Early experimental results of a novel delivery carrier, which may allow simple bladder instillation of Botulinum Toxin A as effectively as direct detrusor muscle injection
Sumit Dave, MD, MSc, FRCSC1, Mohamed G. Elshatoury, M.B.B.Ch, MSc2, Ling De Young, MSc1, Husain Alenezi, SBU1, Sohrab Ali, M.D, MSc1, Arjang Yazdani, M.D, FRCSC1, Eva Turley, M.D, PhD, FRCSC1.
1Western University, London, ON, Canada, 2Western Uniersity, London, ON, Canada.
Botulinum toxin A (BoNT/A) is a biological neurotoxin that inhibits acetylcholine release by cleaving cytosolic SNAP-25 protein resulting in bladder muscle relaxation. BoNT/A injection into the detrusor muscle has been established as an effective management option for treating neurogenic detrusor overactivity. This study assesses a novel mechanism for BoNT/A delivery using simple bladder instillation without bladder muscle injection by utilizing a specially formulated hyaluronic acid- phospholipid carrier that could potentially allow BoNT/A delivery across the urothelium.
60 female Sprague Dawley rats were divided into 6 groups. Rats in control groups 1-3 received BoNT/A (10 U)-saline instillation, HA-PE (0.5g)-saline instillation and BoNT/A (5 U) intra-detrusor injections respectively. Rats in treatment group 4 received Alexa Fluor®594-labelled BoNT/A (10 U)-HA-PE (0.5g) instillation, group 5 rats received BoNT/A (5 U) -HA-PE instillation with varying doses of HA-PE (0.2-0.5 g) for 60 minutes instillation time while group 6 rats had BoNT/A (10 U) with varying doses of HA-PE (0.2-0.5g) instilled for 30 minutes. Urodynamic studies under anesthesia were done at baseline and at 2-weeks following initial procedure after 1% acetic acid instillation for 30 minutes to assess response to treatment using the following parameters: maximum bladder pressure during filling (MP) and Inter-contraction intervals (ICI). Harvested bladders from all groups were assessed for SNAP 25 staining, while group 4 bladders were examined for Alexa594 fluorescence using confocal microscopy.
Bladders harvested from Group 3 rats that received our current treatment standard of direct intra detrusor injections showed minimal SNAP-25 activity with a mean staining percentage of 7.29±5.03 .Group 1 & 2 control rats showed extensive SNAP-25 activity (21.83±6.48 and 24.61±12.34% respectively). Group 5 rats that received higher HA-PE dose (0.5g versus 0.3g) and underwent a 60- minute instillation time showed comparable SNAP-25 staining percentage to Group 3 (12.4 ±12.27 & 7.29±5.03%, p value- 1.0). Group 6 rats, which had a shorter instillation time of 30 minutes showed fairly extensive SNAP- 25 activity despite higher BoNT/A dose (27.18±8.51). Confocal microscopy of group 4 animals confirmed the presence of Alexa594 fluorescence across the urothelium. Baseline urodynamic parameters were not significantly different between groups (p=1.0). There was no difference in the MP between groups after acetic acid instillation (p=0.2). Group 5 animals that received higher HA-PE (0.5g) for 1-hour instillation showed minimal change in ICI after acetic acid compared to baseline (Baseline: 11.75±4.34& Post acetic acid:11.25±6.99), which was comparable to ICI in group 3 rats ( 13±11.44 & 16.83±10.24).
Histological evidence of SNAP-25 cleavage, detection of Alexa594 in bladder tissue and limited evidence of urodynamic response to acetic acid, support that HA-PE may be an efficient carrier to deliver BoNT/A across the urothelium. A higher dose of HA-PE (0.5g) and longer instillation time (60 minutes) may be required to allow BoNT/A translocation. Further experiments with larger animals are required to confirm these novel findings and study the dose response of higher HA-PE dose and instillation times.
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