Potential Orphan Drug Therapy of Intravesical Liposomal Onabotulinumtoxin-A for Ketamine-Induced Cystitis by Mucosal Protection and Anti-inflammation in a Rat Model

Ketamine abusers may develop ulcerative cystitis and severe lower urinary tract symptoms, which is a medical dilemma. Recently, researchers have found the endemic of ketamine-induced cystitis worldwide. The intravesical administration of liposome-encapsulated onabotulinumtoxinA (Lipotoxin) might facilitate the healing of the damaged urothelium from liposomes, and reduce the urinary symptoms by onabotulinumtoxinA-induced chemo-denervation. Using female Sprague-Dawley rats, we investigated the effects of Lipotoxin on ketamine-induced cystitis. Functional magnetic resonance imaging, metabolic cage study, and cystometry were conducted. Paraffin-embedded sections were stained. The bladder mucosa and muscle proteins were assessed through Western blotting. We observed that repeated intravesical Lipotoxin instillation could improve suburothelial hemorrhage, recover the urothelial tight junction and adhesion proteins (zonula occludens-1 and E-cadherin), ensure less substance P in the urothelium, inhibit the overexpression of inflammatory mediators (IL-6, TNF-α, nuclear NF-κB, and COX-2) in the detrusor, suppress the upregulation of the mucosal TRPV1 and detrusor M2-mAChR, and ameliorate bladder overactivity in the ketamine-treated rats. These data reveal the mechanisms underlying the action of Lipotoxin in ketamine-induced cystitis of rats, which provide a basis of Lipotoxin for further treating ketamine-induced cystitis in humans.


Mucosal protection and chemical denervation by Lipotoxin instillation, as revealed by histology and Western blotting.
shows the histological features of ketamine-associated bladder damage and effects of bladder Lipotoxin instillation. Compared to controls, the ketamine group showed markedly high red blood cell debris under the suburothelium, faint immunolabeling of the zonula occludens (ZO)-1 (tight junction protein), significantly reduced expression of E-cadherin (adhesion protein), and enhanced immunolabeling of substance P in urothelium. In contrast, the ketamine/Lipotoxin group did not. Moreover, the ketamine/Lipotoxin group showed reduced SNAP25 expression in the detrusor, indicating BoNT-A action. Intercontractile interval (min) 16.7 ± 2.2 11.1 ± 0.6* 15.5 ± 0.8

Response of inflammatory mediators and neuroceptors to ketamine insults and Lipotoxin treatment. Figures 3 and 4 present representative results of Western blotting and statistical comparisons of
protein expression in the bladder mucosa and detrusor of the three groups. Compared with the control group, the ketamine group showed significant overexpression of the inflammatory mediators in the detrusor, including interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, nuclear nuclear factor (NF)-κB, and cyclooxygenase (COX)-2, as well as neuroreceptors in the bladder mucosal transient receptor potential vanilloid 1 (TRPV1) receptor, and M 2 , and M 3 -muscarinic acetylcholine receptors (mAChRs) in the detrusor. However, the ketamine/Lipotoxin group showed no significant differences compared with the control, except for higher IL-1β and M 3 -mAChR expression levels in the detrusor.

Discussion
Our findings reveal that repeated intravesical Lipotoxin instillation can ameliorate KIC in rats. Lipotoxin instillation can provide the dual effects of mucosal protection and chemical denervation for treating ketamine-induced micturition symptoms and bladder overactivity, mucosal damage, and inflammation. Compared with the control group, the ketamine group showed a stronger activation of the PAG area in fMRI, despite an empty bladder, and significantly increased micturition frequency and cystometric bladder overactivity; however, the ketamine/ Lipotoxin group did not. Daily ketamine injection caused suburothelial hemorrhage, reduced urothelial tight junction (ZO-1) and adhesion protein (E-cadherin), and enhanced substance P spread over the urothelium in the rat bladders. The ketamine/Lipotoxin group exhibited sparse suburothelial hemorrhage, improved mucosal tight junction and adhesion protein loss, and less substance P spread in the urothelium, in addition to exhibiting decreased SNAP25 in the detrusor. Furthermore, the mechanisms underlying the action of Lipotoxin in treating KIC may involve the modulation of inflammatory mediators and neuroceptors in rat bladders, and disruption of central sensitization, as revealed by reduced fMRI signals in the brain. The ketamine group had significantly increased inflammatory mediators, namely IL-1β, IL-6, TNF-α, nuclear NF-κB, and COX-2, in the detrusor. Moreover, this group showed significant overexpression of bladder mucosal TRPV1 receptor, and detrusor M 2-and M 3 -mAChRs. However, the ketamine/Lipotoxin group had insignificant changes from controls, except for IL-1β and M 3 -mAChR overexpression in the detrusor. These results demonstrate that repeated Lipotoxin instillation may have the potential in treating KIC by ameliorating bladder overactivity, ensuring mucosal protection, inhibiting detrusor inflammation, modulating the aberrant vesical neurotransmission, and inhibiting central sensitization. KIC is notorious for its severe urinary symptoms and ulcerative cystitis 2 . At present, the best KIC animal model available was established by Chuang et al. 9,17,18 , representing the micturition behavior in and bladder pathology 2,19 . In the current study, we observed that the bladder instillation of Lipotoxin reduced vesical afferent inputs in the PAG area, micturition frequency and bladder overactivity in ketamine-treated rats. These beneficial effects might be a result of the Lipotoxin-induced dual effects of mucosal protection and chemical denervation 11 . Micturition behavior involves the interaction of the bladder with the central nervous system. The neural circuitry in supraspinal control system, particularly in the PAG area, plays an important role in the occurrence of urgency 20,21 . The vesical afferent signals emerge into the PAG area. The PAG area signals can enable understanding the peripheral sensory input from the bladder and be a reference of "afferent noise" intensity 21,22 . In the current study, the Lipotoxin-induced inhibition of central sensitization might provide evidence demonstrating that intravesical Lipotoxin instillation can abate bladder afferent noise caused by KIC insults and avoid the possible neural plasticity in urgent sensation 23 .
Repeated liposomes instillation may result in mucosal protection effects in ketamine-treated rat bladders. Our results reveal sparse suburothelial hemorrhage, improved urothelial tight junction and adhesion protein damage, and less substance P spread in the bladder of rats in the ketamine/Lipotoxin group. Liposomes have been reported to protect the bladder from irritating urine solutes and promote the healing of urothelial damage [24][25][26] . Tyagi et al. reported that liposomes instilled in a rat's bladder formed a coating on the urothelium surface and blocked the irritation of the submucosal afferents 24 . Thus, this liposome coating may cover the injured urothelium and block the direct toxic damage induced by ketamine and its metabolites on the bladder of ketamine/Lipotoxin-treated rats. Reduced submucosal afferent excitation after liposomes instillation might be reflected in the less spread of substance P over the urothelium in this study. Certainly, C-fiber denervation caused by BoNT-A may contribute to this phenomenon in Lipotoxin-treated rats 11,15 . Chuang et al. 9 reported that daily ketamine injection could cause denuded urothelium and suburothelial hemorrhage in rats on day 14. Our fMRI results reveal stronger activation of the PAG area in ketamine-treated rats on day 14. However, less bladder mucosal damage was observed in the ketamine/Lipotoxin group on day 28. We believe that liposomes account for the direct repair of bladder urothelial deficits, such as the recovery of the tight junction and adhesion protein in the ketamine/Lipotoxin group.
Our study supports the notion that bladder instillation of Lipotoxin can deliver BoNT-A into rats' detrusor and cause chemo-denervation, which modulates the inflammatory mediators and aberrant vesical neuroreceptors of chemical cystitis in rats 11 . In this study, detrusor SNAP25 decreased in the ketamine/Lipotoxin group. Meanwhile, Lipotoxin suppressed the upregulation of bladder mucosal TRPV1 receptor and detrusor M 2 -mAChR, and inhibited the overexpression of IL-6, TNFα, COX-2 and nuclear NF-κB proteins in the detrusor of ketamine-treated rats. Direct toxic damage caused by ketamine and its metabolites is considered to induce a cascade of bladder pathology of KIC, namely bladder barrier dysfunction, neurogenic inflammation, and COX-mediated inflammation 2 . After toxic substances activate vesical C-fibers, substance P release plays a key role in inflammatory response initiation 27 . Juan et al. reported that NF-κB translocation into the nucleus could activate COX-2 mediated inflammation and fibrosis of KIC 17 . Through Lipotoxin instillation and its action on C-fiber denervation 11,15 , BoNT-A may suppress mucosal TRPV1 receptor upregulation and prevent substance P release in ketamine-treated rat bladders. Through such mechanisms, Lipotoxin could inhibit the neurogenic inflammation and modulate a series of inflammatory mediators, such as IL-6, TNF-α, nuclear NF-κB and COX-2 in the detrusor of rats in the ketamine/Lipotoxin group. Moreover, the cholinergic denervation effect of BoNT-A can suppress the upregulation of detrusor M 2 -mAChR in ketamine/Lipotoxin-treated rats, as our results and a previous report 15 . Thus, Lipotoxin treatment can improve the aberrant neurotransmission and associated inflammatory response of KIC. This is the first report of the promise of Lipotoxin in KIC treatment; however, the study has some limitations. This study did not include a control group of liposomes administration alone, which would limit us to speculate on the added effect of botulinum toxin and the mechanisms as to how lipotoxin is causing changes in both mucosa and smooth muscle layer. Although we demonstrated the therapeutic effects of Lipotoxin in a rat model of KIC, the results cannot be completely generalized to humans. Furthermore, the dose-effect relationship should be determined before we can apply Lipotoxin for human treatment.

Conclusion
Our study demonstrates that repeated Lipotoxin bladder instillation can ameliorate the ketamine-induced bladder overactivity and chronic inflammation in rats by inducing mucosal protection and chemical denervation. Furthermore, repeated Lipotoxin treatment could improve suburothelial hemorrhage, recover urothelial tight junction and adhesion proteins, ensure less substance P in the urothelium, inhibit the overexpression of inflammatory mediators (IL-6, TNF-α, nuclear NF-κB, and COX-2) in the detrusor, and suppress the upregulation of the mucosal TRPV1 and detrusor M 2 -mAChR in the ketamine group. Our results reveal the mechanisms underlying the action of Lipotoxin in KIC, which can facilitate future clinical trials on an orphan drug.

Materials and Methods
This study was conducted in accordance with the guidelines of National Research Council, USA, and the Animal Protection Law by the Council of Agriculture of the Republic of China. The experimental protocol was approved by the Institutional Animal Ethics Committee of Chang Gung Memorial Hospital (permit number: 2014121104). Invasive procedures were performed under anesthesia, and every effort was made to minimize both the suffering and number of animals used in the experiments.
Sixty female Sprague-Dawley rats (BioLASCO Taiwan Co., Ltd., Taipei, Taiwan; weight: 200-250 g) were randomly allocated to three groups (n = 20) and subjected to an experimental course of 28 days. They were maintained in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International under temperature control (24 ± 0.5 °C) and a 12:12-h light-dark cycle. The groups were as follows: i) a control group (0.9% saline), ii) a ketamine group (25 mg/kg/day ketamine, intraperitoneal injection) 9 , and iii) a ketamine/Lipotoxin group (ketamine plus intravesical instillation of Lipotoxin [0.8 ml] retained for 1-h on days 14 and 21, under 2-3% isoflurane anesthesia with mechanical ventilator). Lipotoxin was prepared as previously reported 28 : liposomes (Lipella, Pennsylvania) 10 mg/ml mixed with onabotulinumtoxinA (Allergan, California) 20 U/ml.

Resting-state fMRI. Under intramuscular zoletil (50 mg/kg) injection anesthesia and bladder emptying
with a polyethelence-50 catheter through the urethra, four rats in each group were used to evaluate the activation of PAG matter on days 0, 14, and 28. After emptying the bladder, the rats were prepared for MRI scanning less than 5 min elapsed. The fMRI was performed using a 9.4-T horizontal-bore animal MR scanning system (Biospec 94/20, Bruker, Ettingen, Germany). This scanning system comprises a self-shielded magnet with a 20 cm clear bore and a BGA-12S gradient insert (inner diameter: 12 cm) that offers a maximal gradient strength of 675mT/m with transmitter only coil and receiver rat brain surface array coil for signal detection from the head of the rat.
According to orientation of landmark structures from the sagittal images 21 Figure S1).
By using identical spatial dimensions as in the T2-weighted axial reference imaging, we acquired functional images through the echo-planar imaging (EPI) sequence with the following parameters: field of view (FOV) = 25.0 × 25.0 mm; matrix size = 96 × 96; spatial resolution = 260 × 260 μm; slice thickness = 1.0 mm; effective spectral bandwidth = 250000 Hz; echo time (TE) = 20.0 ms; repetition time (TR) = 2000ms; segment = 1; number of averages = 1; total acquisition time = 4 min. One hundred twenty EPI volumes were acquired for each run. Rats were in resting state during all imaging sessions. Images were preprocessed using conventional procedures: registration to a segmented rat brain atlas and motion correction with SPM8 (Wellcome Department of Cognitive Neurology, London, UK), spatial smoothing (FWHM = 1 mm), regressions of motion parameters and white matter/ventricle signals, and band-pass filtering (0.002-0.1 Hz). Resting-state fMRI images were viewed as single intensity in the PAG region of the rats' brains were referred to the standard anatomical atlas 29 and analyzed with ImageJ 1.51. The procedures to localize the PAG area were shown in Supplementary Figure S1.
Metabolic cage. On day 23, 12 rats in each group were placed in individual 3701M081 metabolic cage (Tecniplast, Buguggiate, Italy), as previously reported 30 . After a 24-h familiarization period, a known volume of water was filled in the animal drinking bottles. Both micturition frequency and urine output were determined using a cup fitted to an FT-104 force transducer (iWox/CB Sciences, Inc., Dover, NH, USA). The volume of liquid consumed and urine production were measured for 3 days. Urine samples were subjected to ketamine and norketamine assays through liquid chromatography-mass spectrometry.
Cystometry. Rats were anesthetized with subcutaneous urethane (1.0 gm/kg) on day 28. A polyethelence-50 catheter was inserted in the urethra and was connected through a T-tube to a pressure transducer and a microinjection pump (infros AG, CH-4130, Bottmingen, Switzerland). Room temperature saline was infused into the bladder at a rate of 0.08 ml/ml and cystometrography was recorded using a Gould polygraph (RS3400; Gould, Cleveland, OH). After starting the saline infusion, we waited at least 30 min for voiding patterns to stabilize. Thus, reproducible micturition cycles were recorded for a 1-h period. Bladder morphology and immunohistochemical study. To characterize morphological changes in rats' bladders, paraffin sections were subjected to routine hematoxylin and eosin staining and immunohistochemical analyses.
Determination of protein levels in bladder mucosa and muscle layer. Bladder bodies were divided into mucosa and detrusor layers by a microdissection technique 31 . Interested detrusor inflammatory markers of KIC were investigated, namely IL-β, IL-6, TNF-α, Nerve growth factor, NF-κB and COX2 9,19,32 . Mucosal neuroceptors involved in the urothelial afferent signaling were evaluated, such as TRPV1, P2X 2 and P2X 3 receptors 33 . Also, the detrusor neuroceptors engaged in detrusor contraction were examined, such as M 2-and M 3 -mAchRs and P2X 1 receptor 33 . The Western immunoblotting was performed as previously described 30 . In brief, alternative samples from each group were homogenized on ice in CelLytic TM MT cell lysis buffer (Sigma-Aldrich) containing a protease inhibitor. The total protein was measured using the Pierce 660-nm protein assay (Thermo, Waltham, MA, USA). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using the Laemmli buffer system. Nuclear/Cytosol Fractionation Kit (BioVision, Inc., Milpitas, CA, USA) was used for preparing cytosolic and nuclear extracts and Western blot analysis of NF-κB translocation according to the manufacturer's instructions.
Antibodies Statistical Analysis. All data are presented as the mean ± standard error. Data were subjected to 1-way ANOVA and multiple comparisons by Dunnett's test. For all statistical tests, p < 0.05 was considered significant.
Data available statement. The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.