JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 JPET FastThis Forward. article has not Published been copyedited on andNovember formatted. The 10, final 2005 version as mayDOI:10.1124/jpet.105.094508 differ from this version. JPET #94508
Advantages for Transdermal over Oral Oxybutynin to Treat Overactive
Bladder: Muscarinic Receptor Binding, Plasma Drug Concentration and
Salivary Secretion
Downloaded from
TOMOMI OKI, AYAKO TOMA-OKURA & SHIZUO YAMADA
jpet.aspetjournals.org
at ASPET Journals on October 9, 2021
Department of Pharmacokinetics and Pharmacodynamics and Center of Excellence Program in the 21st Century, School of Pharmaceutical Sciences, University of Shizuoka (TO, AO, SY),
Shizuoka, Japan.
1 Copyright 2005 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
A running title : Advantages of Transdermal over Oral Oxybutynin
Correspondence to: Shizuo Yamada, Ph.D., Department of Pharmacokinetics and
Pharmacodynamics and Center of Excellence Program in the 21st Century, School of
Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
(Tel: +81-54-264-5631, Fax: +81-54-264-5635, E-mail: [email protected])
Downloaded from Text pages: 23 pages (title page to reference page)
Tables : 4
Figures: 6 jpet.aspetjournals.org
References: 34
Abstract: 248 words
Introduction: 433 words at ASPET Journals on October 9, 2021
Discussion: 1486 words
ABBREVIATIONS: [3H]NMS: [N-methyl-3H]scopolamine methyl chloride, DEOB:
N-desethyl-oxybutynin, Kd: apparent dissociation constant, Bmax: maximum number of binding sites
Recommended section: Neuropharmacology
2 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
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
3 3 (Kd) for specific [N-methyl- H]scopolamine ([ H]NMS) binding in the rat bladder, submaxillary Downloaded from gland, heart and colon, compared with control values. Concomitantly, submaxillary gland and
3 heart showed a significant decrease in maximal number of binding sites (Bmax) for [ H]NMS
binding which lasted until 24 h. Transdermal application of oxybutynin caused a dose-dependent jpet.aspetjournals.org
3 increases in Kd values for specific [ H]NMS binding in rat tissues. The increment of Kd 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 at ASPET Journals on October 9, 2021 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.
3 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
Introduction
An overactive bladder, characterized by symptoms of increased frequency of micturition, urgency and urge incontinence, is very common in the geriatric population, a group which rapidly increases in number (Bulmer and Abrams, 2000). 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 since parasympathetic innervation is the predominant stimulus for bladder contraction (Andersson, 1993). However, the Downloaded from 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 jpet.aspetjournals.org agents in patients with overactive bladder stem from blockade of muscarinic receptors in the bladder and other tissues, respectively. In order to reduce or even eliminate systemic
anticholinergic adverse effects of oxybutynin, novel anticholinergic agents and dosage forms at ASPET Journals on October 9, 2021 have been currently developed which 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 controlled release dosage form of oxybutynin (OROS) 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 improves significantly 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-desethyl-oxybutynin (DEOB) has been suggested to contribute to the 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.
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Our previous studies have revealed that comparative characterization of extent and duration of receptor binding at target and non-target tissues after clinical route of drug administration is 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 Downloaded from after transdermal application of oxybutynin compared with the oral administration.
jpet.aspetjournals.org at ASPET Journals on October 9, 2021
5 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
Methods
Materials. [N-methyl-3H]scopolamine methyl chloride ([3H]NMS, 3.03 TBq/mmol) was purchased from Dupont-NEN Co. Ltd. (Boston, MA, USA). Oxybutynin hydrochloride, its active metabolite (N-desethyl-oxybutynin: DEOB), the transdermal therapeutic system for oxybutynin free base (CS-801) 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 Downloaded from 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 cm2 and contains 33.6 µmol (12 mg) of oxybutynin free base. All
other chemicals were purchased from commercial sources. jpet.aspetjournals.org
Animals. Male Sprague-Dawley rats (Charles River Japan, Inc.) weighing about 250-350 g were used in this study. Rats were housed with a 12 h light-dark cycle and fed laboratory food and water
ad libitum. at ASPET Journals on October 9, 2021
Administration of Oxybutynin and DEOB. For oral administration of oxybutynin, rats were fasted for 16 h and then given oral oxybutynin hydrochloride (127 µmol/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) 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 self-adherent wrap (Coban, made in U.S.A. by 3M
Health Care). The vehicles were applied transdermally to the control rats. Furthermore, oxybutynin
(7.61 µmol/kg) and DEOB (8.20 µmol/kg) were injected to the femoral vein of rats under temporary anesthesia with diethyl ether.
Tissue Preparation. At various times (oral: 1 to 24 h, transdermal: 2 to 24 h-application, intravenous : 1 h) after drug administration, rats were killed by taking the blood from the descending
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aorta under temporary anesthesia with diethyl ether, and the tissues were then perfused with cold saline from the aorta. Then, the bladder, submaxillary gland, heart and colon 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 40000×g 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 Downloaded from (homogenization and suspension) after drug administration. Yamada et al. (1980) have previously shown that the dissociation of antagonists from receptor sites at 4℃ was extremely slow. Therefore,
to minimize the dissociation of oxybutynin from the receptor sites, all steps in the preparation were jpet.aspetjournals.org
3 performed at 4℃. In fact, there was no large difference in [ H]NMS binding parameters (Kd and Bmax) between single washing and double washing of rat bladder homogenates at 4℃, 3 h after oral
administration of oxybutynin hydrochloride (127 µmol/kg). Protein concentrations were measured by at ASPET Journals on October 9, 2021 the method of Lowry et al. (1951).
Muscarinic Receptor Binding Assay. The binding assay for muscarinic receptors was performed using [3H]NMS as previously described (Oki et al., 2004). The homogenates (70-350 µg protein) of rat tissues were incubated with different concentrations (0.06-1.5 nM) of [3H]NMS in 30 mM
Na+/HEPES buffer (pH 7.5). Incubation was carried out for 60 min at 25℃. The reaction was terminated by rapid filtration (Cell Harvester, Brandel Co., Gaithersburg, MD, U.S.A.) 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 2,5-diphenyloxazole, 0.3 g
1,4-bis[2-(5-phenyloxazolyl)]benzene), and the 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 µM atropine. All assays were conducted in duplicate.
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Measurement of Plasma Concentrations of Oxybutynin and DEOB. The measurement of the plasma concentrations of oxybutynin and DEOB was performed in rats following oral administration of oxybutynin hydrochloride (127 µmol/kg) and transdermal oxybutynin (1 patch/body), as described above. After drug administration, rats were killed by 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℃ until analysis. Concentrations of oxybutynin and DEOB in plasma were determined by gas chromatography and mass spectrometry (GC/MS). Briefly, a plasma sample Downloaded from 2 2 (0.1-0.5 ml) was mixed with internal standard ([ H13]oxybutynin・HCl and [ H13]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 1500×g for 5 min, the supernatant was evaporated to dryness jpet.aspetjournals.org under reduced pressure. The residue was dissolved in 100 µL of CH3CN, and 0.5-1 µL 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 at ASPET Journals on October 9, 2021 a LASER JET 4 printer (Hewlett Packard Co.). Chromatographic separation was carried out using a
15 m×0.25 mm i. d.×0.25 µm film UA+-1 HT (FRONTIER LAB LTD.). The carrier gas was helium at a flow rate of 1.0 mL/min. Oven temperature was held at 150℃ for 1 min, then programmed from 150℃ to 220℃ at 20℃/min for first ramp, from 220℃ to 260℃ at 10℃/min for second ramp, and from 260℃ to 300℃ at 30℃/min for third ramp. It was held at 300℃ for 2 min before returning to the initial starting temperature of 150℃. The injection temperature was
200℃. Fragmentation was accomplished by electron impact at 70eV ionizing voltage and 300 µA ionizing current. Selected ion monitoring was performed at m/z 342 (oxybutynin), m/z 355 (internal
2 2 standard: [ H13]oxybutynin), m/z 189 (DEOB), m/z 200 (internal standard: [ H13]DEOB). The limit 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 µmol/kg). Any saliva remaining in the oral cavity was removed
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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 (i.v.) 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 Downloaded from 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. Additionally, the effects of oral
and transdermal administration of oxybutynin on salivary secretion induced by cumulative doses jpet.aspetjournals.org
(0.04 to 123 µmol/kg, i.v.) of pilocarpine in rats given at 5-min intervals were examined.
Data Analysis. Analysis of [3H]NMS binding data was performed as described previously
(Yamada et al., 1980). The apparent dissociation constant (Kd) and maximal number of binding sites at ASPET Journals on October 9, 2021
3 (Bmax) for [ H]NMS were estimated by Rosenthal analysis of the saturation data.
Statistical analysis of the data was performed by a one-way analysis of variance (ANOVA), followed by Dunnett’s test for multiple comparison. The data were expressed as mean±S.E..
Statistical significance was accepted at P<.05.
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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 µmol/kg), there was a
3 significant increase in the Kd values for specific [ H]NMS binding in each tissue of rats compared with the corresponding control values (Table 1). The increases in the Kd 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 Kd Downloaded from values at 3 h produced by oral oxybutynin was significantly (P<.01) greater in the submaxillary gland than in other tissues. There was little significant increase in the Kd values in all these tissues at 12
and 24 h later. In the submaxillary gland and heart of oxybutynin-administered rats, there was a jpet.aspetjournals.org significant (submaxillary gland: 26.7-56.8 %, heart: 13.5-19.5 %) reduction in the Bmax values for
[3H]NMS binding from 1 or 3 to 24 h after oral administration, compared with the corresponding
control values. at ASPET Journals on October 9, 2021
The transdermal application of oxybutynin at 1 patch (33.6 µmol)/body for 2, 4 and 12 h brought
3 about a time-dependent increase in the Kd values for specific [ H]NMS binding in each tissue compared with the corresponding control value, and no further increase was seen at 24 h following application (Table 2). The increase in the Kd values was significant after 2 to 24 h-application in the submaxillary gland and colon and after 4 or 12 to 24 h-application in the heart and bladder. The rate of enhancement in the Kd values after transdermal application of oxybutynin for 2 to 24 h was significantly (P<.01) greater in the submaxillary gland (226-547 %), compared with the bladder
(90.4-102 %), heart (22.2-36.7 %) and colon (38.8-101 %). There was little change in the Bmax value in each tissue by transdermal oxybutynin.
At 11 h after the removal of patch following 12 h-application of transdermal oxybutynin, there was
3 no longer significant change in the Kd values for specific [ H]NMS binding in the bladder (control:
179±3 nM, n=4, oxybutynin: 215±16 nM, n=3) and submaxillary gland (control: 115±3 nM,
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oxybutynin: 144±12 nM).
Following the transdermal application of oxybutynin at 0.5 (16.8 µmol), 1 (33.6 µmol) and 2 (67.2
µmol) patches/body for 24 h, there was a tendency toward a dose-dependent increase in the Kd values for [3H]NMS binding in the submaxillary gland, heart and colon, while the bladder showed a maximum enhancement in the Kd values already at 1 patch/body-application of oxybutynin (Table 3).
The Bmax value at 2 patches/body was significantly increased in the bladder and significantly decreased in the submaxillary gland. Downloaded from Effects of Intravenous Injection of Oxybutynin and DEOB on Muscarinic Receptors in Rat
Tissues. At 1 h after i.v. injection of oxybutynin (7.61 µmol/kg), there was a significant increase in
3 the Kd values for specific [ H]NMS binding in the bladder, submaxillary gland, heart and colon, jpet.aspetjournals.org compared with the corresponding control values (Table 4). A similar degree of the enhancement in the Kd values was also seen by i.v. injection of a comparable dose (8.20 µmol/kg) of DEOB in rat
tissues, except the submaxillary gland that showed a significantly (P<.05) greater (17.7-fold) increase at ASPET Journals on October 9, 2021 than the case (13.6-fold) by the oxybutynin injection. Both oxybutynin and DEOB produced greatest increase in the Kd values in the submaxillary gland among rat tissues. The i.v. injection of oxybutynin
3 had little significant effect on the Bmax values for specific [ H]NMS binding in each tissue, while the injection of DEOB reduced significantly (34.2 %) the Bmax values 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 µmol/kg) reached the maximum levels (oxybutynin: 100±44 nM, DEOB: 107±46 nM, n=8) at 1 h, and then they declined rapidly
(Fig. 2A). The plasma concentrations of oxybutynin at 3 (n=9) and 12 h (n=7) were 50.1±8.9 and
1.52±0.73 nM, respectively, and the concentration of DEOB at 3 h was 83.6±19.5 nM
The plasma concentration of oxybutynin after transdermal application of oxybutynin at 1 patch
(33.6 µmol)/body for 2, 4, 12 and 24 h in rats were 70.8±18.9, 81.0±16.6, 187±20 and 169±18
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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.
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 59.4±
7.5, 169±18 and 370±50 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. Downloaded from The plasma concentration of oxybutynin at 1 h after i.v. injection of this drug (7.61 µmol/kg) in rats was 280±64 nM (n=3), and that of DEOB at 1 h after i.v. injection of this metabolite (8.20
µmol/kg) was 505±46 nM (n=3). In the plasma of rats injected with oxybutynin, DEOB was very jpet.aspetjournals.org 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 at ASPET Journals on October 9, 2021 oral cavity of rats at 10 min intervals until 60 min after the stimulation due to i.v. injection of pilocarpine (4.09 µmol/kg). The whole saliva of control rats reached a maximum 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 688±62, 376±53, 251±36, 156±30, 97.5±20.3 and 46.7
±8.4 mg (n=11), respectively. As shown in Fig. 4, the weights of whole saliva secreted by the first 10 min 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 µmol/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 extremely low levels (6.8±6.8 and 0.8±0.8 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 139±27 and 155±37 mg. As shown in Fig. 4B, transdermal
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application of oxybutynin at 1 patch (33.6 µmol)/body for 4, 12 and 24 h decreased significantly the pilocarpine-stimulated salivation, but the respective weights (154±88, 124±29 and 146±79 mg, respectively, n=4) of whole saliva secreted in these rats were significantly (P<.05) larger than the values at 1 and 3 h after oral oxybutynin (Fig. 4A).
Fig. 5 shows the recovery of pilocarpine-stimulated salivation in rats after oral and transdermal administration of oxybutynin. The pilocarpine-stimulated whole saliva (487±43 mg, n=4) after the 11 h-removal of patch following 12 h-transdermal application of oxybutynin was significantly Downloaded from (P<.001) larger than the whole saliva (139±27 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 jpet.aspetjournals.org pilocarpine-stimulated secretion of whole saliva were examined. The pilocarpine-stimulated secretion of whole saliva was measured 3 h after oral oxybutynin (127 µmol/kg) and after 12 h-application of transdermal oxybutynin (33.6 µmol), when showed maximum enhancement of Kd at ASPET Journals on October 9, 2021 in the submaxillary gland (Tables 1 and 2). Oral administration of oxybutynin shifted largely dose-response curves for pilocarpine (0.04-123 µmol/kg)-stimulated salivation to the right with the concomitant reduction (65.5 %) of maximum response, while 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.
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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 oxybutynin compared with oral oxybutynin was characterized by simultaneously measuring tissue [3H]NMS binding, plasma concentrations of oxybutynin and Downloaded from 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).
3 There was a significant increase in Kd values for [ H]NMS binding in the bladder, submaxillary jpet.aspetjournals.org gland, heart and colon of rats 1 and 3 h after oral administration of oxybutynin (127 µmol/kg), compared with the control values. Similarly, transdermal application of oxybutynin (1 patch: 33.6
3 µmol/body) produced a significant increase in Kd values for [ H]NMS binding in these tissues. at ASPET Journals on October 9, 2021
Given that an increase in Kd 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 Kd value in each tissue was dependent on transdermal application time of 2-12 h, reaching a maximum level at the 12 h-application which was maintained by further 24
3 h-application. Also, the enhancement of Kd values for [ H]NMS binding in the bladder and submaxillary gland following transdermal oxybutynin was promptly disappeared by the removal of patch. These data suggest that transdermally administered oxybutynin binds to muscarinic receptors in rat tissues at 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 Kd values for specific
[3H]NMS binding in the submaxillary gland, heart and colon of rats, while the bladder showed a
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maximum enhancement by 1 patch/body application. Consequently, transdermal application of oxybutynin at 1 patch/body may be sufficient to cause maximum 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 by potentiating the blockade of muscarinic receptors in non-target 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). Downloaded from 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
3 in Bmax values for specific [ H]NMS binding only in the rat submaxillary gland and heart occurred at jpet.aspetjournals.org
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 Bmax
3 values for [ H]NMS binding in rat tissues. Thus, oral but not transdermal oxybutynin produced at ASPET Journals on October 9, 2021 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
15 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
in rats receiving transdermal oxybutynin, and further pilocarpine-induced salivation was promptly recovered by the removal of patch (Fig. 5). Additionally, 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 unsurmountable antagonism agrees well with a sustained decrease of [3H]NMS binding sites (Bmax) 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 Downloaded from 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 jpet.aspetjournals.org 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 Kd values, muscarinic receptor binding activity of both oral at ASPET Journals on October 9, 2021 and transdermal oxybutynin was greatest in the submaxillary gland, followed by the bladder, colon and heart. Our recent data with M1 to M5 subtype knockout mice have shown that M3 subtype is expressed predominantly in the submaxillary gland and moderately in the prostate and bladder, whereas M2 subtype is major subtype present in the bladder, heart and colon (our unpublished observation). Since oxybutynin exerted higher affinity to M3 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 M3 subtype selectivity.
It is known that oxybutynin is rapidly absorbed from gastrointestinal tract and 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
16 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
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 maximum level at 1 h followed by a rapid decline. The plasma concentration of transdermal oxybutynin in rats increased time-dependently until 12 h-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 Downloaded from 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 jpet.aspetjournals.org salivary gland than in the bladder (Waldeck et al., 1997; Oki et al., 2005; Maruyama et al., in press), 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 at ASPET Journals on October 9, 2021
3 of Kd values for specific [ 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 Bmax 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 long-lasting occupation of muscarinic receptors in the exocrine gland. Such 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). Additionally, it is also assumed that a steep increase in the plasma concentration of oxybutynin and DEOB after oral oxybutynin causes unsurmountable
17 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
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 first-pass 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 Downloaded from 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 jpet.aspetjournals.org transdermal over oral oxybutynin in the therapy of overactive bladder.
at ASPET Journals on October 9, 2021
18 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
Acknowledgements
We thank to Mr. Akihiro Kawashima and Dr. Masayuki Uchida (Meiji Milk Products Co. Ltd.,
Odawara, Japan) for providing valuable information. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 9, 2021
19 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
References
Abrams P, Freeman R, Anderstrom C and Mattiasson A (1998) Tolterodine, a new
antimuscarinic agent: as effective but better tolerated than oxybutynin in patients with an
overactive bladder. Br J Pharmacol 81: 801-810.
Andersson KE (1993) Pharmacology of lower urinary tract smooth muscles and penile erectile
tissues. Pharmacol Rev 45: 253-308.
Anderson RU, Mobley D, Blank B, Saltzstein D, Susset J and Brown JS (1999) Once daily Downloaded from
controlled versus immediate release oxybutynin chloride for urge urinary incontinence. J Urol
161: 1809-1812. jpet.aspetjournals.org Appell RA, Chancellor MG, Zobrist RH, Thomas H and Sanders SW (2003) Pharmacokinetics,
metabolism, and saliva output during transdermal and extended-release oral oxybutynin
administration in healthy subjects. Mayo Clin Proc 78: 696-702. at ASPET Journals on October 9, 2021 Beauchamp HT, Chang RSL, Sigel PKS and Gibson RE (1995) In vivo receptor occupancy of the
angiotensin II receptor by nonpeptide antagonists: relationship to in vitro affinities and in vivo
pharmacologic potency. J Pharmacol Exp Ther 272: 612-618.
Bulmer P and Abrams P (2000) The overactive bladder. Rev Contemp Pharmacother 11: 1-11.
Chapple CR (2000) Muscarinic receptor antagonists in the treatment of overactive bladder. Urol
55: 33-46.
Davila GW, Daugherty CA and Sanders SW for the transdermal oxybutynin study group (2001)
A short-term, multicenter, randomized double-blind dose titiration study of the efficacy and
anticholinergic side effects of transdermal compared to immediate release oral oxybutynin
treatment of patients with urge urinary incontinence. J Urol 166:140-145.
Dmochowski RR, Sand PK, Zinner NR, Gittelman MC, Davila GW, and Sanders SW, for the
trandermal oxybutynin study group (2003) Comparative efficacy and safety of transdermal
20 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
oxybutynin and oral tolterodine versus placebo in previously treated patients with urge and
mixed urinary incontinence. Urol 62:237-242.
Douchamps J, Derenne F, Stoickis A, Gangji D, Juvent M and Herchuelz A (1988) The
pharmacokinetics of oxybutynin in man. Eur J Clin Pharmacol 35: 515-520.
Gupta SK and Sathyan G (1999) Pharmacokinetics of an oral once-a-day controlled-release
oxybutynin formulation compared with immediate-release oxybutynin. J Clin Pharmacol 39:
289-296. Downloaded from
Hughes KM, Lang JCT, Lazare R, Gordon D, Stanton SL, Malone-Lee J and Geraint M (1992)
Measurement of oxybutynin and its N-desethylmetabolite in plasma, and its application to jpet.aspetjournals.org pharmacokinetic studies in young, elderly and frail elderly volunteers. Xenobio 22: 859-869.
Ikeda K, Kobayashi S, Suzuki M, Miyata K, Takeuchi M, Yamada T and Honda K (2002) M3 receptor antagonism by the novel antimuscarinic agent solifenacin in the urinary bladder and at ASPET Journals on October 9, 2021 salivary gland. Naunyn Schmiedebergs Arch Pharmacol 366: 97-103.
Lowry OH, Rosebrough NJ, Farr AL and Randall RJ (1951) Protein measurement with the Folin
phenol reagent. J Biol Chem 193: 265-275.
Maruyama S, Oki T, Otsuka A, Shinbo H, Ozono S, Kageyama S, Mikami Y, Araki I, Takeda M,
Masuyama K and Yamada S Human muscarinic receptor binding characteristics of
antimuscarinic agents to treat overactive bladder. J Urol (in press)
Mayuzumi K, Watanabe K, Tamaru K and Kasama T (1986) Effects of oxybutynin hydrochloride
on the urinary bladder and urethra in in situ preparations. Folia Pharmacol Jpn 87: 557-571.
Nilvebrant L and Sparf B (1982) Different affinities of some anticholinergic drugs between the
parotid gland and ileum. Scand J Gastroenterol 72: 69-77.
Nilvebrant L, Andersson KE, Gillberg PG, Stahl M and Sparf B (1997) Tolterodine—a new
bladder-selective antimuscarinic agent. Eur J Pharmacol 327: 195-207.
21 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
Ohkura T, Yamada S, Deguchi Y, Kimura R, Matsushima H, Higuchi S, Inagaki O, Honda K and
Takenaka T (1998) Ex vivo occupancy by tamslosin of α1-adrenoceptors in rat tissues in relation to the plasma concentration. Life Sci 63: 2147-2155.
Oki T, Kimura R, Saito M, Miyagawa I and Yamada S (2004) Demonstration of bladder selective
muscarinic receptor binding by intravesical oxybutynin to treat overactive bladder. J Urol
172: 2059-2064.
Oki T, Kawashima A, Uchida M and Yamada S (2005) In vivo demonstration of muscarinic Downloaded from receptor binding activity of N-desethyl-oxybutynin, active metabolite of oxybutynin. Life Sci
76: 2445-2456. jpet.aspetjournals.org Resnick NM and Yalla SV (1985) Management of urinary incontinence in the elderly. New Eng J
Med 313: 800-805.
Sathyan G, Chancellor MB and Gupta SK (2001) Effect of OROSⓇ controlled-release delivery at ASPET Journals on October 9, 2021 on the pharmacokinetics and pharmacodynamics of oxybutynin chloride. Br J Clin
Pharmacol 52: 409-417.
Terai T, Deguchi Y, Ohtsuka M and Kumada S (1991) Effects of the anticholinergic drug
prifinium bromide on urinary bladder contractions in rat in vivo and in guinea-pig in vitro.
Arzneim-forsch/Drug Res 41: 417-420.
Uchida S, Yamada S, Ohkura T, Heshikiri M, Yoshimi A, Shirahase H and Kimura R (1995) The
receptor occupation and plasma concentration of NKY-722, a water-soluble
dihydropyridine-type calcium antagonist, in spontaneously hypertensive rats. Br J Pharmacol
114: 217-223.
Waldeck K, Larsson B and Andersson K-E (1997) Comparison of oxybutynin and its metabolite,
N-desethyl-oxybutynin, in the human detrusor and parotid gland. J Urol 157: 1093-1097.
Yamada S, Yamamura HI and Roeske WR (1980) Characterization of alpha-1 adrenergic
22 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
receptors in the heart using [3H]WB4101: Effect of 6-hydroxydomine treatment. J Pharmacol
Exp Ther 215: 176-185.
Yamada S, Isogai M, Kawabe Y, Takayanagi N, Hayashi E, Tsuji K and Kosuge T (1985) Brain
nicotinic acetylcholine receptors. Biochemical characterization by neosurugatoxin. Mol
Pharmacol 28: 120-127.
Yamada S, Ohkura T, Kimura R and Kawabe K (1998) In vivo receptor binding of novel
α1-adrenoceptor antagonists for treatment of benign prostatic hyperplasia. Life Sci 62: Downloaded from 1585-1589.
Yamada S, Ohkura T, Deguchi Y and Kimura R (1999) In vivo measurement by [3H]tamusulosin jpet.aspetjournals.org of α1 adrenoceptors in rat tissues in relation to the pharmacokinetics. J Pharmacol Exp Ther 289: 1575-1583.
Yamada S, Okura T and Kimura R (2001) In vivo demonstration of α1A-adrenoceptor subtrype at ASPET Journals on October 9, 2021 selectivity of KMD-3213 in rat tissues. J Pharmacol Exp Ther 296: 160-167.
Yamada S, Kusaka T, Urayama A, Kimura R and Watanabe Y (2003) In vitro and ex vivo effects
of a selective nociceptin/orphan FQ(N/OFQ) peptide receptor antagonist, CompB, on specific
binding of [3H]N/OFQ and [35S]GTPγS in rat brain and spinal cord. Br J Pharmacol 139:
1462-1468.
Yarker YE, Goa KL and Fitton A (1995) Oxybutynin. A review of its pharmacodynamic and
pharmacokinetic properties, and its therapeutic use in detrusor instability. Drug & Aging 6:
243-262.
Zobrist RH, Quan D, Thomas HM, Stanworth S and Sanders SW (2003) Pharmacokinetics and
metabolism of transdermal oxybutynin: in vitro and in vio performance of a novel delivary
system. Pharm Res 20: 103-109.
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Footnotes
a) This work was supported in part by a Grant-in-Aid 11672271 for Scientific Research from the
Ministry of Education, Science and Culture of Japan.
b) Reprint requests: Shizuo Yamada, Ph.D., Department of Pharmacokinetics and
Pharmacodynamics and Center of Excellence Program in the 21st Century, School of
Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. Downloaded from
(E-mail: [email protected]) jpet.aspetjournals.org at ASPET Journals on October 9, 2021
24 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
Legends for figures
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 cm2 and containing 33.6 µmol (12 mg) of oxybutynin free base. Oxybutynin was transdermally administered to the shaved backs of rats after peeling off the tab.
Fig. 2. Plasma concentration-time profiles of oxybutynin (●) and DEOB (△) after oral (A) and Downloaded from transdermal (B) administration of oxybutynin in rats. Rats received oral oxybutynin (127 µmol/kg) orally or transdermal oxybutynin (1 patch: 33 µmol/body), and were then sacrificed.
Blood samples were taken from the descending aorta of each rat. Each point represents the jpet.aspetjournals.org mean±S.E. of 7 to 9 (oral oxybutynin) and 9 (transdermal oxybutynin) rats.
Fig. 3. Concentrations of oxybutynin in plasma after transdermal application of oxybutynin at 0.5 at ASPET Journals on October 9, 2021 to 2 patches/body for 24 h in rats. Rats received transdermal oxybutynin at 0.5 (16.8 µmol), 1 (33.6 µmol) or 2 (67.2 µmol) 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 9 (0.5 patch), 9 (1 patch) and 10 (2 patch) rats.
Fig. 4. Effects of oral (A) and transdermal (B) administration of oxybutynin on pilocarpine-induced salivary secretion in rats. Rats received oral oxybutynin (127 µmol/kg) or transdermal oxybutynin (1 patch: 33.6 µmol/body), and the whole salivary secretion was measured for first 10 min after pilocarpine stimulation (4.09 µmol/kg, i.v.). Open columns (A, B): control (vehicle-administered) rats. Solid columns (A) : oral oxybutynin-administrated rats. Dotted columns (B) : transdermal oxybutynin-administered rats. Each column represents the mean±S.E. of 4 (transdermal oxybutynin), 5 (oral oxybutynin) and 6 (control) rats. Asterisks show a significant difference from control values, *P<.05, **P<.01, ***P<.001.
Fig. 5. Recovery of pilocarpine-induced salivary secretion after oral (A) and transdermal (B)
25 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
administration of oxybutynin in rats. (A) Rats received oral oxybutynin (127 µmol/kg), and at 1 h or 12 h later, the whole salivary secretion was measured for first 10 min after pilocarpine stimulation (4.09 µmol/kg, i.v.). (B) Rats received transdermal oxybutynin (1 patch: 33.6 µmol/body) for 12 h, and before and 11 h after the removal of patch, the whole salivary secretion was measured for first 10 min after pilocarpine stimulation (4.09 µmol/kg, i.v.). Open columns (A, B): control (vehicle-administered) rats. Solid columns (A) : oral oxybutynin-administrated rats. Dotted columns (B) : transdermal oxybutynin-administered rats. Each column represents the mean±S.E. of 4 (transdermal oxybutynin), 5 (oral oxybutynin) and 9 (control) rats. Asterisks Downloaded from show a significant difference from control values, *P<.05, ***P<.001.
Fig. 6. Effects of oral and transdermal administration of oxybutynin on dose-response curves of jpet.aspetjournals.org pilocarpine-stimulated salivary secretion in rats. Rats received intravenously cumulative (5 min intervals) doses (0.04-123 µmol/kg) of pilocarpine 3 h after oral administration of oxybutynin (127 µmol/kg) and after 12 h-transdermal application of oxybutynin (1 patch: 33.6 µmol/body),
and the secreted whole saliva were measured. ●: control, ■: oral oxybutynin, ▲: transdermal at ASPET Journals on October 9, 2021 oxybutynin. Each point represents the mean±S.E. of 3 (oral oxybutynin), 3 (transdermal oxybutynin) and 6 (control) rats. Symbols show a significant difference from control value, †††P<.001. Asterisks show a significant difference from the transdermal oxybutynin, *P<.05, **P<.01, **P<.001.
26 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
TABLE 1
3 Kd and Bmax for specific [ 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 µmol/kg of oxybutynin orally, and were then sacrificed 1 to 24 h after administration. Specific binding of [3H]NMS (0.06-1.5 nM) in rat tissues was measured. Values are mean±S.E. of 10 (control) and 4 to 6 (oxybutynin) rats. Values in parentheses represent the Downloaded from fold-increase in Kd values relative to controls. Asterisks show a significant difference from the control values, *P<.05, **P<.01, ***P<.001.
jpet.aspetjournals.org Organs Time after oral oxybutynin Kd Bmax (h) (pM) (fmol/mg protein) Bladder Control 167 ± 5 158 ± 7 1 258 ± 34 (1.54)* 170 ± 34 3 359 ± 61 (2.15)*** 138 ± 14
12 189 ± 5 112 ± 13 at ASPET Journals on October 9, 2021 24 202 ± 10 129 ± 10 Submaxillary gland Control 108 ± 3 126 ± 4 1 377 ± 142 (3.49)* 92.3 ± 9.6* 3 840 ± 166 (7.78)*** 67.8 ± 6.8*** 12 111 ± 8 54.4 ± 5.7*** 24 123 ± 9 63.6 ± 13.6*** Heart Control 270 ± 8 71.1 ± 1.8 1 362 ± 52 (1.34)* 67.0 ± 2.4 3 349 ± 18 (1.29)* 61.5 ± 2.3* 12 245 ± 25 57.2 ± 1.3** 24 253 ± 9 58.6 ± 3.6** Colon Control 152 ± 4 115 ± 9 1 294 ± 28 (1.93)** 119 ± 5 3 370 ± 57 (2.43)*** 120 ± 12 12 191 ± 30 106 ± 7 24 170 ± 8 117 ± 5
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TABLE 2
3 Kd and Bmax for specific [ H]NMS binding in the bladder, submaxillary gland, heart and colon of rats after transdermal application of oxybutynin for 2 to 24 h
Rats received transdermal oxybutynin (1 patch: 33.6 µmol/body) for 2 to 24 h, and were then sacrificed. Specific binding of [3H]NMS (0.06-1.5 nM) in rat tissues was measured. Values are mean±S.E. of 10 (control) and 4 to 5 (oxybutynin) rats. Values in parentheses represent the Downloaded from fold-increase in Kd values relative to controls. Asterisks show a significant difference from the control values, **P<.01, ***P<.001.
jpet.aspetjournals.org Organs Application time Kd Bmax (h) (pM) (fmol/mg protein) Bladder Control 167 ± 5 158 ± 7 2 215 ± 17 157 ± 14 4 247 ± 18 154 ± 20 ± ± 12 337 26 (2.02)*** 161 12 at ASPET Journals on October 9, 2021 24 318 ± 57 (1.90)*** 156 ± 8 Submaxillary gland Control 108 ± 3 126 ± 4 2 393 ± 60 (3.64)*** 123 ± 4 4 352 ± 66 (3.26)*** 117 ± 10 12 699 ± 89 (6.47)*** 108 ± 9 24 689 ± 15 (6.38)*** 120 ± 10 Heart Control 270 ± 8 71.1 ± 1.8 2 301±19 62.7 ± 3.7 4 335 ± 17 (1.24)** 68.4 ± 4.3 12 369 ± 11 (1.37)*** 67.5 ± 4.6 24 330 ± 14 (1.22)** 69.7 ± 0.9 Colon Control 152 ± 4 115 ± 9 2 233 ± 20 (1.53)*** 113 ± 7 4 211 ± 15 (1.39)** 118 ± 6 12 305 ± 22 (2.01)*** 126 ± 12 24 257 ± 17 (1.69)*** 117 ± 5
28 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
TABLE 3
3 Kd and Bmax for specific [ 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 µmol), 1 (33.6 µmol) or 2 (67.2 µmol) patches/body for 24 h, and were then sacrificed. Specific binding of [3H]NMS (0.06-1.5 nM) in rat tissues was measured. Values are mean±S.E. of 10 (control) and 4 to 5 (oxybutynin) rats. Downloaded from Values in parentheses represent the fold-increase in Kd values relative to controls. Asterisks show a significant difference from the control values, *P<.05, **P<.01, ***P<.001.
jpet.aspetjournals.org Organs Doses Kd Bmax (patches/body) (pM) (fmol/mg protein) Bladder Control 167 ± 5 158 ± 7 0.5 218 ± 13 181 ± 9 1 318 ± 57 (1.90)*** 156 ± 8 ± ± 2 302 22 (1.81)** 191 11* at ASPET Journals on October 9, 2021 Submaxillary gland Control 108 ± 3 126 ± 4 0.5 372 ± 61 (3.44)** 112 ± 7 1 689 ± 18 (6.38)*** 120 ± 10 2 1008 ± 97 (9.33)*** 68.7 ± 4.8*** Heart Control 270 ± 8 71.1 ± 1.8 0.5 284 ± 16 63.0 ± 2.3 1 330 ± 14 69.7 ± 0.9 2 451 ± 41 (1.67)*** 67.4 ± 4.0 Colon Control 152 ± 4 115 ± 9 0.5 221 ± 6 (1.45)** 127 ± 3 1 257 ± 17 (1.69)*** 117 ± 5 2 346 ± 24 (2.28)*** 120 ± 6
29 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version. JPET #94508
TABLE 4
3 Kd and Bmax for specific [ 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 µmol/kg) and DEOB (8.20 µmol/kg) intravenously, and at 1 h later, they were sacrificed. Specific binding of [3H]NMS (0.06-2.0 nM) in rat tissues was measured. Values are mean±S.E. of 7 (control) and 4 (oxybutynin, DEOB) rats. Values in Downloaded from parentheses represent the fold-increase in Kd values relative to controls. Asterisks show a significant difference from the control values, **P<.01, ***P<.001.
jpet.aspetjournals.org Organs Drug Kd Bmax (pM) (fmol/mg protein) Bladder Control 176 ± 5 154 ± 16 Oxybutynin 452 ± 83 (2.57)** 140 ± 19 DEOB 390 ± 45 (2.22)** 114 ± 13 ± ± Submaxillary gland Control 96.3 3.6 108 8 at ASPET Journals on October 9, 2021 Oxybutynin 1309 ± 136 (13.6)*** 84.3 ± 3.7 DEOB 1704 ± 96 (17.7)*** 71.1 ± 4.0** Heart Control 230 ± 13 60.8 ± 4.4 Oxybutynin 466 ± 43 (2.03)*** 48.2 ± 2.4 DEOB 451 ± 31 (1.96)*** 52.1 ± 4.2 Colon Control 171 ± 19 96.7 ± 7.6 Oxybutynin 420 ± 10 (2.46)*** 86.9 ± 6.5 DEOB 442 ± 22 (2.58)*** 91.4 ± 7.8
30 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version.
Figure 1
(Top view)
Backing Film Downloaded from (Side view) Matrix Adhesive Layer
Overlapped Release Liner Strip jpet.aspetjournals.org at ASPET Journals on October 9, 2021 JPET Fast Forward. Published on November 10, 2005 as DOI: 10.1124/jpet.105.094508 This article has not been copyedited and formatted. The final version may differ from this version.
Figure 2
250 (A)
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Figure 3
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Figure 4
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Figure 5
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Time after oral administration at ASPET Journals on October 9, 2021 of oxybutynin
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Figure 6
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Pilocarpine doses ( mol/kg, iv) at ASPET Journals on October 9, 2021