYP450 subfamilies probe substrates and their respective metabolic products were developed and validated. The results showed that yonkenafil didn't influence the microsome proteins, CYP450 enzymes and the activity of the main CYP450 subfamilies.5. The influence of the administration routes on the absolute bioavailability of yonkenafil. The early studies of yonkenafil showed that the mean oral bioavailability of yonkenafil in rats was only 29.5%. The total amount of yonkenafil observed in the excretions was only 2.18% of the dosage, which indicated that the poor oral bioavailability of this drug attributed to the hepatic and gastrointestinal first-pass effects. This section aimed at evaluating the pharmacokinetics of yonkenafil in rats after sublingual, oral and intravenous administrations at the same dosage. The results showed that sublingual yonkenafil significantly increased the absolute bioavailability (85.6 %) over oral administration with the advantage of rapid absorption and avoidance of gastrointestinal side effects and first-pass effects. Moreover, the low dose of sublingual administrations was more economical without reducing curative effect.Conclusions: In this thesis, LC/MS/MS methods for the determination of yonkenafil in bio-samples were developed and validated to systemically evaluate the pharmacokinetics and toxicokinetics of yonkenafil. The methods were accurate, rapid, sensitive and selectivity. It could be used to monitor yonkenafil plasma concentrations in patients suffering from ED. |
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