The Pharmacodynamics Research And Safety Evaluation Of Bencycloquidium Bromide

Allergic rhinitis(AR)is one of common diseases in Otolaryngology, the major symptoms of which including rhinorrhea, sneezing, nasal itching, and nasal congestion. AR is a chronic inflammatory disease resulted from the releasement of inflammatory mediators(notably histamine) mediated by IgE after atopic individuals expose to allergen,involving several immunocompetence cells and cytokines.Besides the classic immunological mechanism, AR involves the mechanism of neuroregulation.Autonomic nervous system(ANS)dysfuntion has been proposed as a contibutory factor. Research shows that an exaggerated cholinergic activity occurs in patients with AR, while muscarinic antagonist iprotropium bromide approved to be effective in some symptoms of AR. Today, there is no similar listed preparation as iprotropium bromide in China.Bencycloquidium bromide is a novel muscarinic antagonist,which belongs to type?novel drug and there is no research about it. In order to investigate the effect and action mechanism of bencycloquidium bromide(BCQB)and evaluate the safety of BCQB, studies were performed. Chapter 1 Research on the pharmacodynamics of BCQBMajor experimental methods:The curative effects of BCQB were observed through the model of OVA-sensitized rats and TDI-sensititized guinea pigs,including the behavioral scores?nasal seretions and pathological scores. Also the anti-inflammation and anti-pruritus effect of BCQB were observed by the methods of dimethylbenzene-induced auricular edema,glacial acetic acid-induced augmentation of capillary infiltration in mice,and histamine-induced pruritus in guinea pigs.In rats with AR , the ova-specific IgE in the blood plasma and nasal secretion were analysed by ELISA;In guinea pigs with AR, the concentration of histamine in nasal mucosa were also analysed by ELISA, while the total density of muscarinic receptors and 3 subtypes density of muscarinic receptor in nasal mucosa were determined by radioligand bingding assay(RBA). To investigate the selectivity of BCQB to the subtypes of muscarinic receptor, cell lines expressed m1?m2 and m3 muscarinic receptor were used by the method of RBA. To investigate the pA2 of BCQB,in vitro experiment was performed on the bladder strips of guinea pig.Major finding1?The curative effect of BCQBIn rats and guinea pigs with AR,BCQB could significantly relieve the nasal symptoms, decrease the nasal secretion and allievate the morphological changes of nasal mucosa,which suggested that BCQB has good effect on AR.Furthermore, the results showed that BCQB had significant effect on the dimethylbenzene-induced auricular edema , glacial acetic acid -induced augmentation of capillary infiltration in mice, and histamine-induced pruritus in guinea pigs , which suggested that BCQB contains satisfactory effect on anti-inflammation and anti-pruritus.2?The action mechanism of BCQB2.1Compared with model group,BCQB can remarkably decrease the OVA-specific IgE in blood plasma and nasal secretion of sensitized rats(P?0.01?P<0.05),and can significantly decrease the concentration of histamine and infiltration of eosinophil(P?0.01)in sensitized guinea pigs.Thus,the immunological action of BCQB on the AR may be realized by the regulation of the above links.2.2Compared with model group,BCQB can significantly decrease the muscarinic receptor density in the nasal mucosa of sensitized guinea pig(sP?0.01),specifically as:decreasement of m1 and m3 muscarinic receptor density(P?0.01)?increasement of m2 muscarinic receptor density(P?0.01).M1and m3 muscarinic receptor mainly mediated the nasal secretion and the m2 regulate the releasement of Ach.So the decreased nasal secretion may be resulted from the decreasement of m1 and m3 muscarinic receptor density,and the decreased releasement of Ach may be resulted from the increasement of m2 muscarinic receptor density. This may be the neuroregulation mechanism of BCQB on the AR.2.3In the research of selectivity to 3 muscarinic receptor subtypes, the inhibition capability of PZ was as CHOm1?CHOm3?CHOm2; the inhibition capability of GI was as CHOm2?CHOm3?CHOm1; the inhibition capability of 4-DAMP was as CHOm3?CHOm1?CHOm2, which conform to the pharmacological characteristics of PZ?GI?4-DAMP. So the cell lines can be used for the determination of the drug's selectivity to muscarinic receptor subtypes. The result showed that the inhibition capability of BCQB was as CHOm3?CHOm1?CHOm2,in which the selectivity of BCQB to m1 and m3 muscarinic receptor were close to each other and were above the m2 muscarinic receptor. 2.4In the experiment of bladder strips in vitro, BCQB can put the dose-response curve to the right. The pA2 of BCQB was 7.09±0.057(n=6).Chapter 2 Safety evaluation of BCQBMajor experimental methods:After intranasally administration of BCQB, the effects of BCQB on the central nervous system?respiratory system?cardiovascular system and pupil were observed; Meanwhile, the cutaneous sensitization?mucosa iritation and chronic toxicity of BCQB were also observed.Major finding1?At each time point after intranasally administration, the concentration of BCQB in the mouse brain was low and was below the lower limit of quantitation(0.5405ng/ml)for BCQB,while the concentration of BCQB in the mouse blood plasma was relatively high. This suggests that BCQB can hardly cross the blood brain barrier.As a result,it was observed that there was no effect on the spontaneous activity?pole test?learning and memory ability of mice after intranasally administration of BCQB.2?Compared with vehicle group, there was no obvious influence on the respiratory frequency and amplitude(P>0.05)of anaesthetized dog after intranasally administration of BCQB. Compared with vehicle group, the concentration of phenol red in the bronchus of mice was much lower at 1h,2h in BCQB high dose group, 2h in BCQB medium dose group(P< 0.01).3?There was no obvious difference between vehicle group and each BCQB group for MAP?HR?cardiac rhythm?electrocardiogram (P>0.05)after intranasally administration,which suggest that the side effect of BCQB on the heart was low.4?After 6 month consecutive administration of BCQB.In BCQB high dose group,there was a decreasement of the HGB,HCT,LYM%,[Na]+,and increasement of the BUN,NEUT%,CK at the end of 3 month;There was a decreasement of the body weight, and increasement of the ALT,CRE and coagulation time at the and of 6 month. In BCQB medium dose groups, there was an increasement of CRE at the end of 6 month. While these indexes can return to normal level after the withdrawl of BCQB for 3 weeks.The results suggest that there was no irreversible toxicology after 6 month intranasally administration of BCQB.5?BCQB has no cutaneous sensitization.6?It was observed that there was a decreased diet and mild nasal dryness in BCQB group. But compared with vehicle group, there was no significant pathological changes in BCQB group.7?Compared with vehicle group,BCQB had remarkably effects on the size of the rabbits'pupils(P<0.01)after intranasally administration,but the size of pupils could recover after 2h in the medium and low doses groups of BCQB. There was no effect on the pupillary light-reflex in all groups.ConclusionBCQB administered intranasally is effective in treating AR,which may be resulted from the immunological and neural regulation of allergic reaction by BCQB.BCQB has good selectivity to the subtypes of muscarinic receptor,high safety in central nervous system?cardiovascular system and respiratory system, less mucosa irritation and skin sensitizatin , worthy of being developed to the clinic use.