358 ADTX1, A NEW PEPTIDIC □1-ADRENOCEPTOR ANTAGONIST WITH HIGH AFFINITY AND SELECTIVITY FOR HUMAN □1A-ADRENOCEPTOR SUBTYPE: PHARMACOLOGICAL CHARACTERIZATION

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Clusterin as a novel mediator of benign prostatic hyperplasia Kim J.1, Yanagihara Y.2, Kikugawa T.2, Tanji T.2, Yokoyama Y.2, Freeman M.R.1 Harvard Medical School Children’s Hospital, Dept. of Urology, Boston, United States of America, 2Ehime University, Dept. of Urology, Toon, Japan

1

Introduction & Objectives: To investigate the BPH progression mechanism at the molecular level, we got rat BPH model induced by treatment of phenylephrine. Microarray using ventral prostate (VP) tissues harvested from this rat model indicates the significant increased clusterin expression relative to VP tissues from controls. We found a potential role for clusterin in development of rat BPH. Material & Methods: Adolescent male Wistar rats of age 7 weeks were used for these experiments. L-PE dissolved in saline was injected subcutaneously daily (10 mg/kg/ day) for 28 days. The control group received injections of saline only. The ventral lobe of the rat prostate was extracted on 4, 7, 14, and the 28th. Comparison examination of gene expression by DNA microarray method was used the sample on the 4th. The expression level of Clusterin obtained by array result was checked by real-time RT-PCR, immunohistochemistry staining and Western blot. The function of Clusterin was investigated by using a plasmid and siRNA in rat primary prostate epithelial cell. Moreover, immunohistochemistry was performed using antibodies of TGF-beta and smad-2 which are related factors of clusterin. Results: We could induce prostatic hyperplasia with all characteristic morphological changes compared to control. The morphological changes of atypical prostatic hyperplasia, with piling-up, papillary and cribiform patterns and budding-out of epithelial cells, of rat prostate tissues (AP, DLP and VP) observed in various time points. We found that clusterin expression increased during atypical BPH development in drug-induced rat model. Real time RT-PCR revealed that levels of clusterin were increased 4 and 7 days after PE stimulation. IHC analysis demonstrated that very low level of clusterin distributed cytoplasm in control VP cells while clusterin significantly accumulated at particular intracellular compartments in VP cells from the PE-induced BPH rat model. IHC images demonstrated that TGF-beta and Smad2/3 proteins level were increased in rat model compared to control rat group.



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AdTx1, a new peptidic α1-adrenoceptor antagonist with high affinity and selectivity for human α1A-adrenoceptor subtype: pharmacological characterization Gilles N.1, Lluel P.2, Rekik M.2, Guerard M.2, Palea S.2 1 Cea, Ibitec-S, Simopro, Dept. of Biochemistry, Gif Sur Yvette, France, 2Urosphere, Dept. of Pharmacology, Toulouse, France

Introduction & Objectives: Screening of green mamba venom for natural peptides active on adrenoceptors led to the discovery of AdTx1, a peptide having high affinity and selectivity for α1A-adrenoceptors. The aim of this study was to characterize pharmacologically AdTx1 using functional studies in the isolated prostate and in anesthetized rats. Material & Methods: Membranes from Pichia pastoris expressing α1-adrenoceptor (α1AR) subtypes were used. Strips of dorsal prostate obtained from albino rabbits were placed in organ baths containing oxygenated Krebs-Henseleit solution at 37°C. After 60 min of equilibration, strips were exposed to 30 µM phenylephrine (PHE) to test their viability, then a cumulative concentration-response-curve (CRC) to PHE was performed. After a washout period, strips were incubated with AdTx1 (10, 30 or 100 nM) or its solvent for 180 min and a 2nd CRC to PHE was performed. Effects were expressed as % of response induced by 30 µM PHE.Rats were anesthetized with pentobarbital. Catheters were inserted into the jugular vein for drug administration and into the urethra for measuring intraurethral pressure (IUP). AdTx1 was administered i.v. 30 min before PHE administration. PHE (10-3000 µg/kg, i.v.) was administered as a bolus with a 3 min interval between each dose. Only one dose of AdTx1 was tested in each rat. In each animal and for each PHE dose, IUP increase from baseline was determined. Results are given as mean values ± s.e.m. Oneway ANOVA and Student-Newman-Keuls were used to compare experimental groups. Results: Binding: Prazosin and AdTx1 inhibited 3H-prazosin binding to h-α1aAR with IC50 of 1.0±0.1 nM and 1.1±0.05 nM, respectively. Importantly, AdTx1 had low affinities for the h-α1b AR and r-α1d AR subtypes, with IC50 of 950±110 nM and 1250±250 nM, respectively. Rabbit isolated prostate: In control strips, PHE pEC50 value was 5.28±0.05 and max. effect (EMax) 107.1±2.2%. Pre-incubation with 10 nM AdTx1 had no effect. However, concentrations of 30 and 100 nM, AdTx1 decreased both PHE potency (pEC50 = 4.81±0.14 and 4.27±0.29, respectively) and PHE efficacy (Emax of 58.8±4.3% and 14.0± 2.2%, respectively). Using the displacement obtained at 30 nM AdTx1, a pA2 value of 7.81 was calculated. In vivo results: PHE maximal effect in controls was obtained at 300 µg/kg i.v., producing an increase of IUP up to 22.5±2.6 mmHg. At the lowest dose tested (0.1mg/kg), AdTx1 increased PHE EC50 to 721 µg/ kg, without effect on the maximal IUP. At 0.3 and 1mg/kg, AdTx1 decreased maximal IUP to 12.1±1.7 and 7.8±1.3mmHg, respectively (p<0.05). Moreover, contractions induced by PHE at 150 µg/kg (dose inducing the submaximal effect in controls) were significantly reduced in the presence of every dose of AdTx1 (p<0.01).

Conclusions: We determined that clusterin functions as an important player for BPH development. Our findings provide first evidence of clusterin role in BPH rat model suggesting that blockade of clusterin-TGF-b-Smad2 signaling in prostate cells represents a new therapeutic target for BPH.

Conclusions: AdTx1 is highly selective for h-α1aAR, having 860 and 1100 fold less affinity for h-α1b AR and r-α1d AR, respectively. The antagonistic potency of AdTx1 is very similar to potencies reported for alfuzosin, doxazosin and terazosin on rabbit isolated prostate. However, AdTx1, differently from classical α1AR antagonists, is an insurmountable antagonist since it decreased Emax to PHE. Moreover, in vivo, AdTx1 dose-dependently reduced PHE efficacy. In conclusion AdTx1 is a promising template for the development of new α1-AR antagonists for the treatment of benign prostatic hyperplasia.



O4 FEMALE STRESS URINARY INCONTINENCE Thursday, 19 March, 12.15-13.45, Room K1

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A PSA-activated protoxin (PRX302) administered transperineally to men with symptomatic benign prostatic hyperplasia is well tolerated and exhibits sustained signs of activity

The effect of adaptable removable insoles on pelvic floor muscle activity in healthy female volunteers

Pommerville P.1, Egerdie B.2, Denmeade S.3, Merchant R.4, Buckley T.5, Abi-Habib R.J.6

1

Can-Med, Clinical Research, Victoria, Canada, 2Urology Associates, Urologic Medical Research, Kitchner, Canada, 3Johns Hokins University, Sidney Kimmel Comprehensive Cancer Center, Baltimore, United States of America, 4Protox Therapeutics, Dept. of Development and Regulatory Affairs, Vancouver, Canada, 5University of Victoria, Dept. of Biochemistry, Victoria, Canada, 6Protox Therapeutics, Dept. of Research and Development, Vancouver, Canada

1

Introduction & Objectives: The use of PSA-activated drugs for the treatment of benign prostatic hyperplasia (BPH) is a novel approach. PRX302 is a cytotoxic protoxin that was modified to be cleaved and activated by PSA. Activate PRX302 forms heptamers that insert into the cell membrane forming pores that lead to cell death. We hypothesize that local destruction of the transition zone of the prostate, induced by PRX302, will decrease pressure exerted on the urethra and relieve BPH symptoms. Here we report one-year findings from a Phase I safety and tolerability study of transperineal PRX302 administration in patients with moderate to severe BPH. Material & Methods: A total of 15 patients (mean age 64.8 years) received transperineal injections of PRX302 into the transition zone of the right and left lobes of the prostate under TRUS guidance. Four cohorts of 3 patients each were treated with increasing concentrations of PRX302 (0.75, 2.25, 7.5, and 10.5 μg/ml) at a fixed volume of 250 μl per deposit (6 to 8 deposits per prostate), while an additional cohort received a higher volume per deposit (1.33 ml, 6 deposits per prostate) at the lowest concentration of 0.75 μg/ml. Patients were evaluated using International Prostate Symptoms Scores (IPSS), Quality of Life (QoL) indicators, and prostate volume. Results: We present data of 1, 3, 6, 9 and 12 months post-treatment for 14 patients who completed the study. Because of small cohort sizes, an overall analysis was done irrespective of cohort assignment. At screening, mean IPSS was 19.2±4.6, and decreased, in all cohorts, to overall mean values of 13.9±5.7 at 1 month (p=0.01), 9.9±5.4 at 3 months (p<0.01) and 11.9±4.5 (p<0.01) at 6 months. This decrease was sustained up to 9 months (13.1±5.0, p<0.01) and 1 year post-treatment (12.7±4.6, p<0.01). QoL scores also decreased from 4.6±1.0 at screening to 2.5±1.6 at 1 month, 2.1±1.7 at 3 months, 2.4±1.5 at 6 months, 2.2±1.8 at 9 months and 2.6±1.6 at 12 months (p<0.01, for all time points). Similarly, prostate volume decreased at all time points compared to screening but achieved statistical significance only at the 3 months time point. The 14-fold dose escalation was well tolerated. Maximal tolerated dose (MTD) was not reached and no serious adverse events (SAEs) or Grade 3 or higher AEs were reported. Most AEs were mild to moderate, transient and deemed unrelated to PRX302. Conclusions: Transperineal administration of PRX302 is well tolerated. Symptomatic improvements in IPSS and QOL scores were significant and sustained up to 1 year post-treatment. PRX302 appears to provide lasting symptomatic relief and might constitute a promising treatment for patients with BPH.

Eur Urol Suppl 2009;8(4):210

Cerruto M.A.1, Vedovi E.2, Mantovani W.3, Gozzi I.2, Marangoni E.2, Fumene P.4, Pozzo A.4, Cangemi A.4, Sbarbati A.4, Curti P.1, Zattoni F.1 University of Verona, Urology Clinic, Dept. of Biomedical and Surgical Sciences, Verona, Italy, 2University of Verona, Dept. of Neurological and Visual Sciences, Verona, Italy, 3University of Verona, Dept. of Medicine and Public Health, Verona, Italy, 4University of Verona, Dept. of Morphological-Biomedical Sciences, Verona, Italy

Introduction & Objectives: Previous studies suggested that, although in upright standing an ankles dorsiflexion might improve the resting pelvic floor muscle activity (PFMa), a plantar flexion would seem the best position to facilitate maximal PFM contractions in both in incontinent women. No data are available of the impact of different shoe heels on female PFMa. We carried out this study in order to assess the relationship between PFMa and the use of different removable insoles in standing position and during walking. Material & Methods: Thirty healthy young women, ranging in age from 18 and 28 years participated in testing of PFMa changes in upright position and during walking using specific removable insoles within suitable shoes, allowing either ankles dorsiflexion or plantar-flexion at 5° and 10°. An electromyographic (EMG) biofeedback instrument using surface EMG electrodes has been employed to measure changes in PFMa in standing position and during 5 Km/h walking for 3 minutes with and without pelvic floor muscle (PFM) contractions. Statistical analysis was performed with the Statistical Package for Social Sciences analytical software (version 12.0). The results are shown as median values and interquartile range (IQR). The Wilcoxon signed-ranks test was used to perform pair-wise comparison of the different procedures conducted on each subject. The level of statistical significance was set at p=0.05. Results: In all cases, PFMa, both at rest and during maximal contraction, was significantly greater during walking than in standing up position (p<0.0001) as well as keeping contracted PFM but in plantar flexion at 5° (see table). In static condition (upright standing) resting PFMa was significantly higher wearing 10° plantarflexion insoles than without (p=0.035). Table. Comparison of median resting and maximal PFMa between static and dynamic conditions according to the different insole inclination Insole inclination

Resting PFMa (IQR) mV in static position 10° dorsiflexion (DF10) 75.48 (43.77-148.59) 5° DF (DF5) 77.32 (10.16-142.02) Without insoles (WI) 76.05 (35.43-116.97) 10° plantar flexion (PF10) 85.32 (41.11-144.65) 5° PF (PF5) 74.90 (41.63-184.05) Insole inclination Maximal PFMa (IQR) mV in static position DF10 187.19 (85.52-367.68) DF5 166.91 (79.34-358.84) WI 182.21 (79.20-315.60) PF10 202.27 (82.74-372.15) PF5 186.81 (95.35-418.63)

Resting PFMa (IQR) mV in dynamic position 118.49 (88.34-162.64) 150.56 (92.62-199.79) 132.48 (96.17-163.92) 124.67 (87.89-184.57) 145.43 (78.66-190.25) Maximal PFMa (IQR) mV in dynamic position 225.56 (153.89-501,68) 285.32 (203.88-427.19) 306.76 (199.76-411.23) 305.26 (173.95-459.30) 371.06 (169.34-498.84)

p value 0.003 0.002 <0.0001 0.006 0.022 p value 0.003 0.010 <0.0001 0.007 0.086

Conclusions: Our preliminary results using removable insoles having different plantar inclinations showed that a plantar flexion at 10° (such as with a 4.4 cm high heel) may affect resting PFMa only in static position.