Pharmacological Research, Vol . 24, No . 2, 1991
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EFFECTS OF DIFFERENT DRUGS ON THE CYSTOMETROGRAM IN CONSCIOUS RATS LUCIANO GUARNERI, RITA COVA, PATRIZIA ANGELICO, ENRICO COLLI* and RODOLFO TESTA Pharmacology Department, Research Laboratories, and *Medical Department, Recordati S .p .A ., 20148 Milan, Italy Received in final form 14 February 1991
SUMMARY The effects on urodynamic parameters of i .v . administration of different drugs utilized in the therapy of detrusor instability, have been studied in conscious catheterized rats . Emepronium bromide, oxybutynin and nifedipine affected in a dose-dependent way the micturition pressure (MP), with sporadic changes in bladder volume capacity (BVC) . Terodiline induced significant increases in BVC values in a wide range of doses . These changes, however, were always not dose-dependent . The drug significantly reduced MP only at the higher administered dose (10 mg/kg) . Flavoxate induced increases of bladder capacity (BVC) not dependent on the administered doses, with no changes in micturition pressure (MP) . Indomethacin significantly increased BVC and weakly reduced MP, but the effects were not dose-related . The effects of drugs on BVC were unrelated with the basal value of this parameter, whereas the decrease of MP seems to be related to high basal values before treatment. From a quantitative point of view, cystometrographic recordings in conscious normal rats can provide comparative data among drugs acting on bladder contractility (MP) such as anticholinergics and strong calcium antagonists . KEY WORDS :
cystometry, conscious rat, bladder function, antispasmodics, lower urinary tract .
INTRODUCTION In the last decade, disturbances of the function of the lower urinary tract have attracted an increasing interest and, at present, there are several drugs available for the treatment of the overactive detrusor, as recently reviewed by Andersson [1, 2] . The list includes pure anticholinergic drugs (e .g . propantheline and emepronium bromide), as well as drugs with `direct' effects (e .g . flavoxate and calcium Correspondence to : Rodolfo Testa, Recordati S .p .A ., Via Civitali 1, 21048 Milano, Italy . 1043-6618/91/060175-13/$03 .00/0
© 1991 The Italian Pharmacological Society
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Pharmacological Research, Vol. 24, No . 2, 1991
antagonists), drugs with `mixed' actions (e .g . oxybutynin, terodiline, and imipramine, endowed with both anticholinergic and calcium-antagonistic activity), drugs acting on alpha- and beta-adrenoceptors (e .g . phenoxybenzamine and terbutaline), prostaglandin synthetase inhibitors (e .g . indomethacin, flurbiprofen) and drugs such as baclofen, bromocriptine etc . Although many of these substances increase detrusor capacity and block detrusor contractions in humans [3], a long-term selective and side effect-free treatment is still far from being available . Therefore, the search of new substances for the treatment of urinary incontinence appears an attractive field of research . In order to evaluate the potential effects of new chemical entities on bladder voiding, adequate in vivo animal models are needed. Cystometrographic studies are employed in humans and several animal species to quantitate urodynamic parameters [4-20] . Cystometry involves two phases : (1) the slow filling of the bladder with fluid at a constant rate to measure bladder capacity ; and (2) measures of muscle contractility during the voiding phase . Together, both phases represent the micturition reflexes that control bladder function [21] . Although several papers have appeared concerning the assessment of urodynamic parameters in laboratory animals, relatively few of these reported comparative data on drug effects [22-30] . We have, therefore, studied the effects of some drugs clinically used in the therapy of overactive detrusor, on the urodynamic parameters in conscious rats . Flavoxate and nifedipine were chosen as drugs with `direct' effects, terodiline and oxybutynin as `mixed effects' drugs, emepronium bromide as pure anticholinergic, and indomethacin as inhibitor of prostaglandin synthetase [1, 2] .
MATERIALS AND METHODS Animals and drugs
Male Sprague-Dawley rats [Crl : CD° (SD) BR] of 250-350 g body wt from Charles River Italia were used . Animals were housed with free access to food and water and maintained on forced light-dark cycle at 22-24°C, except during experiment performance . Flavoxate hydrochloride was supplied by Recordati S .p .A ., Italy . The other drugs used were synthesized in our laboratories or were purchased from commercial sources . Animal preparation and surgical procedure
Bladder cannulation was performed according to previously reported methods [9, 29] with minor modifications . Male rats, anaesthetized with Nembutal (30 mg/kg)+chloral hydrate (125 mg/kg) i.p ., were placed in a supine position and an approximately 10 mm midline incision was made in the shaved and cleaned abdominal wall . The urinary bladder was gently freed from adhering tissues, emptied and then cannulated, via an incision at the dome, with a polyethylene cannula (Portex PP30), which was permanently sutured with silk thread . The cannula was exteriorized through a subcutaneous tunnel in the retroscapular area,
Pharmacological Research, Vol . 24, No . 2, 1991
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where it was connected with a plastic adapter to avoid the risk of removal by the animal. The abdominal incision was then sutured and medication with antibiotics (penicillin G 200 000 UI/kg and streptomycin 260 mg/kg i.p .) was performed in order to prevent infection. Since an effect of time after catheter implantation on the cystometrographic parameters has been reported [7], for drug testing rats were rigorously used 1 and 7 days after implantation . Cystometrographic recordings
On the day of experiment, the rats were placed in Bollman's cages ; after a stabilization period of 20 min, the free tip of the cannula was connected to a pressure transducer (Bentley T800) and to a peristaltic pump (Gilson Minipuls, France) for a continuous infusion of warmed saline solution (37 °C) into the urinary bladder, at the constant rate of 0 .1 ml/min . Two urodynamic parameters from the cystometrogram, recorded on a fourchannel polygraph (KO 380, Battaglia Rangoni, Italy), were evaluated : bladder volume capacity (BVC) and micturition pressure (MP) . BVC (in ml) is defined as the bladder volume at the time when detrusor contraction is followed by micturition . M P (in mmHg) is defined as the maximal intravesical pressure induced by the contraction of detrusor during micturition . Basal BVC and MP values were evaluated as mean of the first two cystometrograms . At this point, the infusion was interrupted and drugs were administered intravenously (caudal vein) . Fifteen minutes after drug injection, another two cystometrograms were recorded in each animal . Statistical analysis
All data were expressed as mean±sEM . Statistical significance of the differences in parameter values before and after the treatments was evaluated by Student's ttest for paired data . Differences in parameter values at different days after catheter implantation were evaluated by ANOVA and Dunnett's t-test . Linear regression analyses were performed by using a LOTUS standard package on an IBM PC-AT computer . Owing to the high variability of the parameters, we considered always as significant changes showing a probability P
1 75
EFFECTS OF DIFFERENT DRUGS ON THE CYSTOMETROGRAM IN CONSCIOUS RATS LUCIANO GUARNERI, RITA COVA, PATRIZIA ANGELICO, ENRICO COLLI* and RODOLFO TESTA Pharmacology Department, Research Laboratories, and *Medical Department, Recordati S .p .A ., 20148 Milan, Italy Received in final form 14 February 1991
SUMMARY The effects on urodynamic parameters of i .v . administration of different drugs utilized in the therapy of detrusor instability, have been studied in conscious catheterized rats . Emepronium bromide, oxybutynin and nifedipine affected in a dose-dependent way the micturition pressure (MP), with sporadic changes in bladder volume capacity (BVC) . Terodiline induced significant increases in BVC values in a wide range of doses . These changes, however, were always not dose-dependent . The drug significantly reduced MP only at the higher administered dose (10 mg/kg) . Flavoxate induced increases of bladder capacity (BVC) not dependent on the administered doses, with no changes in micturition pressure (MP) . Indomethacin significantly increased BVC and weakly reduced MP, but the effects were not dose-related . The effects of drugs on BVC were unrelated with the basal value of this parameter, whereas the decrease of MP seems to be related to high basal values before treatment. From a quantitative point of view, cystometrographic recordings in conscious normal rats can provide comparative data among drugs acting on bladder contractility (MP) such as anticholinergics and strong calcium antagonists . KEY WORDS :
cystometry, conscious rat, bladder function, antispasmodics, lower urinary tract .
INTRODUCTION In the last decade, disturbances of the function of the lower urinary tract have attracted an increasing interest and, at present, there are several drugs available for the treatment of the overactive detrusor, as recently reviewed by Andersson [1, 2] . The list includes pure anticholinergic drugs (e .g . propantheline and emepronium bromide), as well as drugs with `direct' effects (e .g . flavoxate and calcium Correspondence to : Rodolfo Testa, Recordati S .p .A ., Via Civitali 1, 21048 Milano, Italy . 1043-6618/91/060175-13/$03 .00/0
© 1991 The Italian Pharmacological Society
1 76
Pharmacological Research, Vol. 24, No . 2, 1991
antagonists), drugs with `mixed' actions (e .g . oxybutynin, terodiline, and imipramine, endowed with both anticholinergic and calcium-antagonistic activity), drugs acting on alpha- and beta-adrenoceptors (e .g . phenoxybenzamine and terbutaline), prostaglandin synthetase inhibitors (e .g . indomethacin, flurbiprofen) and drugs such as baclofen, bromocriptine etc . Although many of these substances increase detrusor capacity and block detrusor contractions in humans [3], a long-term selective and side effect-free treatment is still far from being available . Therefore, the search of new substances for the treatment of urinary incontinence appears an attractive field of research . In order to evaluate the potential effects of new chemical entities on bladder voiding, adequate in vivo animal models are needed. Cystometrographic studies are employed in humans and several animal species to quantitate urodynamic parameters [4-20] . Cystometry involves two phases : (1) the slow filling of the bladder with fluid at a constant rate to measure bladder capacity ; and (2) measures of muscle contractility during the voiding phase . Together, both phases represent the micturition reflexes that control bladder function [21] . Although several papers have appeared concerning the assessment of urodynamic parameters in laboratory animals, relatively few of these reported comparative data on drug effects [22-30] . We have, therefore, studied the effects of some drugs clinically used in the therapy of overactive detrusor, on the urodynamic parameters in conscious rats . Flavoxate and nifedipine were chosen as drugs with `direct' effects, terodiline and oxybutynin as `mixed effects' drugs, emepronium bromide as pure anticholinergic, and indomethacin as inhibitor of prostaglandin synthetase [1, 2] .
MATERIALS AND METHODS Animals and drugs
Male Sprague-Dawley rats [Crl : CD° (SD) BR] of 250-350 g body wt from Charles River Italia were used . Animals were housed with free access to food and water and maintained on forced light-dark cycle at 22-24°C, except during experiment performance . Flavoxate hydrochloride was supplied by Recordati S .p .A ., Italy . The other drugs used were synthesized in our laboratories or were purchased from commercial sources . Animal preparation and surgical procedure
Bladder cannulation was performed according to previously reported methods [9, 29] with minor modifications . Male rats, anaesthetized with Nembutal (30 mg/kg)+chloral hydrate (125 mg/kg) i.p ., were placed in a supine position and an approximately 10 mm midline incision was made in the shaved and cleaned abdominal wall . The urinary bladder was gently freed from adhering tissues, emptied and then cannulated, via an incision at the dome, with a polyethylene cannula (Portex PP30), which was permanently sutured with silk thread . The cannula was exteriorized through a subcutaneous tunnel in the retroscapular area,
Pharmacological Research, Vol . 24, No . 2, 1991
1 77
where it was connected with a plastic adapter to avoid the risk of removal by the animal. The abdominal incision was then sutured and medication with antibiotics (penicillin G 200 000 UI/kg and streptomycin 260 mg/kg i.p .) was performed in order to prevent infection. Since an effect of time after catheter implantation on the cystometrographic parameters has been reported [7], for drug testing rats were rigorously used 1 and 7 days after implantation . Cystometrographic recordings
On the day of experiment, the rats were placed in Bollman's cages ; after a stabilization period of 20 min, the free tip of the cannula was connected to a pressure transducer (Bentley T800) and to a peristaltic pump (Gilson Minipuls, France) for a continuous infusion of warmed saline solution (37 °C) into the urinary bladder, at the constant rate of 0 .1 ml/min . Two urodynamic parameters from the cystometrogram, recorded on a fourchannel polygraph (KO 380, Battaglia Rangoni, Italy), were evaluated : bladder volume capacity (BVC) and micturition pressure (MP) . BVC (in ml) is defined as the bladder volume at the time when detrusor contraction is followed by micturition . M P (in mmHg) is defined as the maximal intravesical pressure induced by the contraction of detrusor during micturition . Basal BVC and MP values were evaluated as mean of the first two cystometrograms . At this point, the infusion was interrupted and drugs were administered intravenously (caudal vein) . Fifteen minutes after drug injection, another two cystometrograms were recorded in each animal . Statistical analysis
All data were expressed as mean±sEM . Statistical significance of the differences in parameter values before and after the treatments was evaluated by Student's ttest for paired data . Differences in parameter values at different days after catheter implantation were evaluated by ANOVA and Dunnett's t-test . Linear regression analyses were performed by using a LOTUS standard package on an IBM PC-AT computer . Owing to the high variability of the parameters, we considered always as significant changes showing a probability P
