Abstract 498: ALA dendrimers to improve diagnostic specificity of fluorescence guided diagnosis of bladder cancer

Abstract

Abstract Fluorescence guided cystoscopy with 5-amino levulinic acid (ALA) or its hexyl derivative (h-ALA, Hexvix®) has been shown to increase bladder cancer diagnosis and offer the possibility to perform a more complete resection, thereby reducing the recurrence and progression rate. However, aspects of this procedure could be further improved. The fast photobleaching of the fluorophore protoporphyrin IX (PpIX) limits the visualization during cystoscopy and the specificity is low. We thus investigated the possibility to use dendrimers ALA in order to overcome these drawbacks. We have previously synthesized a dendrimer containing 18 ALA molecules (18-ALA) that provoked a sustained synthesis of PpIX following IP administration. We investigated the in vitro hydrolysis of ALA, the PpIX synthesis rate as well as the photobleaching rate of equimolar concentrations of ALA, h-ALA and 18-ALA. We further instilled 18-ALA intravesically in rats, bearing orthotopic bladder tumors. Frozen sections were analyzed with fluorescence microscopy. We observed a very slow mono-exponential hydrolysis of the dendrimers (k 6.87 h−1), as opposed to the rather fast and bi-exponential liberation of free ALA from Hexvix® (k1 32.06 h−1, k2 3.88 h−1). This can be attributed to steric hindrance and limited access to esterases. Only ALA esters induce PpIX synthesis after withdrawal of the produg. Due to the slow hydrolysis of dendrimers, a minimal concentration is needed to obtain a sustained release. PpIX synthesis is continuous for the first 24 H for both h-ALA and 18-ALA but rapidly decreases thereafter for h-ALA, whereas PPIX is still synthesized after several days for dendrimers. This is compatible with the very slow but prolonged hydrolysis and production of free ALA. This continuous synthesis of PpIX in itself would thus ensure a more prolonged presence of fluorescence during cystoscopy since the tumor cells having endocytosed the dendrimer keep producing the fluorophore. Furthermore, when comparing in vitro photobleaching of h-ALA and 18-ALA, we observe a slower photobleaching of dendrimer induced PpIX. We futher investigated PpIX synthesis following intravesical instillation of 18-ALA. One hour instillation of dendrimers is sufficient to enable endocytosis of the prodrug but a minimum of 3 H resting time is required to observe fluorescence. Unlike ALA or other derivatives administered to rats, dendrimers induce a tumor vs. normal urothelium fluorescence ratio of 3, with a tumor to muscle ratio superior to 7. Intravesical administration of ALA dendrimers in rats results in a PpIX synthesis which is much more selective towards the tumor as opposed to ALA or h-ALA. In vitro experiments show that there is a prolonged and sustained PpIX synthesis with a reduced photobleaching as compared to h-ALA induced PpIX. These are primary indicators that ALA dendrimers could possibly overcome drawbacks of ALA / h-ALA fluorescence guided cystoscopy Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 498.