DOES PROSTAGLANDIN E1 THERAPY MODIFY THE INTRACAVERNOUS MUSCULATURE?

0022-5347/00/1632-0464/0 THE JOURNAL OF UROLOGY® Copyright © 2000 by AMERICAN UROLOGICAL ASSOCIATION, INC.®

Vol. 163, 464 – 466, February 2000 Printed in U.S.A.

DOES PROSTAGLANDIN E1 THERAPY MODIFY THE INTRACAVERNOUS MUSCULATURE? ¨ ERIC WESPES,* AHMED A. SATTAR, JEAN-CHRISTOPHE NOEL

AND

CLAUDE C. SCHULMAN

From the Departments of Urology and Pathology, Erasme Hospital, Universite´ Libre de Bruxelles, Brussels and Department of Urology, CHU Charleroi, Charleroi, Belgium

ABSTRACT

Purpose: Intracavernous injection of prostaglandin E1 in patients with erectile dysfunction has become widespread. However, to our knowledge long-term side effects on the penile smooth muscles in man have not been documented. We evaluated the histological changes of intracavernous smooth muscle after long-term treatment with prostaglandin E1. Materials and Methods: A total of 10 patients with a mean age of 56.5 years (range 46 to 69) underwent biopsy of the corpora before (5) and after (10 in both corpora) prostaglandin E1 treatment. Between 150 and 250 injections during 3 years were performed, and the dosage varied from 5 to 20 mg. Only 1 patient injected prostaglandin E1 on both sides. Staining with hematoxylin and eosin, and immunohistochemical staining with actin anti-actin of intracavernous smooth muscle and its quantification with computerized image analysis were performed. Results: No histological difference was observed with classic staining. A reduction was noted in the percentage of intracavernous smooth muscle after treatment in 2 of 5 patients on the side of injection (before 35% and 41%, and after 19% and 30%, respectively) but there was no difference with the other corpus cavernosum. No difference was observed in the percentage of intracavernous smooth muscle between both corpora in the 5 patients with biopsies performed only after treatment. Conclusions: Prostaglandin E1 does not seem to alter intracavernous structures. KEY WORDS: impotence, alprostadil, injections

Intracavernous injections of vasoactive drugs in patients with erectile dysfunction have become a widespread and well accepted therapy.1 Prostaglandin E1 is the vasoactive agent of choice because it is efficacious, and the risk of priapism and penile fibrosis is reported to be low.2, 3 In a European multicenter study during a 6-month period 4% of patients (34 of 848) had local fibrotic changes.4 The diagnosis of fibrotic lesions of the penis was based on palpation or ultrasound examination, which does not allow differentiation between fibrotic changes inside the corpora cavernosa and those in the tunica albuginea.5 Moreover, palpation or ultrasound does not distinguish traumatic lesions due to the needle from reactive lesions due to the drug. Using immunohistochemical staining with actin anti-actin and computerized image analysis, we quantified the percentage of smooth muscle fibers in penile biopsies of patients treated with intracavernous prostaglandin E1 injection to determine the histological modifications at long-term followup. MATERIALS AND METHODS

The study included 10 impotent patients 46 to 69 years old (mean age 56.5). Complete blood and hormonal assays were performed to exclude endocrinological disease from diagnosis. Every patient spent at least 1 night at the hospital to evaluate nocturnal penile tumescence using the RigiScan† device. Cavernosometry confirmed that impotent patients with pure venous leak had a flow rate necessary to maintain artificial erection greater than 15 ml. per minute after intracavernous injection of 20 mg. prostaglandin E1. Patients di-

agnosed with arterial disease had insufficient cavernous systolic blood flow velocity. Bulbocavernosus reflex latency was measured to exclude somatic neurological disorder from diagnosis. Using the biopsy gun system, all patients underwent penile biopsy in the balanopreputial groove on the dorsolateral side of the shaft of the penis under local anesthesia.6 In 5 patients penile biopsy was performed before treatment. The penis is stretched by the left hand and biopsy is performed on the left corpus cavernosum. All patients underwent penile biopsies after treatment on both sides. Following biopsy removal, cavernous tissue specimens were immediately fixed in 10% formalin phosphate buffer solution. The fixed tissue sections were processed through xylene embedded in paraffin and sectioned at 5 mm. A tissue section was used for hematoxylin and eosin staining, and examined by light microscopy to evaluate the cavernous tissue pathologically, and 1 section was used for immunohistochemistry staining with actin anti-actin to determine the smooth muscle cells. Immunohistochemical staining was done according to the biotinstreptavidin-peroxidase technique. The sections were incubated for 15 minutes with 2% (volume in volume) hydrogen peroxide solution to block endogenous peroxidase activity, and processed for immunohistochemistry. After incubation with 1:20 normal sheep serum the sections were incubated overnight (14 to 18 hours) with mouse anti-human anti-actin antibodies diluted 1:50. After rinsing the sections were incubated for 30 minutes at room temperature successively with sheep anti-mouse immunoglobulin biotin diluted to 1:500 and streptavidin coupled to peroxidase 1:1,000 for 30 minutes. Peroxidase activity was indicated by 10 mg./10 ml., 39,3-diaminobenzidine hydrochloride, pH 7.4, containing 0.01% (volume in volume) hydrogen peroxide for 5 to 10 minutes at room temperature. Quantitative analysis of cavernous smooth muscle cells

Accepted for publication August 13, 1999. Presented at annual meeting of American Urological Association, San Diego, California, May 30 –June 4, 1998. * Requests for reprints: Bd. Paul Janson 92, 6000 Charleroi, Brussells. † Dacomed, Minneapolis, Minnesota. 464

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Percent cavernous smooth muscle content before and after prostaglandin E1 intracavernous injection of different dosages in patients with psychogenic and organic impotence Pt. Age

Origin Impotence

No. Doses

Dosage (mg.)

53 Psychogenic 6250 64 Psychogenic 6150 53 Psychogenic 6150 46 Psychogenic 6200 66 Psychogenic 6200 53 Vasculogenic 6360 61 Vasculogenic 6200 65 Vasculogenic 6150 68 Vasculogenic 6150 69 Vasculogenic 6150 Side of injection was right in all but 1 patient who performed

% Smooth Muscle Content Before Injection Lt.

6 41 6 40 5 10 15 15 20 35 20 27 20 25 20 injection on both sides.

was done with an image analyzer system combined with a light microscope equipped with a video camera. Images were interactively discriminated and measurements were performed on the resulting binary images. The percentage of smooth muscle cells for each image results from the quantification of the difference between the gray levels on the digitized image. At least 20 different fields (3400) in the cavernous tissue were examined from each tissue section. Mean percentage of cavernous smooth cells content was determined. RESULTS

Results and their interpretation are depicted in the table. Of 10 patients 5 (50%) had psychogenic impotence as no organic abnormalities were observed and 5 (50%) had arterial lesions without corporeal veno-occlusive dysfunction. All patients except 1 injected prostaglandin E1 only in 1 corpus cavernosum. The number of injections varied from 150 to 360 during 3 years. The dosage was 5 to 15 mg. for psychogenic and 15 to 20 mg. for organic impotence cases. Previously we analyzed the normal percentage of cavernous smooth muscle content in different age groups from 2 to 72 years and observed that the lowest percentage of cavernous muscle fibers that corresponded to our study age group was 37%.7 Thus, this percentage was considered as the cutoff for normal. Of the 5 cases with pretreatment biopsies 2 with psychogenic impotence had greater than 37% and 3 with organic impotence had less than 37% smooth muscle cells. In these cases posttreatment biopsies showed no difference in quantification except in 1 psychogenic and 1 organic case on the side of injection. Of the patients with only posttreatment biopsies those with psychogenic impotence had greater than 37% and those with organic impotence had less than 37% smooth muscle cells. In these patients no difference was observed between the 2 corpora whereas the injection was always performed on the same side. In the patient who performed injections on both sides no difference was observed. No increase greater than the 37% cutoff was observed in the patients with organic impotence after treatment. DISCUSSION

The reasons for fibrotic changes of the cavernosal tissue after intracavernous vasoactive drug injection are still unclear. Several authors assume that there is an association among injection frequency, the injection procedure, patient predisposition and osmolarity or concentration of the injected drug.8 Others have found no correlation in regard to the number of injections, dose per injection or total dose of prostaglandin E1.9 The most important problem results from the fact that diagnosis is made only by clinical examination. Penile fibrosis before any therapeutical approach seems to be much more common in impotent men than is generally recognized.8 Plaques, constriction and deformity or fibrotic changes have been found in 10% to 20% of impotent patients

After Injection Rt.

After Injection Lt.

30 41 41 42 52 35 19 26 25 23

42 44 39 44 53 32 36 28 24 25

before intracavernosal injection therapy.8 Therefore, it is important that the penis is examined methodically before therapy is initiated, and any fibrotic changes should be carefully documented. However, to our knowledge no histological study of men exists in the literature on prostaglandin E1 intracavernous injection therapy. Aboseif et al found no fibrotic lesions in monkeys after prostaglandin E1 injections, whereas histological changes have been observed in the monkey penis after papaverine injections.10 The fibrotic lesions should result from the product rather than the method of injection, and was previously demonstrated in rabbits.11 The reduced risk of fibrosis development with prostaglandin E1 could also be due to the higher pH value of the injection.10, 12 We do not believe that the observed reduction of smooth muscle cells was due to prostaglandin E1. Lesions existed only at the penile site of injections and not in the other corpus cavernosum, whereas the drug diffused into both corpora. We previously demonstrated that the proportional results of a small Biopty‡ gun biopsy are representative of the entire penile body.13 Pretreatment biopsies were performed in the left corpus cavernosum and, thus, could not skew the reduced percentage of smooth muscle cells observed in the right corpus cavernosum after treatment. Fibrotic lesions would result from traumatic needle punctures. Penile fibrosis would begin as vascularitis in the subtunical tissues and continue as chronic inflammation, leading to perivascular fibrosis and dense plaque formation as described in Peyronie’s disease.14 The fact that half of the fibrotic alterations disappeared in impotent patients treated with papaverine or prostaglandin E1 intracavernous injection after temporary discontinuation of self-injection therapy or improvement of the individual injection technique seems to agree with this hypothesis.15, 16 On the other hand, Moreland showed that hypoxia in patients with impotence of vascular origin induced the expression of transforming growth factor b1 which produces collagen synthesis in the corpus cavernosum and, therefore, fibrosis of the penis.17 Prostaglandins seem to be able to suppress this collagen synthesis in primary cultures of human corpus cavernosum smooth muscle cells and may have potential to prevent fibrotic lesions associated with vasculogenic impotence. Intracavernous prostaglandin E1 injection would result in oxygenation of the corpora cavernosa and perhaps remodeling of the penile connective tissue. We observed no increase in the percentage of smooth cells. It is well known that what is observed in vitro cannot always be applied in vivo.18 Improvement in the erectile quality in patients treated with intracavernous prostaglandin E1 injection would be due to psychogenic effects rather than modifications of the intracavernous structures. CONCLUSIONS

While our study represents a limited number of patients, we conclude that penile fibrosis with intracavernous prosta‡ Bard Urological, Covington, Georgia.

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PROSTAGLANDIN E1 THERAPY AND INTRACAVERNOUS MUSCULATURE

glandin E1 injection seems to result from traumatic lesions caused by the needle and not from alterations of the drug. Prostaglandin E1 intracavernous injection does not seem to influence growth factor activation in vivo and, therefore, does not decrease the percentage of collagen tissue which could be used for remodeling penile smooth musculature. REFERENCES

1. Krane, R. J., Goldstein, I. and Saenz de Tejada, I.: Impotence. N Engl J Med, 321: 1648, 1989 2. Lakin, M. M., Montague, D. K., Medendorp, S. V. et al: Intracavernous injection therapy: analysis of results and complications. J Urol, 143: 1138, 1990 3. Fallon, B.: Intracavernous injection therapy for male erectile dysfunction. Urol Clin North Am, 22: 833, 1995 4. The European Alprostadil Study Group: The long-term safety of alprostadil (prostaglandin-E1) in patients with erectile dysfunction. Br J Urol, 82: 538, 1998 5. Virag, R., Nollet, F., Greco, E. et al: Dynamic echography of the penis in the follow-up of impotent patients treated with intracavernous injections. Br J Urol, 72: 809, 1993 6. Wespes, E., Depierreux, M. and Schulman, C. C.: Use of Biopty gun for corpus cavernosum biopsies. Eur Urol, 18: 81, 1990 7. Wespes, E., Moreira de Goes, P. and Schulman, C. C.: Agerelated changes in the quantification of the intracavernous smooth muscles in potent men. J Urol, suppl., 159: 99, abstract 379, 1998 8. Chew, K. K., Stuckey, B. G. A., Earle, C. M. et al: Penile fibrosis in intracavernosal prostaglandin E1 injection therapy for erectile dysfunction. Int J Impot Res, 9: 225, 1997 9. Chen, R. N., Lakin, M. M., Montague, D. K. et al: Penile scarring with intracavernous injection therapy using prostaglandin E1: a risk factor analysis. J Urol, 155: 138, 1996 10. Aboseif, S. R., Breza, J., Bosch, R. J. L. H. et al: Local and systemic effects of chronic intracavernous injection of papaverine, prostaglandin E1, and saline in primates. J Urol, 142: 403, 1989 11. Stackl, W., Loupal, G. and Holzmann, A.: Intracavernous injection of vasoactive drugs in the rabbit. Urol Res, 16: 455, 1988 12. Samaha, A. M., Jr. and Seidmon, E. J.: Effects of phentolamine on pH of papaverine. J Urol, 139: 256A, abstract 374, 1988 13. Wespes, E., Moreira de Goes, P. and Schulman, C.: Vascular impotence: focal or diffuse penile disease. J Urol, 148: 1435, 1992 14. Vande Berg, J. S., Devine, C. J., Horton, C. E. et al: Peyronie’s disease: an electron microscopic study. J Urol, 126: 333, 1981 15. Porst, H.: Penile fibrosis in intracavernosal prostaglandin E1 injection therapy for erectile dysfunction (editorial comment). Int J Impot Res, 9: 229, 1997 16. Virag, R., Shoukry, K., Floresco, J. et al: Intracavernous selfinjection of vasoactive drugs in the treatment of impotence: 8-year experience with 615 cases. J Urol, 145: 287, 1991 17. Moreland, R. B.: Is there a role of hypoxemia in penile fibrosis: a

viewpoint presented to the Society for the Study of Impotence. Int J Impot Res, 10: 113, 1998 18. Melman, A.: Is there a role of hypoxemia in penile fibrosis (editor’s note). Int J Impot Res, 10: 113, 1998 EDITORIAL COMMENT The authors present an interesting yet controversial study of changes in the intracavernosal musculature from injection therapy using prostaglandin E1. Previous studies have documented intracavernous smooth muscle fibrosis and other changes from papaverine injection but fibrotic changes are rare, occurring in only 4% of patients treated in long-term studies. Clearly the identification of fibrosis previously has been clinical with gross measurements by palpation and patient report only. However, it is well recognized that this fibrosis occurs in some areas of the corpora cavernosa while others are spared. The authors evaluated patients treated with prostaglandin E1 not only clinically, but also histologically with corpus cavernosum biopsies. Controversy has continued regarding the etiology of fibrosis of the corpus cavernosum in patients using papaverine. Is this fibrosis caused by medication, solution pH, or trauma from injection or repeat needle insertions? The authors make a strong case that prostaglandin E1 is safer than papaverine. However, the incidence of corpus cavernosum fibrosis with papaverine is low, occurring in approximately only 4% of patients. Absolute comparisons cannot be made with this small number of patients. However, it is noteworthy that 2 patients demonstrated improved intracavernosal smooth muscle concentrations following injection therapy but one must ask whether biopsies were performed in the areas of injection and whether there was selection bias in biopsy location. Laboratory studies have strongly suggested that prostaglandin E1 and papaverine have different safety profiles. These studies were carefully performed and clear in their conclusions and recommendations. However, because of low numbers and possible selection bias the current study does not conclusively prove difference in fibrosis with these 2 agents in human corpora cavernosa. These caveats do not lessen the importance of this study. The authors for the first time have, to my knowledge, followed patients with pretreatment and posttreatment biopsies of the corpora cavernosa. It is evident that no global changes occurred in the intracavernous smooth muscle from long-term injection of prostaglandin E1. In fact, there is a suggestion that erections and perhaps oxygenation will improve smooth muscle concentrations in some patients. Furthermore, this study strongly suggests that the fibrosis experienced by some patients on intracavernous injection therapy is more likely caused by needle trauma and not the medications. These conclusions are important in counseling our patients who choose intracavernous injection therapy for longterm treatment of erectile dysfunction. Culley C. Carson, III Division of Urology University of North Carolina Chapel Hill, North Carolina