Urodynamic and Immunohistochemical Evaluation of Intravesical Capsaicin Delivery Using Thermosensitive Hydrogel and Liposomes

0022-5347/04/1711-0483/0 THE JOURNAL OF UROLOGY® Copyright © 2004 by AMERICAN UROLOGICAL ASSOCIATION

Vol. 171, 483– 489, January 2004 Printed in U.S.A.

DOI: 10.1097/01.ju.0000102360.11785.d7

URODYNAMIC AND IMMUNOHISTOCHEMICAL EVALUATION OF INTRAVESICAL CAPSAICIN DELIVERY USING THERMOSENSITIVE HYDROGEL AND LIPOSOMES PRADEEP TYAGI, MICHAEL B. CHANCELLOR,* ZHENHUA LI, WILLIAM C. DE GROAT,† NAOKI YOSHIMURA,‡ MATTHEW O. FRASER§ AND LEAF HUANG¶, 㛳 From the Center of Pharmacogenetics, School of Pharmacy, Departments of Pharmacology and Urology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania

ABSTRACT

Purpose: Aqueous insolubility of the vanilloids such as capsaicin is a major disincentive in their intravesical therapy of detrusor hyperreflexia. We sought to overcome the delivery of this hydrophobic neurotoxin by entrapping it in a lipid bilayer of positively charged multilamellar lipid vesicles (liposomes) or in a hydrophobic polymer matrix of thermosensitive hydrogel. Materials and Methods: Liposomes, hydrogel and 30% ethanol/normal saline were prepared with or without 1 mM capsaicin (0.5 ml) and administered intravesically for 30 minutes to 7 groups of age matched, normal female adult Sprague-Dawley rats under halothane anesthesia. At 48 hours after intravesical instillation cystometric studies were performed using urethane anesthesia (0.04 ml per minute). The animals were subsequently sacrificed and whole bladders were harvested for histology and immunohistochemistry. Results: In normal urethane anaesthetized rats capsaicin in 30% ethanol and liposomes completely blocked micturition reflexes. Capsaicin in hydrogel did not completely block the micturition reflex but it significantly decreased bladder contraction frequency compared with vehicle controls. The results of cystometry with capsaicin in liposomes and capsaicin in 30% ethanol correlated with a significant decrease in calcitonin gene-related peptide staining of afferent nerves in the bladder wall. Photographs taken after hematoxylin and eosin staining of the bladder treated with liposomes and hydrogel in the absence of capsaicin did not reveal any adverse histological changes. There were significant histological changes in bladders treated with 30% ethanol alone. Conclusions: In comparison with 30% ethanol liposomes are a superior vehicle for the intravesical administration of capsaicin, producing comparable efficacy with less tissue damage. Hydrogel can also serve as safe alternative option for capsaicin delivery. KEY WORDS: bladder; rats, Sprague-Dawley; liposomes; capsaicin; hydrogel

Capsaicin, the pungent chemical in red pepper, has been used in urology to treat voiding dysfunction and bladder pain.1 The action of capsaicin results from its action on vanilloid receptors (VR-1), which are expressed on afferent nerves and urothelial cells.2 VR-1 activation produces a biphasic response with initial stimulation driven by the influx of Na⫹ and Ca2⫹ ion, and delayed desensitization underlying the paradoxical analgesia produced by capsaicin.3, 4 C-fiber afferents involved in the micturition reflex are believed to be silent under normal conditions but they are activated after bladder irritation and spinal cord injury, causing neurogenic detrusor overactivity.5 Successful treatment of neurogenic in-

continence with intravesical capsaicin or its ultrapotent nonpungent analogue resinferatoxin1, 6 has revealed the role of capsaicin sensitive C-fibers in the triggering of bladder hyperactivity and bladder pain.6, 7 Chemically capsaicin is 8-methyl-N-vanilyl-6-nonenamide, a derivative of homovanillic acid with prominent features. Namely an aromatic ring, an amide bond and a hydrophobic side chain in the chemical structure of capsaicin make it a highly hydrophobic molecule that is insoluble in water (fig. 1). Therefore, for intravesical application capsaicin is usually formulated in normal saline solution (NSS) using 30% ethanol.8 This vehicle has been reported to aggravate the adverse histological changes such as epithelium thinning and submucosal edema produced by capsaicin.9 Thus, there is a need for better and safer vehicles for the water insoluble vanilloids. The undesirable use of ethanol to dissolve vanilloids prompted the current investigation into the potential of hydrogel and liposomes as alternative vehicles to NSS. The hydrophobic nature of capsaicin makes it amenable for entrapment in the lipid bilayer of liposomes. The success of creams and gels as vehicles of capsaicin in topical treatment of peripheral neuropathy and herpes zoster neuralgia guided us in considering hydrogel as an alternative vehicle for intravesical capsaicin.10 A polymer suitable for intravesical administration of capsaicin should be in the fluid state at instillation and change to the gel state after instillation. The triblock thermosensitive polymer polyethylene glycol (PEG)-

Accepted for publication July 11, 2003. Supported by the Fishbein Family CURE-IC. * Financial interest and/or other relationship with Ortho McNeil, Pfizer, ICOS, Lilly, Watson, Cook and Yamanouchi. † Financial interest and/or other relationship with Abbott Pharmaceuticals, Pharmacia, Roche Bioscience, Pfizer, Glaxo SmithKline and Redordati Pharmaceuticals. ‡ Financial interest and/or other relationship with Taiho Pharmaceuticals, Kyowa Hakko, Pfizer UK, Eisai Pharmaceuticals and Dynogen. § Financial interest and/or other relationship with Dynogen Pharmaceuticals. ¶ Financial interest and/or other relationship with BioMet and Conrex Pharmaceuticals. 㛳 Correspondence: Center for Pharmacogenetics, School of Pharmacy, University of Pittsburgh, 633 Salk Hall, Pittsburgh, Pennsylvania 15213 (telephone: 412-648-9667; FAX: 412-648-1664; e-mail: [email protected]). 483

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Statistical analysis. Quantitative data are expressed as the mean ⫾ SE. Multiple comparisons among the different groups were analyzed by single-factor ANOVA, followed by post hoc comparisons with the Newman Keuls test using Prism, version 3.0 (GraphPad Software, San Diego, California). Differences among groups were considered significant at p ⬍0.05. FIG. 1. Chemical structure of capsaicin showing aromatic ring, amide bond and hydrophobic side chain.

polylactic-co-glycolic acid (PLGA)-PEG selected for our experiments converts to gel due to sol-gel transformation at body temperature.11 The polymer accomplishes the twin goals of semisolid consistency after it is inside the bladder and the ability to be injected through a PE-50 (Clay-Adams, Parsippany, New Jersey) catheter. METHODS

Preparation of liposome. Liposomes were prepared as previously reported from our laboratory.12 Phosphatidylcholine, cholesterol and capsaicin in a 2:1:1 mole ratio were coated inside the glass tube and dried in a vacuum overnight to form a thin film inside the tube. The lipids were hydrated with normal saline the next day to form multilamellar vesicles. Preparation of hydrogel. The required amount of PEGPLGA-PEG polymer was dispersed in 0.1 M phosphate buffer, pH 7.4, to form a 30% weight per volume aqueous dispersion at room temperature. Aqueous dispersion of the polymer was prepared by constant vortex at room temperature. The prepared polymer dispersion was then added to the glass tube containing 1 mM capsaicin with stock solution solvent ethanol previously removed by air drying. Preparation of ethanolic solution. Capsaicin was added from its stock solution to normal saline containing 30% ethanol to produce 1 mM capsaicin solution. Instillation of drugs. Capsaicin (1 mM) entrapped in lipid bilayer of liposome and dispersed in the polymer or dissolved in ethanolic saline was instilled intravesically into female Sprague-Dawley rats weighing 200 to 300 gm (8 per group) under halothane anaesthesia. The volume of intravesical instillation was 0.5 ml for each formulation of capsaicin. Subsequent to instillation the urethra was ligated to prevent evacuation and allow enough time for gel formation in the bladder. Dwell time for all instillations was 30 minutes. Subsequently the bladders were emptied by pressing the lower abdomen and then washed with 0.5 ml saline. All animals were also treated with Pen-strep (HyClone, Logan, Utah) antibiotic (30 mg/kg subcutaneously) to prevent infection. Cystometry. Animals were anesthetized with urethane (1.2 gm/kg subcutaneously) before transurethral cystometry, 48 hours after intravesical instillation. PE-50 tubing was inserted into the bladder through the urethra. Using a 3-way stopcock the catheter was connected to a pressure transducer for recording intravesical pressure and to a syringe pump for infusing saline into the bladder. The catheter system was filled with 0.9% weight per volume saline. After the bladder was emptied a cystometrogram (CMG) was performed by filling with a constant infusion (0.04 ml per minute) of saline. The amplitude, pressure threshold and frequency of reflex bladder contractions per minute were recorded. Measurements in each animal represented the average of 3 to 5 bladder contractions. Histopathological analysis. After cystometry whole bladders were harvested, fixed in 10% buffered formalin and cryopreserved. Tissue blocks were blind coded and sectioned (20 ␮m) for hematoxylin and eosin staining, and immunohistochemistry for calcitonin gene-related peptide (CGRP) staining.

RESULTS

Effects of intravesical capsaicin on CMG. In cystometric studies of rats under urethane anesthesia with 30% ethanol and liposome as vehicles capsaicin was able to produce a blockade of the micturition reflex (fig. 2, F and G). The absence of periodic bladder contractions in the CMG represented blockade of the micturition reflex following capsaicin treatment and the raised plateau in intravesical pressure reflected urinary retention. Mean bladder contraction frequency was considered zero for such animals, which was observed in 6 and 4 rats in the capsaicin treated groups using 30% ethanol in NSS and liposomes, respectively (8 per group). The remaining rats in the capsaicin treated groups using ethanol in NSS or liposomes showed decreased mean bladder contraction frequency with no significant difference between the 2 groups (0.01 ⫾ 0.006 vs 0.01 ⫾ 0.007, p ⬎0.05). Figure 2 shows representative CMG tracings from various groups. Hydrogel capsaicin significantly decreased mean bladder contraction frequency compared with hydrogel alone (0.10 ⫾ 0.021 vs 0.25 ⫾ 0.033) and a similar significant difference in mean bladder contraction frequency was observed in ethanol in NSS treated groups in the presence and absence of capsaicin (0.01 ⫾ 0.00654 vs 0.12 ⫾ 0.021, fig. 3). However, liposome treated groups failed to show a significant difference in mean bladder contraction frequency in the presence and absence of capsaicin (0.01 ⫾ 0.007 vs 0.08 ⫾ 0.025). The mean bladder contraction frequency of hydrogel treated rats in the absence of capsaicin was lower but not significantly different from that in saline treated rats (0.25 ⫾ 0.033 vs 0.28 ⫾ 0.02491, p ⬎0.05). Other CMG parameters, such as the pressure threshold and amplitude of bladder contractions, were not affected by capsaicin treatment in various vehicles (data not shown). Gross bladder morphology. None of the animals in our study showed any signs of urinary tract infection after instillation of the intravesical solutions. However, bladders treated with 30% ethanol showed sign of severe redness throughout the bladder tissue, which turned to bleeding within the walls and ulceration at the bladder lumen in the presence of capsaicin, in marked contrast to untreated bladders (fig. 4, A, C and D). Only blood vessel dilatation at the bladder dome was visible in other vehicle treated groups in the absence of capsaicin, which become more prominent in the presence of capsaicin (fig. 4, B and E to H). Immunohistochemistry. CGRP staining was performed to assess CGRP depletion by capsaicin. We found that capsaicin caused significant depletion of CGRP with liposomes and ethanol as vehicles (fig. 5, B and D). However, capsaicin in hydrogel failed to produce the depletion observed in the liposome and ethanol treatment groups (fig. 5, F). Effect on bladder histology. Hydrogel and liposomes without capsaicin demonstrated similar bladder mucosal histology (fig. 6, C and E). Intravesical instillation of ethanol alone revealed distinct histological changes, including thinning and denuding of the epithelium, submucosal edema and vascular congestion (fig. 6, A). These changes were further aggravated by capsaicin and acute mucosal injury was visible in the capsaicin plus 30% ethanol in normal saline treatment group (fig. 6, B). On comparison histological changes produced by capsaicin delivered using liposome and hydrogel appeared mild with urothelium remaining intact in presence of capsaicin (fig. 6, C to F).

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FIG. 2. Representative CMG tracings in various groups. A, periodic micturition events in normal saline treated rats under urethane anesthesia. B and C, dissimilar effects of ethanol and liposomes on bladder afferents by decrease in bladder contraction frequency in 30% ethanol and liposomes treated rats, respectively, in absence of capsaicin. D and E, decrease in bladder contraction frequency in presence of capsaicin in hydrogel treated rats in absence and presence of capsaicin, respectively. F and G, complete blockade of micturition reflex in rats treated with liposomes and 30% ethanol, respectively, in presence of capsaicin. Raised plateau of bladder contraction pressure reflects urinary retention.

FIG. 3. Bladder contraction frequency 48 hours after administration of capsaicin in various vehicles. All capsaicin treated groups showed significant difference from saline treated group (p ⬍0.05). Hydrogel in absence of capsaicin vs saline, ethanol vs liposome in presence of capsaicin and liposome in presence vs absence of capsaicin were not significant (p ⬎0.05).

DISCUSSION

Traditional anticholinergic therapies for hyperactive bladder target the efferent branch of the micturition reflex. Treatment of the afferent branch of the micturition reflex with C-fiber neurotoxin capsaicin is an attractive alternative that

would avoid systemic anticholinergic side effects. The role of capsaicin sensitive bladder afferents in micturition control and bladder irritation is collectively supported by close apposition of VR-1 expressing fibers with bladder smooth muscle cells as well as urothelial expression of VR-1.2, 13 Intravesical instillation of capsaicin and resiniferatoxin for the treatment of detrusor overactivity not only decreases the problem of systemic neurological toxicity, which is an intrinsic property of neurotoxins,14 but also eliminates their significant first pass effect.15 The currently accepted vehicle for intravesical capsaicin is 30% ethanol in saline. Unfortunately the ethanol vehicle alone was observed to be just as irritating to the bladder mucosa as capsaicin in a study in spinal cord injured patients.16 The pain and autonomic dysreflexia reported by some patients during and sometimes after instillations has hindered the wider application of the clinical use of intravesical vanilloid. In the current study normal adult female rats were used to investigate the efficacy of capsaicin entrapped in the lipid bilayer of liposomes or multilamellar vesicles and in the hydrophobic matrix of thermosensitive hydrogel. Micturition in such rats with an intact neuraxis depends on a spinobulbospinal reflex activated by A␦ bladder afferents and their use as an animal model for the comparison of efficacy for capsaicin formulations under urethane anesthesia is based on extensive information provided by groups at various laboratories. Maggi et al reported that the facilitatory action of capsaicin sensitive nerves on the micturition threshold is more evident in anesthetized than in awake rats and capsaicin resistant bladder afferents are more sensitive to the depressant action of urethane than capsaicin sensitive afferents.17 Electrophysiological experiments showed that the micturition reflex triggered by bladder distention during

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FIG. 4. Dissecting microscope photographs show gross bladder morphology of previously fixed rat bladders 48 hours after administration of capsaicin in various vehicles. A, untreated bladder. B, saline treated bladder. C and D, 30% ethanol in NSS treatment group in absence and presence of 1 mM capsaicin, respectively. E and F, liposome treated group in the absence and presence of 1 mM capsaicin, respectively. G and H, hydrogel treated group in absence and presence of 1 mM capsaicin, respectively. Reduced from ⫻1.

CMG performed using urethane anesthesia requires activation of a supraspinal reflex pathway.18 A recent study of Chuang et al showed that the afferent limb of the micturition reflex under urethane anesthesia can be blocked by intravesical administration of resiniferatoxin, another vanilloid drug.19 Given this knowledge of the micturition control in normal rats under urethane anesthesia, we expected to see blockade of the micturition reflex as a successful end point of capsaicin delivery from various vehicles. Moreover, by using normal rats we can avoid the variable influence of disease on the

uptake of capsaicin from the different delivery systems. Damaged urothelium is more permeable than normal urothelium and it is possible that pathological conditions induce variation in a study designed to examine capsaicin uptake from various vehicles. Aqueous solutions of poly(ethylene glycol)-b-poly (DL-lactic acid-co-glycolic acid)-b-poly(ethylene glycol) PEG-PLGAPEG triblock copolymers form a free flowing sol at room temperature, which becomes a gel at body temperature.20 The amphiphilic nature of the polymer used in our study might be helpful in the permeation of drug substances

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FIG. 5. Photographs of CGRP staining in rat bladder 48 hours after administration of capsaicin in various vehicles. Primary rabbit polyclonal antibody bound to CGRP containing nerve fibers was localized by cyanine-3 fluorescent labeled secondary goat antibody. CGRP fibers were visible as bright red lines against dull red tissue autofluorescence (arrows). A, untreated control. B, 30% ethanol in NSS treatment group in presence of 1 mM capsaicin. C and D, liposome treated groups in absence and presence of 1 mM capsaicin, respectively. E and F, hydrogel treated group in absence and presence of 1 mM capsaicin, respectively. Reduced from ⫻10.

through biological membranes. The slow erosion of this material in aqueous environments gives it a biodegradable and ultimately a more biocompatible nature. We chemically modified this polymer to improve the robustness of the hydrogel to withstand urine constituents such as urea and electrolytes. We compared the effect of hydrogel and liposomes containing capsaicin with that of the usual vehicle (30% alcohol in saline) as a positive control in our study. The 3 vehicles (liposomes, hydrogel and 30% ethanolic saline) were also tested in our study in the absence of any capsaicin. CMGs done 48 hours following intravesical instillation demonstrated that liposomal capsaicin was as successful as ethanolic capsaicin in blocking the micturition reflex in urethane anesthetized normal rats. In a previous study a large dose of capsaicin (50 mg/kg subcutaneously) elicited an acute block of bladder activity that persisted for 8 to 15 hours.21 The longer duration of the capsaicin effect (48 hours) reported in the current study might be explained by the high local concentration of capsaicin following intravesical administration, which would also

prevent the first pass effect. The prolonged desensitizing effect of capsaicin in the current series corroborates the earlier report documenting initial excitation produced by capsaicin following its intravesical delivery using liposomes of similar composition.22 As previously noted at our laboratory,12 liposomes alone in the absence of capsaicin were able to decrease bladder contraction frequency in normal rats and in a bladder injury model. Due to the lack of any previous studies in the literature on the interaction of liposomes with urothelial surface at this time we can only speculate on this observation in normal rats. Perhaps liposome treatment produces some alteration in the biophysical properties of urothelium, thereby, affecting the afferent branch of the micturition reflex. Compared with hydrogel and saline treated animals 30% ethanol in NSS in the absence of capsaicin decreased bladder contraction frequency as well as produced adverse histological changes in the bladder. Possible damage to the afferents located near the urothelium by ethanol might explain the CMG and histological observations. Hydrogel with capsaicin was effective in decreasing bladder contraction frequency compared with vehicle controls of

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FIG. 6. Photographs of H & E staining of rat bladder hours after administration of capsaicin in various vehicles. Lumen side of bladder faces upward (arrow). A and B, 30% ethanol in NSS treatment group in absence and presence of 1 mM capsaicin, respectively. Note partial to complete urothelial denudation and vascular congestion. C and D, liposome treated group in absence and presence of 1 mM capsaicin, respectively. Note normal appearance of urothelium. E and F, hydrogel in absence and presence of 1 mM capsaicin, respectively. Note intact urothelium. Reduced from ⫻10.

hydrogel and liposomes alone but it failed to produce overflow incontinence, a feature observed when capsaicin was delivered using 30% ethanol and liposomes.23 The difference seen in capsaicin delivery in hydrogel treated animals may be related to tight binding of capsaicin with hydrophobic chains of the polymer, rendering it unavailable for an immediate effect. However, this study was done in normal bladders and it would be interesting to see how the biocompatible and emollient nature of liposomes and hydrogel affect capsaicin activity in a rat model of cystitis compared with 30% ethanol. At the cellular level capsaicin acts by releasing CGRP stored in afferent fibers24 and it was reported to produce reduction in suburothelial nerve densities in the bladder of patients with detrusor hyperreflexia. It may explain its prolonged beneficial effect in these patients.4 Capsaicin delivered in liposome and ethanol vehicles was able to decrease CGRP staining in bladders removed following the completion of CMG studies. Hematoxylin and eosin staining of formalin fixed bladder sections revealed a disruptive effect of the existing ethanolic vehicle on the urothelium even in the absence of capsaicin. Our results agree with those reported previously9 and they explain the discomfort felt by patients following intravesical administration in an ethanol vehicle.

CONCLUSIONS

The findings in our study support that liposomes are a superior vehicle for the intravesical administration of capsaicin compared with 30% ethanol in terms of safety, while they are comparable in efficacy. The evidence in favor of liposomal formulation of capsaicin in this preclinical study argues for clinical translation of this study, which is yet to be determined. Hydrogel can be a safe alternative option for capsaicin delivery. REFERENCES

1. Chancellor, M. B. and de Groat, W. C.: Intravesical capsaicin and resiniferatoxin therapy: spicing up the ways to treat the overactive bladder. J Urol, 162: 3, 1999 2. Birder, L. A., Kanai, A. J., de Groat, W. C., Kiss, S., Nealen, M. L., Burke, N. E. et al: Vanilloid receptor expression suggests a sensory role for urinary bladder epithelial cells. Proc Natl Acad Sci USA, 98: 13396, 2001 3. Craft, R. M., Cohen, S. M. and Porreca, F.: Long-lasting desensitization of bladder afferents following intravesical reiniferatoxin and capsaicin in the rat. Pain, 61: 317, 1995 4. Dasgupta, P., Chandiramani, V. A., Beckett, A., Scaravilli, F. and Fowler, C. J.: The effect of intravesical capsaicin on the suburothelial innervation in patients with detrusor hyperreflexia. BJU Int, 85: 238, 2000

CAPSAICIN DELIVERY USING HYDROGEL AND LIPOSOMES 5. Cruz, F.: Vanilloid receptor and detrusor instability. Urology, suppl., 59: 51, 2002 6. Fowler, C. J., Beck, R. O., Gerrard, S., Betts, C. D. and Fowler, C. G.: Intravesical capsaicin for treatment of detrusor hyperreflexia. J Neurol Neurosurg Psychiatry, 57: 169, 1994 7. de Seze, M., Wiart, L., de Seze, M. P., Joseph, P. A., Brochet, B., Ferriere, J. M. et al: Reiterated intravesical instillation of capsaicin in neurogenic detrusor hyperreflexia: a 5-years experience of 100 instillations. Ann Readapt Med Phys, 44: 514, 2001 8. Giannantoni, A., Di Stasi, S. M., Stephen, R. L., Navarra, P., Scivoletto, G., Mearini, E. et al: Intravesical capsaicin versus resiniferatoxin in patients with detrusor hyperreflexia: a prospective randomized study. J Urol, 167: 1710, 2002 9. Byrne, D. S., Das, A., Sedor, J., Huang, B., Rivas, D. A., Flood, H. J. et al: Effect of intravesical capsaicin and vehicle on bladder integrity in control and spinal cord injured rats. J Urol, 159: 1074, 1998 10. Jensen, P. G. and Larson, J. R.: Management of painful diabetic neuropathy. Drugs Aging, 18: 737, 2001 11. Li, Z., Ning, W., Wang, J., Choi, A., Lee, P. Y., Tyagi, P. and Huang, L.: Controlled gene delivery system based on thermosensitive biodegradable hydrogel. Pharm Res, 20: 884, 2003 12. Fraser, M. O., Chuang, Y. C., Tyagi, P., Yokoyama, T., Yoshimura, N., Huang, L. et al: Intravesical liposome administration—a novel treatment for hyperactive bladder in the rat. Urology, 61: 656, 2003 13. Avelino, A., Cruz, C., Nagy, I. and Cruz, F.: Vanilloid receptor 1 expression in the rat urinary tract. Neuroscience, 109: 787, 2002 14. Ritter, S. and Dinh, T. T.: Age-related changes in capsaicininduced degeneration in rat brain. J Comp Neurol, 318: 103, 1992 15. Donnerer, J., Amann, R., Schuligoi, R. and Lembeck, F.: Absorption and metabolism of capsaicinoids following intragastric

16.

17.

18.

19.

20.

21.

22.

23.

24.

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administration in rats. Naunyn Schmiedebergs Arch Pharmacol, 342: 357, 1990 de Seze, M., Wiart, L., Joseph, P. A., Dosque, J. P., Mazaux, J. M. and Barat, M.: Capsaicin and neurogenic detrusor hyperreflexia: a double-blind placebo-controlled study in 20 patients with spinal cord lesions. Neurourol Urodyn, 17: 513, 1998 Maggi, C. A. and Conte, B.: Effect of urethane anesthesia on the micturition reflex in capsaicin-treated rats. J Auton Nerv Syst, 30: 247, 1990 Mallory, B., Steers, W. D. and De Groat, W. C.: Electrophysiological study of micturition reflexes in rats. Am J Physiol, 257: R410, 1989 Chuang, Y. C., Fraser, M. O., Yu, Y., Beckel, J. M., Seki, S., Nakanishi, Y. et al: Analysis of the afferent limb of the vesicovascular reflex using neurotoxins, resiniferatoxin and capsaicin. Am J Physiol Regul Integr Comp Physiol, 281: R1302, 2001 Jeong, B., Bae, Y. H., Lee, D. S. and Kim, S. W.: Biodegradable block copolymers as injectable drug-delivery systems. Nature, 388: 860, 1997 Cheng, C. L., Ma, C. P. and de Groat, W. C.: Effects of capsaicin on micturition and associated reflexes in rats. Am J Physiol, 265: R132, 1993 Tyagi, P., Lu, S. H., Huang, L., de Groat, W. C., Chancellor, M. B. and Fraser, M. O.: Liposomal delivery of vanilloids for intravesical treatment of hyperactive bladder. In: XIVth World Congress of Pharmacology Meeting Abstracts, vol. 44, No. 2, p. 26.6, abstract 30, 2002 Santicioli, P., Maggi, C. A. and Meli, A.: The effect of capsaicin pretreatment on the cystometrograms of urethane anesthetized rats. J Urol, 133: 700, 1985 Tucci, P., Evandri, M. G. and Bolle, P.: Tachykinin-independent activity of capsaicin on in-vitro lamb detrusor. J Pharm Pharmacol, 54: 1111, 2002