Liu Qinggang, Gao Yi, Cong Huiling, Liao Limin
Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China.
Department of Urology, China Rehabilitation Research Center, Beijing, China.
Front Pharmacol. 2023 Jul 24;14:1214145. doi: 10.3389/fphar.2023.1214145. eCollection 2023.
Intradetrusor injection of botulinum toxin A (BTX-A) is an effective treatment for overactive bladder (OAB). However, the occurrence of adverse events associated with BTX-A injection therapy hinders its acceptance among patients and its clinical promotion. Intravesical instillation of BTX-A offers a promising alternative to injection therapy for treating OAB. Nevertheless, due to the presence of the bladder permeability barrier (BPB) and the high molecular weight of BTX-A, direct instillation is unable to penetrate the bladder urothelium. This study aims to investigate the safety and feasibility of ultrasound-assisted intravesical delivery of BTX-A and its potential benefits in a rat model of bladder hyperactivity induced by acetic acid instillation. Hengli BTX-A and microbubbles (MB) were mixed and prepared as a novel complex. The size distribution and zeta potentials of the complex were measured. On day 1, rats' bladders were instilled with 1 mL of saline, BTX-A (20 U in 1 mL), MB, or MB-BTX-A (20 U in 1 mL) complex with or without ultrasound (US) exposure (1 MHz, 1.5 W/cm, 50% duty cycle, sonication for 10 s with a 10-s pause for a total of 10 min). The instillations were maintained for 30 min. After 7 days, cystometry was performed by filling the bladder with saline and 0.3% acetic acid (AA). Bladders were collected, weighed, and processed for immunoblotting, enzyme-linked immunosorbent assay (ELISA), histologic, and immunofluorescence analyses. Expression and distribution of SNAP-25 and SNAP-23 were assessed using Western blot and immunofluorescence. Calcitonin gene-related peptide (CGRP) in the bladder was detected using ELISA. Intercontraction intervals (ICI) decreased by 72.99%, 76.16%, and 73.96% in rats pretreated with saline, BTX-A, and US + MB, respectively. However, rats treated with US + MB + BTX-A showed a significantly reduced response to AA instillation (57.31% decrease in ICI) without affecting amplitude, baseline pressure, or threshold pressure. Rats treated with US + MB + BTX-A exhibited increased cleavage of SNAP-25 and CGRP expression compared to the control group. Ultrasound-assisted intravesical delivery of BTX-A, with the assistance of MB cavitation, led to cleavage of SNAP-25, inhibition of calcitonin gene-related peptide release from afferent nerve terminals, and amelioration of acetic acid-induced bladder hyperactivity. These results support ultrasound-assisted intravesical delivery as an efficient non-injection method for administering BTX-A.
膀胱逼尿肌内注射A型肉毒毒素（BTX-A）是治疗膀胱过度活动症（OAB）的一种有效方法。然而，与BTX-A注射治疗相关的不良事件的发生阻碍了其在患者中的接受度及其临床推广。膀胱内灌注BTX-A为治疗OAB提供了一种有前景的注射治疗替代方法。然而，由于膀胱通透性屏障（BPB）的存在以及BTX-A的高分子量，直接灌注无法穿透膀胱尿路上皮。本研究旨在探讨超声辅助膀胱内递送BTX-A的安全性和可行性及其在乙酸灌注诱导的膀胱功能亢进大鼠模型中的潜在益处。将衡力BTX-A与微泡（MB）混合并制备成一种新型复合物。测量了该复合物的大小分布和zeta电位。在第1天，向大鼠膀胱内灌注1 mL生理盐水、BTX-A（1 mL中含20 U）、MB或MB-BTX-A（1 mL中含20 U）复合物，有或没有超声（US）暴露（1 MHz，1.5 W/cm，50%占空比，超声处理10 s，停顿10 s，共10 min）。灌注持续30 min。7天后，通过向膀胱内灌注生理盐水和0.3%乙酸（AA）进行膀胱内压测量。收集膀胱，称重，并进行免疫印迹、酶联免疫吸附测定（ELISA）、组织学和免疫荧光分析。使用蛋白质免疫印迹法和免疫荧光法评估SNAP-25和SNAP-23的表达和分布。使用ELISA检测膀胱中的降钙素基因相关肽（CGRP）。分别用生理盐水、BTX-A和US + MB预处理的大鼠的收缩间期（ICI）分别降低了72.99%、76.16%和73.96%。然而，用US + MB + BTX-A治疗的大鼠对AA灌注的反应显著降低（ICI降低57.31%），而不影响幅度、基线压力或阈值压力。与对照组相比，用US + MB + BTX-A治疗的大鼠表现出SNAP-25的裂解增加和CGRP表达增加。在MB空化的辅助下，超声辅助膀胱内递送BTX-A导致SNAP-25的裂解、抑制传入神经末梢降钙素基因相关肽的释放，并改善了乙酸诱导的膀胱功能亢进。这些结果支持超声辅助膀胱内递送作为一种有效的非注射方法来施用BTX-A。
