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Effect of 2-week combination therapy with the luteinizing hormone-releasing hormone (LHRH) agonist [d -Trp6, des-Gly-NH210]LHRH ethylamide and the antiandrogen flutamide on prostate structure and steroid levels in the dog
Molecular and Cellular Endocrinology, 67 (1989) 131-138 Elsevier Scientific Publishers Ireland, Ltd.
131
MOLCEL 02166
Effect of 2-week combination therapy with the lute'mizing hormone-releasing hormone (LHRH) agonist [D-Trp6, des-Gly-NH21°]LHRH ethylamide and the antiandrogen flutamide on prostate structure and steroid levels in the dog Daniel Lacoste, Donald Dub6, Alain B61angerand Fernand Labrie Medical Research Council Group in Molecular Endocrinology, Research Centre, Laval University Medical Centre, Quebec GI V 4G2, Canada
(Received 1 August 1989; accepted 14 August 1989)
Key words: Prostate; Dihydrotestosterone; Androgen; Antiandrogen; Flutamide; Lut"emizing hormone-releasing hormone agonist; [D-Trpe, des-Gly-NH21°]lute'mizin8hormone-releasing hormone ethylamide; Combination therapy
Summary Treatment of adult dogs for 15 days with the luteinizing hormone-releasing hormone (LHRH) agonist [D-Trpe, des-Gly-HH21°]LHRH ethylamide (50/tg daily, s.c.) causes a 41.6~ inhibition of prostatic weight while a 55~ inhibition is achieved when the antiandrogen flutamide (250 mg, twice daily) is given in association with the LHRI-I agonist (p < 0.01). At histology, the glandular acini in the prostate of animals treated with the combination therapy are more atrophied than with either treatment used alone. A 2-week treatment with the LHRH agonist is characterized by two distinct phases, namely a stimulation of testicular androgen secretion between days 0 and 8 followed by an inhibition betw'eet~ days 9 and 15. During the inhibitory phase, the concentration of all testicular steroids progressively decreased to castration levels, thus indicating that the differences observed at the prostatic level at 2 weeks are due to the inhibition of androgen action by the antiandrogen flutamide during the period which precedes complete chemical castration by the LHRH agonist. Flutamide administered alone had no effect on plasma or prostatic steroid levels measured after 2 weeks and it did not interfere with the potent and generalized inhibitory effect of the LHRH agonist on the serum and prostatic concentration of all steroids measured. Since dihydrotestosterone (DH'I) is the active androgen in the prostatic tissue, it is of major interest to observe that treatment with the LHRH agonist or orchiectomy alone or in combination with flutamide caused a decrease in the intraprostatic level of DHT from 4.7 + 1.2 ng/g tissue in intact control dogs to the lower limits of detection of the assay (0.3 ng/g) while flutamide administered alone was less efficient with a value of 1.1 + 0.3 ng D H T / g tissue. The present data demonstrate that the addition of the antiandrogen flutamide to treatment with an LHRH agonist permits a more rapid and complete regression of the prostate in the dog through more efficient and rapid blockade of testicular androgen action.
Introduction Address for correspondence: Daniel Lacoste, Medical Research Council Group in Molecular Endocrinology, Research Centre, Laval University Medical Centre, Quebec GIV 4G2,
Canada.
Soon after discovery that the agonists of luteinizing hormone-releasing hormone (LHRH)
0303-7207/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland, Ltd.
132
block testicuiar androgen biosynthesis in the rat (Auclair e~ al., 1977; Labrie et al., 1978), it was found ~at chemical castration can be efficiently and easily achieved with the same peptides in adult men without side effects other than those related to hypoandrogenism, namely hot flashes and a decrease or loss of potency and libido (Labrie et al., 1980, 1982, 1985a; Faure et al., 1982; Allen et al., 1983; Wenderoth and Jacobi, 1984; The Leuprolide Study Group, 1984). Since the observatio~ of Huggins and his colleagues in 1941 (Huggins and Hedges, 1941), the standard treatment of advanced prostatic cancer has been the removal or blockade of testicular androgens by orchiectomy and treatment with estrogens, respectively (Nesbit and Baum, 1950; Jordan et al., 1977). Since orchiectomy is psychologically unacceptable by many patients while the administration of estrogens causes serious and frequently lethal cardiovascular complications (Byar, 1970; Glashan and Robinson, 1981), treatment with LHRH agonists offers an alternative to orchiectomy and should certainly replace estrogen therapy. However, as observed in the first patient with prostate cancer treated with an LHRH agonist (Labrie et al., 1980), one limitation to the use of LHRH agonists alone is the transient rise in serum testosterone and dihydrotestosterone levels during the first 5-8 clays of treatment with the accompanying risks of disease flare or exacerbation of the disease in a significant proportion of patients (Waxman et al., 1983; Kahan et al., 1984; The Leuprolide Study Group, 1984). In order to avoid this problem, we have developed an approach which combines an LHRH agonist with an inidbitor of androgen binding to its receptor (Neri et al,, 1972; Poyet and Labrie, 1985; Simard et al., 1986) administered at the start of therapy of prostate cancer (Labrie et al., 1982, 1985b, 1986, 1987a, b). Since a transient rise in testicular androgen secretion during the early phase of treatment with an LHRH ,agonist is likely to exert a significant influence on the most significant parameters of androgen action, namely prostatic weight, glandular morphology as well as steroid content, we have studied the effect of treatment with the LHRH agonist [D-Trpe, des-GIy-NH21°]LHRH ethylamide and flutamide each administered alone or
in association compared to castration or castration + flutamide on histological structures of the prostate gland as well as on the levels of a large series of steroids in the serum and in the prostate. In order to assess the residual influence of the LHRH-A stimulation phase of androgens on the glandular morphology, we chose a 2-week treatment time interval, as supported by the observation of a stimulation of argine esterase, an androgen-dependent secreted protein of epithelial origin (Frenette, 1987). The dog has been chosen for these studies since the changes in testicular steroidogenesis induced in this species by LHRH agonist treatment (Tremblay et al., 1984; Tremblay and B61anger, 1984) closely resemble those observed in adult men (Labrie et al., 1986). Materials and methods
Chemicals The LHRH agonist [D-Trp6, des-GIy-NH21°]-LHRH ethylamide (LHRH-A) was obtained from Bachen, Torrance, CA, U.S.A., while the pure nonsteroidal antiandrogen flutamide (Euflex) was supplied by Schering Canada, Montreal. All other chemicals and reference steroid standards were of analytical grade from Anachemia and BDH. Animals Thirty adult mongrel male dogs weighing 20-25 kg were selected for this study. Animals were house individually and fed tap water and dog chow ad libitum. Treatments ~roups of six animals received 50/~g LHRHA/day administered subcutaneously in 0.5 ml 1~ gelatin-0.9~ NaCI, flutamide (Flu), 250 mg orally, twice daily, or the combination LHRH-A + flutamide. Similar groups of animals were surgically castrated and received placebo or flutamide. Before treatment, dogs were anesthetized under halothane and the volume of the prostate was measured in three dimensions with calipers. Animals were treated for 15 days. Blood samples were collected from all dogs via the cephalic vein on days - 2 and - 1 (control value), 0 (beginning of treatment, immediately prior to, as well as 3 h and 8 h after the administration of the drugs
133
on days 1, 2, 3, 4, 5, 6, 8, 10, 13 and 15. Blood samples were kept on ice before collecting plasma by centrifugation. Plasma samples were stored at - 2 0 ° C until assayed for steroid content. At the end of the experiment, the dogs were sacrificed, the prostates were immediately removed and measured in three dimensions with calipers. A small portion of each prostate was fixed by immersion in Bouin's fluid for 3 days and then embedded in paraffin. Tissue sections were cut at 7 igm and stained with hematoxylin and eosin. The remaining part of the gland was frozen on dry ice and stored at - 2 0 ° C until assayed for steroid content.
Steroid radioimmunoassays Steroid concentrations were measured by radioimmunoassays (RIA) following diethyl ether extraction and separation on LH-20 columns as described in detail elsewhere (Btlanger et al., 1980). The data are presented as the mean :i: SE~vl. Radioimmanoassay data were analyzed using a program based on model II of Rodbard and Lewald (1970).
Statistical analyses Statistical significance was measured according to the multiple-range test of Duncan-Kramer (1956).
Results As illustrated in Fig. 1, daily subcutaneous administration of 50 /Lg of the LHRH agonist [D-Trp6, des.GIy-NH21°]LHRH ethylamide for 15 days caused a 41.6% inhibition of prostate weight while a 55.0% inhibition was achieved by addition of the antiandrogen flutamide (250 rag, twice daily, per os) to the LHRH agonist administration (p < 0.01). Orchiectomy alone and in combination with flutamide caused 64.0% and 59.0% inhibitions, respectively, while flutamide alone led to a 56.7% reduction in prostatic weight. In order to better understand tne changes in prostatic volume, it is then of interest to examine in detail the concentration of androgens, their precursors and metabolites in the prostatic tissue as well as in the plasma of the same animals. As illustrated in Fig. 2, the intraprostatic levels of
too
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/2/#/
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Fig. 1. Effect of treatment for 15 days with the LHRH agonist [D-Trp6, des-GIy-NH21°]LHRH ethylamide (50 /tg, subcutaneously daily), flutamide (250 mg twice daily per os), the combinations LHRH agonist+flutamide, orchiectomy o:" orchiectomy+flutamide on the volume of the prostate (six animals per group). Data are presented as means + SEM.
progesterone are reduced below detection limits by all treatments while no significant effect is observed on the concentration of the three other C-21 steroids (pregnenolone, 17-OH-pregnenolone and 17-OH-progesterone). While there is no significant effect of flutamide alone on either dS-diol or A4-dione prostatic levels, treatment with LHRH-A, surgical castration, LHRH-A + flutamide or castration + flutamide causes a 50% decrease in the co:,centrations of AS-diol and A4-dione while the intraprostatic testosterone (T) concentration is reduced by 85% (Fig. 2). The most important and striking effect of treatme.-*, ;,s, however, that on the h,tl'epro~tatic concentration of dihydrotestosterone (DHT) which is reduced from 4.7:1:1.2 ng/g tissue in intact animals to the lower limits of detection of the assay (0.3 ng/g tL- ~e) in all the treatment group~,. However, when taken alone, flutamide increases dehydroepiandrosterone (DHEA), 3a-diol and 3/3-diol prostatic content well above control levels and decreases prostatic T and DHT levels from 1.9 :t: 0.2 ng/g to 0.9 to 0.1 ng/g and from 4.7 :i: 1.2 ng/g to 1.1:1:0.3 n g / g of tissue, respectively. During the inhibitory phase of LHRH agonist action (between days 9 and 15) (Fig. 3), the areas under the plasma concentration curves expressed in percentage of the intact group taken as control
134 17-0H nR~'.GNENOLONE
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Fig, 3. Effect of 15-day treatment as described in Fig. 1 on the p+~sma steroid levels measured between days 0-8 (open columns) and 9-15 (sbadc~ columns).
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B: LHRH
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(100%) show that the plasma steroid levels of DHEA, aS-diol, progesterone, androstenedione, testosterone, dihydrotestosterone, 3a-diol and 3/]-
diol reach castration or near-castration levels during this period. At 15 days, the plasma concentration of all these steroids is not different from the
136
values measured after castration. However, during ,_he stimulatory phase (between days 0 and 8), it can be observed that animals treated with the LHRH agonist alone or in combination with flutamide have 30-50~ higher plasma levels of DHEA, AS-diol, progesterone, androstenedione, testosterone and DHT. On the other hand, flutamide alone or in combination with the LHRH agonist does not affect steroid levels during the same period of treatment. Castration alone or in association with flutamide reduces plasma steroid levels of pregnenolone, progesterone and 17-OHprogesterone to about 30~ of control while the levels of DHEA, AS-diol, testosterone, DHT, 3adiol and 3~-diol are reduced to approximately 10~ of control. Surprisingly, plasma 17-OH-pregnenolone levels were increased 15 days after surgical or medical castration. The histology of the glandular acini in the prostate of intact animals showed a well-differentiated epithelium with primary and secondary papillae in the glandular lumen. The glandular cells lining those papillae had a well-developed apical pole (Fig. 4A). The prostate of animals of all other groups showed acini with various degrees of regression of the apical pole of glandular cells, disappearance of secondary papillae and a decrease in the length of primary papillae in the lumen of the acini. The surface area of the apical pole in glandular cells was 30-50~ reduced in the animals treated with the LHRH agonist (Fig. 48) or flutamide (Fig. 4C). Castration plus flutamide, castration alone and treatment with the LHRH agonist and flutamide caused an almost complete disappearance of the apical pole of the glandular cells (Fig. 4D, E, F). Moreover, the primary papillae of the epithelium were markedly reduced in number and length in the prostatic tissue from animals treated with flutamide and the LHRH agonist or orchiectomy (Fig. 4D, F). Simultaneously with the atrophy of the glands, there was a relative increase in stromal volume, this phenomenon being of more amplitude in the animals treated with the combination therapy. Discussion The present data clearly demonstrate that despite the presence of higher serum levels of androgens during the first day~ of treatment with
the LHRH agonist, a maximal reduction in prostatic weight undistinguishable from that achieved by surgical castration is observed after 15 days of treatment with the antiandrogen flutamide associated with the LHRH agonist [D-Trp6, des-G!yNH21°]LHRH ethylamide. Such findings are in agreement with our previous data showing a rapid and marked fall in serum prostatic acid phosphatase (PAP) following combination therapy with the same LHRH agonist and antiandrogen in prostate cancer patients (Labrie et al., 1987a). However, while serum PAP levels closely reflect changes in tumor cell activity, no data were pre~iously available on the influence of an LHRH agonist alone and the combination of an antiandrogen and LHRH agonist on direct parameters of prostatic growth, especially prostatic volume and histological features. Since dog adrenals do not contribute to the total androgens pool as indicated by the absence of effect of ACTH on plasma DHEA levels in castrated dogs (Tremblay, 1984), it is expected that any medical treatment which eliminates or neutralizes the androgens of testicular origin will have the same maximal inhibitory effect as orchiectomy on prostatic weight. Moreover, since complete chemical castration with LHRH agonists is achieved after approximately 2 weeks, the differences observed during the early days of treatment will disappear at later time intervals. We have thus chosen the 15-day interval to perform the present study. In fact, in a recent study performed at 21 days, we have found similar inhibitory effects on dog prostate weight after treatment with the LHRH agonist [D-Trp~, des-GIyNH,1°]LHRH ethylamide alone or in combination with flutamide (Lacoste et al., 1989). Although differences are likely to be greater at earlier time intervals, especially during the stimulatory period, the present data show that the differences are still present after 15 days of treatment (Fig. 1) while they have disappeared 7 days later (Lacoste et al., 1989). The present data show that the inhibition of prostate weight induced by treatment with the LHRH agonist alone was 41.6~ while the addition of flutamide enhanced the inhibition by a further 13.4% (p _<0.05) to reach an inhibitory effect of 55%. Following treatment with flutamide alone,
137
the inhibition of prostate weight after 2 weeks of treatment was not significantly different from the values observed in the animals orchiectomized or in the animals receiving the combination orchiectomy + flutamide. It is likely, however, as mentioned above, that measurements at earlier time intervals could have shown different efficacies between the treatments used. Concomitantly with the inhibition of prostate weight observed after 2 weeks of treatment, there was an alteration in the morphology of the glandular acini. The primary and secondary papillae of the acini and the apical pole of the glandular cells showed atrophy following therapy. These histological structures are recognized as markers of the secretory activity of the prostate (Brandes, 1966). The acini were found to be more atrophied after LHRH agonist + flutamide or c'astration + flutamide treatments compared to either treatment used alone. This observation suggests that more rapid regression follows more complete androgen deprivation. Androgen receptors have been reported in almost all the acini of human benign prostatic hyperplasia (BPH) (Barrack, unpubfished observation). We believe that the results of the present study could be extended to human prostate cancer since the human prostate is also highly sensitive to androgen deprivation (Lab~ie e~
al., 1980, 1986). It is well known that fiutamide competes with androgens for their receptors (Simard, 1986), occupancy of the receptor by the antiandrogen leading to an inhibition of the negative feedback of androgens on luteinizing hormone (LH) secretion, especially in man (Knuth, 1984). The same phenomenon is not observed in the dog. In fact, the present study shows that plasma androgen levels are not affected by fiutamide administered alone. Such a difference between species can possibly be explained by variable access of the antiandrogen to the hypothalamic site(s) of negative feedback action of androgens. It should be mentioned, however, that the present data demonstrate the relative efficacies of treatment with an LHRH agonist used alone and in combination with flutamide on the growth of normal prostatic tissue. Using the androgen-sensitive mouse mammary carcinoma, Shionogi SC-115, we have recently found that androgen sensitivity is
progressively lost in the presence of low concentrations of androgens and that such development of androgen insensitivity can be prevented or at least delayed by simultaneous treatment with flutamide (Luthy and Labrie, 1986). In other word~% in addition to delaying the regression of prostate cancer, the use of LHRH agonists alone (as well as orchiectomy alone) could well induce the development of treatment insensitivity and thus compromise antiandrogen therapy applied at a later stage (Labrie et al., 1988a). It should also be mentioned that androgen-sensitive cancer shows a wide range of sensitivities to androgens. Using the Sbionogi carcinoma cell line SC-115, we have found clones having a 1250-fold range of sensitivities to DHT (Labrie and Vei!leux, 1986). One clone was so sensitive to androgens that its KD value of stimulation by DHT was 0.008 ng/ml. Although under the present experimental conditions, there were no significant differences in prostatic weight induced by the addition of flutamide to orchiectomy, it is expected that tumors having a higher sensitivity to androgens (or requiring a smaller amount of androgens for growth) would respond differently to the various forms of treatment. While the blockade achieved by castration or flutamide alone is sufficient to block the growth of the normal prostatic cells in an animal specially having no adrenal androgens, cancer cells having a higher than normal sensitivity to androgens escape partial androgen blockade and need more efficient blockade before showing tumor regression (Labrie and Veilleux, 1985; Labrie et al., 1986, 1987a, b; 1988a, b). In mean, one must always take into consideration the fact that the adrenal glands contribute to about 50~ of DHT present in the prostatic tissue (Labrie et al., 1987b; 1988c). Thus, while in the dog, a species having no significant adrenal secretion of androgens, orchiectomy has a maximal effect on prostate weight, ~imultaneous blockade of the secretion or action of adrenal androgens would be required to obtain a similar maximal effect on prostate growth in men. The advantage of the present model is that detailed study of the efficacy of different treatments on testicular androgen secretion and/or action can be performed independently from adrenal androgens.
138 References Allen, J.M., O'Shea, J.P., Mashiter, K., Williams, G. and Broom, S.R. (1987) Lancet ii, 413. Auclair, C., Kelly, P.A., Coy, D.H., Schally, A.V. and Labrie, F. (1977) Endocrinology 101, 1890-1893. B~langer, A., Caron, S. and Picard, V. (1980) J. Steroid Biochem. 13, 185-180. Brandes, D. (1966) Int. Rev. Cytol. 20, 207-276. Byar, D.P. (1970) Cancer 32, 1126-1130. Faure, N., Labrie, F., Lemay, A., B61anger, A., Gourdeau, Y., Laroche, B, and Robert, G. (1982) Fertil. Steril. 37, 416-424. Frenette, G., Dub6, J.Y., Lacoste, D. and Tremblay, R.R. (1987) Prostate 10, 145-152. Glashan, R.W. and Robinson, M.R.G. (1981) Br. J. Urol. 53, 624-630. Huggins, C. and Hodges, C.V. (1941) Cancer Res. 1, 293-297. Jordan, Jr., W.P., Blackard, C.E. and Byar, D.P. (, 977) South. Med. J. 70, 1411-1413. Kahan, A., Delrieu, F., Amor, B., Chiche, R. and Steg, A. (19P';) Lancet i, 971-972. Khutl~, U.A., Nano, R. and Nieschla8, E. (1984) J. Clin. Endocrinol. Metal). 59, 963-969. Kramer, C.Y. ~1956) Biometrics 12, 307-310. Labrie, F. and Veilleux, R. (1986) Prostate 8, 293-300. Labrie, F., Aucl~r, C~, Cusan, L., Kelly, P.A., Pelletier, G. and Ferland, L. ~978) in Endocrine Approach to Male Contraception (Ha~s~,n, V., ed.), Int. J. Androl. (Suppl. 2), 303-308. Labrie, F., B61~ger, A., Cusan, L., S~guin, C., Pelletier, G., Kelly, P.A., J:,efebvre. F.A., Lemay, A. and Raynaud, J.P. (1980) J. Androi. 1, ~ - 2 2 8 . Labrie, F., Dupom, A., ~lanser, A., Cusan, L., Lacourci~re, Y., Monfett¢, G., Laberge, J.G., Emond, J.P., Fazekas, A.T.A., Ra)'r~aud, J.P. and Husson, J.M. (1982) J. Clin. Invest. Med. 5, 267-275. Labrie, F., Dupcmt, A. and i~langer, A. (1985a) in Important Advan~ in O~colo~ (De Vita, Jr., V.T., Hellman, S. and kosenberg, S,A., eds.), pp. 193-217, J.B. Lippincott, Philadelphia, PA. Labrie~ F., Dupont, ~ , B61anger, A., Lachance, R. and Gigu~re, ~. (]~RSb~ Br. Med. J. 291, 369-370. L~,~i~., F., Dupont, A., B61anger, A., St-Arnaud, R., Gigu~re, ~., Lacourci~re, Y., Emond, J.P. and Monfette, G. (1986) ~-odocr. Rev. 7, 67-74. La~c, F., Dupont, A. and B~langer, A. (1987a) in
Genitourinary Cancer (Ratliff, T.L. and Catalona, W.J., eds.), pp. 157-200, Martinus Nijhoff, Boston, MA. Labrie, F., Dupont, A., B61anger, A. and Lachance, R. (1987b) J. Urol. 138, 804-806. Labrie, F., Veilleux, R. and Fournier, A. (1988a) J. Natl. Cancer Inst. 80, 1138-1147. Labrie, F., Dupont, A., Gigu~re, M., Borsanyi, J.P., Lacourci~re, Y., Monfette, G., Emond, J. and Bergeron, N. (1988b) Br. Med. J. 61, 341-346. Labrie, F., B61anger, A., Veilleux, R., Lacoste, D., Labrie, C., Marchetti, B., Poulin, R., Dupont, A., Cusan, L. and Luthy, I. (1988c) in Bailli~re's Clinical Oncology (B. Furr and L. Denis, eds.), Vol. 2, pp. 597-619, Saunders, London. Lacoste, D., St-Arnaud, R., B61anger, A. and Labrie, F. (1988) Mol. Cell. Endocrinol. 56, 141-147. Lacoste, D., Caron, S., B61anger, A. and Labrie, F. (1989) J. Steroid Biochem. 33, 233-242. Luthy, I. and Labrie, F. (1986) Prostate 10, 89-94. Neri, R., Florance, K., Koziol, P. and Van Cleave, S. (1972) Endocrinology 91, 427-437. Nesbit, R.M. and Baum, W.C. (~950) J. Am. Med. Assoc. 143, 317-320. Poyet, P. and Labrie, F. (1985) Mol. Cell. Endocrinol. 42, 283-288. Rodbard, D. and Lewald, J.E. (1970) in 2rtd Karolinska Symposium on Research Methods in Reproductive Endocrinology (Diczfalusy, E., ed.), pp. 79-103, Bogtrykleriet Forum, Copenhagen. Simard, J., Luthy, I., Guay, J., B61anger, A. and Labrie, F. (1986) Mol. Cell. Endocrinol. 44, 261-270. The LeuproUde Study Group (1984) New Engl. J. Med. 311, 1281-1286. Tremblay, Y. and B61anger, A. (1984a) Contraception 30, 483 -497. Tremblay, Y. and B61anger, A. (1984b) Steroids 44, 57-65. Tremblay, Y., B~langer, A,, Labrie, F., Frenette, G., Dub6, J.Y. and Tremblay, R.R. (1984) Prostate 5, 424-432. Waxman, J.H., Wass, J.A.H., Hendry, W.F., Whitfleld, H.N., Besser, G.M., Malpas, J.H., Wags, J.A.H., Hendry, W.F., Whitfield, H.N., Besser, G.M., Malpas, J.S. and Oliver, R.T. (1983) Br. Med. J. 286, 1309-1312. Wenderoth, U.K. and Jacobi, G.H. (1984) in LRHH and its Analogues: Basic and Clinical Aspects (Labrie, F., B61anget, A. and Dupont, A., eds.), pp. 349-357, Excerpta Medica, New York.
131
MOLCEL 02166
Effect of 2-week combination therapy with the lute'mizing hormone-releasing hormone (LHRH) agonist [D-Trp6, des-Gly-NH21°]LHRH ethylamide and the antiandrogen flutamide on prostate structure and steroid levels in the dog Daniel Lacoste, Donald Dub6, Alain B61angerand Fernand Labrie Medical Research Council Group in Molecular Endocrinology, Research Centre, Laval University Medical Centre, Quebec GI V 4G2, Canada
(Received 1 August 1989; accepted 14 August 1989)
Key words: Prostate; Dihydrotestosterone; Androgen; Antiandrogen; Flutamide; Lut"emizing hormone-releasing hormone agonist; [D-Trpe, des-Gly-NH21°]lute'mizin8hormone-releasing hormone ethylamide; Combination therapy
Summary Treatment of adult dogs for 15 days with the luteinizing hormone-releasing hormone (LHRH) agonist [D-Trpe, des-Gly-HH21°]LHRH ethylamide (50/tg daily, s.c.) causes a 41.6~ inhibition of prostatic weight while a 55~ inhibition is achieved when the antiandrogen flutamide (250 mg, twice daily) is given in association with the LHRI-I agonist (p < 0.01). At histology, the glandular acini in the prostate of animals treated with the combination therapy are more atrophied than with either treatment used alone. A 2-week treatment with the LHRH agonist is characterized by two distinct phases, namely a stimulation of testicular androgen secretion between days 0 and 8 followed by an inhibition betw'eet~ days 9 and 15. During the inhibitory phase, the concentration of all testicular steroids progressively decreased to castration levels, thus indicating that the differences observed at the prostatic level at 2 weeks are due to the inhibition of androgen action by the antiandrogen flutamide during the period which precedes complete chemical castration by the LHRH agonist. Flutamide administered alone had no effect on plasma or prostatic steroid levels measured after 2 weeks and it did not interfere with the potent and generalized inhibitory effect of the LHRH agonist on the serum and prostatic concentration of all steroids measured. Since dihydrotestosterone (DH'I) is the active androgen in the prostatic tissue, it is of major interest to observe that treatment with the LHRH agonist or orchiectomy alone or in combination with flutamide caused a decrease in the intraprostatic level of DHT from 4.7 + 1.2 ng/g tissue in intact control dogs to the lower limits of detection of the assay (0.3 ng/g) while flutamide administered alone was less efficient with a value of 1.1 + 0.3 ng D H T / g tissue. The present data demonstrate that the addition of the antiandrogen flutamide to treatment with an LHRH agonist permits a more rapid and complete regression of the prostate in the dog through more efficient and rapid blockade of testicular androgen action.
Introduction Address for correspondence: Daniel Lacoste, Medical Research Council Group in Molecular Endocrinology, Research Centre, Laval University Medical Centre, Quebec GIV 4G2,
Canada.
Soon after discovery that the agonists of luteinizing hormone-releasing hormone (LHRH)
0303-7207/89/$03.50 © 1989 Elsevier Scientific Publishers Ireland, Ltd.
132
block testicuiar androgen biosynthesis in the rat (Auclair e~ al., 1977; Labrie et al., 1978), it was found ~at chemical castration can be efficiently and easily achieved with the same peptides in adult men without side effects other than those related to hypoandrogenism, namely hot flashes and a decrease or loss of potency and libido (Labrie et al., 1980, 1982, 1985a; Faure et al., 1982; Allen et al., 1983; Wenderoth and Jacobi, 1984; The Leuprolide Study Group, 1984). Since the observatio~ of Huggins and his colleagues in 1941 (Huggins and Hedges, 1941), the standard treatment of advanced prostatic cancer has been the removal or blockade of testicular androgens by orchiectomy and treatment with estrogens, respectively (Nesbit and Baum, 1950; Jordan et al., 1977). Since orchiectomy is psychologically unacceptable by many patients while the administration of estrogens causes serious and frequently lethal cardiovascular complications (Byar, 1970; Glashan and Robinson, 1981), treatment with LHRH agonists offers an alternative to orchiectomy and should certainly replace estrogen therapy. However, as observed in the first patient with prostate cancer treated with an LHRH agonist (Labrie et al., 1980), one limitation to the use of LHRH agonists alone is the transient rise in serum testosterone and dihydrotestosterone levels during the first 5-8 clays of treatment with the accompanying risks of disease flare or exacerbation of the disease in a significant proportion of patients (Waxman et al., 1983; Kahan et al., 1984; The Leuprolide Study Group, 1984). In order to avoid this problem, we have developed an approach which combines an LHRH agonist with an inidbitor of androgen binding to its receptor (Neri et al,, 1972; Poyet and Labrie, 1985; Simard et al., 1986) administered at the start of therapy of prostate cancer (Labrie et al., 1982, 1985b, 1986, 1987a, b). Since a transient rise in testicular androgen secretion during the early phase of treatment with an LHRH ,agonist is likely to exert a significant influence on the most significant parameters of androgen action, namely prostatic weight, glandular morphology as well as steroid content, we have studied the effect of treatment with the LHRH agonist [D-Trpe, des-GIy-NH21°]LHRH ethylamide and flutamide each administered alone or
in association compared to castration or castration + flutamide on histological structures of the prostate gland as well as on the levels of a large series of steroids in the serum and in the prostate. In order to assess the residual influence of the LHRH-A stimulation phase of androgens on the glandular morphology, we chose a 2-week treatment time interval, as supported by the observation of a stimulation of argine esterase, an androgen-dependent secreted protein of epithelial origin (Frenette, 1987). The dog has been chosen for these studies since the changes in testicular steroidogenesis induced in this species by LHRH agonist treatment (Tremblay et al., 1984; Tremblay and B61anger, 1984) closely resemble those observed in adult men (Labrie et al., 1986). Materials and methods
Chemicals The LHRH agonist [D-Trp6, des-GIy-NH21°]-LHRH ethylamide (LHRH-A) was obtained from Bachen, Torrance, CA, U.S.A., while the pure nonsteroidal antiandrogen flutamide (Euflex) was supplied by Schering Canada, Montreal. All other chemicals and reference steroid standards were of analytical grade from Anachemia and BDH. Animals Thirty adult mongrel male dogs weighing 20-25 kg were selected for this study. Animals were house individually and fed tap water and dog chow ad libitum. Treatments ~roups of six animals received 50/~g LHRHA/day administered subcutaneously in 0.5 ml 1~ gelatin-0.9~ NaCI, flutamide (Flu), 250 mg orally, twice daily, or the combination LHRH-A + flutamide. Similar groups of animals were surgically castrated and received placebo or flutamide. Before treatment, dogs were anesthetized under halothane and the volume of the prostate was measured in three dimensions with calipers. Animals were treated for 15 days. Blood samples were collected from all dogs via the cephalic vein on days - 2 and - 1 (control value), 0 (beginning of treatment, immediately prior to, as well as 3 h and 8 h after the administration of the drugs
133
on days 1, 2, 3, 4, 5, 6, 8, 10, 13 and 15. Blood samples were kept on ice before collecting plasma by centrifugation. Plasma samples were stored at - 2 0 ° C until assayed for steroid content. At the end of the experiment, the dogs were sacrificed, the prostates were immediately removed and measured in three dimensions with calipers. A small portion of each prostate was fixed by immersion in Bouin's fluid for 3 days and then embedded in paraffin. Tissue sections were cut at 7 igm and stained with hematoxylin and eosin. The remaining part of the gland was frozen on dry ice and stored at - 2 0 ° C until assayed for steroid content.
Steroid radioimmunoassays Steroid concentrations were measured by radioimmunoassays (RIA) following diethyl ether extraction and separation on LH-20 columns as described in detail elsewhere (Btlanger et al., 1980). The data are presented as the mean :i: SE~vl. Radioimmanoassay data were analyzed using a program based on model II of Rodbard and Lewald (1970).
Statistical analyses Statistical significance was measured according to the multiple-range test of Duncan-Kramer (1956).
Results As illustrated in Fig. 1, daily subcutaneous administration of 50 /Lg of the LHRH agonist [D-Trp6, des.GIy-NH21°]LHRH ethylamide for 15 days caused a 41.6% inhibition of prostate weight while a 55.0% inhibition was achieved by addition of the antiandrogen flutamide (250 rag, twice daily, per os) to the LHRH agonist administration (p < 0.01). Orchiectomy alone and in combination with flutamide caused 64.0% and 59.0% inhibitions, respectively, while flutamide alone led to a 56.7% reduction in prostatic weight. In order to better understand tne changes in prostatic volume, it is then of interest to examine in detail the concentration of androgens, their precursors and metabolites in the prostatic tissue as well as in the plasma of the same animals. As illustrated in Fig. 2, the intraprostatic levels of
too
VOLUMEOFPHOSTATE
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Fig. 1. Effect of treatment for 15 days with the LHRH agonist [D-Trp6, des-GIy-NH21°]LHRH ethylamide (50 /tg, subcutaneously daily), flutamide (250 mg twice daily per os), the combinations LHRH agonist+flutamide, orchiectomy o:" orchiectomy+flutamide on the volume of the prostate (six animals per group). Data are presented as means + SEM.
progesterone are reduced below detection limits by all treatments while no significant effect is observed on the concentration of the three other C-21 steroids (pregnenolone, 17-OH-pregnenolone and 17-OH-progesterone). While there is no significant effect of flutamide alone on either dS-diol or A4-dione prostatic levels, treatment with LHRH-A, surgical castration, LHRH-A + flutamide or castration + flutamide causes a 50% decrease in the co:,centrations of AS-diol and A4-dione while the intraprostatic testosterone (T) concentration is reduced by 85% (Fig. 2). The most important and striking effect of treatme.-*, ;,s, however, that on the h,tl'epro~tatic concentration of dihydrotestosterone (DHT) which is reduced from 4.7:1:1.2 ng/g tissue in intact animals to the lower limits of detection of the assay (0.3 ng/g tL- ~e) in all the treatment group~,. However, when taken alone, flutamide increases dehydroepiandrosterone (DHEA), 3a-diol and 3/3-diol prostatic content well above control levels and decreases prostatic T and DHT levels from 1.9 :t: 0.2 ng/g to 0.9 to 0.1 ng/g and from 4.7 :i: 1.2 ng/g to 1.1:1:0.3 n g / g of tissue, respectively. During the inhibitory phase of LHRH agonist action (between days 9 and 15) (Fig. 3), the areas under the plasma concentration curves expressed in percentage of the intact group taken as control
134 17-0H nR~'.GNENOLONE
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(100%) show that the plasma steroid levels of DHEA, aS-diol, progesterone, androstenedione, testosterone, dihydrotestosterone, 3a-diol and 3/]-
diol reach castration or near-castration levels during this period. At 15 days, the plasma concentration of all these steroids is not different from the
136
values measured after castration. However, during ,_he stimulatory phase (between days 0 and 8), it can be observed that animals treated with the LHRH agonist alone or in combination with flutamide have 30-50~ higher plasma levels of DHEA, AS-diol, progesterone, androstenedione, testosterone and DHT. On the other hand, flutamide alone or in combination with the LHRH agonist does not affect steroid levels during the same period of treatment. Castration alone or in association with flutamide reduces plasma steroid levels of pregnenolone, progesterone and 17-OHprogesterone to about 30~ of control while the levels of DHEA, AS-diol, testosterone, DHT, 3adiol and 3~-diol are reduced to approximately 10~ of control. Surprisingly, plasma 17-OH-pregnenolone levels were increased 15 days after surgical or medical castration. The histology of the glandular acini in the prostate of intact animals showed a well-differentiated epithelium with primary and secondary papillae in the glandular lumen. The glandular cells lining those papillae had a well-developed apical pole (Fig. 4A). The prostate of animals of all other groups showed acini with various degrees of regression of the apical pole of glandular cells, disappearance of secondary papillae and a decrease in the length of primary papillae in the lumen of the acini. The surface area of the apical pole in glandular cells was 30-50~ reduced in the animals treated with the LHRH agonist (Fig. 48) or flutamide (Fig. 4C). Castration plus flutamide, castration alone and treatment with the LHRH agonist and flutamide caused an almost complete disappearance of the apical pole of the glandular cells (Fig. 4D, E, F). Moreover, the primary papillae of the epithelium were markedly reduced in number and length in the prostatic tissue from animals treated with flutamide and the LHRH agonist or orchiectomy (Fig. 4D, F). Simultaneously with the atrophy of the glands, there was a relative increase in stromal volume, this phenomenon being of more amplitude in the animals treated with the combination therapy. Discussion The present data clearly demonstrate that despite the presence of higher serum levels of androgens during the first day~ of treatment with
the LHRH agonist, a maximal reduction in prostatic weight undistinguishable from that achieved by surgical castration is observed after 15 days of treatment with the antiandrogen flutamide associated with the LHRH agonist [D-Trp6, des-G!yNH21°]LHRH ethylamide. Such findings are in agreement with our previous data showing a rapid and marked fall in serum prostatic acid phosphatase (PAP) following combination therapy with the same LHRH agonist and antiandrogen in prostate cancer patients (Labrie et al., 1987a). However, while serum PAP levels closely reflect changes in tumor cell activity, no data were pre~iously available on the influence of an LHRH agonist alone and the combination of an antiandrogen and LHRH agonist on direct parameters of prostatic growth, especially prostatic volume and histological features. Since dog adrenals do not contribute to the total androgens pool as indicated by the absence of effect of ACTH on plasma DHEA levels in castrated dogs (Tremblay, 1984), it is expected that any medical treatment which eliminates or neutralizes the androgens of testicular origin will have the same maximal inhibitory effect as orchiectomy on prostatic weight. Moreover, since complete chemical castration with LHRH agonists is achieved after approximately 2 weeks, the differences observed during the early days of treatment will disappear at later time intervals. We have thus chosen the 15-day interval to perform the present study. In fact, in a recent study performed at 21 days, we have found similar inhibitory effects on dog prostate weight after treatment with the LHRH agonist [D-Trp~, des-GIyNH,1°]LHRH ethylamide alone or in combination with flutamide (Lacoste et al., 1989). Although differences are likely to be greater at earlier time intervals, especially during the stimulatory period, the present data show that the differences are still present after 15 days of treatment (Fig. 1) while they have disappeared 7 days later (Lacoste et al., 1989). The present data show that the inhibition of prostate weight induced by treatment with the LHRH agonist alone was 41.6~ while the addition of flutamide enhanced the inhibition by a further 13.4% (p _<0.05) to reach an inhibitory effect of 55%. Following treatment with flutamide alone,
137
the inhibition of prostate weight after 2 weeks of treatment was not significantly different from the values observed in the animals orchiectomized or in the animals receiving the combination orchiectomy + flutamide. It is likely, however, as mentioned above, that measurements at earlier time intervals could have shown different efficacies between the treatments used. Concomitantly with the inhibition of prostate weight observed after 2 weeks of treatment, there was an alteration in the morphology of the glandular acini. The primary and secondary papillae of the acini and the apical pole of the glandular cells showed atrophy following therapy. These histological structures are recognized as markers of the secretory activity of the prostate (Brandes, 1966). The acini were found to be more atrophied after LHRH agonist + flutamide or c'astration + flutamide treatments compared to either treatment used alone. This observation suggests that more rapid regression follows more complete androgen deprivation. Androgen receptors have been reported in almost all the acini of human benign prostatic hyperplasia (BPH) (Barrack, unpubfished observation). We believe that the results of the present study could be extended to human prostate cancer since the human prostate is also highly sensitive to androgen deprivation (Lab~ie e~
al., 1980, 1986). It is well known that fiutamide competes with androgens for their receptors (Simard, 1986), occupancy of the receptor by the antiandrogen leading to an inhibition of the negative feedback of androgens on luteinizing hormone (LH) secretion, especially in man (Knuth, 1984). The same phenomenon is not observed in the dog. In fact, the present study shows that plasma androgen levels are not affected by fiutamide administered alone. Such a difference between species can possibly be explained by variable access of the antiandrogen to the hypothalamic site(s) of negative feedback action of androgens. It should be mentioned, however, that the present data demonstrate the relative efficacies of treatment with an LHRH agonist used alone and in combination with flutamide on the growth of normal prostatic tissue. Using the androgen-sensitive mouse mammary carcinoma, Shionogi SC-115, we have recently found that androgen sensitivity is
progressively lost in the presence of low concentrations of androgens and that such development of androgen insensitivity can be prevented or at least delayed by simultaneous treatment with flutamide (Luthy and Labrie, 1986). In other word~% in addition to delaying the regression of prostate cancer, the use of LHRH agonists alone (as well as orchiectomy alone) could well induce the development of treatment insensitivity and thus compromise antiandrogen therapy applied at a later stage (Labrie et al., 1988a). It should also be mentioned that androgen-sensitive cancer shows a wide range of sensitivities to androgens. Using the Sbionogi carcinoma cell line SC-115, we have found clones having a 1250-fold range of sensitivities to DHT (Labrie and Vei!leux, 1986). One clone was so sensitive to androgens that its KD value of stimulation by DHT was 0.008 ng/ml. Although under the present experimental conditions, there were no significant differences in prostatic weight induced by the addition of flutamide to orchiectomy, it is expected that tumors having a higher sensitivity to androgens (or requiring a smaller amount of androgens for growth) would respond differently to the various forms of treatment. While the blockade achieved by castration or flutamide alone is sufficient to block the growth of the normal prostatic cells in an animal specially having no adrenal androgens, cancer cells having a higher than normal sensitivity to androgens escape partial androgen blockade and need more efficient blockade before showing tumor regression (Labrie and Veilleux, 1985; Labrie et al., 1986, 1987a, b; 1988a, b). In mean, one must always take into consideration the fact that the adrenal glands contribute to about 50~ of DHT present in the prostatic tissue (Labrie et al., 1987b; 1988c). Thus, while in the dog, a species having no significant adrenal secretion of androgens, orchiectomy has a maximal effect on prostate weight, ~imultaneous blockade of the secretion or action of adrenal androgens would be required to obtain a similar maximal effect on prostate growth in men. The advantage of the present model is that detailed study of the efficacy of different treatments on testicular androgen secretion and/or action can be performed independently from adrenal androgens.
138 References Allen, J.M., O'Shea, J.P., Mashiter, K., Williams, G. and Broom, S.R. (1987) Lancet ii, 413. Auclair, C., Kelly, P.A., Coy, D.H., Schally, A.V. and Labrie, F. (1977) Endocrinology 101, 1890-1893. B~langer, A., Caron, S. and Picard, V. (1980) J. Steroid Biochem. 13, 185-180. Brandes, D. (1966) Int. Rev. Cytol. 20, 207-276. Byar, D.P. (1970) Cancer 32, 1126-1130. Faure, N., Labrie, F., Lemay, A., B61anger, A., Gourdeau, Y., Laroche, B, and Robert, G. (1982) Fertil. Steril. 37, 416-424. Frenette, G., Dub6, J.Y., Lacoste, D. and Tremblay, R.R. (1987) Prostate 10, 145-152. Glashan, R.W. and Robinson, M.R.G. (1981) Br. J. Urol. 53, 624-630. Huggins, C. and Hodges, C.V. (1941) Cancer Res. 1, 293-297. Jordan, Jr., W.P., Blackard, C.E. and Byar, D.P. (, 977) South. Med. J. 70, 1411-1413. Kahan, A., Delrieu, F., Amor, B., Chiche, R. and Steg, A. (19P';) Lancet i, 971-972. Khutl~, U.A., Nano, R. and Nieschla8, E. (1984) J. Clin. Endocrinol. Metal). 59, 963-969. Kramer, C.Y. ~1956) Biometrics 12, 307-310. Labrie, F. and Veilleux, R. (1986) Prostate 8, 293-300. Labrie, F., Aucl~r, C~, Cusan, L., Kelly, P.A., Pelletier, G. and Ferland, L. ~978) in Endocrine Approach to Male Contraception (Ha~s~,n, V., ed.), Int. J. Androl. (Suppl. 2), 303-308. Labrie, F., B61~ger, A., Cusan, L., S~guin, C., Pelletier, G., Kelly, P.A., J:,efebvre. F.A., Lemay, A. and Raynaud, J.P. (1980) J. Androi. 1, ~ - 2 2 8 . Labrie, F., Dupom, A., ~lanser, A., Cusan, L., Lacourci~re, Y., Monfett¢, G., Laberge, J.G., Emond, J.P., Fazekas, A.T.A., Ra)'r~aud, J.P. and Husson, J.M. (1982) J. Clin. Invest. Med. 5, 267-275. Labrie, F., Dupcmt, A. and i~langer, A. (1985a) in Important Advan~ in O~colo~ (De Vita, Jr., V.T., Hellman, S. and kosenberg, S,A., eds.), pp. 193-217, J.B. Lippincott, Philadelphia, PA. Labrie~ F., Dupont, ~ , B61anger, A., Lachance, R. and Gigu~re, ~. (]~RSb~ Br. Med. J. 291, 369-370. L~,~i~., F., Dupont, A., B61anger, A., St-Arnaud, R., Gigu~re, ~., Lacourci~re, Y., Emond, J.P. and Monfette, G. (1986) ~-odocr. Rev. 7, 67-74. La~c, F., Dupont, A. and B~langer, A. (1987a) in
Genitourinary Cancer (Ratliff, T.L. and Catalona, W.J., eds.), pp. 157-200, Martinus Nijhoff, Boston, MA. Labrie, F., Dupont, A., B61anger, A. and Lachance, R. (1987b) J. Urol. 138, 804-806. Labrie, F., Veilleux, R. and Fournier, A. (1988a) J. Natl. Cancer Inst. 80, 1138-1147. Labrie, F., Dupont, A., Gigu~re, M., Borsanyi, J.P., Lacourci~re, Y., Monfette, G., Emond, J. and Bergeron, N. (1988b) Br. Med. J. 61, 341-346. Labrie, F., B61anger, A., Veilleux, R., Lacoste, D., Labrie, C., Marchetti, B., Poulin, R., Dupont, A., Cusan, L. and Luthy, I. (1988c) in Bailli~re's Clinical Oncology (B. Furr and L. Denis, eds.), Vol. 2, pp. 597-619, Saunders, London. Lacoste, D., St-Arnaud, R., B61anger, A. and Labrie, F. (1988) Mol. Cell. Endocrinol. 56, 141-147. Lacoste, D., Caron, S., B61anger, A. and Labrie, F. (1989) J. Steroid Biochem. 33, 233-242. Luthy, I. and Labrie, F. (1986) Prostate 10, 89-94. Neri, R., Florance, K., Koziol, P. and Van Cleave, S. (1972) Endocrinology 91, 427-437. Nesbit, R.M. and Baum, W.C. (~950) J. Am. Med. Assoc. 143, 317-320. Poyet, P. and Labrie, F. (1985) Mol. Cell. Endocrinol. 42, 283-288. Rodbard, D. and Lewald, J.E. (1970) in 2rtd Karolinska Symposium on Research Methods in Reproductive Endocrinology (Diczfalusy, E., ed.), pp. 79-103, Bogtrykleriet Forum, Copenhagen. Simard, J., Luthy, I., Guay, J., B61anger, A. and Labrie, F. (1986) Mol. Cell. Endocrinol. 44, 261-270. The LeuproUde Study Group (1984) New Engl. J. Med. 311, 1281-1286. Tremblay, Y. and B61anger, A. (1984a) Contraception 30, 483 -497. Tremblay, Y. and B61anger, A. (1984b) Steroids 44, 57-65. Tremblay, Y., B~langer, A,, Labrie, F., Frenette, G., Dub6, J.Y. and Tremblay, R.R. (1984) Prostate 5, 424-432. Waxman, J.H., Wass, J.A.H., Hendry, W.F., Whitfleld, H.N., Besser, G.M., Malpas, J.H., Wags, J.A.H., Hendry, W.F., Whitfield, H.N., Besser, G.M., Malpas, J.S. and Oliver, R.T. (1983) Br. Med. J. 286, 1309-1312. Wenderoth, U.K. and Jacobi, G.H. (1984) in LRHH and its Analogues: Basic and Clinical Aspects (Labrie, F., B61anget, A. and Dupont, A., eds.), pp. 349-357, Excerpta Medica, New York.