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DEMONSTRATION OF BLADDER SELECTIVE MUSCARINIC RECEPTOR BINDING BY INTRAVESICAL OXYBUTYNIN TO TREAT OVERACTIVE BLADDER
0022-5347/04/1725-2059/0 THE JOURNAL OF UROLOGY® Copyright © 2004 by AMERICAN UROLOGICAL ASSOCIATION
Vol. 172, 2059 –2064, November 2004 Printed in U.S.A.
DOI: 10.1097/01.ju.0000138472.16876.8d
DEMONSTRATION OF BLADDER SELECTIVE MUSCARINIC RECEPTOR BINDING BY INTRAVESICAL OXYBUTYNIN TO TREAT OVERACTIVE BLADDER TOMOMI OKI, RYOHEI KIMURA, MOTOAKI SAITO, IKUO MIYAGAWA
AND
SHIZUO YAMADA*
From the Department of Biopharmaceutical Sciences and COE Program in the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka (TO, RK, SY) and Department of Urology, Tottori University Faculty of Medicine (MS, IM), Yonago, Japan
ABSTRACT
Purpose: The current study was done to elucidate the in vivo mechanism of action of intravesical instillation of oxybutynin to treat overactive bladder. Materials and Methods: In rats receiving oral and intravesical oxybutynin we measured muscarinic receptors in the bladder and other tissues by radioligand binding assay using [3H]NMS ([N-methyl-3H]scopolamine methyl chloride) with the simultaneous measurement of plasma concentrations of oxybutynin and its active metabolite N-desethyl-oxybutynin. Pilocarpine induced salivary secretion was also measured. Results: Following oral administration of oxybutynin there was a significant increase in the apparent dissociation constant (Kd) for specific [3H]NMS binding in the bladder, submaxillary gland, heart and colon of rats at 1 and 3 hours with a consistent decrease in the maximal number of binding sites (Bmax) in the submaxillary gland. Furthermore, a marked and prolonged decrease in pilocarpine induced salivary secretion in rats was observed by oral oxybutynin. In contrast, intravesical instillation of oxybutynin produced a significant increase in Kd for specific [3H]NMS binding in the bladder of rats at 0.5 to 4 hours later and also in the submaxillary gland only at 0.5 hours later. The enhancement in Kd was much larger and longer lasting in the bladder than in the submaxillary gland. Moreover, intravesical oxybutynin had little muscarinic receptor binding activity in the heart and colon, and little significant suppression of pilocarpine induced salivation in rats. The plasma concentrations of oxybutynin and N-desethyl-oxybutynin were much higher in rats receiving oxybutynin orally than intravesically. Conclusions: Intravesical oxybutynin in rats may cause selective binding of bladder muscarinic receptors via a direct local effect, while oral oxybutynin may exert predominant binding of salivary gland receptors. KEY WORDS: bladder; administration, intravesical; oxybutynin; receptors, muscarinic; saliva
Urinary incontinence and overactive bladder are important and common conditions that have received little general medical attention. Oral anticholinergic agents such as oxybutynin are effective in many patients with these disorders but there are some patients who do not respond to oral medication or who experience intolerable systemic side effects of these drugs.1, 2 The systemic side effects of oxybutynin are suggested to be caused by the high plasma level of its active metabolite, N-desethyl-oxybutynin (DEOB).1, 2 There is increasing evidence that the intravesical instillation of oxybutynin, which decreases the plasma level of DEOB compared with that of oral administration of oxybutynin, is effective therapy for overactive bladder.1⫺7 In addition, intravesical oxybutynin has been shown to cause few systemic adverse events.3, 5–7 Despite these clinical successes to our knowledge the exact mechanism of action of intravesical oxybutynin remains unknown. Although oxybutynin is known to have mixed anticholinergic and spasmolytic effects, and possibly local anesthetic properties, the in vivo mechanism to suppress bladder contractility has yet to be fully elucidated. Also,
controversy still exists as to whether the efficacy of intravesical oxybutynin results from a local or a systemic effect. Although many studies have dealt with the pharmacological effects and in vitro receptor binding affinity of anticholinergic agents,8, 9 little information has been published on in vivo muscarinic receptor binding characteristics, such as the extent and duration at the target, and nontarget tissues following oral and intravesical administration of anticholinergic agents. In general the results of in vitro receptor binding studies of drugs may not necessarily guarantee in vivo pharmacological specificity such as organ selectivity because they do not consider a number of pharmacokinetic and pharmacodynamic factors.10 Therefore, in this study we clarified the in vivo mechanism of action of intravesical instillation of oxybutynin for the treatment of overactive bladder. We simultaneously measured muscarinic receptor binding in the bladder and other tissues, plasma levels of oxybutynin and DEOB, and salivary secretion in rats receiving intravesical and oral oxybutynin. MATERIALS AND METHODS
Accepted for publication May 7, 2004. Supported by a Grant-in-Aid for Scientific Research (C)(2)(No. Materials.We used [3H]NMS ([N-methyl-3H]scopolamine 15591703) from the Ministry of Education, Science, Sports and Cul- methyl chloride) (PerkinElmer Life Sciences, Inc., Boston, ture of Japan. Massachusetts) (3.03 TBq/mmol), oxybutynin hydrochloride * Correspondence: 52–1 Yada, Shizuoka 422-8526, Japan (telephone: ⫹81–54-264 –5631; FAX: ⫹81–54-264 –5635; e-mail: yamada@ys7. and DEOB hydrochloride. Animals. Male Sprague-Dawley rats (Charles River Japan, u-shizuoka-ken.ac.jp). 2059
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Inc., Atsugi, Japan) weighing about 250 to 350 gm were housed in the laboratory with free access to food (normal chow) and water, and maintained on a 12-hours dark/light cycle in a room at controlled mean temperature ⫾ SE (24C ⫾ 1C) and humidity (55% ⫾ 5%). Administration of oxybutynin. Rats were fasted for 16 hours and then received oxybutynin orally (127 mol/kg) dissolved in distilled water. Control animals received vehicle alone. For intravesical oxybutynin instillation rats were anesthetized with pentobarbital (121 mol/kg intraperitoneally). Through a midline incision in the abdomen the bladder was exposed and emptied of urine by sucking through a syringe connected to a 30 gauge needle. Oxybutynin (76.1 nmol/0.2 ml per rat) dissolved in physiological saline was instilled into the bladder by injecting it directly through a syringe connected to the needle. Oxybutynin solution was kept in the bladder for 30 minutes by tying the urethral openings with a thread, after which the bladder was evacuated by taking out the thread. Control animals received physiological saline alone. This study was done according to the guideline approved by the experimental animal ethical committee, University of Shizuoka. Tissue preparation. After oral and intravesical oxybutynin administration rats were exsanguinated by taking the blood from the descending aorta under temporary anesthesia with diethyl ether. The tissues were perfused with cold saline from the aorta. The bladder, submaxillary gland, heart and colon were then dissected, and fat and blood vessels were removed by trimming. The tissues were minced with scissors and homogenized using a Polytron homogenizer (Kinematica AG, Lucerne, Switzerland) in 19 volumes of ice-cold 30 mM Na⫹/ HEPES buffer (pH 7.5). The homogenates were then centrifuged at 40,000 ⫻ gravity for 20 minutes. The resulting pellet was finally resuspended in 29 volumes of ice-cold buffer for the binding assay. In the ex vivo experiment there was a possibility that oxybutynin might dissociate in part from receptor sites during tissue preparation (homogenization and suspension) after drug administration. Yamada et al have previously reported that the dissociation of antagonists from receptor sites at 4C is extremely slow.11 Therefore, to minimize the dissociation of oxybutynin from receptor sites all steps for the preparation were performed at 4C. In fact, there was not a large difference in [3H]NMS binding parameters (Kd and Bmax) between single washing and double washing of rat bladder homogenates at 4C 3 hours after oral administration of oxybutynin (127 mol/kg). Protein concentrations were measured according to the method of Lowry et al.12 Rat plasma was isolated from blood by centrifugation and stored at – 80C until drug analysis. Muscarinic receptor binding assay. The binding assay for muscarinic receptors was performed using [3H]NMS, as previously described.13 Homogenates (70 to 350 g protein) of rat tissues were incubated with different concentrations (0.06 to 2.0 nM) of [3H]NMS in 30 mM Na⫹/HEPES buffer (pH 7.5). Incubation was done for 60 minutes at 25C. The reaction was terminated by rapid filtration using a Cell Harvester (Brandel Co., Gaithersburg, Maryland) through GF/B glass fiber filters (Whatman, Brentford, United Kingdom) and the filters were then rinsed 3 times with 3 ml ice-cold buffer. Tissue bound radioactivity was extracted from the filters by placing them overnight by immersion in scintillation fluid (2 l toluene, 1 l Triton X-100, 15 gm 2,5-diphenyloxazole and 0.3 gm 1,4-bis[2-(5-phenyloxazolyl)]benzene) and radioactivity was determined by a liquid scintillation counter. Specific [3H]NMS binding was determined experimentally from the difference between counts in the absence and presence of 1 M atropine. All assays were performed in duplicate. Measurement of plasma concentrations of oxybutynin and DEOB. The concentrations of oxybutynin and DEOB in rat plasma were determined by gas chromatography and mass spectrometry (GC/MS). Briefly, a plasma sample (0.1 to 0.5 ml.)
was mixed with internal standard ([2H13]oxybutynin⬘HCl and [2H13]DEOB⬘HCl). After being alkalinized with 0.5 ml 0.5 M carbonate buffer (pH 9.5) it was extracted with 6 ml n-hexane. After centrifugation at 1,500 ⫻ gravity for 5 minutes the supernatant was evaporated until dry under decreased pressure. The residue was dissolved in 100 l CH3CN and 0.5 to 1 l was injected into the GC/MS system. The GC/MS system consisted of a 5890 Series II gas chromatograph, a 5792 Series mass selective detector, a 7673 GC/SFC injector, a Vectra 486/66U computer and a Laser JET 4 printer (Hewlett Packard Co., Palo Alto, California). Chromatographic separation was done using 15 m ⫻ 0.25 mm inner diameter ⫻ 0.25 m UA⫹-1 HT film (Frontier Lab, Ltd., Fukushima, Japan). The carrier gas was helium at a flow rate of 1.0 ml per minute Oven temperature was held at 150C for 1 minute and then programmed from 150C to 220C at 20C per minute for the first ramp, 220C to 260C at 10C per minute for the second ramp and 260C to 300C at 30C per minute for the third ramp. It was held at 300C for 2 minutes before returning to the initial starting temperature of 150C. Injection temperature was 200C. Fragmentation was accomplished by electron impact at 70 eV ionizing voltage and 300 A ionizing current. Select ion monitoring was performed at m/z 342 (oxybutynin), m/z 355 (internal standard [2H13]oxybutynin), m/z 189 (DEOB) and m/z 200 (internal standard [2H13]DEOB). The limit of detection of oxybutynin and DEOB in plasma was 1.40 and 3.04 nM, respectively. Measurement of salivary secretion. Rats were anesthetized by intraperitoneal administration of pentobarbital (121 mol/kg). Immediately after wiping the saliva remaining in the oral cavity with a cotton ball all saliva in the cavity was collected by absorbing it into 3 balls for 10 minutes. The cotton ball was then immediately weighed on an electric balance to prevent moisture loss. The weight of secreted saliva was estimated as the difference in cotton ball weight before vs after application into the oral cavity. The effects of oral and intravesical oxybutynin on pilocarpine induced salivary secretion in rats were examined. Pilocarpine (4.09 mol/kg dissolved in physiological saline) was intravenously administered 1, 3 and 12 hours after oral oxybutynin administration as well as 0.5, 2 and 4 hours after intravesical instillation and all saliva was collected for 10 minutes. In another experiment the effect of intravesical instillation of oxybutynin on salivary secretion by cumulative doses (0.04 to 12.3 mol/kg intravenously) of pilocarpine in rats was examined. The secretion of whole saliva was measured in rats receiving cumulative doses of pilocarpine at every 5 minutes. Data analysis. Analysis of [3H]NMS binding data were performed as described previously.11 The apparent dissociation constant (Kd) and maximal number of binding sites (Bmax) for [3H]NMS were estimated by Rosenthal analysis of saturation data. In the analysis of salivary secretion data pilocarpine potency was expressed as pD2 (negative logarithm of the molar dose of the agonist producing 50% of the maximum response). Statistical analysis of the data was performed by 1-way ANOVA, followed by Dunnett’s test for multiple comparisons with p ⬍0.05 considered statistically significant. RESULTS
Muscarinic receptors in rat tissues. The effects of oral and intravesical administration of oxybutynin on specific [3H]NMS binding in rat tissues were investigated. Figure 1 shows Scatchard plots of [3H]NMS binding in the bladder and submaxillary gland of control and oxybutynin administered rats. One and/or 3 hours after oral administration of oxybutynin (127 mol/kg) there was a significant increase in Kd values for specific [3H]NMS binding in each rat tissue compared with corresponding control values (fig. 1, table 1). Increased rates in the bladder and submaxillary gland at 1
MUSCARINIC RECEPTOR BINDING AFTER INTRAVESICAL OXYBUTYNIN
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FIG. 1. Scatchard plots of specific [3H]NMS binding in rat bladder and submaxillary gland after oral (A) and intravesical (B) oxybutynin administration. Rats received oxybutynin orally (127 mol/kg) and intravesically (76.1 nmol/0.2 ml per rat) and 0.5 to 4 hours (h) later specific [3H]NMS binding in homogenate of each dissected tissue was measured. Points represents mean of 3 (oxybutynin) and 5 (control) rats.
hour were 66.7% and 295%, and those at 3 hours were 113% and 775%, respectively. Thus, the enhancement of Kd values produced by oxybutynin administration was much greater in the submaxillary gland than in the bladder of rats (p ⬍0.01 at 3 hours). There was little increase in Kd values in these tissues 12 hours later. In the submaxillary gland there was a consistent (25.1% to 59.3%) decrease in Bmax values for [3H]NMS binding 1 to 12 hours after oral administration compared with control values. Similarly oral oxybutynin administration caused a significant increase in the Kd value for [3H]NMS binding in the rat heart and colon with a significant decrease in the Bmax value in the heart at 12 hours. Increased rates in the heart and colon at 1 hour were 44.6% and 113%, and those at 3 hours were 34.8% and 159%, respectively. At 0.5, 2 and 4 hours after intravesical instillation of oxybutynin (76.1 nmol/0.2 ml per rat) there was a significant increase in the Kd value for specific [3H]NMS binding in the bladder of rats compared with control values (966%, 133% and 59.9%, respectively, table 1). In the submaxillary gland there was a significant (168%) increase in the Kd value for [3H]NMS binding only 0.5 hours after intravesical oxybutynin instillation. Thus, the enhancement in Kd values produced by oxybutynin instillation was significantly greater in the bladder than in the submaxillary gland (p ⬍0.001). On the other hand, Bmax values for [3H]NMS binding 0.5 to 12 hours after intravesical oxybutynin instillation in the submaxillary gland were unaltered. Furthermore, intravesical oxybutynin instillation had little effect on Kd and Bmax values for [3H]NMS binding in the heart and colon of rats. Plasma levels of oxybutynin and DEOB. Figure 2 shows the
plasma concentration-time profiles of oxybutynin and DEOB in rats after oral and intravesical administration of oxybutynin. Plasma oxybutynin and DEOB concentrations reached maximum levels (about 91 nM) 1 hour after oral administration of oxybutynin (127 mol/kg) and then they rapidly decreased (fig. 2, A). In 3 or 4 preparations 0.5, 2 and 4 hours after intravesical instillation of oxybutynin (76.1 nmol/0.2 ml per rat) mean plasma oxybutynin concentrations were 12.4 ⫾ 2.8, 3.88 ⫾ 0.25 and 0.87 ⫾ 0.50 nM, respectively (fig. 2, B). DEOB was barely detectable in rat plasma 0.5 to 12 hours after intravesical oxybutynin instillation. Salivary secretion.The amount of pooled saliva of anesthetized rats secreted during every 10 minutes was the highest, comprising 42.6% of total saliva secreted for 60 minutes, within the first 10 minutes after intravenous injection of pilocarpine (4.09 mol/kg) and thereafter it gradually decreased (fig. 3). Table 2 shows the effects of oral and intravesical administration of oxybutynin on pilocarpine induced salivary secretion in rats (first 10 minutes). The secretory response of saliva caused by pilocarpine in control rats was nicely reproducible because similar amounts of pooled saliva were secreted during the first 10 minutes after pilocarpine simulation at each time of 1, 3 and 12 hours (oral) as well as 0.5, 2 and 4 hours (intravesical). Thus, pooled control data are presented. One to 12 hours after oral administration of oxybutynin (127 mol/kg) there was a significant decrease in the amount of pilocarpine induced saliva secretion compared with that in control rats. In particular the amount of saliva secreted 1 and 3 hours later was extremely low and secretion
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TABLE 1. Kd and Bmax for specific [3H]NMS binding in rat bladder, submaxillary gland, heart and colon after oral and intravesical oxybutynin Hrs After Administration
Mean Kd ⫾ SE (pM)
Mean Bmax ⫾ SE (fmol/mg protein)
Oral Bladder: Control 1 3 12 Submaxillary gland: Control 1 3 12 Heart: Control 1 3 12 Colon: Control 1 3 12
165 ⫾ 5 275 ⫾ 42 351 ⫾ 85* 188 ⫾ 7
157 ⫾ 8 189 ⫾ 40 131 ⫾ 17 101 ⫾ 7
110 ⫾ 3 435 ⫾ 183† 962 ⫾ 159‡ 104 ⫾ 4
124 ⫾ 4 92.9 ⫾ 13.5† 61.7 ⫾ 4.5‡ 50.5 ⫾ 6.1‡
267 ⫾ 8 386 ⫾ 65† 360 ⫾ 20† 240 ⫾ 30
71.7 ⫾ 1.9 66.6 ⫾ 3.4 63.0 ⫾ 2.5 56.7 ⫾ 1.7*
149 ⫾ 4 318 ⫾ 21* 386 ⫾ 78‡ 162 ⫾ 12 Intravesical
106 ⫾ 4 120 ⫾ 7 112 ⫾ 12 105 ⫾ 9
Bladder: Control 172 ⫾ 9 140 ⫾ 7 0.5 1,834 ⫾ 212‡ 125 ⫾ 20 2 400 ⫾ 34‡ 123 ⫾ 7 4 275 ⫾ 13* 149 ⫾ 9 12 205 ⫾ 12 126 ⫾ 5 Submaxillary gland: Control 148 ⫾ 12 120 ⫾ 6 0.5 396 ⫾ 55‡ 132 ⫾ 4 2 179 ⫾ 10 125 ⫾ 10 4 165 ⫾ 9 124 ⫾ 10 12 149 ⫾ 22 124 ⫾ 15 Heart: Control 298 ⫾ 8 83.7 ⫾ 3.5 0.5 297 ⫾ 14 82.2 ⫾ 6.7 2 304 ⫾ 20 86.3 ⫾ 2.8 4 302 ⫾ 5 80.2 ⫾ 4.3 12 304 ⫾ 20 83.6 ⫾ 4.1 Colon: Control 181 ⫾ 7 143 ⫾ 7 0.5 323 ⫾ 100 145 ⫾ 12 2 188 ⫾ 9 144 ⫾ 12 4 202 ⫾ 13 147 ⫾ 11 12 189 ⫾ 6 125 ⫾ 14 Three to 9 rats per group received oxybutynin orally (127 mol/kg) and intravesically (76.1 nmol/0.2 ml per rat), were sacrificed 0.5 to 12 hours after administration, and specific binding of [3H]NMS (0.06 to 2.0 nM) in rat tissues was measured. * Significantly different vs control (p ⬍0.01). † Significantly different vs control (p ⬍0.05). ‡ Significantly different vs control (p ⬍ 0.001).
12 hours later showed some recovery but it was only 22% of the control level. On the other hand, the intravesical instillation of oxybutynin (76.1 nmol/0.2 ml per rat) had little significant effect on pilocarpine (4.09 mol/kg) induced salivary secretion 0.5, 2 and 4 hours later. Furthermore, we measured saliva secretion at cumulative doses (0.04 to 12.3 mol/kg intravenously) of pilocarpine 0.5 hours after intravesical oxybutynin instillation in rats. This time (0.5 hours) was chosen because there was significant (168%) enhancement of Kd values for [3H]NMS binding in the submaxillary gland (table 1). The cumulative intravenous injection of pilocarpine at these doses induced dose dependent salivary secretion in rats (fig. 4). Intravesical oxybutynin instillation tended to attenuate salivary secretion only at low pilocarpine doses (0.41 and 1.23 mol/kg intravenously) but the extent of suppression was statistically insignificant. In fact, pD2 values without and with intravesical oxybutynin were 5.86 ⫾ 0.10 and 5.79 ⫾ 0.07, respectively.
FIG. 2. Plasma concentration-time profiles of oxybutynin (filled circles) and DEOB (open circles) after oral (A) and intravesical (B) oxybutynin administration in rats. Rats received oxybutynin orally (127 mol/kg) and intravesically (76.1 nmol/0.2 ml per rat) and 0.5 to 12 hours (h) later blood samples were taken from aorta of each rat. Points represents mean ⫾ SE of 3 to 8 rats.
FIG. 3. Time course of pilocarpine induced salivary secretion in rats. Rats received pilocarpine (4.09 mol/kg) intravenously and weight of pooled saliva secreted during each 10 minutes was measured periodically at 0 to 60 minutes. Columns represents mean ⫾ SE in 11 rats.
DISCUSSION
In the current study we simultaneously measured muscarinic receptor binding in tissues, including the bladder and
MUSCARINIC RECEPTOR BINDING AFTER INTRAVESICAL OXYBUTYNIN TABLE 2. Effects of oral and intravesical administration of oxybutynin on pilocarpine induced salivary secretion in rats Hrs After Administration
Mean Salivary Secretion ⫾ SE (mg)
Control 632 ⫾ 53 Oral route: 1 6.76 ⫾ 6.76 3 0.80 ⫾ 0.80 12 139 ⫾ 27 p Value vs control ⬍0.001 Control 493 ⫾ 12 Intravesical route: 0.5 440 ⫾ 32 2 500 ⫾ 76 4 492 ⫾ 18 Three to 5 rats per group received oxybutynin orally (127 mol/kg) and intravesically (76.1 nmol/0.2 ml per rat), and pooled saliva secreted during the first 10 minutes after intravenous stimulation of pilocarpine (4.09 mol/kg) was measured.
FIG. 4. Effects of intravesical oxybutynin administration on doseresponse curve of pilocarpine induced salivary secretion in rats. Rats received intravenously cumulative doses (0.04 to 12.3 mol/kg) pilocarpine before (filled circles) and 0.5 hours after (open circles) intravesical oxybutynin instillation (76.1 nmol/0.2 ml per rat) and saliva secretion was measured. Salivary secretion caused by each pilocarpine dose is expressed as percent of maximal control response to pilocarpine (12.3 mol/kg). Points represents mean ⫾ SE of 3 (oxybutynin) and 5 (control) rats.
submaxillary gland, plasma levels of oxybutynin and DEOB, and salivary secretion in rats after intravesical oxybutynin instillation, in comparison with the results obtained after oral administration of this drug. The technique of ex vivo and in vivo receptor binding has been demonstrated to be useful for predicting the potency, organ selectivity and duration of action of drugs in relation to their pharmacokinetic and pharmacodynamic profiles.10, 14 Thus, the ex vivo receptor binding technique was used to characterize simultaneously muscarinic receptor binding in the bladder, submaxillary gland, heart and colon of rats after oral and intravesical administration of oxybutynin. The oral and intravesical doses of oxybutynin used were based on pharmacological doses of oxybutynin for decreasing detrusor contractility in rat cystometry studies.15, 16 There was a significant increase in Kd values for specific [3H]NMS binding in the bladder, submaxillary gland, heart and colon of rats 1 and 3 hours after oral oxybutynin administration compared with each control value. These data suggest that oral oxybutynin binds uniformly to muscarinic receptors in all rat tissues examined. In the submaxillary gland the increase in Kd values was greatest among 4 tissues and concomitantly a long lasting decrease in Bmax values was observed. Inasmuch as the significant decrease in Bmax values in radioligand binding studies has been generally shown to indicate insurmountable antagonism,17 it is anticipated that oral oxybutynin produces prolonged binding of muscarinic receptor in the submaxillary gland.
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Intravesical oxybutynin instillation produced a significant increase in Kd values for specific [3H]NMS binding in the bladder of rats 0.5, 2 and 4 hours later, and also in the submaxillary gland only 0.5 hours later compared with control values. Kd value enhancement was much larger and longer lasting in the bladder than in the submaxillary gland. Furthermore, there were few changes in [3H]NMS binding parameters in the heart and colon following intravesical oxybutynin administration. Therefore, it is suggested that intravesically instilled oxybutynin, unlike oral administration, binds selectively to muscarinic receptors in the rat bladder. Yokoyama et al noted that the same intravesical dose of oxybutynin as in the current study produced a significant decrease in detrusor contraction pressure on cystometrograms of conscious rats, which lasted 3 to 5 hours after instillation.16 Therefore, it can be presumed that the significant binding of muscarinic receptors in rat bladder by intravesical oxybutynin is pharmacologically relevant in terms of the functional blockade of these receptors. Oral oxybutynin was shown to bind to muscarinic receptors in the submaxillary gland, heart and colon as well as in the bladder and binding activity was greatest in the submaxillary gland. In agreement with this observation pilocarpine induced salivation in rats was almost completely abolished by oral oxybutynin and it had recovered by only 22% even 12 hours later. Thus, the relatively selective and prolonged binding of salivary muscarinic receptors by oral oxybutynin may contribute to the high incidence of dry mouth in clinical settings. The possibility is considered that the appearance of the side effect after oral oxybutynin is more largely caused by the active metabolite DEOB rather than by oxybutynin because the area under the plasma concentration time curve for DEOB in humans after oral oxybutynin was considerably larger (5 to 11-fold) than that for the parent compound.18 Furthermore, this assumption is strengthened by the fact that the higher receptor binding affinity in the human parotid gland was exerted by DEOB than oxybutynin.19 Hence, DEOB may contribute largely to the marked and sustained suppression of pilocarpine induced salivation after oral oxybutynin because the area under the curve of the plasma concentration of this metabolite was greater than that of oxybutynin (fig. 2, A). To date much attention has been given to the in vivo exact mechanism of the action of intravesical oxybutynin to suppress bladder contractility, that is whether the efficacy of intravesical oxybutynin for improving bladder dysfunction is a pure antimuscarinic blocking effect, whether spasmolytic and local anesthetic properties are involved or whether efficacy results from a local or a systemic effect. Although most previous pharmacological and pharmacokinetic studies appear to substantiate the idea that the suppression of bladder contractility by intravesical oxybutynin may be attributable to the local direct blockade of muscarinic receptors in the detrusor muscle,1, 2, 9 direct evidence for this hypothesis is still lacking. The data obtained here are suggestive of a difference concerning mechanisms of action for oral and intravesical oxybutynin in relaxing the detrusor muscle. Intravesical oxybutynin at the pharmacologically relevant dose showed much greater binding activity to rat bladder muscarinic receptors compared with oral oxybutynin, as demonstrated by an approximately 5 times greater increase in peak Kd values for bladder [3H]NMS binding after intravesical oxybutynin compared with oral oxybutynin (table 1) although plasma concentrations of oxybutynin and DEOB after oral oxybutynin were approximately 9 times higher at the peak level and showed a slower elimination rate (fig. 2). Taken together it should be emphasized that intravesical oxybutynin may work exclusively via a local anticholinergic effect on rat detrusor muscles. Support for this predominant direct effect on bladder receptors has been furnished by the previous finding that in beagle dogs given intravesical oxybutynin after bladder augmentation the tissue concentration of this
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MUSCARINIC RECEPTOR BINDING AFTER INTRAVESICAL OXYBUTYNIN
drug was 52-fold higher in the ileal bladder than in the intact ileum.20 However, it must be kept in mind that a systemic effect by oxybutynin absorbed into the bloodstream may appear at higher intravesical doses because there was transient binding activity of submaxillary gland muscarinic receptors by the pharmacological dose of intravesical oxybutynin, as shown by a significant increase in the Kd value for [3H]NMS at 0.5 hours (table 1). Further detailed experiments are required to resolve this issue fully. Recent functional studies suggest that muscarinic receptors are located not only on bladder detrusor smooth muscles, but also on the urothelium.21⫺23 In fact, Hawthorn et al observed greater muscarinic receptor density in pig bladder urothelium than in detrusor smooth muscle.21 Thus, there is a possibility that the binding of oxybutynin and [3H]NMS in the rat bladder measured in the current study may reflect partly binding to the urothelium.
8.
9.
10.
11.
12.
CONCLUSIONS
Intravesical oxybutynin binds selectively to rat bladder muscarinic receptors, possibly via a direct local effect, while oral oxybutynin may exert a predominant binding of salivary receptors. Thus, simultaneous analysis of muscarinic receptor binding in target and nontarget tissues, pharmacokinetics and pharmacological effects after systemic and local administration of anticholinergic drugs may be powerful tool with which to analyze the exact in vivo mechanism of action of these drugs under physiological and pathological conditions. A. Kawashima, T. Yamaji, N. Murata and Dr. M. Uchida, Meiji Milk Products Co., Ltd. provided technical assistance. Oxybutynin hydrochloride and DEOB hydrochloride were provided by Meiji Milk Products Co., Ltd., Odawara, Japan. All other chemicals were purchased from commercial sources.
13.
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15.
16.
17.
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22.
23.
of overactive bladder with modified intravesical oxybutynin choloride. Neurourol Urodyn, 19: 683, 2000 Nilvebrant, L. and Sparf, B.: Dicyclomine, benzhexol and oxybutynin distinguish between subclasses of muscarinic binding sites. Eur J Pharmacol, 123: 133, 1986 Yarker, Y. E., Goa, K. L. and Fitton, A.: Oxybutynin. A review of its pharmacodynamic and pharmacokinetic properties, and its therapeutic use in detrusor instability. Drugs Aging, 6: 243, 1995 Yamada, S., Uchida, S. and Kimura R.: Integration of pharmacokinetics and pharmacodynamics in drug development—in vivo receptor occupancy by calcium antagonist and angiotensin II receptor antagonist. Curr Topics Pharmacol, 4: 239, 1998 Yamada, S., Yamamura, H. I. and Roeske, W. R.: Characterization of alpha-1 adrenergic receptors in the heart using [3H]WB4101: effect of 6-hydroxydomine treatment. J Pharmacol Exp Ther, 215: 176, 1980 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J.: Protein measurement with the Folin phenol reagent. J Biol Chem, 193: 265, 1951 Ehlert, F. J. and Tran, L. P.: Regional distribution of M1, M2 and non-M1, non-M2 subtypes of muscarinic binding sites in rat brain. J Pharmacol Exp Ther, 255: 1148, 1990 Uchida, S., Yamada, S., Ohkura, T., Heshikiri, M., Yoshimi, A., Shirahase, H. et al: The receptor occupation and plasma concentration of NKY-722, a water-soluble dihydropyridine-type calcium antagonist, in spontaneously hypertensive rats. Br J Pharmacol, 114: 217, 1995 Terai, T., Deguchi, Y., Ohtsuka, M. and Kumada, S.: Effects of the anticholinergic drug prifinium bromide on urinary bladder contractions in rat in vivo and in guinea-pig in vitro. Arzneimittelforschung, 41: 417, 1991 Yokoyama, O., Ishiura, Y., Nakamura, Y. and Ohkawa, M.: Urodynamic effects of intravesical oxybutynin chloride in conscious rats. J Urol 155: 768, 1996 Yamada, S., Isogai, M., Kagawa, Y., Takayanagi, N., Hayashi, E., Tsuji, K. et al: Brain nicotinic acetylcholine receptors. Biochemical characterization by neosurugatoxin. Mol Pharmacol, 28: 120, 1985 Hughes, K. M., Lang, J. C., Lazare, R., Gordon, D., Stanton, S. L., Malone-Lee, J. et al: Measurement of oxybutynin and its N-desethyl metabolite in plasma, and its application to pharmacokinetic studies in young, elderly and frail elderly volunteers. Xenobiotica, 22: 859, 1992 Waldeck, K., Larsson, B. and Andersson, K.-E: Comparison of oxybutynin and its active metabolite, N-desethyl-oxybutynin, in the human detrusor and parotid gland. J Urol, 157: 1093, 1997 Mohler, J. L.: Relaxation of intestinal bladders by intravesical oxybutynin chloride. Neurourol Urodyn, 9: 179, 1990 Hawthorn, M. H., Chapple, C. R., Cock, M. and Chess-Williams, R.: Urothelium-derived inhibitory factor(s) influences on detrusor muscle contractility in vitro. Br J Pharmacol, 129: 416, 2000 Chess-Williams, R.: Muscarinic receptors of the urinary bladder: detrusor, urothelial and prejunctional. Auton Autacoid Pharmacol, 22: 133, 2002 Yoshida, M., Miyamae, K., Iwashita, H., Otani, M. and Inadome, A.: Management of detrusor dysfunction in the elderly: changes in acetylcholine and adenosine triphosphate release during aging. Urology, 63: 17, 2004
Vol. 172, 2059 –2064, November 2004 Printed in U.S.A.
DOI: 10.1097/01.ju.0000138472.16876.8d
DEMONSTRATION OF BLADDER SELECTIVE MUSCARINIC RECEPTOR BINDING BY INTRAVESICAL OXYBUTYNIN TO TREAT OVERACTIVE BLADDER TOMOMI OKI, RYOHEI KIMURA, MOTOAKI SAITO, IKUO MIYAGAWA
AND
SHIZUO YAMADA*
From the Department of Biopharmaceutical Sciences and COE Program in the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka (TO, RK, SY) and Department of Urology, Tottori University Faculty of Medicine (MS, IM), Yonago, Japan
ABSTRACT
Purpose: The current study was done to elucidate the in vivo mechanism of action of intravesical instillation of oxybutynin to treat overactive bladder. Materials and Methods: In rats receiving oral and intravesical oxybutynin we measured muscarinic receptors in the bladder and other tissues by radioligand binding assay using [3H]NMS ([N-methyl-3H]scopolamine methyl chloride) with the simultaneous measurement of plasma concentrations of oxybutynin and its active metabolite N-desethyl-oxybutynin. Pilocarpine induced salivary secretion was also measured. Results: Following oral administration of oxybutynin there was a significant increase in the apparent dissociation constant (Kd) for specific [3H]NMS binding in the bladder, submaxillary gland, heart and colon of rats at 1 and 3 hours with a consistent decrease in the maximal number of binding sites (Bmax) in the submaxillary gland. Furthermore, a marked and prolonged decrease in pilocarpine induced salivary secretion in rats was observed by oral oxybutynin. In contrast, intravesical instillation of oxybutynin produced a significant increase in Kd for specific [3H]NMS binding in the bladder of rats at 0.5 to 4 hours later and also in the submaxillary gland only at 0.5 hours later. The enhancement in Kd was much larger and longer lasting in the bladder than in the submaxillary gland. Moreover, intravesical oxybutynin had little muscarinic receptor binding activity in the heart and colon, and little significant suppression of pilocarpine induced salivation in rats. The plasma concentrations of oxybutynin and N-desethyl-oxybutynin were much higher in rats receiving oxybutynin orally than intravesically. Conclusions: Intravesical oxybutynin in rats may cause selective binding of bladder muscarinic receptors via a direct local effect, while oral oxybutynin may exert predominant binding of salivary gland receptors. KEY WORDS: bladder; administration, intravesical; oxybutynin; receptors, muscarinic; saliva
Urinary incontinence and overactive bladder are important and common conditions that have received little general medical attention. Oral anticholinergic agents such as oxybutynin are effective in many patients with these disorders but there are some patients who do not respond to oral medication or who experience intolerable systemic side effects of these drugs.1, 2 The systemic side effects of oxybutynin are suggested to be caused by the high plasma level of its active metabolite, N-desethyl-oxybutynin (DEOB).1, 2 There is increasing evidence that the intravesical instillation of oxybutynin, which decreases the plasma level of DEOB compared with that of oral administration of oxybutynin, is effective therapy for overactive bladder.1⫺7 In addition, intravesical oxybutynin has been shown to cause few systemic adverse events.3, 5–7 Despite these clinical successes to our knowledge the exact mechanism of action of intravesical oxybutynin remains unknown. Although oxybutynin is known to have mixed anticholinergic and spasmolytic effects, and possibly local anesthetic properties, the in vivo mechanism to suppress bladder contractility has yet to be fully elucidated. Also,
controversy still exists as to whether the efficacy of intravesical oxybutynin results from a local or a systemic effect. Although many studies have dealt with the pharmacological effects and in vitro receptor binding affinity of anticholinergic agents,8, 9 little information has been published on in vivo muscarinic receptor binding characteristics, such as the extent and duration at the target, and nontarget tissues following oral and intravesical administration of anticholinergic agents. In general the results of in vitro receptor binding studies of drugs may not necessarily guarantee in vivo pharmacological specificity such as organ selectivity because they do not consider a number of pharmacokinetic and pharmacodynamic factors.10 Therefore, in this study we clarified the in vivo mechanism of action of intravesical instillation of oxybutynin for the treatment of overactive bladder. We simultaneously measured muscarinic receptor binding in the bladder and other tissues, plasma levels of oxybutynin and DEOB, and salivary secretion in rats receiving intravesical and oral oxybutynin. MATERIALS AND METHODS
Accepted for publication May 7, 2004. Supported by a Grant-in-Aid for Scientific Research (C)(2)(No. Materials.We used [3H]NMS ([N-methyl-3H]scopolamine 15591703) from the Ministry of Education, Science, Sports and Cul- methyl chloride) (PerkinElmer Life Sciences, Inc., Boston, ture of Japan. Massachusetts) (3.03 TBq/mmol), oxybutynin hydrochloride * Correspondence: 52–1 Yada, Shizuoka 422-8526, Japan (telephone: ⫹81–54-264 –5631; FAX: ⫹81–54-264 –5635; e-mail: yamada@ys7. and DEOB hydrochloride. Animals. Male Sprague-Dawley rats (Charles River Japan, u-shizuoka-ken.ac.jp). 2059
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MUSCARINIC RECEPTOR BINDING AFTER INTRAVESICAL OXYBUTYNIN
Inc., Atsugi, Japan) weighing about 250 to 350 gm were housed in the laboratory with free access to food (normal chow) and water, and maintained on a 12-hours dark/light cycle in a room at controlled mean temperature ⫾ SE (24C ⫾ 1C) and humidity (55% ⫾ 5%). Administration of oxybutynin. Rats were fasted for 16 hours and then received oxybutynin orally (127 mol/kg) dissolved in distilled water. Control animals received vehicle alone. For intravesical oxybutynin instillation rats were anesthetized with pentobarbital (121 mol/kg intraperitoneally). Through a midline incision in the abdomen the bladder was exposed and emptied of urine by sucking through a syringe connected to a 30 gauge needle. Oxybutynin (76.1 nmol/0.2 ml per rat) dissolved in physiological saline was instilled into the bladder by injecting it directly through a syringe connected to the needle. Oxybutynin solution was kept in the bladder for 30 minutes by tying the urethral openings with a thread, after which the bladder was evacuated by taking out the thread. Control animals received physiological saline alone. This study was done according to the guideline approved by the experimental animal ethical committee, University of Shizuoka. Tissue preparation. After oral and intravesical oxybutynin administration rats were exsanguinated by taking the blood from the descending aorta under temporary anesthesia with diethyl ether. The tissues were perfused with cold saline from the aorta. The bladder, submaxillary gland, heart and colon were then dissected, and fat and blood vessels were removed by trimming. The tissues were minced with scissors and homogenized using a Polytron homogenizer (Kinematica AG, Lucerne, Switzerland) in 19 volumes of ice-cold 30 mM Na⫹/ HEPES buffer (pH 7.5). The homogenates were then centrifuged at 40,000 ⫻ gravity for 20 minutes. The resulting pellet was finally resuspended in 29 volumes of ice-cold buffer for the binding assay. In the ex vivo experiment there was a possibility that oxybutynin might dissociate in part from receptor sites during tissue preparation (homogenization and suspension) after drug administration. Yamada et al have previously reported that the dissociation of antagonists from receptor sites at 4C is extremely slow.11 Therefore, to minimize the dissociation of oxybutynin from receptor sites all steps for the preparation were performed at 4C. In fact, there was not a large difference in [3H]NMS binding parameters (Kd and Bmax) between single washing and double washing of rat bladder homogenates at 4C 3 hours after oral administration of oxybutynin (127 mol/kg). Protein concentrations were measured according to the method of Lowry et al.12 Rat plasma was isolated from blood by centrifugation and stored at – 80C until drug analysis. Muscarinic receptor binding assay. The binding assay for muscarinic receptors was performed using [3H]NMS, as previously described.13 Homogenates (70 to 350 g protein) of rat tissues were incubated with different concentrations (0.06 to 2.0 nM) of [3H]NMS in 30 mM Na⫹/HEPES buffer (pH 7.5). Incubation was done for 60 minutes at 25C. The reaction was terminated by rapid filtration using a Cell Harvester (Brandel Co., Gaithersburg, Maryland) through GF/B glass fiber filters (Whatman, Brentford, United Kingdom) and the filters were then rinsed 3 times with 3 ml ice-cold buffer. Tissue bound radioactivity was extracted from the filters by placing them overnight by immersion in scintillation fluid (2 l toluene, 1 l Triton X-100, 15 gm 2,5-diphenyloxazole and 0.3 gm 1,4-bis[2-(5-phenyloxazolyl)]benzene) and radioactivity was determined by a liquid scintillation counter. Specific [3H]NMS binding was determined experimentally from the difference between counts in the absence and presence of 1 M atropine. All assays were performed in duplicate. Measurement of plasma concentrations of oxybutynin and DEOB. The concentrations of oxybutynin and DEOB in rat plasma were determined by gas chromatography and mass spectrometry (GC/MS). Briefly, a plasma sample (0.1 to 0.5 ml.)
was mixed with internal standard ([2H13]oxybutynin⬘HCl and [2H13]DEOB⬘HCl). After being alkalinized with 0.5 ml 0.5 M carbonate buffer (pH 9.5) it was extracted with 6 ml n-hexane. After centrifugation at 1,500 ⫻ gravity for 5 minutes the supernatant was evaporated until dry under decreased pressure. The residue was dissolved in 100 l CH3CN and 0.5 to 1 l was injected into the GC/MS system. The GC/MS system consisted of a 5890 Series II gas chromatograph, a 5792 Series mass selective detector, a 7673 GC/SFC injector, a Vectra 486/66U computer and a Laser JET 4 printer (Hewlett Packard Co., Palo Alto, California). Chromatographic separation was done using 15 m ⫻ 0.25 mm inner diameter ⫻ 0.25 m UA⫹-1 HT film (Frontier Lab, Ltd., Fukushima, Japan). The carrier gas was helium at a flow rate of 1.0 ml per minute Oven temperature was held at 150C for 1 minute and then programmed from 150C to 220C at 20C per minute for the first ramp, 220C to 260C at 10C per minute for the second ramp and 260C to 300C at 30C per minute for the third ramp. It was held at 300C for 2 minutes before returning to the initial starting temperature of 150C. Injection temperature was 200C. Fragmentation was accomplished by electron impact at 70 eV ionizing voltage and 300 A ionizing current. Select ion monitoring was performed at m/z 342 (oxybutynin), m/z 355 (internal standard [2H13]oxybutynin), m/z 189 (DEOB) and m/z 200 (internal standard [2H13]DEOB). The limit of detection of oxybutynin and DEOB in plasma was 1.40 and 3.04 nM, respectively. Measurement of salivary secretion. Rats were anesthetized by intraperitoneal administration of pentobarbital (121 mol/kg). Immediately after wiping the saliva remaining in the oral cavity with a cotton ball all saliva in the cavity was collected by absorbing it into 3 balls for 10 minutes. The cotton ball was then immediately weighed on an electric balance to prevent moisture loss. The weight of secreted saliva was estimated as the difference in cotton ball weight before vs after application into the oral cavity. The effects of oral and intravesical oxybutynin on pilocarpine induced salivary secretion in rats were examined. Pilocarpine (4.09 mol/kg dissolved in physiological saline) was intravenously administered 1, 3 and 12 hours after oral oxybutynin administration as well as 0.5, 2 and 4 hours after intravesical instillation and all saliva was collected for 10 minutes. In another experiment the effect of intravesical instillation of oxybutynin on salivary secretion by cumulative doses (0.04 to 12.3 mol/kg intravenously) of pilocarpine in rats was examined. The secretion of whole saliva was measured in rats receiving cumulative doses of pilocarpine at every 5 minutes. Data analysis. Analysis of [3H]NMS binding data were performed as described previously.11 The apparent dissociation constant (Kd) and maximal number of binding sites (Bmax) for [3H]NMS were estimated by Rosenthal analysis of saturation data. In the analysis of salivary secretion data pilocarpine potency was expressed as pD2 (negative logarithm of the molar dose of the agonist producing 50% of the maximum response). Statistical analysis of the data was performed by 1-way ANOVA, followed by Dunnett’s test for multiple comparisons with p ⬍0.05 considered statistically significant. RESULTS
Muscarinic receptors in rat tissues. The effects of oral and intravesical administration of oxybutynin on specific [3H]NMS binding in rat tissues were investigated. Figure 1 shows Scatchard plots of [3H]NMS binding in the bladder and submaxillary gland of control and oxybutynin administered rats. One and/or 3 hours after oral administration of oxybutynin (127 mol/kg) there was a significant increase in Kd values for specific [3H]NMS binding in each rat tissue compared with corresponding control values (fig. 1, table 1). Increased rates in the bladder and submaxillary gland at 1
MUSCARINIC RECEPTOR BINDING AFTER INTRAVESICAL OXYBUTYNIN
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FIG. 1. Scatchard plots of specific [3H]NMS binding in rat bladder and submaxillary gland after oral (A) and intravesical (B) oxybutynin administration. Rats received oxybutynin orally (127 mol/kg) and intravesically (76.1 nmol/0.2 ml per rat) and 0.5 to 4 hours (h) later specific [3H]NMS binding in homogenate of each dissected tissue was measured. Points represents mean of 3 (oxybutynin) and 5 (control) rats.
hour were 66.7% and 295%, and those at 3 hours were 113% and 775%, respectively. Thus, the enhancement of Kd values produced by oxybutynin administration was much greater in the submaxillary gland than in the bladder of rats (p ⬍0.01 at 3 hours). There was little increase in Kd values in these tissues 12 hours later. In the submaxillary gland there was a consistent (25.1% to 59.3%) decrease in Bmax values for [3H]NMS binding 1 to 12 hours after oral administration compared with control values. Similarly oral oxybutynin administration caused a significant increase in the Kd value for [3H]NMS binding in the rat heart and colon with a significant decrease in the Bmax value in the heart at 12 hours. Increased rates in the heart and colon at 1 hour were 44.6% and 113%, and those at 3 hours were 34.8% and 159%, respectively. At 0.5, 2 and 4 hours after intravesical instillation of oxybutynin (76.1 nmol/0.2 ml per rat) there was a significant increase in the Kd value for specific [3H]NMS binding in the bladder of rats compared with control values (966%, 133% and 59.9%, respectively, table 1). In the submaxillary gland there was a significant (168%) increase in the Kd value for [3H]NMS binding only 0.5 hours after intravesical oxybutynin instillation. Thus, the enhancement in Kd values produced by oxybutynin instillation was significantly greater in the bladder than in the submaxillary gland (p ⬍0.001). On the other hand, Bmax values for [3H]NMS binding 0.5 to 12 hours after intravesical oxybutynin instillation in the submaxillary gland were unaltered. Furthermore, intravesical oxybutynin instillation had little effect on Kd and Bmax values for [3H]NMS binding in the heart and colon of rats. Plasma levels of oxybutynin and DEOB. Figure 2 shows the
plasma concentration-time profiles of oxybutynin and DEOB in rats after oral and intravesical administration of oxybutynin. Plasma oxybutynin and DEOB concentrations reached maximum levels (about 91 nM) 1 hour after oral administration of oxybutynin (127 mol/kg) and then they rapidly decreased (fig. 2, A). In 3 or 4 preparations 0.5, 2 and 4 hours after intravesical instillation of oxybutynin (76.1 nmol/0.2 ml per rat) mean plasma oxybutynin concentrations were 12.4 ⫾ 2.8, 3.88 ⫾ 0.25 and 0.87 ⫾ 0.50 nM, respectively (fig. 2, B). DEOB was barely detectable in rat plasma 0.5 to 12 hours after intravesical oxybutynin instillation. Salivary secretion.The amount of pooled saliva of anesthetized rats secreted during every 10 minutes was the highest, comprising 42.6% of total saliva secreted for 60 minutes, within the first 10 minutes after intravenous injection of pilocarpine (4.09 mol/kg) and thereafter it gradually decreased (fig. 3). Table 2 shows the effects of oral and intravesical administration of oxybutynin on pilocarpine induced salivary secretion in rats (first 10 minutes). The secretory response of saliva caused by pilocarpine in control rats was nicely reproducible because similar amounts of pooled saliva were secreted during the first 10 minutes after pilocarpine simulation at each time of 1, 3 and 12 hours (oral) as well as 0.5, 2 and 4 hours (intravesical). Thus, pooled control data are presented. One to 12 hours after oral administration of oxybutynin (127 mol/kg) there was a significant decrease in the amount of pilocarpine induced saliva secretion compared with that in control rats. In particular the amount of saliva secreted 1 and 3 hours later was extremely low and secretion
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TABLE 1. Kd and Bmax for specific [3H]NMS binding in rat bladder, submaxillary gland, heart and colon after oral and intravesical oxybutynin Hrs After Administration
Mean Kd ⫾ SE (pM)
Mean Bmax ⫾ SE (fmol/mg protein)
Oral Bladder: Control 1 3 12 Submaxillary gland: Control 1 3 12 Heart: Control 1 3 12 Colon: Control 1 3 12
165 ⫾ 5 275 ⫾ 42 351 ⫾ 85* 188 ⫾ 7
157 ⫾ 8 189 ⫾ 40 131 ⫾ 17 101 ⫾ 7
110 ⫾ 3 435 ⫾ 183† 962 ⫾ 159‡ 104 ⫾ 4
124 ⫾ 4 92.9 ⫾ 13.5† 61.7 ⫾ 4.5‡ 50.5 ⫾ 6.1‡
267 ⫾ 8 386 ⫾ 65† 360 ⫾ 20† 240 ⫾ 30
71.7 ⫾ 1.9 66.6 ⫾ 3.4 63.0 ⫾ 2.5 56.7 ⫾ 1.7*
149 ⫾ 4 318 ⫾ 21* 386 ⫾ 78‡ 162 ⫾ 12 Intravesical
106 ⫾ 4 120 ⫾ 7 112 ⫾ 12 105 ⫾ 9
Bladder: Control 172 ⫾ 9 140 ⫾ 7 0.5 1,834 ⫾ 212‡ 125 ⫾ 20 2 400 ⫾ 34‡ 123 ⫾ 7 4 275 ⫾ 13* 149 ⫾ 9 12 205 ⫾ 12 126 ⫾ 5 Submaxillary gland: Control 148 ⫾ 12 120 ⫾ 6 0.5 396 ⫾ 55‡ 132 ⫾ 4 2 179 ⫾ 10 125 ⫾ 10 4 165 ⫾ 9 124 ⫾ 10 12 149 ⫾ 22 124 ⫾ 15 Heart: Control 298 ⫾ 8 83.7 ⫾ 3.5 0.5 297 ⫾ 14 82.2 ⫾ 6.7 2 304 ⫾ 20 86.3 ⫾ 2.8 4 302 ⫾ 5 80.2 ⫾ 4.3 12 304 ⫾ 20 83.6 ⫾ 4.1 Colon: Control 181 ⫾ 7 143 ⫾ 7 0.5 323 ⫾ 100 145 ⫾ 12 2 188 ⫾ 9 144 ⫾ 12 4 202 ⫾ 13 147 ⫾ 11 12 189 ⫾ 6 125 ⫾ 14 Three to 9 rats per group received oxybutynin orally (127 mol/kg) and intravesically (76.1 nmol/0.2 ml per rat), were sacrificed 0.5 to 12 hours after administration, and specific binding of [3H]NMS (0.06 to 2.0 nM) in rat tissues was measured. * Significantly different vs control (p ⬍0.01). † Significantly different vs control (p ⬍0.05). ‡ Significantly different vs control (p ⬍ 0.001).
12 hours later showed some recovery but it was only 22% of the control level. On the other hand, the intravesical instillation of oxybutynin (76.1 nmol/0.2 ml per rat) had little significant effect on pilocarpine (4.09 mol/kg) induced salivary secretion 0.5, 2 and 4 hours later. Furthermore, we measured saliva secretion at cumulative doses (0.04 to 12.3 mol/kg intravenously) of pilocarpine 0.5 hours after intravesical oxybutynin instillation in rats. This time (0.5 hours) was chosen because there was significant (168%) enhancement of Kd values for [3H]NMS binding in the submaxillary gland (table 1). The cumulative intravenous injection of pilocarpine at these doses induced dose dependent salivary secretion in rats (fig. 4). Intravesical oxybutynin instillation tended to attenuate salivary secretion only at low pilocarpine doses (0.41 and 1.23 mol/kg intravenously) but the extent of suppression was statistically insignificant. In fact, pD2 values without and with intravesical oxybutynin were 5.86 ⫾ 0.10 and 5.79 ⫾ 0.07, respectively.
FIG. 2. Plasma concentration-time profiles of oxybutynin (filled circles) and DEOB (open circles) after oral (A) and intravesical (B) oxybutynin administration in rats. Rats received oxybutynin orally (127 mol/kg) and intravesically (76.1 nmol/0.2 ml per rat) and 0.5 to 12 hours (h) later blood samples were taken from aorta of each rat. Points represents mean ⫾ SE of 3 to 8 rats.
FIG. 3. Time course of pilocarpine induced salivary secretion in rats. Rats received pilocarpine (4.09 mol/kg) intravenously and weight of pooled saliva secreted during each 10 minutes was measured periodically at 0 to 60 minutes. Columns represents mean ⫾ SE in 11 rats.
DISCUSSION
In the current study we simultaneously measured muscarinic receptor binding in tissues, including the bladder and
MUSCARINIC RECEPTOR BINDING AFTER INTRAVESICAL OXYBUTYNIN TABLE 2. Effects of oral and intravesical administration of oxybutynin on pilocarpine induced salivary secretion in rats Hrs After Administration
Mean Salivary Secretion ⫾ SE (mg)
Control 632 ⫾ 53 Oral route: 1 6.76 ⫾ 6.76 3 0.80 ⫾ 0.80 12 139 ⫾ 27 p Value vs control ⬍0.001 Control 493 ⫾ 12 Intravesical route: 0.5 440 ⫾ 32 2 500 ⫾ 76 4 492 ⫾ 18 Three to 5 rats per group received oxybutynin orally (127 mol/kg) and intravesically (76.1 nmol/0.2 ml per rat), and pooled saliva secreted during the first 10 minutes after intravenous stimulation of pilocarpine (4.09 mol/kg) was measured.
FIG. 4. Effects of intravesical oxybutynin administration on doseresponse curve of pilocarpine induced salivary secretion in rats. Rats received intravenously cumulative doses (0.04 to 12.3 mol/kg) pilocarpine before (filled circles) and 0.5 hours after (open circles) intravesical oxybutynin instillation (76.1 nmol/0.2 ml per rat) and saliva secretion was measured. Salivary secretion caused by each pilocarpine dose is expressed as percent of maximal control response to pilocarpine (12.3 mol/kg). Points represents mean ⫾ SE of 3 (oxybutynin) and 5 (control) rats.
submaxillary gland, plasma levels of oxybutynin and DEOB, and salivary secretion in rats after intravesical oxybutynin instillation, in comparison with the results obtained after oral administration of this drug. The technique of ex vivo and in vivo receptor binding has been demonstrated to be useful for predicting the potency, organ selectivity and duration of action of drugs in relation to their pharmacokinetic and pharmacodynamic profiles.10, 14 Thus, the ex vivo receptor binding technique was used to characterize simultaneously muscarinic receptor binding in the bladder, submaxillary gland, heart and colon of rats after oral and intravesical administration of oxybutynin. The oral and intravesical doses of oxybutynin used were based on pharmacological doses of oxybutynin for decreasing detrusor contractility in rat cystometry studies.15, 16 There was a significant increase in Kd values for specific [3H]NMS binding in the bladder, submaxillary gland, heart and colon of rats 1 and 3 hours after oral oxybutynin administration compared with each control value. These data suggest that oral oxybutynin binds uniformly to muscarinic receptors in all rat tissues examined. In the submaxillary gland the increase in Kd values was greatest among 4 tissues and concomitantly a long lasting decrease in Bmax values was observed. Inasmuch as the significant decrease in Bmax values in radioligand binding studies has been generally shown to indicate insurmountable antagonism,17 it is anticipated that oral oxybutynin produces prolonged binding of muscarinic receptor in the submaxillary gland.
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Intravesical oxybutynin instillation produced a significant increase in Kd values for specific [3H]NMS binding in the bladder of rats 0.5, 2 and 4 hours later, and also in the submaxillary gland only 0.5 hours later compared with control values. Kd value enhancement was much larger and longer lasting in the bladder than in the submaxillary gland. Furthermore, there were few changes in [3H]NMS binding parameters in the heart and colon following intravesical oxybutynin administration. Therefore, it is suggested that intravesically instilled oxybutynin, unlike oral administration, binds selectively to muscarinic receptors in the rat bladder. Yokoyama et al noted that the same intravesical dose of oxybutynin as in the current study produced a significant decrease in detrusor contraction pressure on cystometrograms of conscious rats, which lasted 3 to 5 hours after instillation.16 Therefore, it can be presumed that the significant binding of muscarinic receptors in rat bladder by intravesical oxybutynin is pharmacologically relevant in terms of the functional blockade of these receptors. Oral oxybutynin was shown to bind to muscarinic receptors in the submaxillary gland, heart and colon as well as in the bladder and binding activity was greatest in the submaxillary gland. In agreement with this observation pilocarpine induced salivation in rats was almost completely abolished by oral oxybutynin and it had recovered by only 22% even 12 hours later. Thus, the relatively selective and prolonged binding of salivary muscarinic receptors by oral oxybutynin may contribute to the high incidence of dry mouth in clinical settings. The possibility is considered that the appearance of the side effect after oral oxybutynin is more largely caused by the active metabolite DEOB rather than by oxybutynin because the area under the plasma concentration time curve for DEOB in humans after oral oxybutynin was considerably larger (5 to 11-fold) than that for the parent compound.18 Furthermore, this assumption is strengthened by the fact that the higher receptor binding affinity in the human parotid gland was exerted by DEOB than oxybutynin.19 Hence, DEOB may contribute largely to the marked and sustained suppression of pilocarpine induced salivation after oral oxybutynin because the area under the curve of the plasma concentration of this metabolite was greater than that of oxybutynin (fig. 2, A). To date much attention has been given to the in vivo exact mechanism of the action of intravesical oxybutynin to suppress bladder contractility, that is whether the efficacy of intravesical oxybutynin for improving bladder dysfunction is a pure antimuscarinic blocking effect, whether spasmolytic and local anesthetic properties are involved or whether efficacy results from a local or a systemic effect. Although most previous pharmacological and pharmacokinetic studies appear to substantiate the idea that the suppression of bladder contractility by intravesical oxybutynin may be attributable to the local direct blockade of muscarinic receptors in the detrusor muscle,1, 2, 9 direct evidence for this hypothesis is still lacking. The data obtained here are suggestive of a difference concerning mechanisms of action for oral and intravesical oxybutynin in relaxing the detrusor muscle. Intravesical oxybutynin at the pharmacologically relevant dose showed much greater binding activity to rat bladder muscarinic receptors compared with oral oxybutynin, as demonstrated by an approximately 5 times greater increase in peak Kd values for bladder [3H]NMS binding after intravesical oxybutynin compared with oral oxybutynin (table 1) although plasma concentrations of oxybutynin and DEOB after oral oxybutynin were approximately 9 times higher at the peak level and showed a slower elimination rate (fig. 2). Taken together it should be emphasized that intravesical oxybutynin may work exclusively via a local anticholinergic effect on rat detrusor muscles. Support for this predominant direct effect on bladder receptors has been furnished by the previous finding that in beagle dogs given intravesical oxybutynin after bladder augmentation the tissue concentration of this
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MUSCARINIC RECEPTOR BINDING AFTER INTRAVESICAL OXYBUTYNIN
drug was 52-fold higher in the ileal bladder than in the intact ileum.20 However, it must be kept in mind that a systemic effect by oxybutynin absorbed into the bloodstream may appear at higher intravesical doses because there was transient binding activity of submaxillary gland muscarinic receptors by the pharmacological dose of intravesical oxybutynin, as shown by a significant increase in the Kd value for [3H]NMS at 0.5 hours (table 1). Further detailed experiments are required to resolve this issue fully. Recent functional studies suggest that muscarinic receptors are located not only on bladder detrusor smooth muscles, but also on the urothelium.21⫺23 In fact, Hawthorn et al observed greater muscarinic receptor density in pig bladder urothelium than in detrusor smooth muscle.21 Thus, there is a possibility that the binding of oxybutynin and [3H]NMS in the rat bladder measured in the current study may reflect partly binding to the urothelium.
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CONCLUSIONS
Intravesical oxybutynin binds selectively to rat bladder muscarinic receptors, possibly via a direct local effect, while oral oxybutynin may exert a predominant binding of salivary receptors. Thus, simultaneous analysis of muscarinic receptor binding in target and nontarget tissues, pharmacokinetics and pharmacological effects after systemic and local administration of anticholinergic drugs may be powerful tool with which to analyze the exact in vivo mechanism of action of these drugs under physiological and pathological conditions. A. Kawashima, T. Yamaji, N. Murata and Dr. M. Uchida, Meiji Milk Products Co., Ltd. provided technical assistance. Oxybutynin hydrochloride and DEOB hydrochloride were provided by Meiji Milk Products Co., Ltd., Odawara, Japan. All other chemicals were purchased from commercial sources.
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