Tomomi Oki, Ayako Toma-Okura, and Shizuo Yamada

Transcription

1 /06/ $20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 316, No. 3 Copyright 2006 by The American Society for Pharmacology and Experimental Therapeutics 94508/ JPET 316: , 2006 Printed in U.S.A. Advantages for Transdermal over Oral Oxybutynin to Treat Overactive Bladder: Muscarinic Receptor Binding, Plasma Drug Concentration, and Salivary Secretion Tomomi Oki, Ayako Toma-Okura, and Shizuo Yamada Department of Pharmacokinetics and Pharmacodynamics and Center of Excellence Program in the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan Received August 22, 2005; accepted November 9, 2005 An overactive bladder, characterized by symptoms of increased frequency of micturition, urgency, and urge incontinence, is very common in the geriatric population, a group that rapidly increases in number (Wein and Rovner, 2002). Urge urinary incontinence is one of the most common types of incontinence in geriatric patients (Resnick and Yalla, 1985). Anticholinergic agents, such as oxybutynin, are widely used to treat overactive bladder because parasympathetic innervation is the predominant stimulus for bladder contraction (Andersson, 1993). However, the use of oxybutynin is often limited by the systemic side effects, such as dry mouth, blurred vision, constipation, and tachycardia, which present frequently as serious problems in patients receiving oral oxybutynin (Yarker et al., 1995). The therapeutic and unwanted effects of anticholinergic agents in patients with This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Article, publication date, and citation information can be found at doi: /jpet ABSTRACT To clarify pharmacological usefulness of transdermal oxybutynin in the therapy of overactive bladder, we have characterized muscarinic receptor binding in rat tissues with measurement of plasma concentrations of oxybutynin and its metabolite N- desethyl-oxybutynin (DEOB) and salivation after transdermal oxybutynin compared with oral route. At 1 and 3 h after oral administration of oxybutynin, there was a significant increase in apparent dissociation constant (K d ) for specific [N-methyl- 3 H]scopolamine ([ 3 H]NMS) binding in the rat bladder, submaxillary gland, heart, and colon compared with control values. Concomitantly, submaxillary gland and heart showed a significant decrease in maximal number of binding sites (B max ) for [ 3 H]NMS binding, which lasted until 24 h. Transdermal application of oxybutynin caused dose-dependent increases in K d values for specific [ 3 H]NMS binding in rat tissues. The increment of K d values by transdermal oxybutynin was dependent on the application time. Plasma concentrations of oxybutynin and DEOB peaked at 1 h after oral oxybutynin. In contrast, plasma concentrations of oxybutynin increased slowly, depending on the transdermal application time of this drug until 12 h. Suppression of pilocarpine-induced salivation in rats due to transdermal oxybutynin was significantly weaker and more reversible than that by oral oxybutynin, which abolished salivary secretion. The present study has shown that transdermal oxybutynin binds significantly to rat bladder muscarinic receptors without producing both long-lasting occupation of exocrine receptors and cessation of cholinergic salivation evoked by oral oxybutynin. Thus, the present study provides further pharmacological basis for advantage of transdermal over oral oxybutynin in the therapy of overactive bladder. overactive bladder stem from blockade of muscarinic receptors in the bladder and other tissues, respectively. To reduce or even eliminate systemic anticholinergic adverse effects of oxybutynin, novel anticholinergic agents and dosage forms have been currently developed that may exhibit pharmacological selectivity in the bladder relative to the salivary gland (Nilvebrant et al., 1997; Abrams et al., 1998; Anderson et al., 1999; Gupta and Sathyan, 1999; Chapple, 2000; Davila et al., 2001; Ikeda et al., 2002). In fact, it has been shown that the oxybutynin chloride extended-release dosage form (Ditropan XL; ALZA Corp., Palo Alto, CA) exhibits lower rate of dry mouth than the immediate release form of oxybutynin in patients with overactive bladder (Anderson et al., 1999; Sathyan et al., 2001). It is also worth noting that transdermal therapeutic system of oxybutynin significantly improves anticholinergic adverse effects of oral oxybutynin in patients with overactive bladder (Davila et al., 2001; Dmochowski et al., 2003). Although, in the novel dosage form of oxybutynin, low plasma concentration of its active metabolite N-desethyloxybutynin (DEOB) has been suggested to contribute to the ABBREVIATIONS: DEOB, N-desethyl-oxybutynin; [ 3 H]NMS, [N-methyl- 3 H]scopolamine methyl chloride; GC, gas chromatography; MS, mass spectrometry. 1137

2 1138 Oki et al. low incidence of anticholinergic side effects (Davila et al., 2001; Dmochowski et al., 2003), the underlying pharmacological mechanism for the advantage of transdermal over oral oxybutynin is unknown. Our previous studies have revealed that comparative characterization of extent and duration of receptor binding at target and nontarget tissues after clinical route of drug administration is an extremely powerful way to elucidate in vivo pharmacological specificity, such as organ selectivity, efficacy, and safety, in relation to the pharmacokinetics and pharmacodynamics (Uchida et al., 1995; Ohkura et al., 1998; Yamada et al., 1998, 1999, 2001; Oki et al., 2004). Therefore, in the present study, we measured simultaneously muscarinic receptor binding in tissues, including the bladder and submaxillary gland, plasma levels of oxybutynin and DEOB, and saliva output in rats after transdermal application of oxybutynin compared with the oral administration. Materials and Methods Materials. [N-methyl- 3 H]Scopolamine methyl chloride ([ 3 H]NMS; 3.03 TBq/mmol) was purchased from DuPont-NEN Co. Ltd. (Boston, MA). Oxybutynin hydrochloride, its active metabolite DEOB, the transdermal therapeutic system for oxybutynin free base (CS-801; Sankyo Co. Ltd., Tokyo, Japan), and its placebo were donated by Meiji Milk Products Co. Ltd. (Odawara, Japan) and Sankyo Co. Ltd. (Tokyo, Japan). Transdermal therapeutic system of oxybutynin is composed of three layers that consist of backing film, matrix adhesive layer (containing oxybutynin free base), and an overlapping release liner strip. This system is applied to the skin after peeling off the tab (Fig. 1). One patch covers an area of 13 cm 2 and contains 33.6 mol (12 mg) of oxybutynin free base. All other chemicals were purchased from commercial sources. Animals. Male Sprague-Dawley rats (Charles River Japan, Inc., Kanagawa, Japan) weighing approximately 250 to 350 g were used in this study. Rats were housed with a 12-h light/dark cycle and fed laboratory food and water ad libitum. Administration of Oxybutynin and DEOB. For oral administration of oxybutynin, rats were fasted for 16 h and then given oral oxybutynin hydrochloride (127 mol/kg) suspended in distilled water. Control animals received vehicle alone. For the transdermal application of oxybutynin, the back hairs of rats were shaved with an electroshaver (BRAUN Dual 500, Braun, Kronberg, Germany) after cutting hair with electric clipper under temporary anesthesia with diethyl ether. On the next day, transdermal oxybutynin (0.5, 1, and 2 patches/body) was applied to the rats with the shaved back and then the rats were covered with Transpore surgical tape and selfadherent wrap (Coban; 3M Health Care, Tokyo, Japan). The vehicles were applied transdermally to the control rats. Furthermore, oxybutynin (7.61 mol/kg) and DEOB (8.20 mol/kg) were injected to the femoral vein of rats under temporary anesthesia with diethyl ether. Fig. 1. Transdermal therapeutic system of oxybutynin. Transdermal therapeutic system of oxybutynin is composed of three layers that consist of backing film, matrix-after-adhesive layer (containing oxybutynin free base), and an overlapping release liner strip. One patch covers an area of 13 cm 2 containing 33.6 mol (12 mg) of oxybutynin free base. Oxybutynin was transdermally administered to the shaved backs of rats after peeling off the tab. Tissue Preparation. At various times (oral, 1 24 h; transdermal, 2 24 h-application; intravenous, 1 h) after drug administration, rats were killed by taking the blood from the descending aorta under temporary anesthesia with diethyl ether, and the tissues were then perfused with cold saline from the aorta. The bladder, submaxillary gland, heart, and colon then were dissected, and fat and blood vessels were removed by trimming. The tissues were minced with scissors and homogenized in a Kinematica Polytron homogenizer in 19 volumes of ice-cold 30 mm Na /HEPES buffer (ph 7.5). The homogenates were then centrifuged at 40,000g for 20 min. The resulting pellet was finally suspended in buffer for the binding assay. In the ex vivo experiment, there was a possibility that oxybutynin might dissociate in part from the receptor sites during tissue preparation (homogenization and suspension) after drug administration. Yamada et al. (1980) have previously shown that the dissociation of antagonists from receptor sites at 4 C was extremely slow. Therefore, to minimize the dissociation of oxybutynin from the receptor sites, all steps in the preparation were performed at 4 C. In fact, there was no large difference in [ 3 H]NMS binding parameters [apparent dissociation constant (K d ) and maximal number of binding sites (B max )] between single washing and double washing of rat bladder homogenates at 4 C, 3 h after oral administration of oxybutynin hydrochloride (127 mol/kg). Protein concentrations were measured by the method of Lowry et al. (1951). Muscarinic Receptor Binding Assay. The binding assay for muscarinic receptors was performed using [ 3 H]NMS as described previously (Oki et al., 2004). The homogenates ( g of protein) of rat tissues were incubated with different concentrations ( nm) of [ 3 H]NMS in 30 mm Na /HEPES buffer (ph 7.5). Incubation was carried out for 60 min at 25 C. The reaction was terminated by rapid filtration (Cell Harvester; Brandel Co., Gaithersburg, MD) through Whatman GF/B glass fiber filters, and the filters were then rinsed three times with 3 ml of ice-cold buffer. Tissue-bound radioactivity was extracted from the filters overnight by immersion in scintillation fluid [21 toluene, 11 Triton X-100, 15 g of 2,5-diphenyloxazole, and 0.3 g of 1,4-bis[2-(5-phenyloxazolyl)]benzene]), and the radioactivity was determined by a liquid scintillation counter. Specific [ 3 H]NMS binding was determined experimentally from the difference between counts in the absence and presence of 1 M atropine. All assays were conducted in duplicate. Measurement of Plasma Concentrations of Oxybutynin and DEOB. The measurement of the plasma concentrations of oxybutynin and DEOB was performed in rats after oral administration of oxybutynin hydrochloride (127 mol/kg) and transdermal oxybutynin (1 patch/body), as described above. After drug administration, rats were killed by the withdrawal of blood from the descending aorta under anesthesia with diethyl ether. Plasma was isolated from blood by centrifugation. The plasma was stored at 80 C until analysis. Concentrations of oxybutynin and DEOB in plasma were determined by gas chromatography (GC) and mass spectrometry (MS). In brief, a plasma sample ( ml) was mixed with internal standard ([ 2 H 13 ]oxybutynin HCl and [ 2 H 13 ]DEOB HCl) and, after being alkalinized with 0.5 ml of 0.5 M carbonate buffer (ph 9.5), it was extracted with 6 ml of n-hexane. After centrifugation at 1500g for 5 min, the supernatant was evaporated to dryness under reduced pressure. The residue was dissolved in 100 l ofch 3 CN, and 0.5 to 1 l was injected into the GC/MS system. The GC/MS system consisted of a 5890 SERIESII 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, CA). Chromatographic separation was carried out using a 15 m 0.25 mm i. d m film UA -1 HT (Frontier Lab Ltd., Fukushima, Japan). The carrier gas was helium at a flow rate of 1.0 ml/min. Oven temperature was held at 150 C for 1 min and then programmed from 150 to 220 C at 20 C/min for first ramp, from 220 to 260 C at 10 C/min for second ramp, and from 260 to 300 C at 30 C/min for third ramp. It was held at 300 C for 2 min before returning to the initial starting temperature of 150 C. The injection

3 Advantages of Transdermal over Oral Oxybutynin 1139 temperature was 200 C. Fragmentation was accomplished by electron impact at 70-eV ionizing voltage and 300- A ionizing current. Selected ion monitoring was performed at m/z 342 (oxybutynin), m/z 355 (internal standard, [ 2 H 13 ]oxybutynin), m/z 189 (DEOB), and m/z 200 (internal standard, [ 2 H 13 ]DEOB). The limits of detection of oxybutynin and DEOB in plasma were 1.40 and 3.04 nm, respectively. Measurement of Salivary Secretion. Rats were anesthetized with the intraperitoneal administration of pentobarbital (121 mol/ kg). Any saliva remaining in the oral cavity was removed with a cotton ball before measuring the whole saliva collected in the cavity for a 10-min period. The saliva was absorbed into three to five cotton balls for 10 min, and the balls were weighed on an electric balance immediately after the collection period to prevent moisture loss. To examine the effects of oral and transdermal administration of oxybutynin on pilocarpine-induced salivary secretion, pilocarpine (4.09 mol/kg, dissolved in physiological saline) was administered intravenously 1 to 24 h after oral administration of oxybutynin and immediately after the transdermal application for 2 to 24 h and the whole saliva were collected at 10-min intervals. The weight of whole saliva secreted in the oral cavity was estimated as the difference between weight of cotton ball before and after the intravenous injection of pilocarpine. In addition, the effects of oral and transdermal administration of oxybutynin on salivary secretion induced by cumulative doses (0.04 to 123 mol/kg i.v.) of pilocarpine in rats given at 5-min intervals were examined. Data Analysis. Analysis of [ 3 H]NMS binding data were performed as described previously (Yamada et al., 1980). The K d and B max for [ 3 H]NMS were estimated by Rosenthal analysis of the saturation data. Statistical analysis of the data were performed by a one-way analysis of variance followed by Dunnett s test for multiple comparison. The data were expressed as mean S.E. Statistical significance was accepted at P Results Effects of Oral and Transdermal Administration of Oxybutynin on Muscarinic Receptors in Rat Tissues. At 1 and 3 h after oral administration of oxybutynin (127 mol/kg), there was a significant increase in the K d values for specific [ 3 H]NMS binding in each tissue of rats compared with the corresponding control values (Table 1). The increases in the K d values in the bladder, submaxillary gland, heart, and colon at 1 h were 54.5, 249, 34.1, and 93.4%, respectively, and those at 3 h were 115, 678, 29.3, and 143%, respectively. Thus, the magnitude of enhancement in the K d values at 3 h produced by oral oxybutynin was significantly (P 0.01) greater in the submaxillary gland than in other tissues. There was little significant increase in the K d values in all of these tissues at 12 and 24 h later. In the submaxillary gland and heart of oxybutynin-administered rats, there was a significant (submaxillary gland, %; heart, %) reduction in the B max values for [ 3 H]NMS binding from 1 or 3 to 24 h after oral administration compared with the corresponding control values. The transdermal application of oxybutynin at 1 patch (33.6 mol)/body for 2, 4, and 12 h brought about a time-dependent increase in the K d values for specific [ 3 H]NMS binding in each tissue compared with the corresponding control value, and no further increase was seen at 24 h after application (Table 2). The increase in the K d values was significant after 2 to 24 h of application in the submaxillary gland and colon and after 4 or 12 to 24 h of application in the heart and bladder. The rate of enhancement in the K d values after transdermal application of oxybutynin for 2 to 24 h was significantly (P 0.01) greater in the submaxillary gland ( %) compared with the bladder ( %), heart ( %), and colon ( %). There was little change in the B max value in each tissue by transdermal oxybutynin. At 11 h after the removal of patch following a 12 h-application of transdermal oxybutynin, there was no longer significant change in the K d values for specific [ 3 H]NMS binding in the bladder (control, nm, n 4; oxybutynin, nm, n 3) and submaxillary gland (control, nm; oxybutynin, nm). After the transdermal application of oxybutynin at 0.5 (16.8 mol), 1 (33.6 mol), and 2 (67.2 mol) patches/body for 24 h, there was a tendency toward a dose-dependent increase TABLE 1 K d and B max for specific 3 H NMS binding in the bladder, submaxillary gland, heart, and colon of rats at 1 to 24 h after oral administration of oxybutynin Rats received 127 mol/kg oxybutynin orally and were then sacrificed 1 to 24 h after administration. Specific binding of 3 H NMS ( nm) in rat tissues was measured. Values are mean S.E. of 10 (control) and four to six (oxybutynin) rats. Values in parentheses represent the -fold increase in K d values relative to controls. Organs Time after Oral Oxybutynin K d B max h pm fmol/mg protein Bladder Control (1.54)* (2.15)*** Submaxillary gland Control (3.49)* * (7.78)*** *** *** *** Heart Control (1.34)* (1.29)* * ** ** Colon Control (1.93)** (2.43)*** Asterisks show a significant difference from the control values: * P 0.05; ** P 0.01; *** P

4 1140 Oki et al. TABLE 2 K d and B max for specific 3 H NMS binding in the bladder, submaxillary gland, heart, and colon of rats after transdermal application of oxybutynin for2to24h Rats received transdermal oxybutynin (1 patch, 33.6 mol/body) for 2 to 24 h and were then sacrificed. Specific binding of 3 H NMS ( nm) in rat tissues was measured. Values are mean S.E. of 10 (control) and four to five (oxybutynin) rats. Values in parentheses represent the -fold increase in K d values relative to controls. Organs Application Time K d B max h pm fmol/mg protein Bladder Control (2.02)*** (1.90)*** Submaxillary gland Control (3.64)*** (3.26)*** (6.47)*** (6.38)*** Heart Control (1.24)** (1.37)*** (1.22)** Colon Control (1.53)*** (1.39)** (2.01)*** (1.69)*** Asterisks show a significant difference from the control values: ** P 0.01; *** P in the K d values for [ 3 H]NMS binding in the submaxillary gland, heart, and colon, whereas the bladder showed a maximal enhancement in the K d values already at 1-patch/body application of oxybutynin (Table 3). The B max value at 2 patches/body was significantly increased in the bladder and significantly decreased in the submaxillary gland. Effects of Intravenous Injection of Oxybutynin and DEOB on Muscarinic Receptors in Rat Tissues. At1h after i.v. injection of oxybutynin (7.61 mol/kg), there was a significant increase in the K d values for specific [ 3 H]NMS binding in the bladder, submaxillary gland, heart, and colon compared with the corresponding control values (Table 4). A similar degree of the enhancement in the K d values was also seen by i.v. injection of a comparable dose (8.20 mol/kg) of DEOB in rat tissues, with the exception of the submaxillary gland that showed a significantly (P 0.05) greater (17.7- fold) increase than the case (13.6-fold) by the oxybutynin injection. Both oxybutynin and DEOB produced the greatest increase in the K d values in the submaxillary gland among rat tissues. The i.v. injection of oxybutynin had little significant effect on the B max values for specific [ 3 H]NMS binding in each tissue, whereas the injection of DEOB reduced (34.2%) the B max values significantly in the submaxillary gland but not in other tissues. Concentrations of Oxybutynin and DEOB in Rat Plasma. The plasma concentrations of oxybutynin and DEOB after oral administration of oxybutynin (127 mol/kg) reached the maximal levels (oxybutynin, nm; DEOB, nm, n 8) at 1 h, and then they declined rapidly (Fig. 2A). The plasma concentrations of oxybutynin at TABLE 3 K d and B max for specific 3 H NMS binding in the bladder, submaxillary gland, heart, and colon of rats after transdermal application of oxybutynin at 0.5 to 2 patches/body for 24 h Rats received transdermal oxybutynin at 0.5 (16.8 mol), 1 (33.6 mol), or 2 (67.2 mol) patches/body for 24 h and were then sacrificed. Specific binding of 3 H NMS ( nm) in rat tissues was measured. Values are mean S.E. of 10 (control) and four to five (oxybutynin) rats. Values in parentheses represent the -fold increase in K d values relative to controls. Organs Doses K d B max patches/body pm fmol/mg protein Bladder Control (1.90)*** (1.81)** * Submaxillary gland Control (3.44)** (6.38)*** (9.33)*** *** Heart Control (1.67)*** Colon Control (1.45)** (1.69)*** (2.28)*** Asterisks show a significant difference from the control values: * P 0.05; ** P 0.01; *** P

5 Advantages of Transdermal over Oral Oxybutynin 1141 TABLE 4 K d and B max for specific 3 H NMS binding in the bladder, submaxillary gland, heart, and colon of rats 1 h after i.v. injection of oxybutynin and DEOB Rats received oxybutynin (7.61 mol/kg) and DEOB (8.20 mol/kg) intravenously, and at 1 h later, they were sacrificed. Specific binding of 3 H NMS ( nm) in rat tissues was measured. Values are mean S.E. of seven (control) and four (oxybutynin, DEOB) rats. Values in parentheses represent the -fold increase in K d values relative to controls. Organs Drug K d B max pm fmol/mg protein Bladder Control Oxybutynin (2.57)** DEOB (2.22)** Submaxillary gland Control Oxybutynin (13.6)*** DEOB (17.7)*** ** Heart Control Oxybutynin (2.03)*** DEOB (1.96)*** Colon Control Oxybutynin (2.46)*** DEOB (2.58)*** Asterisks show a significant difference from the control values: **, P 0.01; ***, P Transdermal application of oxybutynin at 0.5, 1, and 2 patches/body for 24 h in rats showed a dose-dependent increase in the plasma concentration of oxybutynin as shown by the values of , , and nm (n 9 10), respectively (Fig. 3). DEOB was not detectable in the plasma of rats after transdermal application of oxybutynin at 0.5, 1, and 2 patches/body. The plasma concentration of oxybutynin at 1 h after i.v. injection of this drug (7.61 mol/kg) in rats was nm (n 3), and that of DEOB at 1 h after i.v. injection of this metabolite (8.20 mol/kg) was nm (n 3). In the plasma of rats injected with oxybutynin, DEOB was very little detected. Effects of Oral and Transdermal Administration of Oxybutynin on Rat Salivary Secretion. To examine the time-dependent change in salivation, we measured whole saliva secreted in the oral cavity of rats at 10-min intervals until 60 min after stimulation due to i.v. injection of pilocarpine (4.09 mol/kg). The whole saliva of control rats reached a maximal level for first 10 min after the stimulation of pilocarpine, as the weights of whole saliva secreted for each 10 min after the pilocarpine injection were , , , , , and mg Fig. 2. Plasma concentration-time profiles of oxybutynin (F) and DEOB ( ) after oral (A) and transdermal (B) administration of oxybutynin in rats. Rats received oxybutynin orally (127 mol/kg) or transdermally (1 patch, 33.6 mol/body) and were then sacrificed. Blood samples were taken from the descending aorta of each rat. Each point represents the mean S.E. of seven to nine (oral oxybutynin) and nine (transdermal oxybutynin) rats. 3(n 9) and 12 h (n 7) were and nm, respectively, and the concentration of DEOB at 3 h was nm The plasma concentration of oxybutynin after transdermal application of oxybutynin at 1 patch (33.6 mol)/body for 2, 4, 12, and 24 h in rats were , , , and nm (n 9), respectively (Fig. 2B). Thus, the maximal plasma concentration was seen after the 12-h application of oxybutynin and there was little further increase in the plasma concentration of oxybutynin after a longer application of 24 h. Fig. 3. Concentrations of oxybutynin in plasma after transdermal application of oxybutynin at 0.5 to 2 patches/body for 24 h in rats. Rats received transdermal oxybutynin at 0.5 (16.8 mol), 1 (33.6 mol), or 2 (67.2 mol) patches/body for 24 h and were then sacrificed. Blood samples were taken from the descending aorta of rat. Each column represents the mean S.E. of nine (0.5 patch), nine (1 patch), and 10 (2 patches) rats.

6 1142 Oki et al. Fig. 4. Effects of oral (A) and transdermal (B) administration of oxybutynin on pilocarpine-induced salivary secretion in rats. Rats received oral oxybutynin (127 mol/kg) or transdermal oxybutynin (1 patch, 33.6 mol/ body), and the whole-salivary secretion was measured for the first 10 min after pilocarpine stimulation (4.09 mol/kg i.v.). Open columns (A and B), control (vehicle-administered) rats; solid columns (A), oral oxybutyninadministrated rats; dotted columns (B), transdermal oxybutynin-administered rats. Each column represents the mean S.E. of four (transdermal oxybutynin), five (oral oxybutynin), and six (control) rats. Asterisks show a significant difference from control values (, P 0.05;, P 0.01;, P 0.001). Fig. 5. Recovery of pilocarpine-induced salivary secretion after oral (A) and transdermal (B) administration of oxybutynin in rats. A, rats received oral oxybutynin (127 mol/kg), and at 1 or 12 h later, the wholesalivary secretion was measured for the first 10 min after pilocarpine stimulation (4.09 mol/kg i.v.). B, rats received transdermal oxybutynin (1 patch, 33.6 mol/body) for 12 h, and before and 11 h after the removal of patch, the whole-salivary secretion was measured for the first 10 min after pilocarpine stimulation (4.09 mol/kg i.v.). Open columns (A and B), control (vehicle-administered) rats; solid columns (A), oral oxybutyninadministrated rats; dotted columns (B), transdermal oxybutynin-administered rats. Each column represents the mean S.E. of four (transdermal oxybutynin), five (oral oxybutynin), and nine (control) rats. Asterisks show a significant difference from control values (, P 0.001). (n 11), respectively. As shown in Fig. 4, the weights of whole saliva secreted by the first 10 min of pilocarpine stimulation in control rats showed little significant difference, even by the repetitive agonist stimulation at 1 to 24 h. At 1 to 24 h after oral administration of oxybutynin (127 mol/kg), there was a marked decrease in the whole saliva compared with the value of control rats (Fig. 4A). In particular, the whole saliva at 1 and 3 h were at extremely low levels ( and mg, respectively, n 5), and the pilocarpine-stimulated salivation even after 12 and 24 h was markedly attenuated as shown by the respective values of and mg. As shown in Fig. 4B, transdermal application of oxybutynin at 1 patch (33.6 mol)/body for 4, 12, and 24 h decreased the pilocarpine-stimulated salivation significantly; however, the respective weights (154 88, , and mg, respectively, n 4) of whole saliva secreted in these rats were significantly (P 0.05) larger than the values at 1 and 3 h after oral oxybutynin (Fig. 4A). Figure 5 shows the recovery of pilocarpine-stimulated salivation in rats after oral and transdermal administration of oxybutynin. The pilocarpine-stimulated whole saliva ( mg, n 4) after the 11-h removal of patch following 12-h transdermal application of oxybutynin was significantly (P 0.001) larger than the whole saliva ( mg, n 5) stimulated by pilocarpine at 12 h after oral administration of oxybutynin. Furthermore, the effects of oral and transdermal oxybutynin on the dose-response curves for pilocarpine-stimulated secretion of whole saliva were examined. The pilocarpinestimulated secretion of whole saliva was measured 3 h after oral oxybutynin (127 mol/kg) and after 12-h application of transdermal oxybutynin (33.6 mol) when shown maximal enhancement of K d in the submaxillary gland (Tables 1 and 2). Oral administration of oxybutynin largely shifted doseresponse curves for pilocarpine-stimulated ( mol/ kg) salivation to the right with the concomitant reduction (65.5%) of maximal response, whereas transdermal oxybutynin showed only rightward shift of the curves (Fig. 6). The extent of shift was much smaller in the transdermal oxybutynin compared with the oral oxybutynin. The salivary secretion by each dose of pilocarpine was significantly greater in the transdermal oxybutynin-administered rats than in the oral oxybutynin-administered rats. Discussion A number of previous studies have substantiated the usefulness of ex vivo and in vivo receptor binding assay to clarify the potency, organ selectivity, and duration of action of drugs in relation to their pharmacokinetics and pharmacodynamics (Beauchamp et al., 1995; Uchida et al., 1995; Ohkura et al., 1998; Yamada et al., 1998, 1999, 2001). In the present study, ex vivo muscarinic receptor binding of transdermal oxybuty-

7 Fig. 6. Effects of oral and transdermal administration of oxybutynin on dose-response curves of pilocarpine-stimulated salivary secretion in rats. Rats received intravenously cumulative (5-min intervals) doses ( mol/kg) of pilocarpine 3 h after oral administration of oxybutynin (127 mol/kg) and after 12-h transdermal application of oxybutynin (1 patch, 33.6 mol/body), and the secreted whole saliva were measured. F, control; f, oral oxybutynin; Œ, transdermal oxybutynin. Each point represents the mean S.E. of three (oral oxybutynin), three (transdermal oxybutynin), and six (control) rats. Symbols show a significant difference from control value (, P 0.001). Asterisks show a significant difference from the transdermal oxybutynin (, P 0.05;, P 0.01). nin compared with oral oxybutynin was characterized by simultaneously measuring tissue [ 3 H]NMS binding, plasma concentrations of oxybutynin and DEOB, and salivary secretion in rats. The oral dose of oxybutynin chosen was expected to produce significant pharmacological effects in the urinary bladder (Mayuzumi et al., 1986; Terai et al., 1991). There was a significant increase in K d values for [ 3 H]NMS binding in the bladder, submaxillary gland, heart, and colon of rats 1 and 3 h after oral administration of oxybutynin (127 mol/kg) compared with the control values. Likewise, transdermal application of oxybutynin (1 patch, 33.6 mol/body) produced a significant increase in K d values for [ 3 H]NMS binding in these tissues. Given that an increase in K d values for radioligand in drug-pretreated tissues in this type of assay usually indicates competition between the agent and radioligand for same binding sites (Yamada et al., 2003), these data indicate that oral and transdermal oxybutynin may undergo significant binding to muscarinic receptors in rat tissues. The increase in K d value in each tissue was dependent on transdermal application time of 2 to 12 h, reaching a maximal level at the 12-h application, which was maintained by an additional 24-h application. In addition, the enhancement of K d values for [ 3 H]NMS binding in the bladder and submaxillary gland after transdermal oxybutynin promptly disappeared by the removal of patch. These data suggest that transdermally administered oxybutynin binds to muscarinic receptors in rat tissues at a slow and reversible manner. Transdermal application of oxybutynin at 0.5, 1, and 2 patches/body for 24 h produced a dose-dependent increase in K d values for specific [ 3 H]NMS binding in the submaxillary gland, heart, and colon of rats, whereas the bladder showed maximal enhancement by 1 patch/body application. Consequently, transdermal application of oxybutynin at 1 patch/ body may be sufficient to cause maximal blockade of bladder muscarinic receptors and, in the treatment of overactive bladder, transdermal oxybutynin at relatively high doses may enhance the incidence of side effects, such as dry mouth, Advantages of Transdermal over Oral Oxybutynin 1143 by potentiating the blockade of muscarinic receptors in nontarget tissues without further therapeutic effects. In fact, oxybutynin use in patients with overactive bladder is limited by dose-related dry mouth (Yarker et al., 1995; Chapple, 2000). Striking difference was seen between oral and transdermal routes of administration in the effect on muscarinic receptors in the submaxillary gland and heart. With oral oxybutynin, significant decrease in B max values for specific [ 3 H]NMS binding only in the rat submaxillary gland and heart occurred at 1 and 3 h later and the effect was maintained even at 12 and 24 h later. Interestingly, transdermal application of oxybutynin for 2 to 24 h, unlike oral oxybutynin, produced little reduction in B max values for [ 3 H]NMS binding in rat tissues. Thus, oral but not transdermal oxybutynin produced extremely long-lasting reduction of muscarinic receptor sites in the submaxillary gland and heart, suggesting noncompetitive blockade of muscarinic receptors due to oral oxybutynin. Such antagonism might be due to slowly dissociating blockade by oral oxybutynin of muscarinic receptors in these tissues, as demonstrated previously by in vitro and in vivo receptor binding studies (Yamada et al., 1985, 1999). The observed difference between oral and transdermal oxybutynin in ex vivo muscarinic receptor binding characteristics to the submaxillary gland has been further realized by the mode of antagonism against cholinergic salivation. The pilocarpine-stimulated salivation in rats was completely abolished 1 and 3 h after oral oxybutynin, followed by the marked and sustained suppression of salivation. In contrast, transdermal application of oxybutynin for 2 to 24 h suppressed significantly pilocarpine-stimulated salivation but it did not abolish secretory response. In other words, rapid cessation of salivation induced by oral oxybutynin did not occur in rats receiving transdermal oxybutynin and further pilocarpine-induced salivation was promptly recovered by the removal of patch (Fig. 5). In addition, in dose-response curves of pilocarpine-induced salivation, the antagonism by oral oxybutynin, unlike transdermal oxybutynin, was not simply competitive in that it suppressed markedly maximal response by pilocarpine. Such insurmountable antagonism agrees well with a sustained decrease of [ 3 H]NMS binding sites (B max ) in the submaxillary gland after oral but not transdermal oxybutynin. Thus, it is worth noting that the mode and time course of inhibition of pilocarpine-stimulated salivation after oral and transdermal oxybutynin are in accord with those of exocrine muscarinic receptor binding. Taken together, it is possible that significant difference in exocrine muscarinic receptor binding characteristics under in vivo condition underlies partly lower incidence of severe dry mouth due to transdermal than oral oxybutynin in patients with overactive bladder (Davila et al., 2001; Dmochowski et al., 2003). Based on the magnitude of increases in K d values, muscarinic receptor binding activity of both oral and transdermal oxybutynin was greatest in the submaxillary gland, followed by the bladder, colon, and heart. Our recent data with M 1 to M 5 subtype knockout mice have shown that M 3 subtype is expressed predominantly in the submaxillary gland and moderately in the prostate and bladder, whereas M 2 subtype is major subtype present in the bladder, heart, and colon (T. Oki, et al., unpublished observation). Because oxybutynin

8 1144 Oki et al. exerted higher affinity to M 3 subtype (Nilvebrant and Sparf, 1982; Nilvebrant et al., 1997; Oki et al., 2005), the greater receptor binding activity of this drug in the submaxillary gland may therefore reflect M 3 subtype selectivity. It is known that oxybutynin is rapidly absorbed from gastrointestinal tract and that its major pathway of elimination is hepatic metabolism (Douchamps et al., 1988) and that plasma concentration of DEOB in human after oral administration is much higher than that of oxybutynin (Hughes et al., 1992). In fact, DEOB, as well as oxybutynin, exhibits potent relaxation of human detrusor smooth muscles mainly through the blockade of muscarinic receptors (Waldeck et al., 1997). Therefore, it is considered that DEOB makes significant contribution to the pharmacological effect of oxybutynin. In the present study, similar concentrations of oxybutynin and DEOB were detected in the plasma of rats after oral oxybutynin, reaching a maximal level at 1 h followed by a rapid decline. The plasma concentration of transdermal oxybutynin in rats increased time dependently until 12 h of application and dose dependently at 0.5 to 2 patches/body. On the other hand, little DEOB was detected in the plasma of rats receiving transdermal oxybutynin. These data indicate that transdermal application of oxybutynin produces a slow increase in the plasma concentration of oxybutynin and that this route of administration of oxybutynin efficiently avoids first-pass effect as reported in healthy subjects (Appell et al., 2003; Zobrist et al., 2003). Based on previous reports that muscarinic receptor binding affinity of DEOB was greater in the salivary gland than in the bladder (Waldeck et al., 1997; Oki et al., 2005; Maruyama et al., 2006), it is considered that DEOB may be responsible for long-lasting occupation of exocrine muscarinic receptors after oral oxybutynin. In fact, significantly greater enhancement by DEOB of K d values for specific [ 3 H]NMS binding in the rat submaxillary gland was seen after the i.v. injection of approximately equimolar doses of oxybutynin and DEOB. Furthermore, significant decrease of B max values in the submaxillary gland was observed only by the injection of DEOB. These data suggest that DEOB occupies muscarinic receptors in the exocrine gland with a greater extent and for a longer period than oxybutynin. As described above, similar concentration of DEOB as oxybutynin was detected 1 and 3 h after oral oxybutynin in rats, but little of this metabolite was detected after transdermal oxybutynin. Taken together, these data suggest that DEOB contributes, at least in part, to the longlasting occupation of muscarinic receptors in the exocrine gland. Such an idea is consistent with clinical observation that lower plasma DEOB during oxybutynin treatment may be closely associated with greater saliva output in healthy subjects (Sathyan et al., 2001; Appell et al., 2003). In addition, it is also assumed that a steep increase in the plasma concentration of oxybutynin and DEOB after oral oxybutynin causes insurmountable antagonism against muscarinic receptors and cholinergic salivation by exposing these exocrine receptors to extremely high concentrations of both agents. Consequently, it is possible that pharmacokinetic characteristics, such as substantially lower fluctuation in plasma oxybutynin and avoidance of firstpass effect by transdermal oxybutynin, brings about significant difference of exocrine muscarinic receptor binding characteristics from that in oral oxybutynin, leading to the lower incidence of dry mouth. In conclusion, the present study has shown that transdermal oxybutynin leads to a significant degree of binding to rat bladder muscarinic receptors without causing both long-lasting occupation of muscarinic receptors in the submaxillary gland and abolishment of salivation evoked by oral oxybutynin. Thus, the present study provides further pharmacological evidence for advantage of transdermal over oral oxybutynin in the therapy of overactive bladder. Acknowledgments We thank Akihiro Kawashima and Dr. Masayuki Uchida (Meiji Milk Products Co. Ltd., Odawara, Japan) for providing valuable information. 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