Stereoselective Effects And Mechanisms Of Doxazosin Enantiomers On Lower Urinary Tract In The Rat And Guinea Pig

Lower urinary tract symptoms (LUTS) and benign prostatic hyperplasia (BPH) are the most common urogenital disorders in ageing male. Patients with BPH/LUTS are suffering serious troubles on the quality of life, and BPH/LUTS have become a public health problem in the world. Surgical therapy seems to be the best option to treat BPH/LUTS, but it might cause some impairment to patients, such as uroclepsia, erectile dysfunction, retrograde ejaculation or relapse. Accordingly, it is becoming a considerable issue to develop safe and effective therapeutic agents treating the mild and intermediate BPH/LUTS. At present, therapeutic drugs to treat BPH/LUTS includeα1-adrenoceptor antagonists, 5α-reductase inhibitors and phytotherapeutic agents.Doxazosin (DOX), a quinazoline derivative, is a long acting selectiveα1-adrenoceptor antagonist, and racemic-doxazosin (rac-DOX) is the current medication of DOX for the clinical treatment of BPH/LUTS. Several clinical studies have demonstrated that rac-DOX produces a decrease in urethral resistance and an increase in maximal urinary flow rate with a corresponding improvement in lower urinary tract symptoms in BPH patients. However, rac-DOX is limited in the clinical application for BPH/LUTS due to its common adverse effects of dizziness, headache, adynamia and orthostatic hypotension. Therefore, the urinary tract selectivity should be necessarily considered whenα1-adrenoceptor antagonists are used for the BPH/LUTS patients. In chemical structure, rac-DOX has a chiral carbon with two optical isomers, i.e. R- and S-forms. It was reported that S-doxazosin (S-DOX) and R-doxazosin (R-DOX) were prepared using chiral mobile phase HPLC. In a pharmacological study using the human isolated prostate tissue, S-DOX, R-DOX and rac-DOX were showed to have higher affinity for α1-adrenoceptors thanα2-adrenoceptors, and no significant differences inα1-adrenoceptor affinity were found among the three compounds. Furthermore, S-DOX, R-DOX and rac-DOX all competitively antagonized the phenylephrine-induced constractile responses in the human isolated prostate with no significant differences in their pA2 values among S-DOX, R-DOX and rac-DOX. Recently, a study in our laboratory showed that S-DOX, R-DOX and rac-DOX all competitively inhibited the norepinephrine-induced constractile responses in the rabbit thoracic aorta and carotid artery, but the pA2 value of S-DOX was significantly lower than that of R-DOX or rac-DOX, indicating that the affinity of S-DOX againstα1-adrenoceptors was significantly lower than that of R-DOX or rac-DOX in the rabbit isolated blood vessels. Therefore, it is reasonable to suggest that S-DOX is a potentially therapeutic agent improving BPH/LUTS with minor cardiovascular adverse effects.In the present experiments, the effects of intraduodenal administration of S-DOX, R-DOX and rac-DOX on the rat carotid blood pressure and urinary bladder function, and the effects of S-DOX, R-DOX and rac-DOX by different routes of administration on the guinea pig urinary bladder function were observed. A rat model of partial outlet obstruction of urinary bladder was established and the effects of S-DOX, R-DOX and rac-DOX on the detrusor smooth muscle in vitro were investigated. Furthermore, a prostatic hyperplasia model induced by testosterone propionate in the castrated rat was made, and the effects of S-DOX and rac-DOX on prostatic histomorphology, cell proliferation and apoptosis were investigated.PartⅠEffects of intraduodenal administration of doxazosin enantiomers on blood pressure and urinary bladder function in ratsWe observed the effects of intraduodenal administration of S-DOX, R-DOX and rac-DOX on the carotid blood pressure and urinary bladder function in anesthetized rats. The parameters of carotid blood pressure, heart rate, vesical micturition pressure and intercontraction interval in anesthetized rats were recorded with an ADInstruments PowerLab/8sp data recording and analysis system, and the vesical micturition volume was measured, in order to investigate the selective effects of S-DOX, R-DOX and rac-DOX on the carotid blood pressure and urinary bladder function in rats.1 Effects of doxazosin enantiomers on the carotid blood pressure and heart rate in ratsS-DOX, R-DOX and rac-DOX administered intraduodenally (0.1~3.0 mg·kg-1) decreased the systolic blood pressure, diastolic blood pressure and mean arterial blood pressure significantly in the anesthetized rats in a dose-dependent manner. The inhibition of mean arterial pressure by S-DOX, R-DOX and rac-DOX at 1.0 mg·kg-1 was 26.9±8.2 %, 40.5±8.8 % and 43.1±7.7 %, respectively. The ED30 values of decreasing mean arterial blood pressure by S-DOX, R-DOX and rac-DOX were 1.7±0.8, 0.5±0.6 and 0.5±0.4 mg·kg-1. S-DOX had a weaker inhibitory effect on the carotid blood pressure in comparison with R-DOX and rac-DOX (P<0.05), but no significantly different effects were observed between R-DOX and rac-DOX on the carotid blood pressure (P>0.05). rac-DOX produced a significant inhibition on the heart rate at 1.0 and 3.0 mg·kg-1, but S-DOX and R-DOX reduced the heart rate only at 3.0 mg·kg-1.2 Effects of doxazosin enantiomers on the urinary bladder function in ratsS-DOX, R-DOX and rac-DOX administered intraduodenally (0.1~3.0 mg·kg-1) decreased the vesical micturition pressure dose-dependently in the anesthetized rats. The maximal inhibition of vesical micturition pressure by S-DOX, R-DOX and rac-DOX was 13.4±5.7 %, 14.5±11.0 % and 10.9±7.6 %, and their inhibitory potency on the vesical micturition pressure was not significantly different from each other (P>0.05). However, R-DOX, not S-DOX and rac-DOX, decreased the intercontraction interval and vesical micturition volume significantly (P<0.05).These results indicate that S-DOX administered intraduodenally retains the beneficial action on vesical micturition pressure and improves the adverse effects on blood pressure, heart rate and intercontraction interval in comparison with R-DOX and rac-DOX in the anesthetized rats.PartⅡEffects of doxazosin enantiomers by different routes of administration on urinary bladder function in guinea pigsWe observed the effects of intraduodenal and intravenous administration of S-DOX, R-DOX and rac-DOX on the urinary bladder function in anesthetized guinea pigs. The vesical micturition pressure, micturition threshold pressure and intercontraction interval in the anesthetized guinea pigs were recorded using an ADInstruments PowerLab/8sp data recording and analysis system, and the vesical micturition volume was measured, in order to investigate the effects of S-DOX, R-DOX and rac-DOX on the urinary bladder function by different routes of administration.1 Effects of intraduodenal administration of doxazosin enantiomers on the urinary bladder function in guinea pigsS-DOX, R-DOX and rac-DOX administered intraduodenally (0.08~2.4 mg·kg-1) decreased the vesical micturition pressure dose-dependently in the anesthetized guinea pigs. No significantly different effects on the vesical micturition pressure were observed among S-DOX, R-DOX and rac-DOX at 0.8 and 2.4 mg·kg-1 (P>0.05). S-DOX administered intraduodenally did not affect the micturition threshold pressure, intercontraction interval and vesical micturition volume significantly (P>0.05), but R-DOX and rac-DOX significantly depressed the micturition threshold pressure (P<0.05). R-DOX and rac-DOX also markedly decreased the intercontraction interval at 2.4 mg·kg-1 (P<0.05), and reduced the vesical micturition volume at 0.8 and 2.4 mg·kg-1(P<0.05).2 Effects of intravenous administration of doxazosin enantiomers on the urinary bladder function in guinea pigsS-DOX, R-DOX and rac-DOX administered intravenously (0.008~0.8 mg·kg-1) decreased the vesical micturition pressure in a dose-dependent manner in the anesthetized guinea pigs. The maximal inhibition of vesical micturition pressure by S-DOX, R-DOX and rac-doxazosin administered intravenously was 14.9±7.8 %, 19.2±14.4 % and 19.3±9.8 %, and their inhibitory potency on the vesical micturition pressure was not significantly different from each other (P>0.05). S-DOX, R-DOX and rac-DOX administered intravenously increased the intercontraction interval significantly (P<0.05), and S-DOX and R-DOX also increased the vesical micturition volume markedly (P<0.05). None of the three agents administered intravenously affected the micturition threshold pressure significantly (P>0.05).These results indicate that S-DOX, administered intraduodenally or intravenously, produces a decrease in the vesical micturition pressure with less adverse effects on the micturition threshold pressure, intercontraction interval and vesical micturition volume in comparison with R-DOX and rac-DOX in the anesthetized guinea pigs.PartⅢEffects of doxazosin enantiomers on the bladder strips from rats of partial outlet obstruction of urinary bladderWe investigated the effects of S-DOX, R-DOX and rac-DOX on the detrusor smooth muscle preparations from the rats with partial outlet obstruction of urinary bladder. A rat model of partial outlet obstruction of urinary bladder was established and S-DOX, R-DOX or rac-DOX (3.0 mg·kg-1) were administered intragastrically to the respective groups for 2 weeks. After the rats were killed, the detrusor smooth muscle strips were prepared and the responses to carbachol (0.01~100μmol·L-1) and isoprenaline (0.01~300μmol·L-1) in the detrusor smooth muscle preparations were observed.1 Bladder wet weight of rats treated with doxazosin enantiomersThe bladder wet weight (111±18 mg) of the obstructed model group was significantly increased in comparison with the sham-operated rats (89±8 mg, P<0.01). There were no significant changes in the bladder wet weight of the obstructed rats treated with S-DOX, R-DOX or rac-DOX (3.0 mg·kg-1) for 2 weeks in comparison with that of obstructed model group (P>0.05).2 Contractile responses to carbachol in the detrusor smooth muscle preparations from rats treated with doxazosin enantiomers In comparison with sham-operated rats, the contractile responses to carbachol in the detrusor smooth muscle preparations obtained from obstructed model rats were significantly enhanced (P<0.01), and the concentration-response curve for carbachol was shifted to the left in a parallel manner, with an increase in Emax value and a decrease in EC50 value of carbachol (P<0.01). Contractile responses to carbachol in the detrusor smooth muscle preparations obtained from obstructed rats treated with S-DOX, R-DOX or rac-DOX (3.0 mg·kg-1) for 2 weeks were significantly depressed (P<0.01). The Emax and EC50 values of carbachol in the three groups treated with doxazosin enantiomers were significantly different from that in obstructed model group (P<0.01), but not different from that in sham-operated group.3 Relaxant responses to isoprenaline in the detrusor smooth muscle preparations from rats treated with doxazosin enantiomersIn comparison with sham-operated rats, the relaxant responses to isoprenaline in the detrusor smooth muscle preparations obtained from obstructed model rats were significantly enhanced (P<0.01), and the concentration-response curve for isoprenaline was shifted to the left in a parallel manner, with an increase in Emax value and a decrease in EC50 value of isoprenaline (P<0.01). Relaxant responses to isoprenaline in the detrusor smooth muscle preparations obtained from obstructed rats treated with S-DOX, R-DOX or rac-DOX (3.0 mg·kg-1) for 2 weeks were significantly depressed (P<0.01). The Emax and EC50 values of isoprenaline in the three groups treated with doxazosin enantiomers were significantly different from that in obstructed model group (P<0.01), but not different from that in sham-operated group.These results indicate that S-DOX, R-DOX and rac-DOX are able to reverse abnormal increased responses to carbachol and isoprenaline to normal levels in the detrusor smooth muscle preparations obtained from the outlet obstructed bladder of rats, and the potencies of the three agents are not different from each other. PartⅣEffects of S-doxazosin on experimental prostatic hyperplasia in rats: quantitative evaluation of prostatic histomorphologyWe observed the effects of S-DOX and rac-DOX on experimental prostatic hyperplasia in rats using a method of quantitative evaluation of prostatic histomorphology. A prostatic hyperplasia model was established in the castrated rat treated with testosterone propionate, and S-DOX (0.3, 1.0, 3.0 mg·kg-1) and rac-DOX (1.0 mg·kg-1) were administered intragastrically for 30 days. After the rats were killed, the prostate was dissected and weighed, and the prostate volume was measured. The morphological changes in the anterior lobe and posterolateral lobe of the rat prostate were observed under light microscope, and their quantitative measurements were done by an image analysis system.1 Effects of S-DOX on the volume and wet weight of the rat prostateIn comparison with the sham-operated rats, the volume, volume index, wet weight and wet weight index of the prostate in the castrated model rats were significantly increased (P<0.01). In comparison with the castrated model rats, treatment with S-DOX (3.0 mg·kg-1) produced a significant decrease in volume index of the prostate in castrated rats (P<0.05), but S-DOX (0.3, 1.0 mg·kg-1) and rac-DOX (1.0 mg·kg-1) did not affect the volume, volume index, wet weight and wet weight index of the prostate (P>0.05).2 Morphological changes in prostate induced by S-DOX in prostatic hyperplasia of the ratEpithelial cells in the anterior lobe and posterolateral lobe of the prostate in castrated model rats proliferated markedly, and glandular cavities of the prostate became enlarged significantly. In comparison with the castrated model rats, the proliferation of epithelial cells and enlargement of glandular cavities of the anterior lobe of the prostate were inhibited in prostatic hyperplasia of the rats treated with S-DOX (0.3, 1.0, 3.0 mg·kg-1) and rac-DOX (1.0 mg·kg-1). The morphological changes in posterolateral lobe of the prostate in prostatic hyperplasia of the rats treated with S-DOX (0.3, 1.0, 3.0 mg·kg-1) and rac-DOX (1.0 mg·kg-1) were similar to those of the anterior lobe in prostatic hyperplasia of the rats treated with S-DOX (0.3, 1.0, 3.0 mg·kg-1) and rac-DOX (1.0 mg·kg-1).3 Quantitative evaluation of prostatic histomorphology in the prostatic hyperplasia rat treated with S-DOXIn comparison with the sham-operated rats, the maximal diameter, perimeter and area of the glandular cavity as well as the epithelial cell height of the prostate anterior and posterolateral lobes in prostatic hyperplasia of the rats were significantly increased (P<0.01). In comparison with the castrated model rats, S-DOX (1.0, 3.0 mg·kg-1) significantly decreased the maximal diameter, perimeter and area of the glandular cavity as well as the epithelial cell height of the prostate anterior and posterolateral lobes in prostatic hyperplasia of the rats (P<0.05, P<0.01). rac-DOX (1.0 mg·kg-1) significantly decreased the perimeter of the glandular cavity of the prostate posterolateral lobe and the epithelial cell height of the prostate anterior and posterolateral lobes in prostatic hyperplasia of the rats (P<0.05, P<0.01) without effects on maximal diameter and area of the prostate glandular cavity (P>0.05).Results of quantitative evaluation of prostatic histomorphology indicate that the inhibitory effects by S-DOX on prostatic hyperplasia of the prostate anterior and posterolateral lobes of the castrated rat treated with testosterone propionate are more potent than those by rac-DOX.PartⅤEffects of S-doxazosin on cell proliferation and apoptosis of the prostate in prostatic hyperplasia of the ratWe studied the effects of S-DOX and rac-DOX on the cell proliferation and apoptosis of prostate in prostatic hyperplasia of the rat. A prostatic hyperplasia was induced by testosterone propionate in the castrated rat, and S-DOX (0.3, 1.0, 3.0 mg·kg-1) and rac-DOX (1.0 mg·kg-1) were administered intragastrically for 30 days. After the rats were killed, the anterior and posterolateral lobes of prostate were dissected, and the cell cycle phase distribution, apoptotic rate and expression of Bcl-2 and Bax proteins of prostatic cells were detected by FCM. 1 Effects of S-DOX on proliferation and apoptosis of the prostate anterior lobe cells in prostatic hyperplasia of the ratIn comparison with the sham-operated rats, the prostatic cells in S- and G2/M-phase of the cell cycle and the proliferation index of the prostatic cells in the castrated model rats were significantly increased (P<0.05). S-DOX (0.3, 1.0, 3.0 mg·kg-1) and rac-DOX (1.0 mg·kg-1) had no significant influence on the distribution of cell cycle and proliferation index of the prostatic cells in prostatic hyperplasia of the rats. S-DOX (0.3, 1.0, 3.0 mg·kg-1) and rac-DOX (1.0 mg·kg-1), however, produced a slight and significant increase in the apoptotic rates by 7.90±3.91 %, 8.26±4.21 %, 8.33±4.49 % and 8.29±1.59 % (P<0.05, P<0.01) in prostatic hyperplasia of the rats in comparison with the castrated model rats by 4.42±1.08 %.2 Effects of S-DOX on proliferation and apoptosis of the prostate posterolateral lobe cells in prostatic hyperplasia of the ratS-DOX (0.3, 1.0, 3.0 mg·kg-1) and rac-DOX (1.0 mg·kg-1) did not significantly affect the distribution of cell cycle, proliferation index and the apoptotic rate of the prostatic cells in prostatic hyperplasia of the rats in comparison with the castrated model rats (P>0.05).3 Effects of S-DOX on the expression of Bcl-2 and Bax proteins of the prostate anterior lobe cells in prostatic hyperplasia of the ratIn comparison with the sham-operated rats, the fluorescence index of Bcl-2 protein and the Bcl-2/Bax ratio of the prostatic cells in the castrated model rats were significantly increased (P<0.05). S-DOX (0.3, 1.0, 3.0 mg·kg-1) and rac-DOX (1.0 mg·kg-1) did not significantly affect the fluorescence indexes of Bcl-2 and Bax proteins and the Bcl-2/Bax ratio of the prostatic cells in prostatic hyperplasia of the rats in comparison with the castrated model rats (P>0.05).4 Effects of S-DOX on the expression of Bcl-2 and Bax proteins of the prostate posterolateral lobe cells in prostatic hyperplasia of the ratS-DOX (0.3, 1.0, 3.0 mg·kg-1) and rac-DOX (1.0 mg·kg-1) did not significantly affect the fluorescence indexes of Bcl-2 and Bax proteins and the Bcl-2/Bax ratio of the prostatic cells in prostatic hyperplasia of the rats in comparison with the castrated model rats (P>0.05).These results indicate that S-DOX and rac-DOX administered intragastrically produce a slight and significant increase in the apoptotic rate of the prostate anterior lobe cells in prostatic hyperplasia of the rat induced by testosterone propionate without effects on cell proliferation, and the apoptogenic role of S-DOX and rac-DOX may not be involved in a change in expression of Bcl-2 and Bax proteins.ConclusionS-DOX, R-DOX and rac-DOX administered intraduodenally decrease the vesical micturition pressure dose-dependently in the anesthetized rats and guinea pigs, and the inhibitory potencies on the vesical micturition pressure are not significantly different between S-DOX and rac-DOX. S-DOX has less adverse effects on intercontraction interval and vesical micturition volume, and weaker inhibitory effects on blood pressure and heart rate in comparison with R-DOX and rac-DOX.S-DOX, R-DOX and rac-DOX are able to reverse abnormal increased responses to carbachol and isoprenaline to normal levels in the detrusor smooth muscle preparations obtained from the outlet obstructed bladder of rats, and the potencies of the three agents are not different from each other.Histopathological examination findings indicate that S-DOX inhibits the prostatic hyperplasia induced by testosterone propionate in the castrated rat, and its effect is more potent than rac-DOX at the same dose. Promotion of prostatic cell apoptosis is one of the mechanisms of the inhibitory effect of S-DOX and rac-DOX on prostatic hyperplasia.