BXL-628 is a non-hypercalcemic calcitriol analog successfully tested in a phase IIa trial for benign prostate hyperplasia therapy. Because part of low urinary tract symptoms (LUTS) are generated by overactive bladder (OAB) and bladder expresses the calcitriol receptor (VDR), we investigated the BXL-628 effects on bladder contractility and RhoA/ROK signalling activated in OAB, by in vivo (2-weeks; 30 mcg/Kg, Sprague-Dawley, SD, and spontaneously hypertensive rats, SHR) and in vitro (human bladder stromal cells, hBC) experiments. In SD bladder, BXL-628 did not affect maximal responsiveness to carbachol but increased the lag time to reach it, and reduced the maximal relaxant effect of the ROK inhibitor Y-27632. In SHR bladders, that over-express RhoA/ROK pathway and develop OAB, the Y-27632 effect was higher than in normotensive control rats Wistar-Kyoto (WKY). BXL-628 normalized SHR bladder sensitivity to Y-27632 up to WKY, while did not significantly affect RhoA/ROK expression (qRT-PCR) in rat bladder and in hBC. In immunokinase assay BXL-628 significantly decreased ROK activity, by reducing Y-27632 effect, of hBC extracts immunoprecipitated with anti-ROK. In SHR bladders ROK activity was higher than in WKY and significantly reduced by BXL-628. We therefore investigated whether BXL-628 impaired RhoA activation and its membrane translocation in hBC. Confocal microscopy, using pancadherin (a membrane marker) and RhoA immuno-labelling, revealed a reduction of RhoA membrane expression, which indicates its activation state, in BXL-628 treated hBC. Accordingly, BXL-628 significantly reduced the activated, rhotekin-bound, RhoA fraction in hBC (Western blot). Because RhoA is involved in cell motility, we tested the effect of BXL-628 on hBC migration. BXL-628 even at 0.01 pM inhibited cell migration to the same extent as other inhibitors of RhoA (simvastatin, C3 exoenzyme). Calcitriol was effective at higher concentrations (1 nM). In conclusion, in human and rat bladders, BXL-628 inhibits RhoA/ROK signalling, suggesting a novel therapeutic opportunity for OAB and LUTS treatment.
01 - 05 Apr 2006
European Society of Endocrinology