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Vasoactive agents induce cytotoxicity in cultured human penile smooth muscle cells
BASIC SCIENCE
VASOACTIVE AGENTS INDUCE CYTOTOXICITY IN CULTURED HUMAN PENILE SMOOTH MUSCLE CELLS MAHADEVAN RAJASEKARAN, VALERIA PAGNON,
AND
MANOJ MONGA
ABSTRACT Objectives. To evaluate the direct in vitro cytotoxicity of vasoactive agents (papaverine, phentolamine, and prostaglandin E1 [PGE1]) to human penile cavernosal smooth muscle cells. Intracavernous pharmacotherapy with vasoactive agents for male erectile dysfunction has been associated with long-term complications such as a reduction in penile smooth muscle content and fibrosis. Methods. Human penile cavernosal tissue explants (1 to 2 mm3 size) were obtained with proper institutional review board approval from patients undergoing penile prosthesis implantation. Primary culture was initiated in Dulbecco’s modified Eagles medium containing 10% fetal bovine serum, and monolayer cavernosal cells were grown in 48-well tissue culture dishes. At 60% to 80% confluence, cells were labeled overnight with 51Na2CrO4 (1.5 Ci) and then incubated with therapeutic concentrations of papaverine (1.5 to 30 mg/mL), phentolamine (0.5 mg/mL), and PGE1 (5 g/mL) alone, as well as in combination, for 30 minutes at 37°C. At the end of incubation, an aliquot of supernatant was collected in scintillation vials. The release of cell-free chromium in supernatants was determined in a liquid scintillation counter, and results were expressed as the percentage of cytotoxicity. Results. Papaverine induced a significant dose-dependent increase in chromium release from the cavernosal cells. At therapeutic concentrations, papaverine (30 mg/mL) produced up to 60% cytotoxicity; PGE1 (5 g/mL) resulted in 40% toxicity. The combination of papaverine with either PGE1 or phentolamine had a cumulative toxic effect, and maximal toxicity (70%) was observed with the triple combination. Conclusions. Papaverine-induced cytotoxicity to cavernosal smooth muscle cells may contribute to the fibrosis and loss of smooth muscle content associated with the intracavernous pharmacotherapy. Quantitative evaluation of in vitro cytotoxicity in human cavernosal smooth muscle cell culture may be important in the development of new intracavernosal vasoactive agents. UROLOGY 59: 155–158, 2002. © 2002, Elsevier Science Inc.
M
ore than 30 million men in North America have erectile dysfunction.1 Intracavernosal injection of vasoactive agents such as papaverine, phentolamine, and prostaglandin E1 (PGE1) has proved to be an effective therapeutic alternative and important diagnostic adjunct.2 Initial monotherapy with these agents was associated with poor efficacy (phentolamine),3 side effects such as the incidence of priapism (papaverine),4 and severe This study was supported in part by an American Foundation for Urological Diseases Summer Scholarship to Valeria Pagnon. From the Division of Urology, University of California, San Diego, School of Medicine, San Diego, California Reprint requests: Mahadevan Rajasekaran, Ph.D., c/o Janell Poehling, Division of Urology, University of California, San Diego, School of Medicine, 200 West Arbor Drive (8897), San Diego, CA 92037-8897 Submitted: May 14, 2001, accepted (with revisions): September 4, 2001 © 2002, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
painful erections (PGE1).5 These concerns resulted in patient dissatisfaction and significant drop out from pharmacotherapy programs. Although the combination therapy significantly improved the efficacy and reduced the side effects, long-term complications, including a reduction in penile smooth muscle content and corporal fibrosis have been demonstrated.6 The mechanism of vasoactive agent-induced penile fibrosis is not well understood. This study evaluated the direct in vitro cytotoxicity of vasoactive agents (papaverine, phentolamine, and PGE1) on cultured human penile cavernosal smooth muscle cells. MATERIAL AND METHODS CULTURE OF CAVERNOSAL SMOOTH MUSCLE Human penile cavernosal tissue explants were obtained with proper institutional review board approval from patients undergoing penile prosthesis implantation. Patients with a 0090-4295/02/$22.00 PII S0090-4295(01)01482-0 155
history of prior intracavernosal injection therapy were excluded. The penile tissue obtained was placed into culture flasks containing growth medium (Dulbecco’s minimum essential medium containing 20% fetal calf serum), and culture was initiated. The explant was incubated at 37°C. The cells underwent a minimum of two passages before any experiments to achieve stability.7
CHROMIUM RELEASE CYTOTOXICITY ASSAY
Cells (5 ⫻ 104/well) were suspended in Dulbecco’s minimum essential medium with 10% fetal bovine serum and added to the wells of 48-well assay plates (300 L/well). The assay plates were maintained at 37°C in a controlled environment incubator (5% carbon dioxide and 95% air) for 3 days before labeling. Cells were then labeled with 51Na2CrO4 (1.5 Ci/well) and incubated overnight at 37°C. The unincorporated isotope was removed by washing the wells two times with Hank’s balanced salt solution. The cells were then incubated with therapeutic concentrations of papaverine hydrochloride (1.5 to 30 mg/mL), phentolamine (0.5 mg/mL), or PGE1 (5 g/mL) dissolved in 0.9% normal saline solution for 30 minutes at 37°C. Control wells were incubated with the solvent alone. Bi-mix (papaverine [30 mg/mL] with phentolamine [0.5 mg/mL] or PGE1 [0.5 g/mL]) and tri-mix (papaverine 6 mg/mL, phentolamine 0.5 mg/mL, and PGE1 [5 g/ mL]) combinations were also tested. We used both positive (2% Triton-X detergent) and negative controls (0.9% normal saline) in these evaluations. At the end of incubation, an aliquot of supernatant (150 L) from each well was transferred to scintillation vials and filled with scintillation cocktail. Another aliquot was used to check the pH using a pH meter. Radioactive chromium (Cr) release (counts per minute [cpm]) was determined using a liquid scintillation counter and the percentage of maximal cytotoxicity was computed using the formula: % cytotoxicity ⫽ [{(Cr release in experimental well) ⫺ (spontaneous Cr release)}/{(maximum Cr release) ⫺ (spontaneous Cr release)}] ⫻ 100.8 Spontaneous Cr release was obtained from the wells exposed to solvent alone and the maximal Cr release was calculated from the wells exposed to 2% Triton-X.
STATISTICAL ANALYSIS The InStat statistics program (Graph PAD Software, San Diego, Calif) was used to compare the level of cytotoxicity induced by the different vasoactive agents by one-way analysis of variance, followed by a nonparametric Newman-Keuls test. P values of less than 0.05 were considered statistically significant.
FIGURE 1. Cytotoxicity of increasing concentrations of papaverine to human cavernosal smooth muscle cells. Cells were exposed to varying concentrations of papaverine (1.5 to 30 mg/mL) for 30 minutes, and cytotoxicity was determined as described in the Material and Methods section. Controls were incubated in the absence of papaverine under the same conditions. Data represent the mean ⫾ SEM (n ⫽ 3). *P ⬍0.05 compared with control.
imum of 60% with 30 mg/mL (Fig. 1, P ⬍0.05). Cytotoxicity of papaverine appeared at 7.5 mg/mL; lower doses (1.5 mg/mL) resulted in no toxicity. The addition of papaverine solution reduced the pH (4.1) in the wells. However, a pH-dependent effect was not demonstrated, as no cytotoxicity was observed with lower doses of papaverine, despite the change in pH. PGE1 incubation induced a 40% cytotoxicity (P ⬍0.05), and phentolamine addition caused the least cytotoxicity, reaching a maximum of 18% (P ⬎0.05) (Fig. 2). The bi-mix (papaverine and phentolamine) and tri-mix (papaverine, phentolamine, and PGE1) combinations resulted in a higher percentage of cytotoxicity (70%) than did papaverine alone (Fig. 3). COMMENT
RESULTS We used a chromium release assay to evaluate vasoactive agent-induced cytotoxicity. This assay is based on the principle that target cells are labeled with radioactive chromium (sodium chromate— Na2 51CrO4), which is taken up by the cells and retained in the cytoplasm. In the event of target cell damage, radioactive chromium is released into the medium and can be quantified in a liquid scintillation counter. The maximal radioactive chromium release (60,000 to 90,000 cpm) was observed with exposure to 2% Triton-X (positive control); the control wells exhibited negligible (5000 to 6000 cpm) radioactivity. Papaverine incubation resulted in a dose-dependent cytotoxicity, reaching a max156
Intracavernous injections with vasoactive agents such as papaverine, phentolamine, and PGE1 is a well-accepted approach in the treatment and diagnosis of male erectile dysfunction.2 Papaverine is an opium alkaloid without any morphine-like effect. This vasoactive agent relaxes all components of the penile erectile tissue by increasing the concentration of cyclic adenosine monophosphate intracellularly while decreasing the calcium concentration. It primarily affects the cavernosal artery and cavernous smooth musculature.9,10 Virag et al.11 suggested that an optimal dosage of 38 mg resulted in a success rate of 54%. Side effects included priapism and corporal fibrosis in these patients.12 UROLOGY 59 (1), 2002
FIGURE 2. Individual cytotoxicity of papaverine, phentolamine, and PGE1 to human cavernosal smooth muscle cells. Cells were exposed to therapeutic concentrations of vasoactive agents for 30 minutes, and cytotoxicity was determined as described in the Material and Methods section. Controls were incubated in the absence of vasoactive agents under the same conditions. Data represent the mean ⫾ SEM (n ⫽ 3). *P ⬍0.05 compared with control.
Phentolamine acts as a sympathetic alpha-adrenergic antagonist, facilitating a penile erection by relaxation of the arterial circular smooth musculature.9,10 Single-agent therapy with phentolamine has not proved effective. However, Zorgniotti and Lefleur13 reported a 72% success rate using a bimix preparation (papaverine 30 mg plus phentolamine 1 mg). PGE1 is a prostanoid that affects both the smooth erectile musculature and the penile vasculature.9,10 Intracavernosal injection of PGE1 is associated with a 71% success rate.9 Despite these claims of efficacy, concerns such as painful erections and long-term complications of penile fibrosis led to significant patient dropouts from monotherapy programs. Lakin et al.14 reported a 31% fibrosis rate after 1 year of therapy with papaverine and a 25% fibrosis rate was observed with PGE1 use.15 The exact reasons for vasoactive agent-induced fibrotic changes of the cavernosal tissue are still unclear. Previous reports suggest that there is an association among injection frequency, injection procedure, patient predisposition, and the osmolarity or concentration of the injected drug.15 We hypothesized that intracavernosal agents may have a more direct adverse effect on cavernosal tissue. Our studies demonstrated that papaverine and PGE1 are cytotoxic to cultured human cavernosal cells at therapeutic doses. Papaverine as a single agent resulted in the most cytotoxicity, which correlates with the clinical observation that corporal fibrosis is most commonly associated with this agent.13 It is common urologic practice to use dual or triple-agent therapy to improve efficacy and theoretically decrease complications.9,10 However, the reduction of the UROLOGY 59 (1), 2002
FIGURE 3. Cytotoxicity of bi-mix and tri-mix combinations to human cavernosal smooth muscle cells. Cells were exposed to combinations of vasoactive agents for 30 minutes, and cytotoxicity was determined as described. Controls were incubated in the absence of vasoactive drug combinations under the same conditions. Data represent the mean ⫾ SEM (n ⫽ 3). *P ⬍0.05 compared with control.
dosage of papaverine and PGE1 in our study by using multiagent combinations did not decrease the level of cytotoxicity. CONCLUSIONS Our results suggest that papaverine and PGE1 induce significant cytotoxicity to cavernosal smooth muscle cells. This suggests that the use of intracavernous pharmacotherapy may contribute to corporal fibrosis and the loss of smooth muscle content. Quantitative evaluation of in vitro cytotoxicity in human cavernosal smooth muscle cells should be considered as an adjunct to the development of future intracavernosal agents. REFERENCES 1. Feldman HA, Goldstein I, Hatzichristou DG, et al: Impotence and its medical and psychosocial correlates: results of the Massachusetts Male Aging Study. J Urol 151: 54 – 61, 1994. 2. Sexton WJ, Benedict JF, and Jarow JP: Comparison of long-term outcomes of penile prostheses and intracavernosal injection therapy. J Urol 159: 811– 815, 1998. 3. Leungwattanakij S, Flynn V, and Hellstrom WJ: Intracavernosal injection and intraurethral therapy for erectile dysfunction. Urol Clin North Am 28: 343–354, 2001. 4. Lomas GM, and Jarow JP: Risk factors for papaverineinduced priapism. J Urol 147: 1280 –1281, 1992. 5. Fulgham PF, Cochran JS, Denman JL, et al: Disappointing initial results with transurethral alprostadil for erectile dysfunction in a urology practice setting. J Urol 160: 2041– 2046, 1998. 6. Casabe A, Bechara A, Cheliz G, et al: Drop out reasons and complications in self-injection therapy with a triple vasoactive drug mixture in sexual erectile dysfunction. Int J Impot Res 10: 5–9, 1998. 157
7. Dahiya R, Sikka SC, Hellstrom WJ, et al: Phenotypic and cytogenetic characterization of a human corpus cavernosum cell line (DS-1). Biochem Mol Biol Int 30: 559 –569, 1993. 8. Chopra J, Joist JH, and Webster RO: Loss of 51chromium, lactate dehydrogenase, 111indium as indicators of endothelial cell injury. Lab Invest 57: 578 –584, 1987. 9. Fallon B: Intracavernous injection therapy for male erectile dysfunction. Urol Clin North Am 22: 833– 845, 1995. 10. Lugg J, and Rajfer J: Drug therapy for erectile dysfunction. AUA Update 15: 290 –295, 1996. 11. Virag R, Sussman H, Floresco J, et al: Late results on the treatment of neurogenic impotence by self-intracavernous injection of vasoactive drugs. World J Urol 5: 166 –170, 1987.
158
12. Mannig M, and Juenemann KP: Pharmacotherapy of erectile dysfunction, in Hellstrom WJ (Ed): Male Infertility and Sexual Dysfunction. New York, Springer-Verlag, 1997, pp 440 – 451. 13. Zorgniotti AW, and Lefleur RS: Auto-injection of the corpus cavernosum with a vasoactive drug combination of vasculogenic impotence. J Urol 133: 39 – 41, 1985. 14. Lakin MM, Montague DK, VanderBrug-Medendorp S, et al.: Intracavernous injection therapy: analysis of results and complications. J Urol 143: 1138 –1141, 1990. 15. Chew KK, Stuckey BGA, Earle CM, et al: Penile fibrosis in intracavernosal prostaglandin E1 injection therapy for erectile dysfunction. Int J Impot Res 9: 225–229, 1997.
UROLOGY 59 (1), 2002
VASOACTIVE AGENTS INDUCE CYTOTOXICITY IN CULTURED HUMAN PENILE SMOOTH MUSCLE CELLS MAHADEVAN RAJASEKARAN, VALERIA PAGNON,
AND
MANOJ MONGA
ABSTRACT Objectives. To evaluate the direct in vitro cytotoxicity of vasoactive agents (papaverine, phentolamine, and prostaglandin E1 [PGE1]) to human penile cavernosal smooth muscle cells. Intracavernous pharmacotherapy with vasoactive agents for male erectile dysfunction has been associated with long-term complications such as a reduction in penile smooth muscle content and fibrosis. Methods. Human penile cavernosal tissue explants (1 to 2 mm3 size) were obtained with proper institutional review board approval from patients undergoing penile prosthesis implantation. Primary culture was initiated in Dulbecco’s modified Eagles medium containing 10% fetal bovine serum, and monolayer cavernosal cells were grown in 48-well tissue culture dishes. At 60% to 80% confluence, cells were labeled overnight with 51Na2CrO4 (1.5 Ci) and then incubated with therapeutic concentrations of papaverine (1.5 to 30 mg/mL), phentolamine (0.5 mg/mL), and PGE1 (5 g/mL) alone, as well as in combination, for 30 minutes at 37°C. At the end of incubation, an aliquot of supernatant was collected in scintillation vials. The release of cell-free chromium in supernatants was determined in a liquid scintillation counter, and results were expressed as the percentage of cytotoxicity. Results. Papaverine induced a significant dose-dependent increase in chromium release from the cavernosal cells. At therapeutic concentrations, papaverine (30 mg/mL) produced up to 60% cytotoxicity; PGE1 (5 g/mL) resulted in 40% toxicity. The combination of papaverine with either PGE1 or phentolamine had a cumulative toxic effect, and maximal toxicity (70%) was observed with the triple combination. Conclusions. Papaverine-induced cytotoxicity to cavernosal smooth muscle cells may contribute to the fibrosis and loss of smooth muscle content associated with the intracavernous pharmacotherapy. Quantitative evaluation of in vitro cytotoxicity in human cavernosal smooth muscle cell culture may be important in the development of new intracavernosal vasoactive agents. UROLOGY 59: 155–158, 2002. © 2002, Elsevier Science Inc.
M
ore than 30 million men in North America have erectile dysfunction.1 Intracavernosal injection of vasoactive agents such as papaverine, phentolamine, and prostaglandin E1 (PGE1) has proved to be an effective therapeutic alternative and important diagnostic adjunct.2 Initial monotherapy with these agents was associated with poor efficacy (phentolamine),3 side effects such as the incidence of priapism (papaverine),4 and severe This study was supported in part by an American Foundation for Urological Diseases Summer Scholarship to Valeria Pagnon. From the Division of Urology, University of California, San Diego, School of Medicine, San Diego, California Reprint requests: Mahadevan Rajasekaran, Ph.D., c/o Janell Poehling, Division of Urology, University of California, San Diego, School of Medicine, 200 West Arbor Drive (8897), San Diego, CA 92037-8897 Submitted: May 14, 2001, accepted (with revisions): September 4, 2001 © 2002, ELSEVIER SCIENCE INC. ALL RIGHTS RESERVED
painful erections (PGE1).5 These concerns resulted in patient dissatisfaction and significant drop out from pharmacotherapy programs. Although the combination therapy significantly improved the efficacy and reduced the side effects, long-term complications, including a reduction in penile smooth muscle content and corporal fibrosis have been demonstrated.6 The mechanism of vasoactive agent-induced penile fibrosis is not well understood. This study evaluated the direct in vitro cytotoxicity of vasoactive agents (papaverine, phentolamine, and PGE1) on cultured human penile cavernosal smooth muscle cells. MATERIAL AND METHODS CULTURE OF CAVERNOSAL SMOOTH MUSCLE Human penile cavernosal tissue explants were obtained with proper institutional review board approval from patients undergoing penile prosthesis implantation. Patients with a 0090-4295/02/$22.00 PII S0090-4295(01)01482-0 155
history of prior intracavernosal injection therapy were excluded. The penile tissue obtained was placed into culture flasks containing growth medium (Dulbecco’s minimum essential medium containing 20% fetal calf serum), and culture was initiated. The explant was incubated at 37°C. The cells underwent a minimum of two passages before any experiments to achieve stability.7
CHROMIUM RELEASE CYTOTOXICITY ASSAY
Cells (5 ⫻ 104/well) were suspended in Dulbecco’s minimum essential medium with 10% fetal bovine serum and added to the wells of 48-well assay plates (300 L/well). The assay plates were maintained at 37°C in a controlled environment incubator (5% carbon dioxide and 95% air) for 3 days before labeling. Cells were then labeled with 51Na2CrO4 (1.5 Ci/well) and incubated overnight at 37°C. The unincorporated isotope was removed by washing the wells two times with Hank’s balanced salt solution. The cells were then incubated with therapeutic concentrations of papaverine hydrochloride (1.5 to 30 mg/mL), phentolamine (0.5 mg/mL), or PGE1 (5 g/mL) dissolved in 0.9% normal saline solution for 30 minutes at 37°C. Control wells were incubated with the solvent alone. Bi-mix (papaverine [30 mg/mL] with phentolamine [0.5 mg/mL] or PGE1 [0.5 g/mL]) and tri-mix (papaverine 6 mg/mL, phentolamine 0.5 mg/mL, and PGE1 [5 g/ mL]) combinations were also tested. We used both positive (2% Triton-X detergent) and negative controls (0.9% normal saline) in these evaluations. At the end of incubation, an aliquot of supernatant (150 L) from each well was transferred to scintillation vials and filled with scintillation cocktail. Another aliquot was used to check the pH using a pH meter. Radioactive chromium (Cr) release (counts per minute [cpm]) was determined using a liquid scintillation counter and the percentage of maximal cytotoxicity was computed using the formula: % cytotoxicity ⫽ [{(Cr release in experimental well) ⫺ (spontaneous Cr release)}/{(maximum Cr release) ⫺ (spontaneous Cr release)}] ⫻ 100.8 Spontaneous Cr release was obtained from the wells exposed to solvent alone and the maximal Cr release was calculated from the wells exposed to 2% Triton-X.
STATISTICAL ANALYSIS The InStat statistics program (Graph PAD Software, San Diego, Calif) was used to compare the level of cytotoxicity induced by the different vasoactive agents by one-way analysis of variance, followed by a nonparametric Newman-Keuls test. P values of less than 0.05 were considered statistically significant.
FIGURE 1. Cytotoxicity of increasing concentrations of papaverine to human cavernosal smooth muscle cells. Cells were exposed to varying concentrations of papaverine (1.5 to 30 mg/mL) for 30 minutes, and cytotoxicity was determined as described in the Material and Methods section. Controls were incubated in the absence of papaverine under the same conditions. Data represent the mean ⫾ SEM (n ⫽ 3). *P ⬍0.05 compared with control.
imum of 60% with 30 mg/mL (Fig. 1, P ⬍0.05). Cytotoxicity of papaverine appeared at 7.5 mg/mL; lower doses (1.5 mg/mL) resulted in no toxicity. The addition of papaverine solution reduced the pH (4.1) in the wells. However, a pH-dependent effect was not demonstrated, as no cytotoxicity was observed with lower doses of papaverine, despite the change in pH. PGE1 incubation induced a 40% cytotoxicity (P ⬍0.05), and phentolamine addition caused the least cytotoxicity, reaching a maximum of 18% (P ⬎0.05) (Fig. 2). The bi-mix (papaverine and phentolamine) and tri-mix (papaverine, phentolamine, and PGE1) combinations resulted in a higher percentage of cytotoxicity (70%) than did papaverine alone (Fig. 3). COMMENT
RESULTS We used a chromium release assay to evaluate vasoactive agent-induced cytotoxicity. This assay is based on the principle that target cells are labeled with radioactive chromium (sodium chromate— Na2 51CrO4), which is taken up by the cells and retained in the cytoplasm. In the event of target cell damage, radioactive chromium is released into the medium and can be quantified in a liquid scintillation counter. The maximal radioactive chromium release (60,000 to 90,000 cpm) was observed with exposure to 2% Triton-X (positive control); the control wells exhibited negligible (5000 to 6000 cpm) radioactivity. Papaverine incubation resulted in a dose-dependent cytotoxicity, reaching a max156
Intracavernous injections with vasoactive agents such as papaverine, phentolamine, and PGE1 is a well-accepted approach in the treatment and diagnosis of male erectile dysfunction.2 Papaverine is an opium alkaloid without any morphine-like effect. This vasoactive agent relaxes all components of the penile erectile tissue by increasing the concentration of cyclic adenosine monophosphate intracellularly while decreasing the calcium concentration. It primarily affects the cavernosal artery and cavernous smooth musculature.9,10 Virag et al.11 suggested that an optimal dosage of 38 mg resulted in a success rate of 54%. Side effects included priapism and corporal fibrosis in these patients.12 UROLOGY 59 (1), 2002
FIGURE 2. Individual cytotoxicity of papaverine, phentolamine, and PGE1 to human cavernosal smooth muscle cells. Cells were exposed to therapeutic concentrations of vasoactive agents for 30 minutes, and cytotoxicity was determined as described in the Material and Methods section. Controls were incubated in the absence of vasoactive agents under the same conditions. Data represent the mean ⫾ SEM (n ⫽ 3). *P ⬍0.05 compared with control.
Phentolamine acts as a sympathetic alpha-adrenergic antagonist, facilitating a penile erection by relaxation of the arterial circular smooth musculature.9,10 Single-agent therapy with phentolamine has not proved effective. However, Zorgniotti and Lefleur13 reported a 72% success rate using a bimix preparation (papaverine 30 mg plus phentolamine 1 mg). PGE1 is a prostanoid that affects both the smooth erectile musculature and the penile vasculature.9,10 Intracavernosal injection of PGE1 is associated with a 71% success rate.9 Despite these claims of efficacy, concerns such as painful erections and long-term complications of penile fibrosis led to significant patient dropouts from monotherapy programs. Lakin et al.14 reported a 31% fibrosis rate after 1 year of therapy with papaverine and a 25% fibrosis rate was observed with PGE1 use.15 The exact reasons for vasoactive agent-induced fibrotic changes of the cavernosal tissue are still unclear. Previous reports suggest that there is an association among injection frequency, injection procedure, patient predisposition, and the osmolarity or concentration of the injected drug.15 We hypothesized that intracavernosal agents may have a more direct adverse effect on cavernosal tissue. Our studies demonstrated that papaverine and PGE1 are cytotoxic to cultured human cavernosal cells at therapeutic doses. Papaverine as a single agent resulted in the most cytotoxicity, which correlates with the clinical observation that corporal fibrosis is most commonly associated with this agent.13 It is common urologic practice to use dual or triple-agent therapy to improve efficacy and theoretically decrease complications.9,10 However, the reduction of the UROLOGY 59 (1), 2002
FIGURE 3. Cytotoxicity of bi-mix and tri-mix combinations to human cavernosal smooth muscle cells. Cells were exposed to combinations of vasoactive agents for 30 minutes, and cytotoxicity was determined as described. Controls were incubated in the absence of vasoactive drug combinations under the same conditions. Data represent the mean ⫾ SEM (n ⫽ 3). *P ⬍0.05 compared with control.
dosage of papaverine and PGE1 in our study by using multiagent combinations did not decrease the level of cytotoxicity. CONCLUSIONS Our results suggest that papaverine and PGE1 induce significant cytotoxicity to cavernosal smooth muscle cells. This suggests that the use of intracavernous pharmacotherapy may contribute to corporal fibrosis and the loss of smooth muscle content. Quantitative evaluation of in vitro cytotoxicity in human cavernosal smooth muscle cells should be considered as an adjunct to the development of future intracavernosal agents. REFERENCES 1. Feldman HA, Goldstein I, Hatzichristou DG, et al: Impotence and its medical and psychosocial correlates: results of the Massachusetts Male Aging Study. J Urol 151: 54 – 61, 1994. 2. Sexton WJ, Benedict JF, and Jarow JP: Comparison of long-term outcomes of penile prostheses and intracavernosal injection therapy. J Urol 159: 811– 815, 1998. 3. Leungwattanakij S, Flynn V, and Hellstrom WJ: Intracavernosal injection and intraurethral therapy for erectile dysfunction. Urol Clin North Am 28: 343–354, 2001. 4. Lomas GM, and Jarow JP: Risk factors for papaverineinduced priapism. J Urol 147: 1280 –1281, 1992. 5. Fulgham PF, Cochran JS, Denman JL, et al: Disappointing initial results with transurethral alprostadil for erectile dysfunction in a urology practice setting. J Urol 160: 2041– 2046, 1998. 6. Casabe A, Bechara A, Cheliz G, et al: Drop out reasons and complications in self-injection therapy with a triple vasoactive drug mixture in sexual erectile dysfunction. Int J Impot Res 10: 5–9, 1998. 157
7. Dahiya R, Sikka SC, Hellstrom WJ, et al: Phenotypic and cytogenetic characterization of a human corpus cavernosum cell line (DS-1). Biochem Mol Biol Int 30: 559 –569, 1993. 8. Chopra J, Joist JH, and Webster RO: Loss of 51chromium, lactate dehydrogenase, 111indium as indicators of endothelial cell injury. Lab Invest 57: 578 –584, 1987. 9. Fallon B: Intracavernous injection therapy for male erectile dysfunction. Urol Clin North Am 22: 833– 845, 1995. 10. Lugg J, and Rajfer J: Drug therapy for erectile dysfunction. AUA Update 15: 290 –295, 1996. 11. Virag R, Sussman H, Floresco J, et al: Late results on the treatment of neurogenic impotence by self-intracavernous injection of vasoactive drugs. World J Urol 5: 166 –170, 1987.
158
12. Mannig M, and Juenemann KP: Pharmacotherapy of erectile dysfunction, in Hellstrom WJ (Ed): Male Infertility and Sexual Dysfunction. New York, Springer-Verlag, 1997, pp 440 – 451. 13. Zorgniotti AW, and Lefleur RS: Auto-injection of the corpus cavernosum with a vasoactive drug combination of vasculogenic impotence. J Urol 133: 39 – 41, 1985. 14. Lakin MM, Montague DK, VanderBrug-Medendorp S, et al.: Intracavernous injection therapy: analysis of results and complications. J Urol 143: 1138 –1141, 1990. 15. Chew KK, Stuckey BGA, Earle CM, et al: Penile fibrosis in intracavernosal prostaglandin E1 injection therapy for erectile dysfunction. Int J Impot Res 9: 225–229, 1997.
UROLOGY 59 (1), 2002