Early phase 2 (day 15-30) responders Effect of thalidomide on radiation-induced urinary bladder dysfunction J. Kowaliuk1, E. Bozsaky1,2, S. Sarsarshahi1,2, P. Kuess2,3, W. Dörr1,2 1Medical University of Vienna, Department of Radiotherapy- ATRAB - Applied and Translational Radiobiology, Vienna, Austria. 2Medical University of Vienna, Christian Doppler Laboratory for Medical Radiation Research for Radiation Oncology, Vienna, Austria. 3Medical University of Vienna, Department of Radiotherapy, Vienna, Austria. Introduction The urinary bladder represents an important organ at risk in the radiotherapy of pelvic tumors. Exposure to significant radiation doses results in a decrease in bladder capacity. Patients suffer from dysuria, urgency, incontinence, and increased micturition frequency, including nocturia. The radiation response occurs in three distinct phases: (1) a reversible, biphasic early response, (2) a symptom-free latent phase, which can - inversely depending on the radiation dose - last from months to many years and (3) an irreversible late phase eventually resulting in fibrosis. Local inflammatory processes are significantly involved in the pathogenesis, with a potential role of the transcription factor NF-κB. Therefore, thalidomide, a potent NF-κB inhibitor, is studied in a mouse model for its potential to prevent or alleviate bladder dysfunction. Thalidomide mediated NF-κB inhibition clearly reduces radiation-induced functional urinary bladder changes (response: >50 % reduction in bladder capacity at the intravesical pressure of 10 mm Hg). Daily administration from day 0 – day 15 or day 15 – day 30 significantly increased the radiation tolerance. This may influence consequential late changes in the urinary bladder. Methods This preclinical study into the effect of thalidomide on the radiation response of the urinary bladder was performed in a well-established mouse model. Groups of female mice of the C3H/Neu strain were subjected to local single dose irradiation (200 kV, 20 mA) of the urinary bladder with graded doses (14, 17, 19, 21, 24 Gy) in order to generate complete dose-effect curves and corresponding ED50 values. The setup for single dose irradiation is illustrated in Figure 1A. Bladder compliance was determined by transurethral cystotonometry, which is described in Figure 1.B, in 3-day intervals in the early response phase (day 0- day 30 p. irr.) and subsequently at 4-week intervals until day 360. Thalidomide was applied intraperitoneally, at a daily dose of 100 mg/kg in the biphasic early response phase from day 0 to day 15 or day 15 to day 30. 1A 1B Figure 1B: Transurethral cystotonometry for determination of the bladder capacity. The urinary bladder is emptied by insertion of a catheter which is then replaced by a 0.9 % NaCl(aq) filled catheter (red circle). The latter is linked to an infusion pump (green device) and a pressure transducer (red arrow) via three-way tabs. The bladder filled and the pressure volume curves are recorded via a chart recorder. The bladder capacity is assessed by the intravesical volume at 10 and 20 mm Hg. Figure 1A: Setup for single dose irradiation. Group of five mice were anesthetized and placed in a spinal position exposing the urinary bladder (see black rectangles). The bowel was gently pushed away from the irradiation area. The rest of the body was shielded by lead equivalent (upper right corner of the picture) Results 2 percent responding mice percent responding mice percent responding mice Figure 2: Time-course of the radiation - response. The maximum response in the first phase (day 1-15) occurs between day 12 and 15 and on day 21 in the second phase (day 16-30). Systemic thalidomide administration in phase 1 significantly reduces the response frequency from over 60 % to approximately 30 %. Moreover a tendency towards a decreased response in phase 2 is suggested. Thalidomide administration in phase 2 also reduces radiation response in phase 2. The maximum manifests on day 21 where 90 % response are reduced to 25 %. Table: Dose-response curves variables. pdose determines the fit of the regression model and pvs.control the statistical significance between thalidomide treatment groups and control. 3 Conclusions Thalidomide has a clear potential to alleviate radiation-induced urinary bladder function impairment in the early response phase. This indicates the involvement of NF-κB in the pathogenesis of the early radiation-induced changes in urinary bladder function. Early phase 1 (day 1-15) Thalidomide schedule   ED50 [Gy] $ [Gy] 95% C.I. pdose pvs. control Control 20.2 6.7 15.5;41.8 0.038 --- 0-15 38.4 12.9 -/- 0.512 0.0033 15-30 22.2 0.125 (0.0104) Early phase 2 (day 15-30) responders Thalidomide schedule   ED50 [Gy] $ [Gy] 95% C.I. pdose pvs. control Control 18.5 3.4 16.3;20.4 0.001 --- 0-15 23.2 4.0 20.2;59.5 0.033 0.4465 15-30 24.3 7.2 -;- 0.181 0.0343 Figure 3: ED50 values of the control (radiation only) and additional thalidomide applications of phase 1 (day 1-15) and phase 2 (day 16 – 30).