Joseph Janicki

Intravesical Liposomal Tacrolimus Protects against Radiation Cystitis Induced by 3-Beam Targeted Bladder Radiation.

PURPOSE We primarily determined whether the small animal radiation research platform could create a rat radiation cystitis model via targeted bladder irradiation (phase I). The response to treating early phase radiation cystitis in rats with transurethral catheter instillation of liposomal tacrolimus was also examined (phase II). MATERIALS AND METHODS In phase I 16 adult female Sprague Dawley® rats were used. Metabolic urination patterns were analyzed before and after exposure to 20, 30 or 40 Gy radiation. In phase II irradiated rats were randomly assigned to receive a single instillation of saline or liposomal tacrolimus. RESULTS The 40 Gy radiation dose induced statistically significant reductions in the intermicturition interval compared to the lower radiation doses. By approximately 20 minutes 40 Gy radiation caused a significant decrease in the mean intermicturition interval (p < 0.0001). Histological analysis revealed degenerative epithelial changes and urothelial swelling with evidence of pseudocarcinomatous epithelial hyperplasia. Therefore, 40 Gy were chosen for the phase II efficacy study. There was no measurable change in total voided urine volume after irradiation, or after liposomal tacrolimus or saline instillation. Liposomal tacrolimus significantly increased the post-irradiation intermicturition interval by approximately 30 minutes back to baseline (p < 0.001). CONCLUSIONS The radiation cystitis rat model showed a dose dependent decrease in the intermicturition interval without inducing short-term skin or gastrointestinal damage. This study demonstrates that liposomal tacrolimus may be a promising new intravesical therapy for the rare, serious condition of radiation cystitis.

Development of an interstitial cystitis risk score for bladder permeability

Background Interstitial cystitis/bladder pain syndrome (IC) is a multifactorial syndrome of severe pelvic and genitalia pain and compromised urinary function; a subset of IC patients present with Hunner’s lesions or ulcers on their bladder walls (UIC). UIC is diagnosed by cystoscopy, which may be quite painful. The objective of this study was to determine if a calculated Bladder Permeability Defect Risk Score (BP-RS) based on non-invasive urinary cytokines could discriminate UIC patients from controls and IC patients without Hunner’s ulcers. Methods A national crowdsourcing effort targeted IC patients and age-matched controls to provide urine samples. Urinary cytokine levels for GRO, IL-6, and IL-8 were determined using a Luminex assay. Results We collected 448 urine samples from 46 states consisting of 153 IC patients (147 female, 6 male), of which 54 UIC patients (50 females, 4 male), 159 female controls, and 136 male controls. A defined BP-RS was calculated to classify UIC, or a bladder permeability defect etiology, with 89% validity. Conclusions The BP-RS Score quantifies UIC risk, indicative of a bladder permeability defect etiology in a subset of IC patients. The Bladder Permeability Defect Risk Score is the first validated urine biomarker assay for interstitial cystitis/bladder pain syndrome.

Potential Effect of Liposomes and Liposome-Encapsulated Botulinum Toxin and Tacrolimus in the Treatment of Bladder Dysfunction.

Bladder drug delivery via catheter instillation is a widely used treatment for recurrence of superficial bladder cancer. Intravesical instillation of liposomal botulinum toxin has recently shown promise in the treatment of overactive bladder and interstitial cystitis/bladder pain syndrome, and studies of liposomal tacrolimus instillations show promise in the treatment of hemorrhagic cystitis. Liposomes are lipid vesicles composed of phospholipid bilayers surrounding an aqueous core that can encapsulate hydrophilic and hydrophobic drug molecules to be delivered to cells via endocytosis. This review will present new developments on instillations of liposomes and liposome-encapsulated drugs into the urinary bladder for treating lower urinary tract dysfunction.

Intravesical liposome drug delivery and IC/BPS

Intravesical therapy has previously shown to be effective in delaying or preventing recurrence of superficial bladder cancer. This local route of drug administration is now demonstrating promise in the treatment of interstitial cystitis/bladder pain syndrome (IC/BPS) with the benefit of minimal systemic side effects. Liposomes (LPs) are lipid vesicles composed of phospholipid bilayers surrounding an aqueous core. They can incorporate drug molecules, both hydrophobic and hydrophilic, and vastly improve cellular uptake of these drug molecules via endocytosis. Intravesical LPs have therapeutic effects on IC/BPS patients, mainly due to their ability to form a protective lipid film on the urothelial surface and repair the damaged urothelium. This review considers the current status of intravesical LPs and LP mediated drug delivery for the treatment of IC/BPS.

Novel Contrast Mixture Improves Bladder Wall Contrast For Visualizing Bladder Injury

Here, we tested whether combined contrast-enhanced magnetic resonance imaging (CCE-MRI), using a mixture of gadolinium- and iron oxide-based contrast agents, can segment the bladder wall from the bladder lumen. CCE-MRI relies on the differences in particle size and contrast mechanisms of two agents for improved image contrast. Under isoflurane anesthesia, T1-weighted imaging of adult female Sprague-Dawley rat bladder was performed using standard turbospin echo sequences at 7 Tesla, before and after transurethral instillation of 0.3 ml of single-contrast MRI or CCE-MRI composed of 0.4-64 mM of gadolinium chelate (Gd-DTPA/Gadavist) and 5 mM ferumoxytol. Bladder wall contrast was assessed in the control group exposed to saline and in the bladder injury group exposed to 0.5 ml of protamine sulfate (10 mg/ml) for 30 min. CCE-MRI following instillation of 0.4-4 mM Gd-DTPA and 5 mM ferumoxytol mixture achieved segmentation between the bladder lumen and bladder wall. Hyperintensity in the bladder wall combined with hypointensity in the lumen is consistent with the increased diffusion of the dissolved Gd-DTPA and simultaneous localization of the larger nanoparticles of ferumoxytol in the lumen. The normalized hyperintense signal in the bladder wall increased from 0.46 ± 0.07 in control group to 0.73 ± 0.14 in the protamine sulfate-exposed group (P < 0.0001). CCE-MRI following instillation of contrast mixture identifies bladder wall changes likely associated with bladder injury with improved image contrast.

MP31-04 THE POWER OF CROWDSOURCING: NOVEL METHOD FOR DISCOVERY OF URINE BIOMARKERS

quantitative sensory testing, and urine sample collection. Temporal summation to evoked, thermal cutaneous pain was performed with a Medoc Thermal Sensory Analyzer at .4 Hz, a frequency known to elicit C-fiber mediated wind-up in the dorsal horn of the spinal cord. Subjects were asked to rate their pain (0 e 100 VAS) during each of a sequence of 10 brief (.5 second) heat pulses to 49 C. Temporal summation was defined as the difference in pain rating between the maximum and first pain ratings. An individual with a difference in pain ratings or a first pain rating greater than 1 SD above controls after normalization was designated as demonstrating CS. Mid-stream urine samples collected from each patient were subjected to metagenomic sequencing targeting the V3-V4 region of the 16S-rRNA gene. Relative bacterial abundances were compared using the QIIME and the Wald test statistic in the MGLM package among women with and without OAB and CS. RESULTS: 23 patients comprised the study cohort. 6/10 (60%) subjects with OAB demonstrated CS, 2/8 (25%) subjects demonstrating CS did not have OAB. Bacterial abundance differed significantly between patients with and without OAB (Wald test statistic 316, p<0.01) and CS (Wald test statistic 80, p<0.01). Relative bacterial abundances were similar in patients with OAB and CS (Table). Relative to those without OAB and CS, Enterobacteriaceae, Chitinophagaceae and Burkholderiaceae were more abundant in subjects with OAB and CS and Lactobacillaceae and Prevotellaceae less abundant. CONCLUSIONS: C-fiber activation related to elevated CS and alterations in the urinary microbiome may represent a combined mechanism of action for the development of refractory LUTS in some patients that warrants further study.

PD01-08 NOVEL CONTRAST MIXTURE IMPROVES BLADDER WALL CONTRAST FOR VISUALIZING INTERSTITIAL CYSTITIS

INTRODUCTION AND OBJECTIVES: Past attempts at contrast enhanced MRI (CE-MRI) of the bladder have been unable to enhance the image contrast between bladder wall and lumen in order to effectively resolve the bladder wall changes associated with cystitis or malignancy. Here we tested whether combined contrast-enhanced magnetic resonance imaging (CCE-MRI), using a mixture of Gadolinium and iron-oxide based contrast agent (ferumoxytol) is superior to CE-MRI in enhancing the image contrast of bladder wall. Each FDA approved agent in the contrast mixture is constituted of different particle size and have different contrast effects on the spin-echo imaging protocol. METHODS: Under isoflurane anesthesia, T1-weighted imaging of adult female Sprague-Dawley rat bladder was performed using standard turbo spin echo sequences at 7 Tesla, before and after transurethral instillation of 0.3 mL of single contrast (CE-MRI) or combined contrast mixture (CCE-MRI) composed of 0.4-64 mM of gadolinium chelate (Gadavist/Gd-DTPA) and 5 mM ferumoxytol. Bladder wall contrast was assessed in control group exposed to saline and in cystitis group exposed to 0.5 mL of protamine sulfate (10 mg/mL) for 30min RESULTS: CCE-MRI following instillation of 0.4-4 mM gadavist (gadolinium ) and 5 mM ferumoxytol mixture was superior to CE-MRI (instillation of either Gadavist or ferumoxytol) in achieving the maximum contrast between the lumen and bladder wall. T1-relaxation enhancement in bladder lumen by gadavist is masked by the T2 effect from localization of the larger ferumoxytol nanoparticles in the lumen, but the diffusion of gadavist into the lesions caused by protamine is marked by the hyperintense signal in bladder wall. The normalized hyperintensity in the bladder wall increased from 0.46 0.07 in control group to 0.73 0.14 in the protamine group (p < 0.0001). CONCLUSIONS: CCE-MRI following instillation of the contrast mixture is superior to CE-MRI using individual contrast agents in the visualization of bladder wall changes likely associated with cystitis or malignancy. CCE-MRI relies on differences in particle size and contrast mechanisms of gadolinium chelates and ferumoxytol. This novel approach has the potential to distinguish diffuse versus a focal disruption in the bladder wall integrity of interstitial cystitis patients, facilitating accurate diagnosis and improved patient care.

MP29-07 DEVELOPMENT OF THE ULCERATIVE INTERSTITIAL CYSTITIS RISK SCORE (ICUS): A URINE-BASED MULTIPLE PROTEIN ASSAY TO PREDICT ULCERATIVE INTERSTITIAL CYSTITIS

INTRODUCTION AND OBJECTIVES: Ifosfamide-induced hemorrhagic cystitis and bladder hypersensitivity can be difficult to manage when mesna fails to prevent them. Bladder hypersensitivity associated with various forms of cystitis may be refractory to multiple treatment modalities. Prior work suggests interleukin-4 (IL4) alleviates ifosfamide-induced hemorrhagic cystitis and resiniferatoxin (capsaicin receptor agonist)-induced bladder pain. IL4-inducing principle of Schistosoma mansoni eggs (IPSE) is a host modulatory protein that binds immunoglobulins on leukocytes thereby inducing IL4 production and translocates into host nuclei to alter gene transcription. We sought to determine if the S. haematobium homolog of IPSE (H-IPSE) would reduce ifosfamideand resiniferatoxin-induced bladder pathology. METHODS: We cloned and expressed H-IPSE and a nuclear localization sequence (NLS)-deficient mutant H-IPSE (H-IPSE[NLS]). H-IPSE IgE binding was measured by ELISA. H-IPSE activation of IgEbearing basophils was assayed using RSATL8 basophilic reporter cells. Cellular uptake and NLS-dependent nuclear translocation of H-IPSE and H-IPSE(NLS) were confirmed using HTB9 urothelial cells and fluorescence microscopy. We administered IL4, H-IPSE, H-IPSE+antiIL4 antibody, H-IPSE(NLS), or H-IPSE(NLS)+anti-IL4 antibody to mice prior to ifosfamide (with and without mesna) or resiniferatoxin. Negative controls were administered saline only. Positive controls were administered ifosfamide only. Previously published metrics for pain and urinary frequency were interpreted in blinded fashion. Bladder histology was interpreted in blinded fashion. Bladder hemoglobin was quantified using Drabkin0s assay. Bladder gene expression was assessed via realtime PCR. RESULTS: H-IPSE bound IgE in vitro and activated IgE-bearing RSATL8 cells. Nuclear translocation of H-IPSE but not H-IPSE(NLS) was confirmed. H-IPSE was superior to mesna and IL4 in suppressing ifosfamide-induced bladder hemorrhage (IL4-dependent). H-IPSE was comparable to mesna in dampening ifosfamide-triggered pain behaviors (NLS-dependent) and urinary frequency (NLS-dependent). H-IPSE reduced resiniferatoxin-mediated freezing behaviors (IL4and NLSdependent). H-IPSE reduced mRNA expression of proinflammatory mediators and increased expression of uroplakin mRNA. CONCLUSIONS: Our work suggests a uropathogen-derived host modulatory protein has therapeutic effects in bladder disease models.