Effect of very high dose D-leucine6-gonadotropin-releasing hormone proethylamide on the hypothalamic-pituitary testicular axis in patients with prostatic cancer.

Potent synthetic analogs of gonadotropin-releasing hormone produce parodoxical antireproductive effects when administered chronically. These compounds are minimally toxic and may exhibit no plateau of the dose-response curve even at very high doses. These considerations served as the basis for our systematic evaluation of [D-leucine6-desarginine-glycine-NH2(10)]gonadotropin-releasing hormone (GnRH-A) proethylamide in the very high dose range (i.e., 10-fold larger amounts than previously used). In rats given the analog for 12 wk, prostate, testis, and seminal vesicle weights were suppressed to a greater extent with 200 micrograms q.d. than with 40 micrograms q.d. (P less than 0.01 prostate, less than 0.01 testis, less than 0.01 seminal vesicles), indicating dose-response effects in the very high dose range. 200 micrograms of [D-Leu6-des-Gly-NH2(10]-GnRH-A consistently suppressed leutinizing hormone (LH) values at 6 and 12 wk (basal 71 +/- 9.5; 6 wk 34 +/- 3.8; 12 wk 28 +/- 5 ng/ml) whereas 40 micrograms suppressed LH variably (basal 33 +/- 3.8; 6 wk 17 +/- 3.9; 12 wk 32 +/- 5.2). Testosterone fell to 15 +/- 2.4 and 19 +/- 2.0 ng/100 ml in response to 200 micrograms q.d. and to 27 +/- 6.4 and 22 +/- 7.4 ng/100 ml with the 40-micrograms dose. These findings in the rodent prompted treatment of stage D prostate cancer patients with similarly high doses of [D-Leu6-des-Gly-NH2(10)]-GnRH-A. After treatment for 11 wk with 1,000 or 10,000 micrograms/d of the analog, testosterone and dihydrotestosterone levels transiently rose and then fell into the surgically castrate range (testosterone 19 +/- 4.4 ng/100 ml [D-Leu6-des-Gly-NH2(10)]-GnRH-A vs. surgically castrate 11 +/- 0.9 ng/100 ml, P = NS; dihydrotestosterone 15 +/- 1.7 ng/100 ml GnRH-A vs. surgically castrate 15 +/- 4.1 ng/100 ml. P = NS). However, unlike the chronic stimulatory effect on the pituitary at lower doses, very high dose therapy resulted in profound suppression of plasma and urine LH. Plasma levels fell to the limit of assay detectability, whereas the more sensitive urinary assay detected prepubertal levels of excretion (i.e., 64 +/- 8.4 mlU/h). The highly sensitive rat interstitial cell testosterone bioassay for LH also demonstrated a marked decline in LH to undetectable levels in 17/19 subjects. Clinical results with [D-Leu6-des-Gly-NH2(10)]-GnRH-A simulate those achieved by surgical castration in men with prostatic cancer as suggested by available preliminary data.

Adequacy of estrogen suppression with aminoglutethimide and hydrocortisone as treatment of human breast cancer: correlation of hormonal data with clinical responses.

Human breast neoplasms can be divided into hormone-dependent and hormone-independent subtypes. Estrogen is the major hormonal stimulus for growth of the dependent tumors. Failure to respond to estrogen suppression therapy could reflect either an incomplete lowering of estrogens or the hormonal independence of the tumor. To address this issue, we compared the levels of several estrogens and other hormones in women experiencing objective responses (the responders) and disease progression (the progression group) during therapy with the aromatase-steroidogenesis inhibitor, aminoglutethimide, and replacement hydrocortisone. Pretreatment hormonal profiles of the estrogens, and androgens, ketosteroids, thyroxine, polypeptide hormones, and carcinoembryonic antigen did not differ significantly among response groups. During treatment, the levels of all estrogens were suppressed to a similar degree in the progression group and in the responders. Urinary estrone, for example, fell to 16.7 +/- 3.2% of basal in the responders versus 16.3 +/- 3.8% of basal in the progression group. These data suggested that lack of estrogen suppression did not explain the response to treatment in the patients receiving aminoglutethimide-hydrocortisone. This finding differs from our results in a similarly analyzed control group of patients treated with surgical adrenalectomy. Levels of the weak androgens, dehydroepiandrosterone sulfate and androstenedione, were found to be higher in the progression group compared to the responders. This observation could not be explained by differences in duration of treatment between groups. Analysis at 1 to 12 weeks, 13 to 24 weeks, and 25 to 36 weeks after initiating treatment indicated higher androgen levels at each time point in the progression group. In addition, the results were not attributable to differing serum levels of aminoglutethimide among responder groups. While the finding of higher androgen levels in the responder group remains unexplained, this study indicates that incomplete estrogen suppression is not responsible for lack of tumor response in patients with progressive disease during amino-glutethimide-hydrocortisone therapy.

Recovery of hypothalamic-pituitary-adrenal function after long term suppression by aminoglutethimide and hydrocortisone.

Little data are available concerning recovery of adrenal function after prolonged inhibition of steroidogenesis by enzyme inhibitors. Aminoglutethimide (AG), a potent blocker of adrenal steroid biosynthesis, combined with physiological replacement doses of hydrocortisone (HC) is currently being used to treat women with metastatic breast carcinoma. We studied the time-course of recovery of hypothalamic-pituitary-adrenal function after prolonged drug therapy in 10 women. Fifteen hours after stopping AG-HC therapy, 0900 h serum cortisol levels were normal [12.9 +/- 3.4 (SEM) micrograms/100 ml], but increments observed after provocative stimulation were blunted. However, the cortisol responses to both insulin-induced hypoglycemia and cortrosyn stimulation normalized 36 and 42 h after stopping AG and HC therapy. In addition, the concentration of plasma ACTH peaked at 175 +/- 9.3 pg/ml 15 min after the nadir of hypoglycemia. Adrenal histology in two patients who died while on chronic AG and HC therapy showed hypertrophic cells with large amounts of finely vacuolated cytoplasm in the zona fasciculata but no other abnormalities. We conclude that recovery of the hypothalamic-pituitary-adrenal axis is complete within 36 h after discontinuing chronic AG and HC therapy. This is in contrast to the prolonged suppression observed after chronic therapy with pharmacological doses of glucocorticoids.

How effective is surgical adrenalectomy in lowering steroid hormone concentrations?

Surgical adrenalectomy produces objective tumour regression in 50-60% of estrogen receptor-positive women with metastatic breast carcinoma. Additional responses to antiestrogens or further suppression of estrogens with aminoglutethimide after adrenalectomy suggest the possibility of continued adrenal steroid secretion even after surgical ablation. The use of sensitive and specific RIAs allows precise determination of the degree of hormone suppression after adrenalectomy and could provide documentation of nonsuppression or escape from suppression in individual patients. To evaluate the possibility of continued hormone secretion, we measured 14 hormones in 26 postmenopausal women with breast carcinoma before and after adrenalectomy. While the mean levels of androgens were markedly suppressed [dehydroepiandrosterone sulfate (DHEA-S), 99%, androstenedione, 94%; testosterone, 77%; dihydrotestosterone, 73%] after adrenalectomy, estrogen concentrations fell to a much lesser extent (plasma estrone, 73%; urinary estrone, 86%; plasma estradiol, 53%; urinary estradiol, 67%). Examination of data in individual patients revealed incomplete suppression in several women (less than 50% suppression of plasma estradiol in 14 of 25 patients, of urinary estradiol in 4 of 22, and of urinary estrone in 1 of 22). Androgen concentrations also fell incompletely after adrenalectomy in a few patients. Androstenedione concentrations were greater than 2 SD above the group mean in 2 of 23 patients, and in 2 of 25 patients, DHEA-S concentrations were also greater that 2 SD above the group mean. Serial measurements of hormones over a 1- to 3-yr period following surgery revealed escape from suppression over time (i.e. greater than 2-fold increase in hormone levels) in 7 of 26 women. The practical significance of the lack of suppression or of escape from inhibition was assessed by comparing estrogen levels in responders vs. nonresponders to surgical adrenalectomy. Of all steroids measured, greater suppression of only 1 hormone (urinary estrone) was observed in responders vs. nonresponders. These data indicate that adrenalectomy does not uniformly suppress circulating androgen and estrogen levels in postmenopausal patients. Women who initially suppress after adrenalectomy may show recovery of either androgen or estrogen levels with time.

Plasma estrone-sulfate: assessment of reduced estrogen production during treatment of metastatic breast carcinoma.

Highly sensitive and specific estrogen assays are required to monitor the hormonal effects of surgical adrenalectomy or pharmacologic estrogen suppression in postmenopausal women with breast carcinoma. Because the levels of plasma estrone-sulfate are 10-fold higher than its unconjugated counterpart, we developed a radioimmunoassay for estrone-sulfate to quantitate the minimal estrogen concentrations expected under conditions of endocrine gland ablation. After establishing normal ranges, we compared plasma estrone- sulfate levels and urinary conjugated estrone basally and after surgical adrenalectomy or aminoglutethimide (estrogen suppression) therapy in 23 postmenopausal women with breast carcinoma. In response to either therapy, the plasma levels of estrone-sulfate fell by 63.5-79.2% (p less than .01) and conjugated urinary estrone by 85-94.5% (p less than .01) in all study days over a 12-week period. Correlation analyses yielded r values of 0.77-0.94 between conjugated plasma and urinary estrone concentrations in the surgical adrenalectomy and aminoglutethimide-treated groups, respectively. No significant differences in estrone-sulfate levels were observed when comparing spontaneously menopausal and surgically castrate patients.

Triple drug chemotherapy in treatment of prostate adenocarcinoma Nb Pr A.I.-III.

The combination of cyclophosphamide, cis-platinum, and adriamycin has been evaluated in the Nb tumor model system. Triple drug therapy has resulted in marked reduction in tumor volume (P less than 0.01), as well as decreased number of metastases (P less than 0.01). Combination chemotherapy has reduced the metastatic rate, increased complete tumor regression, and reduced the final tumor volume. The androgen insensitive tumor of Nb Pr A.I.-II was evaluated with combinations of BCNU and adriamycin in three cycles. The final tumor volume when compared to controls was significant for all of the agents evaluated (P less than 0.01). A significant decrease in the number of metastases was observed in the triple drug therapy, administered for three cycles, BCNU only, and BCNU and adriamycin. The acid phosphatase content of the treatment group's tumors, when compared to controls was significant with BCNU treatment only.

Suppression of residual oestrogen production with aminoglutethimide in women following surgical hypophysectomy or adrenalectomy.

In postmenopausal women with breast carcinoma, plasma and urinary oestrogens remain detectable following surgical adrenalectomy or hypophysectomy. These residual oestrogens could result from absorption of exogenous steroids, from endogenous production, or from a combination of these two sources. To determine whether endogenous production contributes to this oestrogen pool, we administered a potent steroidogenesis inhibitor, aminoglutethimide (AG), to women with breast carcinoma following hypophysectomy or adrenalectomy. Plasma and urinary oestrogens were measured with radioimmunoassays developed to provide appropriate sensitivity. In five women treated after initial hypophysectomy (hypox), plasma oestrone fell from 66 + 28 pg/ml (hypox) to 9.1 +/- 2.4 pg/ml (hypox and AG) and oestradiol decreased from 8.3 +/- 1.8 pg/ml to 2.5 +/- 0.69 pg/ml. Similar decrements in urine oestrone (U-E1) and ostradiol (U-E2) were observed (U-E1 hypox: 2.25 +/- 0.71 microgram/24 h 0.071 +/- 0.015 microgram/24 h hypox and AG; U-E2 0.47 +/- 0.12 micrograms/24 h hypox to 0.124 +/- 0.015 hypox and AG, P less than 0.05 for all). Similar significant reductions in plasma oestrone and oestradiol were observed in four women treated with aminoglutethimide following surgical adrenalectomy. While the levels of urinary oestrogens also fell in these patients, the differences were not statistically significant. In response to the decrements in oestrogen levels induced by AG, 2/5 women in the post-hypophysectomy group and 2/4 in the post-adrenalectomy group experienced partial objective tumour regression. These observations indicated that the residual oestrogens produced after surgical adrenalectomy or hypophysectomy, even though made in small quantities, were nonetheless biologically active. We conclude that endogenous production of oestrogens in extragonadal and extra-adrenal sites occurs after major surgical endocrine ablation in women with breast carcinoma. Additional exogenous oestrogen sources can not be excluded.

A possible role of endogenous opioids in the control of prolactin and luteinizing-hormone secretion in the human.

We investigated the participation by endogenous opioid peptides in the control of prolactin and gonadotropin secretion in 5 normal men and 6 normal women, and in 4 men and 5 women with persisting hyperprolactinemia following transsphenoidal pituitary microsurgery for prolactinomas. Iv administration of the specific opiate-receptor antagonist, naloxone hydrochloride (0.2 mg/kg bolus), failed to affect serially sampled serum prolactin levels in normal male or female subjects. With prolactinoma patients, naloxone suppressed hyperprolactinemia to 37% and 32% of mean control values in 2 of 4 males, but in none of 6 females. When luteinizing hormone was serially sampled under the same conditions, 5 of 5 normal males (but no female, normal or abnormal) demonstrated a monophasic increase in serum LH concentrations after injection of the antagonist. The LH peak was 55 +/- 4% above basal levels (p less than 0.01). In contrast to normal men, only one of 4 hyperprolactinemic male patients manifested a stimulatory response of LH to naloxone. Among all 20 subjects, none exhibited a change in FSH levels acutely after naloxone. These data suggest that: i) naloxone will not fulfill its postulated role as an ideal therapy of hyperprolactinemia and hypogonadotropism, at least in women; ii) endogenous opioids may participate in the neuroendocrine regulation of LH secretion in the normal human, iii) male-female differences may modify the role of endogenous opioids; and iv) some male patients with hyperprolactinemia exhibit defective opioid-related neuroregulation of LH secretion.

Evidence that brain aromatization regulates LH secretion in the male dog.

A variety of data suggest an independent role for androgens and estrogens in the regulation of luteinizing hormone (LH) secretion in the male. Estrogens, in the male are primarily derived from testicular androgens that are aromatized both in peripheral tissues and in the CNS. Our prior data suggested a pharmacologic regimen that blocked CNS aromatization without lowering peripheral estrogen or testosterone levels. Such experimental conditions would permit assessment of the relative roles of CNS versus peripheral aromatization in the regulation of LH secretion. We utilized this regimen (aminoglutethimide, a potent aromatase inhibitor, and hydrocortisone) in seven adult male dogs for 14 days. Plasma LH rose to castrate levels, 450% above control values on days 7 and 14. These LH increments stimulated similar rises in androstenedione, testosterone, and dihydrotestosterone. In contrast, plasma estrone and estradiol concentrations remained constant. The induction of castrate LH levels without a concomitant fall in peripheral androgens or estrogens is best explained by a block of central aromatization and thus a reduction in local hypothalamic concentrations. We conclude that aromatization in the CNS rather than peripheral tissues is the more important site with respect to LH negative feedback in the male dog.

Aminoglutethimide as treatment of postmenopausal women with advanced breast carcinoma.

Hormone-dependent breast carcinomas respond to deprivation of biologically active estrogens with objectively quantifiable tumor regression. Aminoglutethimide, a known inhibitor of steroid synthesis, is also a potent blocker of the aromatase enzyme and, thus, of estrogen production. We developed an effective regimen to inhibit estrogen production in postmenopausal women using aminoglutethimide and replacement glucocorticoid. One hundred forty-seven women initially received aminoglutethimide and replacement glucocorticoid as treatment of metastatic breast carcinoma. One hundred twenty-nine women are currently evaluable for assessment of clinical and hormonal responses. Thirty-seven percent of unselected women and 49% of estrogen receptor-positive patients experienced objective tumor regression. Responses occurred predominantly in soft tissue (47%) and bone (35%) and lasted 30 +/- 9.1 months for complete and 14 +/- 1.5 months for partial regressions. Plasma and urinary estrogen levels fell equally in responder versus nonresponder groups whereas androgen levels declined less in patients with progressive disease.

Evaluation of methods for perfusing rat testes.

Organ perfusion methods offer a number of advantages in biologic studies but require full characterization before application. Two new methods for perfusing rat testes were characterized and compared with recirculating hemicorpus system. These preparations, selective and isolated testicular perfusion, are nonrecirculating and consequently, allow direct measurement of testosterone secretion. In both systems, testosterone production was a fuction of the dose of human chorionic gonadotropin in the perfusion medium up to 1000 mIU per ml which appeared to be inhibitory. The isolated testis method, in comparison with the selective, is more sensitive to human chorionic gonadotropin, requires less perfusion medium, maintains normal blood flow rates and water content, and is associated with no ischemia at commencement of perfusion. However, this system does not retain normal levels of ATP and GTP after 3 hr of perfusion. Whereas both procedures may be used for studies of testosterone secretion and androgen receptors, the inability to maintain testicular ATP and GTP levels indicates that present methods are not suitable for study of processes dependent upon high energy phosphate metabolism.

Clinical and biochemical effect of aminoglutethimide in the treatment of advanced prostatic carcinoma.

Treatment of male patients with advanced prostatic carcinoma and disease progression after initial endocrine therapy frequently is unsatisfactory. However, approximately 20 per cent of these patients respond to surgical adrenalectomy or hypophysectomy, indicating continued hormonal responsiveness. A total of 25 previously castrated men with stage D carcinoma received 1,000 mg. aminoglutethimide and 40 mg. hydrocortisone daily. The patients were evaluated using the criteria of the National Prostatic Cancer Project. One patient has had a complete response and is in remission after 275 weeks of therapy. A partial response was noted in 4 patients, while the disease was objectively stable in 6. Pre-treatment testosterone and dihydrotestosterone levels were measured in 9 of 25 patients and were significantly reduced statistically during aminoglutethimide therapy (p less than 0.01). Response and drug toxicity are discussed.

Clinical effect of aminoglutethimide, medical adrenalectomy, in treatment of 43 patients with advanced prostatic carcinoma.

The initial treatment of patients with Stage D prostatic carcinoma with orchiectomy or estrogens is successful in giving objective and subjective improvement for variable periods of time. However, after initial endocrine treatment patients generally relapse, and go on to further progression of their disease. However, a subgroup of approximately 22% of these Stage D prostatic cancer patients respond to either surgical adrenalectomy or hypophysectomy, indicating some degree of continued hormonal responsiveness. Forty-three previously castrated patients with Stage D prostatic carcinoma were treated with 1000 mg of aminoglutethimide and 40 mg of hydrocortisone daily and have been evaluated using the criteria of the National Prostatic Cancer Project. Progression of disease after initial hormonal therapy has varied from 3 to 25 months. One patient has had a complete response, and continues in remission after 290 weeks of therapy. Partial objective responses have been observed in 6 patients, and 10 patients have remained objectively stable for an average of 35 weeks in this latter group.

A randomized trial comparing surgical adrenalectomy with aminoglutethimide plus hydrocortisone in women with advanced breast cancer.

We randomized 96 postmenopausal women with metastatic breast carcinoma to receive surgical adrenalectomy or medical therapy with an adrenal inhibitor, aminoglutethimide (AG), plus replacement hydrocortisone. Before randomization, women were stratified according to disease-free interval, site of dominant disease, and estrogen-receptor status. Of 40 evaluable women treated with AG and hydrocortisone, 53 per cent had objective responses, as compared with 45 per cent of 29 women undergoing surgical adrenalectomy (P value not significant). Responses lasted a mean of 17.2 months in the medical group and greater than 17.1 months in the surgical group (not significant). Estrogen levels fell similarly in response to either treatment, whereas AG and hydrocortisone preserved androgen production. A null hypothesis tested the single question asked by this study: "Is surgical adrenalectomy superior to treatment with AG and hydrocortisone?" Rejection at significance levels of P = 0.01 and P = 0.07 for differences of 20 per cent and 10 per cent, respectively, suggested that medical therapy with AG and hydrocortisone may be logically chosen in place of surgical adrenalectomy.