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In Vitro Intravesical Instillation of Anticholinergic, Antispasmodic and Calcium Blocking Agents (Rabbit Whole Bladder Model)
0022-534'7 /89/1416-; 471$02.00/0 141,June
THE ~TOURNAL OF UROLOG'Y
Copyright© 1989 by Wiliiams & Wilkins
Printed in U.S.A.
IN VITRO INTRAVESICAL INSTILLATION OF ANTICHOLINERGIC, ANTISPASMODIC AND CALCIUM BLOCKING AGENTS (RABBIT WHOLE BLADDER MODEL) KUMIKO KATO, SHINICHIRO KITADA, ALEXA CHUN, ALAN J. WEIN
AND
ROBERT M. LEVIN*
From the Division of Urology, University of Pennsylvania School of Medicine, the Philadelphia Veterans Administration Medical Center, Philadelphia, Pennsylvania, and the Department of Urology, Nagoya University School of Medicine, Nagoya, Japan
ABSTRACT
The systemic side effects accompanying oral pharmacotherapy of neurogenic bladder dysfunction present significant drawbacks to this type of therapy. In these studies we investigated the effect of intravesical administration of anticholinergic, antispasmodic and calcium blocking agents on pressure response mediated by field stimulation and bethanechol. We used the rabbit in vitro whole bladder model for these experiments. The bladder from a mature male NZW rabbit was mounted in an organ bath as a whole bladder preparation. After control field stimulation and bethanechol stimulation, 20 ml. of saline containing the specific drug being evaluated was instilled into the bladder. At 30 minute intervals, the responses to field stimulation and bethanechol were determined. Two hours after instillation of 100 uM of each specific drug, the inhibition of the contractile response to bethanechol and field stimulation (as% inhibition) was as follows: oxybutynin (95%/64%), verapamil (85%/81%), atropine (68%/ 31%), diltiazem (47%/39%), and imipramine (44%/47%). Atropine and oxybutynin suppressed the contractile response of the bladder to bethanechol to a much greater extent than that to field stimulation, while verapamil, diltiazem and imipramine suppressed the contractile response to bethanechol and field stimulation to approximately the same extent. Two hours after drug instillation, the intravesical solution was washed out and replaced with saline, but the recovery of the bladder contraction was slow and incomplete. The results of this study suggest that the use of self-intravesical instillation to suppress bladder contractility should be a good therapeutic approach for patients with neurogenic bladder, especially those who are already managed intermittent catheterization. (J. Ural., 141: 1471-1475, 1989) The management of patients with neurogenic bladder has improved dramatically in the last two decades due to the proper application of intermittent catheterization in combination with oral pharmacotherapy to suppress detrusor hyperreflexia. 1 However, oral pharmacotherapy is sometimes distressing for patients because of the presence of systemic side effects. Therefore, it is worthwhile to investigate alternative routes of drug administration to suppress bladder contractility, especially those which are more localized to the urinary bladder. Although intravesical instillation is a common therapeutic approach to superficial bladder tumors, 2 there have been a few reports on this method of administration for the suppression of bladder contractility. 3 - 7 Obrink et al., 3 investigated the symptomatic effect of instillation of emepronium bromide (100 mg., 1 time per week) in patients with urgency. Higson et al. 4 Mattiason et al. (verapamil, 3 X 10-s (1 % lignocaine, 1 M, 1 time) 5 and Brendler et al. (5 mg. oxybutynin, 2 times per day) 6 investigated the effect of intravesical instillation on cystometrograms of patients with detrusor hyperactivity and showed significant increase in bladder capacity. Experimentally, Gotoh et al. 7 reported that there was a significant decrease of bladder contractility to acetylcholine and field stimulation in in vitro and in vivo rabbit bladders by intravesical verapamil (7.5 or 10 mg.). Nevertheless, there have been no papers which compared the effect of intravesical administration between different anticholinergic, antispasmodic and calcium blocking agents. The aim of this study was to investigate the effect of Accepted for publication January 5, 1989. * Requests for reprints: Div. of Urology, 5-Silverstein, 3400 Spruce St., Hospital of the University of Pennsylvania, Philadelphia PA 19104. Supported by the Veterans Administration, NIH grants R0-1-DK2-6508, R0-1-DK 33559, P-50-DK 39257 and the McCabe Fund.
intravesical administration of various drugs used or considered to be used clinically in the treatment of detrusor hyperreflexia on bladder contractile function. using the rabbit in vitro whole bladder model,8 we could evaluate the effect of drug instillation on the ability of the bladder to generate pressure in response to both field stimulation and bethanechol administration. MATERIALS AND METHODS
mature male New Zealand were sedated with an intramusof a ketamine/xylazine mixture 8.3 mg./ml. xylazine). Surgical anesthesia was maintained with intravenous pentobarbital (50 mg./ ml., 0.2 - 0.5 ml.) for 10 minutes. Prior studies have demonstrated that this method of anesthesia has no effect on the response of the in vitro whole bladder to pharmacological agents. The bladder was exposed through a midline incision and excised as low on the urethra as possible. Both ureters and main vessels were tied with 3-0 silk ligature. The excised bladder was emptied and the urethra canulated with a saline-filled electrode-tipped catheter. The whole bladder was mounted in a 300 ml. bath chamber containing Tyrode' s solution (NaCl 125 mM, KC! 2.7 mM, NaH 2P0 4 0.4 mM, CaCI 2 1.8 mM, MgCl2 0.5 mM, NaHC0 3 23.8 mM and glucose 1 mg./ 1 ml.) equilibrated with 95% 0 2 /5% CO 2 and maintained at 37C. Intravesical pressure was continuously monitored using a Statham pressure transducer and recorded on a Grass Polygraph. The signals from the Grass Polygraph were fed into an IBM PC computer via a Keithly model 500 Analog-Digital converter system and the rate of pressure generation was determined and recorded during each field stimulation. 9
1471
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KATO AND ASSOCIATES
Experimental protocol. The bladder was instilled with 20 ml. of saline and allowed to accomodate for 30 minutes. After 30 minutes the bladder was stimulated by electrical field stimulation (80 Volts, 30 Hz, 1 msec). Five minutes later, the bladder was stimulated by 500 µM of bethanechol added to the extravesical fluid. After bethanechol stimulation, the bladder was washed three times with fresh oxygenated Tyrode's solution. The bladder was emptied and instilled with 20 ml. of either fresh saline, or saline containing 10 µMor 100 µM concentration of one of the following drugs: verapamil hydrochloride, diltiazem hydrochloride, imipramine hydrochloride, atropine sulfate or oxybutynin hydrochloride. The pH of drug solutions was between 5.6 and 6.0. All drugs were of the highest purity available, purchased through standard commercial sources. Bladder responses to field stimulation and bethanechol were investigated at 30, 60, 90 and 120 minutes following the intravesical instillation of drugs. At 120 minutes, the bladder was irrigated with saline five times at two minute intervals and instilled with 20 ml. of fresh saline. The same stimulation procedures were performed 30, 60, 90 and 120 minutes after the removal of the drug. Statistical analysis. All values presented in the results represent the mean +/- standard error of the mean. The peak pressure response and the peak rate of pressure development are presented as the percentage of the control response before the instillation of drugs. Statistical significance was determined by Student's t test. A probability level of less than 0.05 was required for statistical significance.
four hour period, the peak pressure response to field stimulation and bethanechol decreased only 3% and 9%, respectively. Intravesical instillation of verapamil and diltiazem (calcium entry blockers) suppressed the contractions induced both by bethanechol and field stimulation in a time-dependent and dose-dependent manner (fig. 1). Verapamil had a greater efficacy than diltiazem. There was no significant difference between the magnitude of suppression of the response to bethanechol and field stimulation by these drugs. The suppression of the peak rate of pressure development by verapamil was proportional to the suppression of the peak pressure response to field stimulation (fig. 2). Diltiazem showed the same tendency. Figure 3 presents the effect of intravesical instillation of imipramine, a tricyclic antidepressant, on the peak pressure response of the whole bladder. The suppression of contractions induced by field stimulation and bethanechol was time-dependent and dose-dependent. At concentrations of 10 µM and 100 µM, imipramine suppressed the response to bethanechol and field stimulation to approximately the same extent. The suppression of peak pressure development by imipramine was significantly greater than the suppression of the rate of pressure generation. Intravesical instillation of atropine and oxybutynin suppressed the response to both bethanechol and field stimulation in a time-dependent manner. At the concentration of 10 µM, EFFECT OF INTRAVESICAL INSTILLATION OF VERAPAMIL ON FIELD STIMULATION T
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Preliminary experiments confirmed that the pressure response to field stimulation could be inhibited almost completely by the addition of tetrodotoxin (1 µM) whereas the response to bethanechol was not affected by tetrototoxin. This demonstrates that field stimulation acts via the release of excitatory neurotransmitters from presynaptic terminals whereas bethanechol acts by directly blocking the post-synaptic muscarinic cholinergic receptors. The peak pressure response of the control bladder (containing 20 ml. of saline) to field stimulation was 11.2 +/- 0.4 cm. H20 and the peak rate of this pressure development was 10.4 +/- 0.5 cm. H 20/sec. The peak pressure response of the control bladder (containing 20 ml. of saline) to 500 µM ofbethanechol was 11.6 +/-0.4 cm. H20. When 20 ml. saline and no other drug was used for intravesical instillation, the responses following both field stimulation and bethanechol were highly reproducible at 30 minute intervals until four hours. Even at the
EFFECT OF INTRAVESICAL INSTILLATION OF VERAPAMIL ON PRESSURE GENERATION
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1473
INTRAVESICAL ADMINISTRATION TO INHIBIT BLADDER CONTRACTILITY
both atropine and oxybutynin suppressed the response to bethanechol to almost the same extent as the response to field stimulation. At 100 µ,M, both drugs suppressed the response to bethanechol to a significantly greater extent than the response to field stimulation (fig. 4). The suppression of the rate of
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EFFECT OF INTRAVESICAL INSTILLATION OF IMIPRAMINE ON FIELD STIMULATION
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EFFECT OF INTRAVESICAL INSTILLATION
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1474
KATO AND ASSOCIATES
TABLE 1.
Suppression of contraction two hr. after intravesical instillation- I 00 µm
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18.9 61.4 52.9 68.7 36.0
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stillation. For comparative purposes the inhibition at two hours is presented in table 1. Only atropine and oxybutynin showed a significant difference between their ability to inhibit contractions to field stimulation and to bethanechol. Recovery of the bladder contraction after removal of the drugs was slow and incomplete. Additional experiments were performed where the intravesical fluid was changed following each stimulation at 30 minute intervals during the recovery phase of the experiments. There was no effect of changing the intravesical fluid on the recovery of the response to stimulation. DISCUSSION
In the last two decades, intermittent catheterization and administration of anticholinergic and antispasmodic agents have contributed greatly to the management of neurogenic bladder by preventing upper tract deterioration and decreasing urinary incontinence. 1 However, commonly used anticholinergics and tricyclic antidepressants are not selective to the urinary bladder, and when orally administered, often cause systemic side effects such as dry mouth, flushed skin, constipation and blurred vision. Although intravesical instillation is a popular method of treatment for bladder carcinoma (Thiotepa, BCG) 2 and interstitial cystitis (DMS0), 10• 11 there have been only a few reports about intravesical administration of antispasmodic agents both clinically 3 - 6 and experimentally. 7 As the rationale for intravesical therapy, it is expected that high concentrations of drug may contact the bladder mucosa for prolonged periods, and that systemic side effects would be minimal because of limited systemic absorption of the drug. In the present study, intravesical instillation of all investigated drugs suppressed the pressure response of the in vitro whole bladder to bethanechol and field stimulation. All drugs showed a time-dependent inhibition requiring 90 to 120 minutes to attain maximal effects. Gotoh et al. 7 also reported a timedependent inhibition of tension response with intravesical verapamil. It seems that the mechanism of action of anticholinergics and antispasmodics is not merely a local anesthetic effect on the bladder mucosa because of the long time period before maximal inhibition was attained. The drug administered intravesically may gradually penetrate the intermuscular spaces throughout the mucosa and directly affect the smooth muscle. As mentioned previously, the response to field stimulation was eliminated by pre-incubation with tetrodotoxin, which means that field stimulation acts through the release of endogenous neurotransmitters from nerve endings. Previous studies have also demonstrated that approximately 50% of the initial pressure response to field stimulation is cholinergic in nature; the remaining 50% is primarily purinergic. 12 In the present study, the intravesical instillation of atropine and oxybutynin, muscarinic-cholinergic antagonists, suppressed the response to bethanechol to a much greater extent than the response to field stimulation. This would be expected since these agents inhibit mainly the cholinergic aspect of field stimulation. Although oxybutynin is known to have not only anticholinergic but also direct smooth muscle relaxant activities, 13 its anticholinergic activity seems to be significantly
stronger than its direct smooth muscle relaxant properties. The suppression of the rate of pressure development by atropine and oxybutynin was significantly smaller than the suppression of the magnitude of pressure development to field stimulation. This might also be related to the fact that both atropine and oxybutynin have little effect on the non-cholinergic elements of field stimulation which make up approximately 50% of the initial response. Intravesical instillation of verapamil or diltiazem suppressed the contractile response to bethanechol and field stimulation to approximately the same extent. Verapamil and diltiazem are calcium entry blockers which selectively block the extracellular Ca2 + influx into cells. 14 The present results indicate that the same calcium ion channels were involved in the response to both field stimulation and direct muscarinic activation. The present demonstration that verapamil had a greater efficacy than diltiazem is consistent with the results of in vitro muscle strip studies by Finkbeiner et al. 15 Some calcium entry blockers are known to have an affinity for alpha-adrenergic receptors and to a lesser extent muscarinic cholinergic receptors. 16• 17 Malkowicz et al.1 8 reported that verapamil was ten times more potent in competing for alpha1 and alpha2 sites in bladder smooth muscle than diltiazem. These factors may contribute to our findings that verapamil was more effective than diltiazem. Verapamil and diltiazem inhibited the peak rate and the magnitude of the pressure development to field stimulation equally. This relationship suggests that both the cholinergic and non-cholinergic elements of field stimulation may be mediated by the same calcium ion channels. Imipramine, a tricyclic antidepressant, is known to have various pharmacological actions including muscarinic antagonism, blockade of norepinephrine uptake, and direct smooth muscle relaxant properties including calcium antagonism. 13• 19• 20 Intravesical instillation of imipramine suppressed the response to bethanechol and field stimulation to approximately the same extent. This was similar to the results of the intravesical calcium entry blockers and different from the effects of the intravesical anticholinergics. These results are consistent with prior studies which demonstrated that direct smooth muscle relaxant properties of imipramine are significantly more potent than its anticholinergic properties. 21 On the other hand, intravesical imipramine inhibited the magnitude of pressure generation induced by field stimulation to a significantly greater extent than it inhibited the rate of pressure generation. This result was similar to the response of the in vitro whole bladder to the extravesical administration of imipramine. 22 The peak rate of pressure development is dependent on several factors including the rate of transmitter release, rate of calcium entry into the muscle, rate of release of intracellular calcium and the rate of the initial response of the muscle elements to the released calcium. The magnitude of pressure development reflects the efficiency, extent and duration to which the smooth muscle elements react to the released calcium. Although we have no direct evidence, our present study is consistent with the concept that imipramine has a greater effect on the degree and duration of the intracellular response of the smooth muscle to calcium than on the rate of calcium entry and immediate rate of response of the smooth muscle elements to the released calcium. There may be some fundamental differences in the effect of intravesical drug administration on the in vitro whole bladder compared to that on the in vivo bladder. For example, the mucosal membrane barrier may be less efficient in the in vitro bladder than in the in vivo bladder because of several intravesical saline washes performed during the in vitro whole bladder studies; this may disrupt the mucin layers. The role of blood circulation in the bladder wall on the effect of intravesical drug administration is currently unknown. Blood circulation may be beneficial to the diffusion of the drug into the detrusor muscle;
INTRAVESICAL ADMINISTRATION TO INHIBIT BLADDER CONTRACTILITY
however, our results along with those of Gotoh et al. 7 showed that intravesical administration of drugs to suppress bladder contractility was effective in the in vitro rabbit whole bladder which had no blood circulation. Further studies are necessary to elucidate these differences. Although the present in vitro study does not address the problem of systemic absorption and the development of side effects on intravesical drug administration, Gotoh et aL 7 showed that there was no change in the cardiovascular status of the rabbit following intravesical administration of verapamiL Higson et aL (lignocaine),4 and Brendler et aL (oxybutynin) 6 reported no systemic side effects with clinical intravesical therapy. Obrink et al. 3 further showed that the drug concentrations in human serum were very low or were not detectable even after a very high dosage of instillation (emepronium bromide 100 mg. per 100 mL saline). These reports support the possibility that intravesical instillation could be a route of administration of anticholinergic and antispasmodic drugs with fewer systemic side effects. Recovery of the bladder contraction after removal of the drugs was slow and incomplete in the present study. This result was consistant with Gotoh's report7 about intravesical verapamiL He speculated that the inhibitory effect lasting after the removal of the drug was due to the action of the drug which remained in the intermuscular spaces. Although clinical reports about intravesical drug administration have not mentioned the long-term time-course of responses, 3- 6 the lasting inhibitory effect might cause urinary retention by suppressing the micturition reflex and limit the usefulness of this method of administration in patients who are unwilling to accept intermittent catheterization. However, intravesical therapy in patients already managed by intermittent catheterization would not be an additional burden. Even if there is a prolonged inhibition of micturition after removal of the drug, this condition can be easily managed by self-catheterization and might further decrease the necessary number of instillations. One point to be kept in mind when considering the clinical utilization of intravesical drug administration is that it is not well known whether the effect of intravesical instillation in the in vivo human bladder is comparable to that in animal experiments. In the present study, we found that instillation of oxybutynin and verapamil was very effective in suppressing bladder contractility in the in vitro rabbit bladder. Gotoh et al. 7 working on the rabbit reported that the suppression of the contractility of the in vivo bladder by intravesical verapamil correlated with that of the in vitro bladder. Clinically, Brendler et al. 6 showed that intravesical instillation of oxybutynin abolished the unstable contractions and increased bladder capacity in patients with neurogenic bladders. However, Mattiason et aL 5 found that intravesical administration of verapamil in patients with detrusor hyperreflexia increased bladder capacity without changing the amplitudes of the first unstable contraction or maximum detrusor contraction. Therefore, in their study, even though intravesical verapamil was therapeutically effective in increasing capacity, bladder contractility was not decreased as it was in the in vitro and in vivo rabbit studies. These findings indicate that one must be cautious in applying data of animal experiments to human clinical trials. Nevertheless, animal studies including the in vitro whole bladder model should be useful in screening potential drugs for intravesical therapy of detrusor hyperreflexia.
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9. 10. lL 12. 13. 14. 15. 16. 17.
18. 19. 20. 21. 22.
a logical and practical approach. Chicago: Year Book Medical Publishers, Inc., pp. 195-261, 1988. Herr, W. H., Laudone, V. P. and Whitmore, W. F. Jr.: An overview of intravesical therapy for superficial bladder tumors. J. UroL, 138: 1363, 1987. Obrink, A. and Bunne, G.: Treatment of urgency by instillation of emepronium bromide in the urinary bladder. Scand. J. UroL NephroL, 12: 215, 1978. Higson, R H., Smith, J. C. and Hills, W.: Intravesical lignocaine and detrusor instability. Brit. J. UroL, 51: 500, 1979. Mattiasson, A., Ekstrom, B. and Andersson, K-E.: Effects of intravesical instillation 1of verap;;i,-µ,il in patients with detrusor hyperactivity. NeurourO:, Urodyrf,' 6: 253, 1987 (Proceedings of the International Contii:tence Sci~iety). Brendler, C. B., Radebaugh, L. C. and Mohler, J. L.: Topical oxybutynin chloride (Ditropan) for relaxation of dysfunctional bladders. J. UroL, 139: 334A, 1988 (AUA Abstract). Gotoh, M., Hassouna, M., Mokhless, L and Elhilali, M. M.: Intravesical instillation of a calcium entry blocker and its effects on detrusor contractility: in vitro and vivo experiments. J. UroL, 135: 1304, 1986. Levin, R M., Brendler, K. and Wein, A. J.: Comparative Pharmacological response of an in vitro whole bladder preparation (rabbit) with the response of isolated smooth muscle strips. Urology, 30: 377, 1983. Levin, R M., Memberg, W., Ruggieri, M. R and Wein, A. J.: Functional effects of in vitro obstruction on the rabbit urinary bladder. J. UroL, 135: 847, 1986. Shirley, S. W., Stewart, B. I-Land Mirelman, S.: Dimethyl sulfoxide in treatment of inflammatory genitourinary disorders. Urology, 11: 215, 1978. Biggers, R. D.: Self-administration of dimethyl sulfoxide (DMSO) for interstitial cystitis. Urology, 28: 10, 1986. Levin, R M., Ruggieri, M. R and Wein, A J.: Functional effects of the purinergic innervation of the rabbit urinary bladder. J. PharmacoL Exp. Ther., 236: 452, 1986. Fredericks, C. M., Green, R L. and Anderson, G. F.: Comparative in vitro effects of imipramine, oxybutynin, and flavoxate on rabbit detrusor. Urology, '7: 487, 1978. Zsoter, T. T. and Ghurch, J. C.: Calcium antagonists; pharmacodynamic effects and mechanism of action. Drugs, 25: 93, 1983. Finkbeiner, A. E.: Effect of extracellular calcium and calcium blocking agents on detrusor contractility: an in vitro study. NeurouroL Urodyn., 2: 245, 1983. Glossman, H. and Hornug, R.: Calcium and potassium channel blockers interact with alpha adrenoceptors. MoL Cell EndocrinoL, 19: 243, 1980. Karliner, J. S., Motulsky, H.J., Dunlap, J., Brown, J. H. and Insel, P. A: Verapamil competitively inhibits alpha 1 adrenergic and muscarinic but not beta adrenergic receptors in rat myocardium. J. Cardiovasc. PharmacoL, 4: 515, 1982. Malkowicz, S. B., Wein, A. J. and Levin, RM.: Calcium antagonist affinity for adrenergic and cholinergic receptors in rabbit urinary bladder. Neurouro. Urodyn., 4: 143, 1985. Hallister, L.: Tricyclic antidepressants. N. Engl. J. Med., 299: 1106, 1978. Malkowicz, S. B., Wein, A. J., Ruggieri, M. Rand Levin, R !VL: Comparison of calcium antagonist properties of antispasmodic agents. J. UroL, 138: 667, 1987. Levin, R M. and Wein, A. J.: Comparative effects of five tricyclic compounds on the rabbit urinary bladder. NeurouroL Urodyn. 3: 127, 1984. Grover, R, Wein, A. J., Ruggieri, M. R and Levin, R M.: Functional effects of imipramine on the rabbit urinary bladder: an in-vitro study. Pharmacology, 37: 148, 1988.
THE ~TOURNAL OF UROLOG'Y
Copyright© 1989 by Wiliiams & Wilkins
Printed in U.S.A.
IN VITRO INTRAVESICAL INSTILLATION OF ANTICHOLINERGIC, ANTISPASMODIC AND CALCIUM BLOCKING AGENTS (RABBIT WHOLE BLADDER MODEL) KUMIKO KATO, SHINICHIRO KITADA, ALEXA CHUN, ALAN J. WEIN
AND
ROBERT M. LEVIN*
From the Division of Urology, University of Pennsylvania School of Medicine, the Philadelphia Veterans Administration Medical Center, Philadelphia, Pennsylvania, and the Department of Urology, Nagoya University School of Medicine, Nagoya, Japan
ABSTRACT
The systemic side effects accompanying oral pharmacotherapy of neurogenic bladder dysfunction present significant drawbacks to this type of therapy. In these studies we investigated the effect of intravesical administration of anticholinergic, antispasmodic and calcium blocking agents on pressure response mediated by field stimulation and bethanechol. We used the rabbit in vitro whole bladder model for these experiments. The bladder from a mature male NZW rabbit was mounted in an organ bath as a whole bladder preparation. After control field stimulation and bethanechol stimulation, 20 ml. of saline containing the specific drug being evaluated was instilled into the bladder. At 30 minute intervals, the responses to field stimulation and bethanechol were determined. Two hours after instillation of 100 uM of each specific drug, the inhibition of the contractile response to bethanechol and field stimulation (as% inhibition) was as follows: oxybutynin (95%/64%), verapamil (85%/81%), atropine (68%/ 31%), diltiazem (47%/39%), and imipramine (44%/47%). Atropine and oxybutynin suppressed the contractile response of the bladder to bethanechol to a much greater extent than that to field stimulation, while verapamil, diltiazem and imipramine suppressed the contractile response to bethanechol and field stimulation to approximately the same extent. Two hours after drug instillation, the intravesical solution was washed out and replaced with saline, but the recovery of the bladder contraction was slow and incomplete. The results of this study suggest that the use of self-intravesical instillation to suppress bladder contractility should be a good therapeutic approach for patients with neurogenic bladder, especially those who are already managed intermittent catheterization. (J. Ural., 141: 1471-1475, 1989) The management of patients with neurogenic bladder has improved dramatically in the last two decades due to the proper application of intermittent catheterization in combination with oral pharmacotherapy to suppress detrusor hyperreflexia. 1 However, oral pharmacotherapy is sometimes distressing for patients because of the presence of systemic side effects. Therefore, it is worthwhile to investigate alternative routes of drug administration to suppress bladder contractility, especially those which are more localized to the urinary bladder. Although intravesical instillation is a common therapeutic approach to superficial bladder tumors, 2 there have been a few reports on this method of administration for the suppression of bladder contractility. 3 - 7 Obrink et al., 3 investigated the symptomatic effect of instillation of emepronium bromide (100 mg., 1 time per week) in patients with urgency. Higson et al. 4 Mattiason et al. (verapamil, 3 X 10-s (1 % lignocaine, 1 M, 1 time) 5 and Brendler et al. (5 mg. oxybutynin, 2 times per day) 6 investigated the effect of intravesical instillation on cystometrograms of patients with detrusor hyperactivity and showed significant increase in bladder capacity. Experimentally, Gotoh et al. 7 reported that there was a significant decrease of bladder contractility to acetylcholine and field stimulation in in vitro and in vivo rabbit bladders by intravesical verapamil (7.5 or 10 mg.). Nevertheless, there have been no papers which compared the effect of intravesical administration between different anticholinergic, antispasmodic and calcium blocking agents. The aim of this study was to investigate the effect of Accepted for publication January 5, 1989. * Requests for reprints: Div. of Urology, 5-Silverstein, 3400 Spruce St., Hospital of the University of Pennsylvania, Philadelphia PA 19104. Supported by the Veterans Administration, NIH grants R0-1-DK2-6508, R0-1-DK 33559, P-50-DK 39257 and the McCabe Fund.
intravesical administration of various drugs used or considered to be used clinically in the treatment of detrusor hyperreflexia on bladder contractile function. using the rabbit in vitro whole bladder model,8 we could evaluate the effect of drug instillation on the ability of the bladder to generate pressure in response to both field stimulation and bethanechol administration. MATERIALS AND METHODS
mature male New Zealand were sedated with an intramusof a ketamine/xylazine mixture 8.3 mg./ml. xylazine). Surgical anesthesia was maintained with intravenous pentobarbital (50 mg./ ml., 0.2 - 0.5 ml.) for 10 minutes. Prior studies have demonstrated that this method of anesthesia has no effect on the response of the in vitro whole bladder to pharmacological agents. The bladder was exposed through a midline incision and excised as low on the urethra as possible. Both ureters and main vessels were tied with 3-0 silk ligature. The excised bladder was emptied and the urethra canulated with a saline-filled electrode-tipped catheter. The whole bladder was mounted in a 300 ml. bath chamber containing Tyrode' s solution (NaCl 125 mM, KC! 2.7 mM, NaH 2P0 4 0.4 mM, CaCI 2 1.8 mM, MgCl2 0.5 mM, NaHC0 3 23.8 mM and glucose 1 mg./ 1 ml.) equilibrated with 95% 0 2 /5% CO 2 and maintained at 37C. Intravesical pressure was continuously monitored using a Statham pressure transducer and recorded on a Grass Polygraph. The signals from the Grass Polygraph were fed into an IBM PC computer via a Keithly model 500 Analog-Digital converter system and the rate of pressure generation was determined and recorded during each field stimulation. 9
1471
1472
KATO AND ASSOCIATES
Experimental protocol. The bladder was instilled with 20 ml. of saline and allowed to accomodate for 30 minutes. After 30 minutes the bladder was stimulated by electrical field stimulation (80 Volts, 30 Hz, 1 msec). Five minutes later, the bladder was stimulated by 500 µM of bethanechol added to the extravesical fluid. After bethanechol stimulation, the bladder was washed three times with fresh oxygenated Tyrode's solution. The bladder was emptied and instilled with 20 ml. of either fresh saline, or saline containing 10 µMor 100 µM concentration of one of the following drugs: verapamil hydrochloride, diltiazem hydrochloride, imipramine hydrochloride, atropine sulfate or oxybutynin hydrochloride. The pH of drug solutions was between 5.6 and 6.0. All drugs were of the highest purity available, purchased through standard commercial sources. Bladder responses to field stimulation and bethanechol were investigated at 30, 60, 90 and 120 minutes following the intravesical instillation of drugs. At 120 minutes, the bladder was irrigated with saline five times at two minute intervals and instilled with 20 ml. of fresh saline. The same stimulation procedures were performed 30, 60, 90 and 120 minutes after the removal of the drug. Statistical analysis. All values presented in the results represent the mean +/- standard error of the mean. The peak pressure response and the peak rate of pressure development are presented as the percentage of the control response before the instillation of drugs. Statistical significance was determined by Student's t test. A probability level of less than 0.05 was required for statistical significance.
four hour period, the peak pressure response to field stimulation and bethanechol decreased only 3% and 9%, respectively. Intravesical instillation of verapamil and diltiazem (calcium entry blockers) suppressed the contractions induced both by bethanechol and field stimulation in a time-dependent and dose-dependent manner (fig. 1). Verapamil had a greater efficacy than diltiazem. There was no significant difference between the magnitude of suppression of the response to bethanechol and field stimulation by these drugs. The suppression of the peak rate of pressure development by verapamil was proportional to the suppression of the peak pressure response to field stimulation (fig. 2). Diltiazem showed the same tendency. Figure 3 presents the effect of intravesical instillation of imipramine, a tricyclic antidepressant, on the peak pressure response of the whole bladder. The suppression of contractions induced by field stimulation and bethanechol was time-dependent and dose-dependent. At concentrations of 10 µM and 100 µM, imipramine suppressed the response to bethanechol and field stimulation to approximately the same extent. The suppression of peak pressure development by imipramine was significantly greater than the suppression of the rate of pressure generation. Intravesical instillation of atropine and oxybutynin suppressed the response to both bethanechol and field stimulation in a time-dependent manner. At the concentration of 10 µM, EFFECT OF INTRAVESICAL INSTILLATION OF VERAPAMIL ON FIELD STIMULATION T
_J
Preliminary experiments confirmed that the pressure response to field stimulation could be inhibited almost completely by the addition of tetrodotoxin (1 µM) whereas the response to bethanechol was not affected by tetrototoxin. This demonstrates that field stimulation acts via the release of excitatory neurotransmitters from presynaptic terminals whereas bethanechol acts by directly blocking the post-synaptic muscarinic cholinergic receptors. The peak pressure response of the control bladder (containing 20 ml. of saline) to field stimulation was 11.2 +/- 0.4 cm. H20 and the peak rate of this pressure development was 10.4 +/- 0.5 cm. H 20/sec. The peak pressure response of the control bladder (containing 20 ml. of saline) to 500 µM ofbethanechol was 11.6 +/-0.4 cm. H20. When 20 ml. saline and no other drug was used for intravesical instillation, the responses following both field stimulation and bethanechol were highly reproducible at 30 minute intervals until four hours. Even at the
EFFECT OF INTRAVESICAL INSTILLATION OF VERAPAMIL ON PRESSURE GENERATION
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1473
INTRAVESICAL ADMINISTRATION TO INHIBIT BLADDER CONTRACTILITY
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1474
KATO AND ASSOCIATES
TABLE 1.
Suppression of contraction two hr. after intravesical instillation- I 00 µm
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Bethanechol
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stillation. For comparative purposes the inhibition at two hours is presented in table 1. Only atropine and oxybutynin showed a significant difference between their ability to inhibit contractions to field stimulation and to bethanechol. Recovery of the bladder contraction after removal of the drugs was slow and incomplete. Additional experiments were performed where the intravesical fluid was changed following each stimulation at 30 minute intervals during the recovery phase of the experiments. There was no effect of changing the intravesical fluid on the recovery of the response to stimulation. DISCUSSION
In the last two decades, intermittent catheterization and administration of anticholinergic and antispasmodic agents have contributed greatly to the management of neurogenic bladder by preventing upper tract deterioration and decreasing urinary incontinence. 1 However, commonly used anticholinergics and tricyclic antidepressants are not selective to the urinary bladder, and when orally administered, often cause systemic side effects such as dry mouth, flushed skin, constipation and blurred vision. Although intravesical instillation is a popular method of treatment for bladder carcinoma (Thiotepa, BCG) 2 and interstitial cystitis (DMS0), 10• 11 there have been only a few reports about intravesical administration of antispasmodic agents both clinically 3 - 6 and experimentally. 7 As the rationale for intravesical therapy, it is expected that high concentrations of drug may contact the bladder mucosa for prolonged periods, and that systemic side effects would be minimal because of limited systemic absorption of the drug. In the present study, intravesical instillation of all investigated drugs suppressed the pressure response of the in vitro whole bladder to bethanechol and field stimulation. All drugs showed a time-dependent inhibition requiring 90 to 120 minutes to attain maximal effects. Gotoh et al. 7 also reported a timedependent inhibition of tension response with intravesical verapamil. It seems that the mechanism of action of anticholinergics and antispasmodics is not merely a local anesthetic effect on the bladder mucosa because of the long time period before maximal inhibition was attained. The drug administered intravesically may gradually penetrate the intermuscular spaces throughout the mucosa and directly affect the smooth muscle. As mentioned previously, the response to field stimulation was eliminated by pre-incubation with tetrodotoxin, which means that field stimulation acts through the release of endogenous neurotransmitters from nerve endings. Previous studies have also demonstrated that approximately 50% of the initial pressure response to field stimulation is cholinergic in nature; the remaining 50% is primarily purinergic. 12 In the present study, the intravesical instillation of atropine and oxybutynin, muscarinic-cholinergic antagonists, suppressed the response to bethanechol to a much greater extent than the response to field stimulation. This would be expected since these agents inhibit mainly the cholinergic aspect of field stimulation. Although oxybutynin is known to have not only anticholinergic but also direct smooth muscle relaxant activities, 13 its anticholinergic activity seems to be significantly
stronger than its direct smooth muscle relaxant properties. The suppression of the rate of pressure development by atropine and oxybutynin was significantly smaller than the suppression of the magnitude of pressure development to field stimulation. This might also be related to the fact that both atropine and oxybutynin have little effect on the non-cholinergic elements of field stimulation which make up approximately 50% of the initial response. Intravesical instillation of verapamil or diltiazem suppressed the contractile response to bethanechol and field stimulation to approximately the same extent. Verapamil and diltiazem are calcium entry blockers which selectively block the extracellular Ca2 + influx into cells. 14 The present results indicate that the same calcium ion channels were involved in the response to both field stimulation and direct muscarinic activation. The present demonstration that verapamil had a greater efficacy than diltiazem is consistent with the results of in vitro muscle strip studies by Finkbeiner et al. 15 Some calcium entry blockers are known to have an affinity for alpha-adrenergic receptors and to a lesser extent muscarinic cholinergic receptors. 16• 17 Malkowicz et al.1 8 reported that verapamil was ten times more potent in competing for alpha1 and alpha2 sites in bladder smooth muscle than diltiazem. These factors may contribute to our findings that verapamil was more effective than diltiazem. Verapamil and diltiazem inhibited the peak rate and the magnitude of the pressure development to field stimulation equally. This relationship suggests that both the cholinergic and non-cholinergic elements of field stimulation may be mediated by the same calcium ion channels. Imipramine, a tricyclic antidepressant, is known to have various pharmacological actions including muscarinic antagonism, blockade of norepinephrine uptake, and direct smooth muscle relaxant properties including calcium antagonism. 13• 19• 20 Intravesical instillation of imipramine suppressed the response to bethanechol and field stimulation to approximately the same extent. This was similar to the results of the intravesical calcium entry blockers and different from the effects of the intravesical anticholinergics. These results are consistent with prior studies which demonstrated that direct smooth muscle relaxant properties of imipramine are significantly more potent than its anticholinergic properties. 21 On the other hand, intravesical imipramine inhibited the magnitude of pressure generation induced by field stimulation to a significantly greater extent than it inhibited the rate of pressure generation. This result was similar to the response of the in vitro whole bladder to the extravesical administration of imipramine. 22 The peak rate of pressure development is dependent on several factors including the rate of transmitter release, rate of calcium entry into the muscle, rate of release of intracellular calcium and the rate of the initial response of the muscle elements to the released calcium. The magnitude of pressure development reflects the efficiency, extent and duration to which the smooth muscle elements react to the released calcium. Although we have no direct evidence, our present study is consistent with the concept that imipramine has a greater effect on the degree and duration of the intracellular response of the smooth muscle to calcium than on the rate of calcium entry and immediate rate of response of the smooth muscle elements to the released calcium. There may be some fundamental differences in the effect of intravesical drug administration on the in vitro whole bladder compared to that on the in vivo bladder. For example, the mucosal membrane barrier may be less efficient in the in vitro bladder than in the in vivo bladder because of several intravesical saline washes performed during the in vitro whole bladder studies; this may disrupt the mucin layers. The role of blood circulation in the bladder wall on the effect of intravesical drug administration is currently unknown. Blood circulation may be beneficial to the diffusion of the drug into the detrusor muscle;
INTRAVESICAL ADMINISTRATION TO INHIBIT BLADDER CONTRACTILITY
however, our results along with those of Gotoh et al. 7 showed that intravesical administration of drugs to suppress bladder contractility was effective in the in vitro rabbit whole bladder which had no blood circulation. Further studies are necessary to elucidate these differences. Although the present in vitro study does not address the problem of systemic absorption and the development of side effects on intravesical drug administration, Gotoh et aL 7 showed that there was no change in the cardiovascular status of the rabbit following intravesical administration of verapamiL Higson et aL (lignocaine),4 and Brendler et aL (oxybutynin) 6 reported no systemic side effects with clinical intravesical therapy. Obrink et al. 3 further showed that the drug concentrations in human serum were very low or were not detectable even after a very high dosage of instillation (emepronium bromide 100 mg. per 100 mL saline). These reports support the possibility that intravesical instillation could be a route of administration of anticholinergic and antispasmodic drugs with fewer systemic side effects. Recovery of the bladder contraction after removal of the drugs was slow and incomplete in the present study. This result was consistant with Gotoh's report7 about intravesical verapamiL He speculated that the inhibitory effect lasting after the removal of the drug was due to the action of the drug which remained in the intermuscular spaces. Although clinical reports about intravesical drug administration have not mentioned the long-term time-course of responses, 3- 6 the lasting inhibitory effect might cause urinary retention by suppressing the micturition reflex and limit the usefulness of this method of administration in patients who are unwilling to accept intermittent catheterization. However, intravesical therapy in patients already managed by intermittent catheterization would not be an additional burden. Even if there is a prolonged inhibition of micturition after removal of the drug, this condition can be easily managed by self-catheterization and might further decrease the necessary number of instillations. One point to be kept in mind when considering the clinical utilization of intravesical drug administration is that it is not well known whether the effect of intravesical instillation in the in vivo human bladder is comparable to that in animal experiments. In the present study, we found that instillation of oxybutynin and verapamil was very effective in suppressing bladder contractility in the in vitro rabbit bladder. Gotoh et al. 7 working on the rabbit reported that the suppression of the contractility of the in vivo bladder by intravesical verapamil correlated with that of the in vitro bladder. Clinically, Brendler et al. 6 showed that intravesical instillation of oxybutynin abolished the unstable contractions and increased bladder capacity in patients with neurogenic bladders. However, Mattiason et aL 5 found that intravesical administration of verapamil in patients with detrusor hyperreflexia increased bladder capacity without changing the amplitudes of the first unstable contraction or maximum detrusor contraction. Therefore, in their study, even though intravesical verapamil was therapeutically effective in increasing capacity, bladder contractility was not decreased as it was in the in vitro and in vivo rabbit studies. These findings indicate that one must be cautious in applying data of animal experiments to human clinical trials. Nevertheless, animal studies including the in vitro whole bladder model should be useful in screening potential drugs for intravesical therapy of detrusor hyperreflexia.
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