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A pure antiandrogen does not interfere with the LHRH agonist-induced blockade of testicular androgen secretion in the dog
Molecular and Cellular Endocrinology, Elsevier Scientific Publishers Ireland,
56 (1988) 141-147 Ltd.
141
MCE 01819
A pure antiandrogen does not interfere with the LHRH agonist-induced blockade of testicular androgen secretion in the dog D. Lacoste, R. St-Amaud, A. BClanger and F. Labrie Medical Research Council Group in Molecular Endocrinology, Luval Universiiy Medical Center, Quebec, Que. GI V 4G2, Canada (Received
Key wordr:
24 July 1987; accepted
Antiandrogen, pure; Disease flare; Prostate gen, intraprostatic; Dibydrotestosterone
cancer;
20 November
[D-Trp6]Luteinizing
1987)
hormone
releasing
hormone
ethylamide;
Andro-
Summary
Daily subcutaneous administration of 50 pg of the luteinizing hormone-releasing hormone (LHRH) agonist [D-T$]LHRH ethylamide in adult dogs causes a transient increase in the serum testosterone (T) concentration which reaches a maximum at 200% above control on days 2-4 of treatment and progressively decreases to 7% of the pretreatment value on day 21, the last time interval studied. After a transient increase, the concentration of serum bioactive luteinizing hormone (LH) was progressively decreased on days 11 and 19, thus suggesting that in analogy with human findings, the loss of LH bioactivity is responsible for the inhibition of testicular steroidogenesis induced in the dog by LHRH agonists. Of major significance is the finding that the changes in serum T levels observed during the first 3 weeks of treatment, as well as the complete inhibition of the intratesticular concentration of sex steroids observed at the end of this period of treatment with the LHRH agonist were not affected by simultaneous administration of flutamide (125 mg per OS every 8 h). Such findings indicate that at the dose used, the LHRH agonist is in full control of gonadotropin secretion, thus completely overcoming feedback influences. Since the administration of the antiandrogen flutamide does not decrease the efficacy of the LHRH agonist as blocker of testicular androgen biosynthesis, the present data support the use of a pure antiandrogen in order to neutralize the effect of the transient rise in testicular androgen secretion which always accompanies the first days of treatment with LHRH agonists in patients with advanced prostate cancer.
Introduction
The unexpected rats with agonists hormone (LHRH) one secretion by
finding that treatment of adult of luteinizing hormone-releasing causes a blockade in testosterthe testes resulting in a loss in
Address for correspondence: D. Lacoste, Medical Research Council Group in Molecular Endocrinology, Laval University Medical Center, Quebec, Que. GlV 4G2, Canada.
0303-7207/88/$03.50
0 1988 Elsevier Scientific
Publishers
Ireland,
prostate weight (Labrie et al., 1978, 1980) opened the possibility of an advantageous replacement for estrogens in the treatment of prostate cancer in men. In fact, since the observation of Huggins and Hodges (1941), the standard treatment of advanced prostate cancer has been the removal or blockade of testicular androgens by orchiectomy or treatment with estrogens, respectively (Nesbit and Baum, 1950; Jordan et al., 1977). While orchiectomy is psychologically unacceptable by Ltd.
142
many patients, estrogens are a frequent cause of serious cardiovascular complications (Byar, 1981; Glashan and Robinson, 1981). As an alternative to these two approaches, it is now well recognized that castration levels of serum testosterone can be achieved by chronic administration of LHRH agonists 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, b, 1986; Wenderoth and Jacobi, 1984). 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 for the treatment of this disease is the transient rise in serum testosterone which occurs during the first 5-8 days of treatment with the accompanying risk of disease flare in a significant proportion of patients (Kahan et al., 1984; Waxman et al., 1985). At later time intervals, complete pituitary desensitization occurs with a loss of circulating bioactive LH (St-Arnaud et al., 1986). Serum testosterone then remains at castration levels for as long as treatment continues (Labrie et al., 1985a, b, 1986). Since antiandrogens neutralize, in a competitive fashion, the action of androgens at the target cell level (Neri and Pee&, 1975; Simard et al., 1986), one approach to prevent the potential harmful effects of the rise in serum androgens observed during the first days of treatment with LHRH agonists is the use of a pure antiandrogen in combination with the gonadotropin-releasing peptide. In order to achieve maximal antiandrogenie effect, it is important, however, to verify that the antiandrogen, due to neutralisation of the inhibitory feedback effect of androgens, does not interfere with the desensitizing action of the LHRH agonist on pituitary LH secretion. Since the dog appears to be the best model to study the inhibitory effect of LHRH agonists on testicular functions (Tremblay et al., 1984; Tremblay and Belanger, 1984), we have analyzed in detail the effect of the pure antiandrogen flutamide on the LHRH agonist-induced changes in testicular steroid secretion in the dog during the first 3 weeks of treatment. The present data show that flutamide does not interfere with the potent desensitizing action of the LHRH agonist [D-
Trp6]LHRH ethyla~de on testicular androgen secretion, thus supporting the combined use of the antiandrogen with an LHRH agonist for the treatment of prostate cancer. Materials and methods
Chemicals The potent LHRH agonist [D-Trpb,des-GlyNHi*]LHRH ethylamide (LHRH-A) and the pure nonsteroidal antiandrogen flutamide (Euflex) were supplied by Schering Canada, Montreal. The purified ovine LH preparation oLH-23 was prepared by Dr. A.F. Parlow, Harbor-UCLA Medical Center, Torrance, CA, and obtained through courtesy of the National Hormone and Pituitary Program, NIAMDD, Bethesda, MD. All other chemicals and reference steroid standards were of analytical grade. Animals Twenty-four adult mongrel male dogs weighing 20-25 kg were used in this study. Animals were housed individually and fed dog chow and tap water ad libitum. Treatments Groups of six animals received either 50 ,ug LHRH-A per day subcutaneously in 1% gelatin0.9% NaCl, 125 mg of flutamide (Flu) given orally every 8 h or the combination LHRH-A + Flu for 21 days. Blood samples were collected from all dogs via the cephalic vein immediately prior to and at 2 h, 4 h and 8 h after ad~nistration of the drugs on days 0 (beginning of treatment), 1, 2, 3, 4, 5, 6, 8, 10, 15, 16, 19 and 21. Blood samples were also taken at the same times on days -2 and -11 (control values). Plasma samples were stored at - 20 o C until assayed. Extraction and purification of steroids Individual prostates were homogenized in 5 ml of methanol and extracted overnight at 4O C. After centrifugation at 2000 x g for 10 min, the pellet was washed with 5 ml of methanol, recentrifuged and the combined supematants evaporated under nitrogen. The residue was then resuspended in 1 ml of water and extracted twice with 5 ml of ether.
143
The organic phase was dried under a stream of nitrogen. Plasma steroids, on the other hand, were extracted twice with 5 ml of ether. LH-20 columns were prepared as follows: 1.7 g of LH-20 and 10 ml of benzene/methanol (85 : 15) were poured into 1 x 20 cm glass columns. After packing and washing with 25 ml of benzenemethanol, the columns were washed with 20 ml of the first solvent system used for elution (isooctane/benzene/methanol, 90 : 5 : 5). Extracts of prostatic and plasma steroids were applied onto the columns and eluted with the same solvent as described (Belanger et al., 1980). Radioimmunoassays
Assays were performed as follows: 500 ~1 of phosphate buffer containing increasing concentrations of unlabeled steroid or unknown were incubated with 200 ~1 of the antibody solution and 200 ~1 of the labeled steroid in phosphate buffer. For assays using 125I-labeled steroid derivatives, 200 ~1 of second antibody in phosphate buffer were also added. Incubation at 4°C was performed for 24 h and, after cent~fugation, bound radioactivity was counted in a LKB gamma counter. For tritiated steroids, after a 24 h incubation at 4OC, 200 ~1 of a dextran-coated charcoal suspension in phosphate buffer was added and the tubes were cent~fuged 10 min later. Unbound radioactivity was counted in a Beckman scintillation counter after addition of Aquasol at a counting efficiency of 458 (Belanger et al., 1980; Carmichael et al., 1980). LH bio~say
Levels of circulating LH biological activity were measured as described using the mouse interstitial cell bioassay (St-Amaud et al., 1986). Bioactivity was calculated using a program based on model II of Rodbard and Lewald (1970). Statistical
analysis
Statistical significance was measured according to the multiple range test of Duncan-Kramer (1956).
RtSUltS As illustrated in Fig. 1.4, no significant diurnal variation of serum testosterone (T) concentration
is observed between 0890 and 17:00 h in control animals. Moreover, flutamide administered alone at the dose of 125 mg every 8 h orally has no significant influence on serum T levels up to at least 21 days of treatment (Fig. 10). It can be seen in Fig. 1B that the first administration of 50 pg of the LHRH agonist [DT$]LHRH ethylamide caused a 1.5-fold increase in serum T (T) measured 3, 6 and 7 h later (P I 0.01). On the second day of treatment, serum T concentrations continued to increase slowly to reach a peak at appro~mately 200% above pretreatment values on day 3 of treatment with a progressive decrease thereafter. Mean T serum values were 60% inhibited on day 8 of treatment with a progressive inhibition to 7% of pretreatment values on day 21, the last time interval studied. When comparing to the effect of LHRH-A alone, it can be seen in Fig. 1C that almost superimposable serum T concentrations are observed when flutamide is administered in combination with the LHRH agonist. Since human studies have demonstrated that a loss of LH bioactivity is responsible for the potent inhibitory action of LHRH agonists on the pituitary gonadal axis (St-Amaud et al., 1986), we have made similar measurements in the present study using the same mouse interstitial cell bioassay. It can be seen in Fig. 2 that after a small stimulation (258, P I 0.05) on day 6 of treatment, the serum levels of bioactive LH decreased to 70 (P s 0.05) and 30% (P I 0.01) of pretreatment values on days 13 and 19 of treatment, respectively. In fact, there was a tendency for flutamide to further decrease the serum levels of bioactive LH at the two later time intervals studied (P < 0.05). It can be seen in Fig. 3 that the addition of flutamide does not interfere with the marked inhibitory effect of the LHRH agonist on the intratesticular levels of a series of steroids measured after 21 days of treatment, namely pregnenolone, 17-OH-pregnenolone, dehydroepiandrosterone, androst-5-ene-3/$17j%diol, androstenedione, testosterone, 17P-estradiol, androstane-3a,l7&diol, androstane-3~,17~-diol and 5ff-d~ydrotestosterone. The testicular concentration of progesterone and 17-OH-progesterone was significantly elevated in the animals receiving flutamide in combination
144 LHRH-A
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,
,
-2
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2
4
6
I
1
,
I
,
,
I
/
8
10
12
14
16
18
20
22
LHRH-A
TC
+ FLUTAMIOE
q
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FLUTAMIDE
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OAYS OF TREATMENT
Fig. 1. Response of serum testosterone to daily subcutaneous administration for 21 days of 50 pg of the LHRH agonist [D-T$]LHRH ethylamide (B), flutamide at the dose of 125 mg three times daily, orally (D) or both drugs in combination (C). Control animals receiving the vehicle alone are shown in (A). Blood samples were drawn at OS:OO, ll:OO, 14:00 and 15:OO h. The LHRH agonist was injected at 0830 h while flutamide was given orally at 08:00, 14:OO and 2090 h. Bars represent SEM.
x
H k-a
1.5
LHRH-A LHRH-A + FLUTAMIDE
/’
0
5
10
DAYS
15
20
with [XI-T$]LHRH ethylamide as compared with those who received the LHRH agonist alone. Flutamide administered alone, on the other hand, had a stimulatory effect on the testicular levels of pregnenolone, 17-OH-pregnenolone, dehydroepiandrosterone, androst-5-ene-3~,17~-diol and androstane-3/3,17@-diol. Administration of the antiandrogen alone, however, had no effect on the intratesticular concentration of the active androgens T and DHT while a significant (P < 0.05) inhibition of testicular levels of 17-OH-progesterone and androstane-3cY,17@-diol was ob___._ ___.__ served. -___i_ Fig. 2. Response of serum bioactive LH to daily subcutaneous administration of 50 pg of the LHRH agonist [D-Tt#]LHRH ethylamide alone or in combination with Butamide administered orally at the dose of 125 mg, three times daily.
DHT
!IFig. 3. Effect of 2 days of treatment with the LHRH agonist [D-T$]LHRH three times daily) or the combination of both drugs on the intratesticular active androgens.
PROSTATE
Since the androgen active at the level of the prostate is ScY-dihydrotestosterone, it is of major interest to see in Fig. 4 that the intraprostatic concentration of DHT decreased from 5.0 + 1.0 rig/g tissue to the lower limit of sensitivity of the assay, namely 0.2 rig/g,, in the prostatic tissue obtained from animals treated with the LHRH agonist alone as well as in those who received the combination therapy with flutamide. When flutamide was administered alone, the intraprostatic concentration of DHT was reduced to 1.1 + 0.3 rig/g tissue (P I 0.01).
6 1
ethylamide (50 pg SC., daily), flutamide (125 mg per OS, levels of a series of steroid precursors, metabolites and
1
1 LIMIT OF DETEC 0 CT
LHRH-A
LHRH -A
FLU
F+LU
Fig. 4. Effect of 21 days of treatment with the LHRH agonist [D-Tt$]LHRH ethylamide (50 gg s.c., daily), flutamide (125 mg per OS, three times daily) or the combination of both drugs on the intraprostatic concentration of 5u-dihydrotestosterone PHT).
Discussion The present data clearly demonstrate that the’ pure antiandrogen flutamide administered in the dog at a dose comparable to that used in the human (Labrie et al., 1986), namely 375 mg per day (approximately 15 mg/kg), has no significant
146
effect on the basal concentration of serum T up to at least 21 days of treatment. In normal adult men, after 2 weeks of treatment with the same antiandrogen (750 mg per day), the serum T concentration was increased by only about 40% (Knuth et al., 1984). The low or absent response of serum T to flutamide administration in men and dog is different from the results obtained in the rat where a 5to g-fold increase in serum T is observed after 2 weeks of treatment (Soderstein et al., 1975; Reznikon et al., 1978; Viguier-Martinez et al., 1983). Such data clearly suggest major species differences in the sensitivity of the hypothalamo-pituitary complex to the negative feedback action of androgens. The above-mentioned data suggest that the hypothalamo-pituitary feedback control of LH secretion in the rat is highly sensitive to the inhibitory feedback action of androgens while, in the dog and man, there is a relatively low sensitivity to androgen feedback. It is also possible that the access and/or action of flutamide at the level of the hypothalamo-pituitary feedback elements show(s) significant species differences. The complete lack of influence of flutamide on the stimulatory (acute) and inhibitory (chronic) phases of LHRH agonist action on testicular androgen secretion observed in the present experiments is in agreement with recent data showing that 2 weeks of daily treatment of adult men with flutamide (750 mg per day) had no significant influence on the LH response to LHRH (Knuth et al., 1984). In that study performed in men, there was even a tendency for a decreased LH response to LHRH in men treated for 8 days with flutamide. We have previously shown that androgens exert a direct inhibitory action at the anterior pituitary level on LHRH-induced LH release (Drouin and Labrie, 1976), the effect of the androgens being at least partially mediated by a decrease in the number of LHRH binding sites in gonadotrophs (Gig&e et al., 1981). Although androgens could well be able to modulate the desensitizing action of smaller doses of an LHRH agonist, the present data clearly show that blockade of the androgen receptor in pituitary gonadotrophs by treatment with flutamide has no influence on the inhibitory action of a maximal dose of the LHRH agonist. In
fact, as clearly illustrated by the present data, the antiandrogen has no detectable influence on either the stimulatory (first 8 days of treatment) or the inhibitory (after 8 days of treatment) periods of LHRH agonist action on LH secretion. Maximal doses of the LHRH agonist are thus able to completely overcome the feedback effects exerted by androgens on gonadotropin secretion. It is of interest to see in Fig. 4 that flutamide, at a dose comparable to that used in the human, leaves approximately 20% of DHT in the dog prostate, the value being decreased from 5.0 f 1.0 to 1.1 f 0.3 rig/g tissue. In intact men, the average concentration of DHT in prostate cancer tissue (Labrie et al., 1985a, b, 1987) is comparable to the value measured in the normal dog prostate in the present experiment. Knowing the affinity of hydroxy-flutamide, the active metabolite of flutamide, for the androgen receptor (Simard et al., 1986) and its tissue concentration, we have calculated that l-2 ng of DHT per g is left in the prostate cancer tissue following treatment of adult men with flutamide at the dose of 250 mg every 8 h. Such data are comparable to the value (1.1 f 0.3 rig/g)) measured in the normal dog prostate during antiandrogen treatment in the present study. Since 1.0-2.0 ng DHT per g tissue is left in the prostate cancer after castration (Geller et al., 1984; Labrie et al., 1987), one can assume that the effects of castration and treatment with flutamide alone should lead to similar clinical results in men with prostate cancer. This suggestion is confirmed by the clinical observations showing that comparable results are obtained in patients with stage D2 prostate cancer following chemical (DES or LHRH agonist alone) or surgical (orchiectomy) castration (Nesbit and Baum, 1950; Jordan et al., 1977; Wenderoth and Jacobi, 1984; Labrie et al., 1987) and treatment with flutamide alone (Sogani et al., 1984). The most likely explanation for this equivalency of results is that castration and treatment with flutamide alone leave equal amounts of DHT in the prostatic cancer tissue. We have recently observed (Labrie et al., 1987b) that the intraprostatic concentration of DHT is decreased below detection limits (I 0.2 rig/g)) in patients receiving the combination therapy with flutamide and an LHRH agonist. It can be noticed that prostatic DHT levels are
147
already reduced below detection limits by treatment with the LHRH agonist alone in the dog (Fig. 4). This finding can be explained by the fact that dog adrenals do not secrete appreciable amounts of precursor androgens and that, consequently, all sources of androgens are practically eliminated after blockade of testicular androgen secretion in this species. References B&anger, A., Caron, S. and Picard, V. (1980) J. Steroid Biothem. 13,180-190. Byar, D.P. (1981) Cancer 32, 1126-1130. Carmichael, R., B&Ianger, A., Cusan, L., SCguin, C., Caron, S. and Labrie, F. (1980) Steroids 36, 383-391. Drouin, J. and Labrie, F. (1976) Endocrinology 98, 1528-1534. Geiler, J., Aibert, J.D., Nachtsheim, D.A. and Loza, DC. (1984) J. Ural. 132, 693-696. Gig&e, V., Lefebvre, F.A. and Labrie, F. (1981) Endocrinology 108, 350-352. Glashan, R.W. and Robinson, M.R.G. (1981) Br. J. Ural. 53, 624-627. Huggins, C. and Hodges, C.V. (1941) Cancer Res. 1, 293-297. Jordan, Jr., W.P., Blackard, C.E. and Byar, D.P. (1977) South. Med. J. 70, 1411-1413. Kahan, A., Delrieu, F., Amor, B., Chiche, R. and Steg, A. (1984) Lancet i, 971-972. Knuth, U.A., Hano, R. and Nieschlag, E. (1984) J. Clin. Endocrinol. Metab. 59, 963-969. Kramer, C.Y. (1956) Biometrics 12, 30’7-310. Labrie, F., Auclair, C., Cusan, L., Kelly, P.A., Pellet&r, G. and Ferland, L. (1978) Int. J. Andrology Suppl. 2, 103-118. Labrie, F., Bblanger, A., Cusan, L., SCguin, C., Pelletier, G., Kelly, P.A., Reeves, J.J., Lefebvre. F.A., Lemay, A. and Raynaud, J.P. (1980) J. Andrology 1, 209-228. Labrie, F., DuPont, A., Belanger, A., Cusan, L., Lacourci&re, Y., Monfette, G., Laberge, J.G., Emond, J.P., Fazekas, A.T.A., Raynaud, J.P. and Husson, J.M. (1982) J. Clin. Invest. Med. 5, 267-275. Labrie, F., DuPont, A., BBanger, A., Lachance, R. and Gigdre, M. (1985a) Br. Med. J. 291, 369-370. Labrie, F., DuPont, A. and Btlanger, A. (1985b) in Important
Advances in Oncology (De Vita, V.T., Hellman, S. and Rosenberg, S.A., eds.), pp. 193-217, Lippincott, Phiiadelphia, PA. Labrie, F., DuPont, A., B&anger, A., St-Amaud, R., Gig&e, M., Lacourcibre, Y., Emond, J. and Monfette, G. (1986) Endocr. Rev. 7,67-74. Labrie, F., Luthy, I., Veilleux, R., Siiard, J., B&anger, A. and DuPont, A. (1987a) in !!&zond International Symposium on Prostatic Cancer (Murphy, S.P., ed.), in press, Paris. Labrie, F., DuPont, A., B&anger, A., Gig&e, M., Borsanyi, J.P., LacourcZre, Y., Emond, J., Monfette, G. and Lachance, R. (1987b) in 1st International Congress of Neuroendocrinology (McCann, S.M., ed.), pp. 1-15, Karger, Ba&. Neri, D.R. and Peets, E.A. (1975) J. Steroid B&hem. 6, 815-819. Nesbit, R.M. and Baum, W.C. (1950) J. Am. Med. Assoc. 143, 317-320. Reznikon, A.G., Demchenko, V.N., Varga, S.V. and Bozkok, Y.M. (1978) Endokrinologie 72,276-284. Rodbard, D. and Lewald, J.E. (1970) in 2nd Karolinska Symposium on Research Methods in Reproductive Endocrinology (Diczfalusy, E., ed), pp. 79-103, Bogtrykleriet Forum, Copenhagen. Simard, J., Luthy, I., Guay, J., Btlanger, A. and Labrie, F. (1986) Mol. Cell. Endocrinol. 44, 261-270. Soderstein, P., Gray, G., Damassa, D.A., Smith, E.R. and Davidson, J.M. (1975) End~~nolo~ 97,1468-1475. Sogani, P.C., Vagaiwala, M.R. and Whitmore, Jr., W.F. (1984) Cancer 54. 744-750. St-Amaud, R., Lachance, R., DuPont, A. and Labrie, F. (1986) J. Clin. Endocrinol. Metab. 63, 297-302. Tremblay, Y. and Bblanger, A. (1984) Contraception 30, 483-497. Tremblay, Y., B&nger, A., Labrie, F., Frenette, G., Dub&, J.Y. and Tremblay, R.R. (1984) Prostate 5, 424-432. Viguier-Martinez. M.C., Hochereau-Reviers, M.J., Barenton, B. and Perreau, C. (1983) Acta Endocrinol. 104, 246-252. Waxman, J.H., Man, A., Hendry, W.F., Whitfield, H.N., Tiptaft, R.C., Paris, A.M.I. and Oliver, R.T. (1985) Br. Med. J. 291,1387-1388. Wenderoth, U.K. and Jacobi, G.H. (1984) in LHRH and its Analogues: Basic and Clinical Aspects (Labrie, F.. BCfanper, A. and DuPont, A., eds.), pp. 349-357, Excerpta Medica, Amsterdam.
56 (1988) 141-147 Ltd.
141
MCE 01819
A pure antiandrogen does not interfere with the LHRH agonist-induced blockade of testicular androgen secretion in the dog D. Lacoste, R. St-Amaud, A. BClanger and F. Labrie Medical Research Council Group in Molecular Endocrinology, Luval Universiiy Medical Center, Quebec, Que. GI V 4G2, Canada (Received
Key wordr:
24 July 1987; accepted
Antiandrogen, pure; Disease flare; Prostate gen, intraprostatic; Dibydrotestosterone
cancer;
20 November
[D-Trp6]Luteinizing
1987)
hormone
releasing
hormone
ethylamide;
Andro-
Summary
Daily subcutaneous administration of 50 pg of the luteinizing hormone-releasing hormone (LHRH) agonist [D-T$]LHRH ethylamide in adult dogs causes a transient increase in the serum testosterone (T) concentration which reaches a maximum at 200% above control on days 2-4 of treatment and progressively decreases to 7% of the pretreatment value on day 21, the last time interval studied. After a transient increase, the concentration of serum bioactive luteinizing hormone (LH) was progressively decreased on days 11 and 19, thus suggesting that in analogy with human findings, the loss of LH bioactivity is responsible for the inhibition of testicular steroidogenesis induced in the dog by LHRH agonists. Of major significance is the finding that the changes in serum T levels observed during the first 3 weeks of treatment, as well as the complete inhibition of the intratesticular concentration of sex steroids observed at the end of this period of treatment with the LHRH agonist were not affected by simultaneous administration of flutamide (125 mg per OS every 8 h). Such findings indicate that at the dose used, the LHRH agonist is in full control of gonadotropin secretion, thus completely overcoming feedback influences. Since the administration of the antiandrogen flutamide does not decrease the efficacy of the LHRH agonist as blocker of testicular androgen biosynthesis, the present data support the use of a pure antiandrogen in order to neutralize the effect of the transient rise in testicular androgen secretion which always accompanies the first days of treatment with LHRH agonists in patients with advanced prostate cancer.
Introduction
The unexpected rats with agonists hormone (LHRH) one secretion by
finding that treatment of adult of luteinizing hormone-releasing causes a blockade in testosterthe testes resulting in a loss in
Address for correspondence: D. Lacoste, Medical Research Council Group in Molecular Endocrinology, Laval University Medical Center, Quebec, Que. GlV 4G2, Canada.
0303-7207/88/$03.50
0 1988 Elsevier Scientific
Publishers
Ireland,
prostate weight (Labrie et al., 1978, 1980) opened the possibility of an advantageous replacement for estrogens in the treatment of prostate cancer in men. In fact, since the observation of Huggins and Hodges (1941), the standard treatment of advanced prostate cancer has been the removal or blockade of testicular androgens by orchiectomy or treatment with estrogens, respectively (Nesbit and Baum, 1950; Jordan et al., 1977). While orchiectomy is psychologically unacceptable by Ltd.
142
many patients, estrogens are a frequent cause of serious cardiovascular complications (Byar, 1981; Glashan and Robinson, 1981). As an alternative to these two approaches, it is now well recognized that castration levels of serum testosterone can be achieved by chronic administration of LHRH agonists 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, b, 1986; Wenderoth and Jacobi, 1984). 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 for the treatment of this disease is the transient rise in serum testosterone which occurs during the first 5-8 days of treatment with the accompanying risk of disease flare in a significant proportion of patients (Kahan et al., 1984; Waxman et al., 1985). At later time intervals, complete pituitary desensitization occurs with a loss of circulating bioactive LH (St-Arnaud et al., 1986). Serum testosterone then remains at castration levels for as long as treatment continues (Labrie et al., 1985a, b, 1986). Since antiandrogens neutralize, in a competitive fashion, the action of androgens at the target cell level (Neri and Pee&, 1975; Simard et al., 1986), one approach to prevent the potential harmful effects of the rise in serum androgens observed during the first days of treatment with LHRH agonists is the use of a pure antiandrogen in combination with the gonadotropin-releasing peptide. In order to achieve maximal antiandrogenie effect, it is important, however, to verify that the antiandrogen, due to neutralisation of the inhibitory feedback effect of androgens, does not interfere with the desensitizing action of the LHRH agonist on pituitary LH secretion. Since the dog appears to be the best model to study the inhibitory effect of LHRH agonists on testicular functions (Tremblay et al., 1984; Tremblay and Belanger, 1984), we have analyzed in detail the effect of the pure antiandrogen flutamide on the LHRH agonist-induced changes in testicular steroid secretion in the dog during the first 3 weeks of treatment. The present data show that flutamide does not interfere with the potent desensitizing action of the LHRH agonist [D-
Trp6]LHRH ethyla~de on testicular androgen secretion, thus supporting the combined use of the antiandrogen with an LHRH agonist for the treatment of prostate cancer. Materials and methods
Chemicals The potent LHRH agonist [D-Trpb,des-GlyNHi*]LHRH ethylamide (LHRH-A) and the pure nonsteroidal antiandrogen flutamide (Euflex) were supplied by Schering Canada, Montreal. The purified ovine LH preparation oLH-23 was prepared by Dr. A.F. Parlow, Harbor-UCLA Medical Center, Torrance, CA, and obtained through courtesy of the National Hormone and Pituitary Program, NIAMDD, Bethesda, MD. All other chemicals and reference steroid standards were of analytical grade. Animals Twenty-four adult mongrel male dogs weighing 20-25 kg were used in this study. Animals were housed individually and fed dog chow and tap water ad libitum. Treatments Groups of six animals received either 50 ,ug LHRH-A per day subcutaneously in 1% gelatin0.9% NaCl, 125 mg of flutamide (Flu) given orally every 8 h or the combination LHRH-A + Flu for 21 days. Blood samples were collected from all dogs via the cephalic vein immediately prior to and at 2 h, 4 h and 8 h after ad~nistration of the drugs on days 0 (beginning of treatment), 1, 2, 3, 4, 5, 6, 8, 10, 15, 16, 19 and 21. Blood samples were also taken at the same times on days -2 and -11 (control values). Plasma samples were stored at - 20 o C until assayed. Extraction and purification of steroids Individual prostates were homogenized in 5 ml of methanol and extracted overnight at 4O C. After centrifugation at 2000 x g for 10 min, the pellet was washed with 5 ml of methanol, recentrifuged and the combined supematants evaporated under nitrogen. The residue was then resuspended in 1 ml of water and extracted twice with 5 ml of ether.
143
The organic phase was dried under a stream of nitrogen. Plasma steroids, on the other hand, were extracted twice with 5 ml of ether. LH-20 columns were prepared as follows: 1.7 g of LH-20 and 10 ml of benzene/methanol (85 : 15) were poured into 1 x 20 cm glass columns. After packing and washing with 25 ml of benzenemethanol, the columns were washed with 20 ml of the first solvent system used for elution (isooctane/benzene/methanol, 90 : 5 : 5). Extracts of prostatic and plasma steroids were applied onto the columns and eluted with the same solvent as described (Belanger et al., 1980). Radioimmunoassays
Assays were performed as follows: 500 ~1 of phosphate buffer containing increasing concentrations of unlabeled steroid or unknown were incubated with 200 ~1 of the antibody solution and 200 ~1 of the labeled steroid in phosphate buffer. For assays using 125I-labeled steroid derivatives, 200 ~1 of second antibody in phosphate buffer were also added. Incubation at 4°C was performed for 24 h and, after cent~fugation, bound radioactivity was counted in a LKB gamma counter. For tritiated steroids, after a 24 h incubation at 4OC, 200 ~1 of a dextran-coated charcoal suspension in phosphate buffer was added and the tubes were cent~fuged 10 min later. Unbound radioactivity was counted in a Beckman scintillation counter after addition of Aquasol at a counting efficiency of 458 (Belanger et al., 1980; Carmichael et al., 1980). LH bio~say
Levels of circulating LH biological activity were measured as described using the mouse interstitial cell bioassay (St-Amaud et al., 1986). Bioactivity was calculated using a program based on model II of Rodbard and Lewald (1970). Statistical
analysis
Statistical significance was measured according to the multiple range test of Duncan-Kramer (1956).
RtSUltS As illustrated in Fig. 1.4, no significant diurnal variation of serum testosterone (T) concentration
is observed between 0890 and 17:00 h in control animals. Moreover, flutamide administered alone at the dose of 125 mg every 8 h orally has no significant influence on serum T levels up to at least 21 days of treatment (Fig. 10). It can be seen in Fig. 1B that the first administration of 50 pg of the LHRH agonist [DT$]LHRH ethylamide caused a 1.5-fold increase in serum T (T) measured 3, 6 and 7 h later (P I 0.01). On the second day of treatment, serum T concentrations continued to increase slowly to reach a peak at appro~mately 200% above pretreatment values on day 3 of treatment with a progressive decrease thereafter. Mean T serum values were 60% inhibited on day 8 of treatment with a progressive inhibition to 7% of pretreatment values on day 21, the last time interval studied. When comparing to the effect of LHRH-A alone, it can be seen in Fig. 1C that almost superimposable serum T concentrations are observed when flutamide is administered in combination with the LHRH agonist. Since human studies have demonstrated that a loss of LH bioactivity is responsible for the potent inhibitory action of LHRH agonists on the pituitary gonadal axis (St-Amaud et al., 1986), we have made similar measurements in the present study using the same mouse interstitial cell bioassay. It can be seen in Fig. 2 that after a small stimulation (258, P I 0.05) on day 6 of treatment, the serum levels of bioactive LH decreased to 70 (P s 0.05) and 30% (P I 0.01) of pretreatment values on days 13 and 19 of treatment, respectively. In fact, there was a tendency for flutamide to further decrease the serum levels of bioactive LH at the two later time intervals studied (P < 0.05). It can be seen in Fig. 3 that the addition of flutamide does not interfere with the marked inhibitory effect of the LHRH agonist on the intratesticular levels of a series of steroids measured after 21 days of treatment, namely pregnenolone, 17-OH-pregnenolone, dehydroepiandrosterone, androst-5-ene-3/$17j%diol, androstenedione, testosterone, 17P-estradiol, androstane-3a,l7&diol, androstane-3~,17~-diol and 5ff-d~ydrotestosterone. The testicular concentration of progesterone and 17-OH-progesterone was significantly elevated in the animals receiving flutamide in combination
144 LHRH-A
o-l
,
/
,
,
-2
0
2
4
6
I
1
,
I
,
,
I
/
8
10
12
14
16
18
20
22
LHRH-A
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+ FLUTAMIOE
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OAYS OF TREATMENT
Fig. 1. Response of serum testosterone to daily subcutaneous administration for 21 days of 50 pg of the LHRH agonist [D-T$]LHRH ethylamide (B), flutamide at the dose of 125 mg three times daily, orally (D) or both drugs in combination (C). Control animals receiving the vehicle alone are shown in (A). Blood samples were drawn at OS:OO, ll:OO, 14:00 and 15:OO h. The LHRH agonist was injected at 0830 h while flutamide was given orally at 08:00, 14:OO and 2090 h. Bars represent SEM.
x
H k-a
1.5
LHRH-A LHRH-A + FLUTAMIDE
/’
0
5
10
DAYS
15
20
with [XI-T$]LHRH ethylamide as compared with those who received the LHRH agonist alone. Flutamide administered alone, on the other hand, had a stimulatory effect on the testicular levels of pregnenolone, 17-OH-pregnenolone, dehydroepiandrosterone, androst-5-ene-3~,17~-diol and androstane-3/3,17@-diol. Administration of the antiandrogen alone, however, had no effect on the intratesticular concentration of the active androgens T and DHT while a significant (P < 0.05) inhibition of testicular levels of 17-OH-progesterone and androstane-3cY,17@-diol was ob___._ ___.__ served. -___i_ Fig. 2. Response of serum bioactive LH to daily subcutaneous administration of 50 pg of the LHRH agonist [D-Tt#]LHRH ethylamide alone or in combination with Butamide administered orally at the dose of 125 mg, three times daily.
DHT
!IFig. 3. Effect of 2 days of treatment with the LHRH agonist [D-T$]LHRH three times daily) or the combination of both drugs on the intratesticular active androgens.
PROSTATE
Since the androgen active at the level of the prostate is ScY-dihydrotestosterone, it is of major interest to see in Fig. 4 that the intraprostatic concentration of DHT decreased from 5.0 + 1.0 rig/g tissue to the lower limit of sensitivity of the assay, namely 0.2 rig/g,, in the prostatic tissue obtained from animals treated with the LHRH agonist alone as well as in those who received the combination therapy with flutamide. When flutamide was administered alone, the intraprostatic concentration of DHT was reduced to 1.1 + 0.3 rig/g tissue (P I 0.01).
6 1
ethylamide (50 pg SC., daily), flutamide (125 mg per OS, levels of a series of steroid precursors, metabolites and
1
1 LIMIT OF DETEC 0 CT
LHRH-A
LHRH -A
FLU
F+LU
Fig. 4. Effect of 21 days of treatment with the LHRH agonist [D-Tt$]LHRH ethylamide (50 gg s.c., daily), flutamide (125 mg per OS, three times daily) or the combination of both drugs on the intraprostatic concentration of 5u-dihydrotestosterone PHT).
Discussion The present data clearly demonstrate that the’ pure antiandrogen flutamide administered in the dog at a dose comparable to that used in the human (Labrie et al., 1986), namely 375 mg per day (approximately 15 mg/kg), has no significant
146
effect on the basal concentration of serum T up to at least 21 days of treatment. In normal adult men, after 2 weeks of treatment with the same antiandrogen (750 mg per day), the serum T concentration was increased by only about 40% (Knuth et al., 1984). The low or absent response of serum T to flutamide administration in men and dog is different from the results obtained in the rat where a 5to g-fold increase in serum T is observed after 2 weeks of treatment (Soderstein et al., 1975; Reznikon et al., 1978; Viguier-Martinez et al., 1983). Such data clearly suggest major species differences in the sensitivity of the hypothalamo-pituitary complex to the negative feedback action of androgens. The above-mentioned data suggest that the hypothalamo-pituitary feedback control of LH secretion in the rat is highly sensitive to the inhibitory feedback action of androgens while, in the dog and man, there is a relatively low sensitivity to androgen feedback. It is also possible that the access and/or action of flutamide at the level of the hypothalamo-pituitary feedback elements show(s) significant species differences. The complete lack of influence of flutamide on the stimulatory (acute) and inhibitory (chronic) phases of LHRH agonist action on testicular androgen secretion observed in the present experiments is in agreement with recent data showing that 2 weeks of daily treatment of adult men with flutamide (750 mg per day) had no significant influence on the LH response to LHRH (Knuth et al., 1984). In that study performed in men, there was even a tendency for a decreased LH response to LHRH in men treated for 8 days with flutamide. We have previously shown that androgens exert a direct inhibitory action at the anterior pituitary level on LHRH-induced LH release (Drouin and Labrie, 1976), the effect of the androgens being at least partially mediated by a decrease in the number of LHRH binding sites in gonadotrophs (Gig&e et al., 1981). Although androgens could well be able to modulate the desensitizing action of smaller doses of an LHRH agonist, the present data clearly show that blockade of the androgen receptor in pituitary gonadotrophs by treatment with flutamide has no influence on the inhibitory action of a maximal dose of the LHRH agonist. In
fact, as clearly illustrated by the present data, the antiandrogen has no detectable influence on either the stimulatory (first 8 days of treatment) or the inhibitory (after 8 days of treatment) periods of LHRH agonist action on LH secretion. Maximal doses of the LHRH agonist are thus able to completely overcome the feedback effects exerted by androgens on gonadotropin secretion. It is of interest to see in Fig. 4 that flutamide, at a dose comparable to that used in the human, leaves approximately 20% of DHT in the dog prostate, the value being decreased from 5.0 f 1.0 to 1.1 f 0.3 rig/g tissue. In intact men, the average concentration of DHT in prostate cancer tissue (Labrie et al., 1985a, b, 1987) is comparable to the value measured in the normal dog prostate in the present experiment. Knowing the affinity of hydroxy-flutamide, the active metabolite of flutamide, for the androgen receptor (Simard et al., 1986) and its tissue concentration, we have calculated that l-2 ng of DHT per g is left in the prostate cancer tissue following treatment of adult men with flutamide at the dose of 250 mg every 8 h. Such data are comparable to the value (1.1 f 0.3 rig/g)) measured in the normal dog prostate during antiandrogen treatment in the present study. Since 1.0-2.0 ng DHT per g tissue is left in the prostate cancer after castration (Geller et al., 1984; Labrie et al., 1987), one can assume that the effects of castration and treatment with flutamide alone should lead to similar clinical results in men with prostate cancer. This suggestion is confirmed by the clinical observations showing that comparable results are obtained in patients with stage D2 prostate cancer following chemical (DES or LHRH agonist alone) or surgical (orchiectomy) castration (Nesbit and Baum, 1950; Jordan et al., 1977; Wenderoth and Jacobi, 1984; Labrie et al., 1987) and treatment with flutamide alone (Sogani et al., 1984). The most likely explanation for this equivalency of results is that castration and treatment with flutamide alone leave equal amounts of DHT in the prostatic cancer tissue. We have recently observed (Labrie et al., 1987b) that the intraprostatic concentration of DHT is decreased below detection limits (I 0.2 rig/g)) in patients receiving the combination therapy with flutamide and an LHRH agonist. It can be noticed that prostatic DHT levels are
147
already reduced below detection limits by treatment with the LHRH agonist alone in the dog (Fig. 4). This finding can be explained by the fact that dog adrenals do not secrete appreciable amounts of precursor androgens and that, consequently, all sources of androgens are practically eliminated after blockade of testicular androgen secretion in this species. References B&anger, A., Caron, S. and Picard, V. (1980) J. Steroid Biothem. 13,180-190. Byar, D.P. (1981) Cancer 32, 1126-1130. Carmichael, R., B&Ianger, A., Cusan, L., SCguin, C., Caron, S. and Labrie, F. (1980) Steroids 36, 383-391. Drouin, J. and Labrie, F. (1976) Endocrinology 98, 1528-1534. Geiler, J., Aibert, J.D., Nachtsheim, D.A. and Loza, DC. (1984) J. Ural. 132, 693-696. Gig&e, V., Lefebvre, F.A. and Labrie, F. (1981) Endocrinology 108, 350-352. Glashan, R.W. and Robinson, M.R.G. (1981) Br. J. Ural. 53, 624-627. Huggins, C. and Hodges, C.V. (1941) Cancer Res. 1, 293-297. Jordan, Jr., W.P., Blackard, C.E. and Byar, D.P. (1977) South. Med. J. 70, 1411-1413. Kahan, A., Delrieu, F., Amor, B., Chiche, R. and Steg, A. (1984) Lancet i, 971-972. Knuth, U.A., Hano, R. and Nieschlag, E. (1984) J. Clin. Endocrinol. Metab. 59, 963-969. Kramer, C.Y. (1956) Biometrics 12, 30’7-310. Labrie, F., Auclair, C., Cusan, L., Kelly, P.A., Pellet&r, G. and Ferland, L. (1978) Int. J. Andrology Suppl. 2, 103-118. Labrie, F., Bblanger, A., Cusan, L., SCguin, C., Pelletier, G., Kelly, P.A., Reeves, J.J., Lefebvre. F.A., Lemay, A. and Raynaud, J.P. (1980) J. Andrology 1, 209-228. Labrie, F., DuPont, A., Belanger, A., Cusan, L., Lacourci&re, Y., Monfette, G., Laberge, J.G., Emond, J.P., Fazekas, A.T.A., Raynaud, J.P. and Husson, J.M. (1982) J. Clin. Invest. Med. 5, 267-275. Labrie, F., DuPont, A., BBanger, A., Lachance, R. and Gigdre, M. (1985a) Br. Med. J. 291, 369-370. Labrie, F., DuPont, A. and Btlanger, A. (1985b) in Important
Advances in Oncology (De Vita, V.T., Hellman, S. and Rosenberg, S.A., eds.), pp. 193-217, Lippincott, Phiiadelphia, PA. Labrie, F., DuPont, A., B&anger, A., St-Amaud, R., Gig&e, M., Lacourcibre, Y., Emond, J. and Monfette, G. (1986) Endocr. Rev. 7,67-74. Labrie, F., Luthy, I., Veilleux, R., Siiard, J., B&anger, A. and DuPont, A. (1987a) in !!&zond International Symposium on Prostatic Cancer (Murphy, S.P., ed.), in press, Paris. Labrie, F., DuPont, A., B&anger, A., Gig&e, M., Borsanyi, J.P., LacourcZre, Y., Emond, J., Monfette, G. and Lachance, R. (1987b) in 1st International Congress of Neuroendocrinology (McCann, S.M., ed.), pp. 1-15, Karger, Ba&. Neri, D.R. and Peets, E.A. (1975) J. Steroid B&hem. 6, 815-819. Nesbit, R.M. and Baum, W.C. (1950) J. Am. Med. Assoc. 143, 317-320. Reznikon, A.G., Demchenko, V.N., Varga, S.V. and Bozkok, Y.M. (1978) Endokrinologie 72,276-284. Rodbard, D. and Lewald, J.E. (1970) in 2nd Karolinska Symposium on Research Methods in Reproductive Endocrinology (Diczfalusy, E., ed), pp. 79-103, Bogtrykleriet Forum, Copenhagen. Simard, J., Luthy, I., Guay, J., Btlanger, A. and Labrie, F. (1986) Mol. Cell. Endocrinol. 44, 261-270. Soderstein, P., Gray, G., Damassa, D.A., Smith, E.R. and Davidson, J.M. (1975) End~~nolo~ 97,1468-1475. Sogani, P.C., Vagaiwala, M.R. and Whitmore, Jr., W.F. (1984) Cancer 54. 744-750. St-Amaud, R., Lachance, R., DuPont, A. and Labrie, F. (1986) J. Clin. Endocrinol. Metab. 63, 297-302. Tremblay, Y. and Bblanger, A. (1984) Contraception 30, 483-497. Tremblay, Y., B&nger, A., Labrie, F., Frenette, G., Dub&, J.Y. and Tremblay, R.R. (1984) Prostate 5, 424-432. Viguier-Martinez. M.C., Hochereau-Reviers, M.J., Barenton, B. and Perreau, C. (1983) Acta Endocrinol. 104, 246-252. Waxman, J.H., Man, A., Hendry, W.F., Whitfield, H.N., Tiptaft, R.C., Paris, A.M.I. and Oliver, R.T. (1985) Br. Med. J. 291,1387-1388. Wenderoth, U.K. and Jacobi, G.H. (1984) in LHRH and its Analogues: Basic and Clinical Aspects (Labrie, F.. BCfanper, A. and DuPont, A., eds.), pp. 349-357, Excerpta Medica, Amsterdam.