Intravesically Applied Chondroitin Sulfate Restores Urothelial Barrier Function in Acid-Damaged Bladder Troy M. Sofinowski MD*, Paul J. Hauser PhD, David D. Buethe MD, John A. Califano MD, Daniel J. Culkin MD, Robert E. Hurst PhD Department of Urology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma INTRODUCTION •Interstitial Cystitis (IC) is a chronic bladder disease characterized by: •the loss of the permeability barrier •reduced or absent glucosaminoglycan (GAG) layer. •Intravesical GAG replenishment therapy has been proposed to restore barrier function. •The GAG chondroitin sulfate (ChS) is used as treatment for IC in Canada and Europe. •This study quantifies ChS binding and restoration of barrier function in acid-damaged rodent bladder. MATERIALS AND METHODS •Rodent bladders (ICR mice, Sprague-Dawley rats) were damaged by exposure to 10 mM HCL for 10 min •Treatments compared: •Negative control (NC) •Acid-damaged positive control (PC) •ChS-treated (20 mg/ml) acid-damaged (CT) •Treatments were visualized by Alcian Blue Immunohistochemistry •Binding was evaluated by quantitative fluorescence microscopy (QFM) using Texas Red-labeled ChS (TR-ChS) (mouse model) •Bladder permeability was measured by quantifying serum concentrations following intravesical instillation of fluorescein and 86 Rb, a potassium mimetic •Titration studies were performed to determine saturation levels and optimal dose of ChS by measuring disappearance of TR-ChS from bladder (rat model) RESULTS 1.Visual examination of bladder sections using Alcian Blue staining demonstrated that acid treatment removes the apical cell layer and the associated GAG layer CONCLUSIONS Intravesically applied ChS binds specifically to damaged bladder and restores urothelial impermeability to levels similar to controls. The restoration of the barrier function by chondroitin sulfate suggests that it may be useful in the treatment of IC. Abstract # 52 NC PC 2. Fluorescence Imaging of TR-ChS Binding – ChS only binds acid-damaged bladder ACKNOWLEDGEMENT Supported by the NIH R01 DK069808 and Stellar Pharmaceuticals, Inc. Negative Control Acid Damaged 4. ChS restores impermeability to acid-damaged bladder • With instillation of 3 x 10 7 dpm, only 0.003% was found in the circulation of rats with intact bladders. A four-fold increase was observed in acid- damaged bladder (p = 0.006). Treatment with ChS restores impermeability in acid-damaged bladder to control levels • Similar results were observed with fluorescein (p = 0.029) 3. Quantitative Image Analysis confirms at each pH examined, ChS binding to acid damaged bladder is significantly higher than control P = 0.006 P = 0.005 P = 0.048 5. Binding of ChS to acid-damaged bladder is reaches saturation Extrapolating to human bladder shows that for a 350 mL capacity bladder, saturation would occur at > 200 mg.

INTRODUCTION Interstitial Cystitis (IC) is a chronic bladder disease characterized by:

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Department of Urology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma

INTRODUCTION•Interstitial Cystitis (IC) is a chronic bladder disease characterized by:

•the loss of the permeability barrier•reduced or absent glucosaminoglycan (GAG) layer.

•Intravesical GAG replenishment therapy has been proposed to restore barrier function.•The GAG chondroitin sulfate (ChS) is used as treatment for IC in Canada and Europe.•This study quantifies ChS binding and restoration of barrier function in acid-damaged rodent bladder.

MATERIALS AND METHODS•Rodent bladders (ICR mice, Sprague-Dawley rats) were damaged by exposure to 10 mM HCL for 10 min•Treatments compared:

•Negative control (NC)•Acid-damaged positive control (PC)•ChS-treated (20 mg/ml) acid-damaged (CT)

•Treatments were visualized by Alcian Blue Immunohistochemistry•Binding was evaluated by quantitative fluorescence microscopy (QFM) using Texas Red-labeled ChS (TR-ChS) (mouse model)•Bladder permeability was measured by quantifying serum concentrations following intravesical instillation of fluorescein and 86Rb, a potassium mimetic•Titration studies were performed to determine saturation levels and optimal dose of ChS by measuring disappearance of TR-ChS from bladder (rat model)

RESULTS1.Visual examination of bladder sections using Alcian Blue staining demonstrated that acid treatment removes the apical cell layer and the associated GAG layer

CONCLUSIONSIntravesically applied ChS binds specifically to damaged bladder and restores urothelial impermeability to levels similar to controls. The restoration of the barrier function by chondroitin sulfate suggests that it may be useful in the treatment of IC.

Abstract #52

NC PC

2. Fluorescence Imaging of TR-ChS Binding – ChS only binds acid-damaged bladder

ACKNOWLEDGEMENTSupported by the NIH R01 DK069808 and Stellar Pharmaceuticals, Inc.

Negative Control

Acid Damaged

4. ChS restores impermeability to acid-damaged bladder• With instillation of 3 x 107 dpm, only 0.003% was found in the

circulation of rats with intact bladders. A four-fold increase was observed in acid-damaged bladder (p = 0.006). Treatment with ChS restores impermeability in acid-damaged bladder to control levels

• Similar results were observed with fluorescein (p = 0.029)

3. Quantitative Image Analysis confirms at each pH examined, ChS binding to acid damaged bladder is significantly higher than control

P = 0.006

P = 0.005

P = 0.048

5. Binding of ChS to acid-damaged bladder is reaches saturation Extrapolating to human bladder shows that for a 350 mL capacity bladder, saturation would occur at > 200 mg.