Androstenedione analogs containing 7α-substituents have proven to be potent inhibitors of aromatase both in vitro and in vivo. Several of these agents have exhibited higher affinity for the enzyme complex than the substrate. In order to examine further the interaction(s) of 7-substituted steroids with aromatase, 7-substituted 4,6-androstadiene-3,17-diones were synthesized and demonstrated competitive inhibition of aromatase activity in human placental microsomes. 7-Substituted 1,4,6-androstatriene-3,17-diones demonstrated mechanism-based inhibition of placental aromatase acitivity. These agents were evaluated for inhibition of aromatase activity in the JAr human choriocarcinoma line. The 7-substituted 4,6-androstadiene-3,17-diones produced dose dependent inhibition of aromatase activity in the cell cultures, with IC50 values ranging from 490 nM to 4.5 μM. However, these agents are less effective when compared to other steroidal inhibitors, such as 7α-thiosubstituted androstenediones. These results on the 7-substituted 4,6-androstadiene-3,17-diones are consistent with the data from biochemical enzyme inhibition studies using human placental aromatase. On the other hand, 7-phenethyl-1,4,6-androstatriene-3,17-dione exhibits greater inhibitory activity, with an IC50 value of 80 nM. Other mechanism-based inhibitors, 7α-(4′-amino)phenylthio-1,4-androstadiene-3,17-dione and 4-hydroxyandrostenedione, also exhibited potent inhibition of aromatase acitivity in JAr cells. In summary, the most effective B-ring modified steroidal aromatase inhibitors are those derivatives that can project the 7-aryl substituent into the 7α-position.

Androstenedione analogs containing 7 alpha-substituents have proven to be potent inhibitors of aromatase both in vitro and in vivo. Several of these agents have exhibited higher affinity for the enzyme complex than the substrate. In order to examine further the interaction(s) of 7-substituted steroids with aromatase, biochemical and pharmacological studies were performed on 7 alpha-thiosubstituted androstenediones and 7-substituted 4,6-androstadiene-3,17-diones. Potent inhibition of aromatase activity in human placental microsomes has been observed with several new 7 alpha-thiosubstituted androstenediones. 7-Benzyl- and 7-phenethyl-4,6-androstadiene-3,17-diones effectively inhibited microsomal aromatase, with apparent Kis ranging from 61 to 174 nM. On the other hand, 7-phenyl-4,6-androstadiene-3,17-dione exhibited poor activity, with an apparent Ki of 1.42 microM. Similar inhibitory activity was observed with reconstituted, purified cytochrome P450Arom preparations. Additionally, these agents were evaluated for inhibition of aromatase activity in two human carcinoma cell lines, the MCF-7 human mammary cancer line and the JAr choriocarcinoma line. The 7 alpha-thiosubstituted androstenediones and 7-substituted 4,6-androstadiene-3,17-diones produced dose-dependent inhibitions of aromatase activity in the cell cultures. The most effective inhibitors were the 7 alpha-substituted androstenediones, with EC50 values ranging from 7.3 to 105 nM. Finally, the JAr cell culture system exhibited prolonged inhibition of aromatase activity following exposure to 7 alpha-APTADD, suggesting enzyme inactivation by this inhibitor. Thus, these agents are effective aromatase inhibitors, and the results encourage further development of this group of medicinal agents for the treatment of estrogen-dependent mammary carcinoma.

Estrogen metabolism was evaluated in freshly isolated kidney and liver microsomes and in primary kidney cell cultures from Syrian hamsters, a potential experimental model for examining the possible role(s) of estrogens in tumor initiation and development. Initial velocity studies of the conversion of estradiol to 2-hydroxyestradiol, as determined by the 3H2O release assay with the substrate [2-3H]estradiol, resulted in similar apparent Kms of estrogen 2-hydroxylase of 2.85 and 6.25 microM for liver and renal microsomes, respectively. The apparent Vmax for freshly prepared liver microsomes was 0.13 nmol.mg-1.min-1, while that for renal microsomes was 0.040 nmol.mg-1.min-1. Evaluation of estrogen metabolism was also performed in primary cell cultures of hamster kidney cells, consisting of 75% epithelial cells. [6,7-3H]Estradiol (10 microM) was incubated for 0, 24 and 48 h in primary kidney cell cultures, and the organic soluble metabolites analyzed by reverse-phase HPLC. The cultures from untreated, castrated hamsters metabolize [3H]estradiol to yield small quantities of estrone and significant amounts of polar metabolites, while no catechol estrogens were isolated. Estrogen metabolism by diethylstilbestrol-treated (DES-treated) hamster kidney cell cultures also provided small quantities of estrone and no evidence of catechol estrogens. Additionally, larger amounts of additional polar metabolites were isolated in the cultures from DES-treated hamsters. Finally, levels of estrogen 2-hydroxylase were detected in these cultures using the 3H2O release assay. Thus, the short-term primary kidney cell cultures from the Syrian hamster are capable of metabolizing estrogens. Furthermore, the enzymatic processes appear to be available for the conversion of any catechol estrogens formed into more polar metabolites. These investigations in intact cells, capable of performing all biochemical processes, complement both in vivo and subcellular biochemical studies and may aid in elucidating the roles of estrogens and estrogen metabolism in the initiation and development of estrogen-induced, estrogen-dependent kidney tumors in the Syrian hamster.

7 alpha-(4'-Amino)phenylthio-1,4-androstadiene-3,17-dione (7 alpha-APTADD), a potent enzyme-activated irreversible inhibitor, was examined in two different human cell culture lines, MCF-7 human mammary carcinoma cells and JAr choriocarcinoma cells. Both the MCF-7 and JAr cell culture systems exhibit aromatase activity, and 7 alpha-APTADD was evaluated for its aromatase-inhibitory activity, for its ability to inactivate the enzyme complex, and for the time course of recovery of enzymatic activity. This inhibitor produced a dose-dependent inhibition of aromatase activity in MCF-7 cells and in JAr cells, with EC50 values of 91 and 7.3 nM, respectively. Two other steroidal inhibitors, 7 alpha-(4'-amino)phenylthio-4-androstene-3,17-dione and 4-hydroxyandrostenedione, produced similar dose-response curves and EC50 values, while the nonsteroidal aminoglutethimide was less effective. Both cell culture systems exhibited prolonged inhibition of aromatase activity following exposure to 7 alpha-APTADD, suggesting enzyme inactivation by this inhibitor. Thus, 7 alpha-APTADD is an effective inhibitor of aromatase in MCF-7 mammary carcinoma cells and in JAr choriocarcinoma cells. These studies encourage further development of this group of medicinal agents for the treatment of estrogen-dependent mammary carcinoma.

Agonist and antagonist activities have been reported for several catechol estrogens given exogenously. Since the metabolic clearance rate for catechol estrogens in the body is very rapid, catechol estrogens produced at other tissues will have minimal effect on breast tissue. Information of the extent of catechol estrogen formation within cells is critical in assessing the overall importance of these estrogen metabolites. Investigations of the conversion of estrogens to catechol estrogens were performed in the MCF-7 human mammary carcinoma cell culture system. Reverse-phase high-performance liquid chromatography (HPLC) analysis demonstrated that very little metabolism of estradiol occurs after 48 h, with only small amounts of estrone, 2-hydroxyestradiol, 2-hydroxyestrone, and estriol being observed. The total amount of 2-hydroxyestrogen products formed from 1 μM estradiol was 406.2 pmol (SD = 60.9) per 3 × 107 cells in 48 h. Similar results were obtained using the simpler radiometric assay for estrogen 2-hydroxylase, which measures the release of 3H2O from [2-3H]estradiol. The effects of inhibitors of estrogen 2-hydroxylase were also examined in MCF-7 cells. 2-Bromoestradiol, 4-bromoestradiol, and 2,4-dibromoestradiol effectively block estrogen 2-hydroxylase in a dose-dependent manner in MCF-7 cultures, with ED50 of approximately 1 μM for each inhibitor. Furthermore, these bromoestrogens bind poorly to estrogen receptors in MCF-7 cells and do not alter cell growth. Thus, in MCF-7 mammary cell cultures, metabolism of estradiol occurs to only a minor degree, and it is unlikely that the levels of catechol estrogens would reach physiologically relevant concentrations in the intact breast cancer cells. In addition, the bromoestrogens are effective inhibitors of estrogen 2-hydroxylase in cell culture systems, bind poorly to the estrogen receptor, and do not exhibit estrogenic effects at concentrations up to 1 μM.

7 alpha-Substituted 4-androstene-3,17-diones are effective inhibitors of aromatase in vitro. The microsomal P-450 enzyme complex has a greater affinity for several of these inhibitors than for the substrate androstenedione, with 7 alpha-(4'-amino)phenylthio-4-androstene-3,17-dione (7 alpha-APTA) being the most potent competitive inhibitor of the series. The effects of 7 alpha-APTA were examined in MCF-7 human mammary carcinoma cell culture. 7 alpha-APTA inhibited aromatase activity in the MCF-7 human mammary carcinoma cell line with an ED50 value of 25.07 nM (+/- 7.71). The inhibitor did not bind to the estrogen receptor of the MCF-7 cells in vitro. In addition, 7 alpha-APTA did not stimulate growth of the MCF-7 cells when compared with controls. No induction of progesterone receptors was observed in MCF-7 cultures grown in the presence of inhibitor at concentrations ranging from 10 pM to 1 microM. Thus, the steroid 7 alpha-APTA is an effective inhibitor of aromatase in intact MCF-7 cells. Furthermore, it does not demonstrate any significant hormonal effects on the cells nor does this agent produce any general toxicological effects.

After years of stagnation in the treatment of chronic haematological malignancies, some interesting agents have emerged which might improve the prognosis of these diseases. For chronic leukaemias of lymphoid lineage, three new chemical agents, all purine analogues, seem to be of particular interest. Pentostatin is a specific inhibitor of ADA and has been shown to be highly efficient in producing CR in patients with HCL. Its relative merit compared with IFN-α for the treatment of HCL is being studied in ongoing randomized trials. Pentostatin is also active in B-CLL and promising activities have been demonstrated in T-or B-PLL and ATCL. Fludarabine is an analogue of adenine which is resistant to the deamination of ADA. It has been reported to be highly active for patients with both pretreated or non-treated B-CLL. CR rates of 13% with overall response rates of 57% can be achieved, even in heavily pretreated patients. Its activity in the other lymphoid malignancies is not yet known. CdA, a substrate analogue of ADA, has also produced encouraging results in B-CLL, HCL and T cell malignancies, and in some patients with just one single course. Thus far, experience with this drug comes from one institution and requires further confirmation.For chronic myeloproliferative diseases, little progress has yet been made. Although IFN-α seems to be active in CML and to result in cytogenetic remissions in bone marrow, a definite advantage of this biological agent over conventional chemotherapy as regards survival and life quality has not yet been proven. Allogeneic bone marrow transplantation is beneficial for those patients who are eligible. No remarkable advances have been made in the treatment of myeloproliferative disorders except for the development of an antiplatelet drug, anagrelide. This agent seems to be highly effective in controlling thrombocytosis. The relative merit of this agent as compared with IFN-α, as well as the impact of this agent on the survival and on life-quality of patients with myeloproliferative disorders, have yet to be defined.

Research efforts over the past several years have focused on the synthesis of competitive and irreversible aromatase inhibitors and examination of these inhibitors in microsomal preparations, in cell culture, and in vivo. Several 7 alpha-substituted androstenediones have demonstrated high affinity for placental aromatase, with apparent Ki's ranging from 1 to 30 nM. Inactivation of aromatase occurred following incubation with alkylating and enzyme-activated irreversible inhibitors. 7 alpha-(4'-Amino)phenylthio-4-androstene-3,17-dione (7 alpha-APTA) exhibits potent inhibitory activity of aromatase in the MCF-7 human mammary carcinoma cell line with an ED50 of approximately 25 nM. The inhibitor did not bind to the estrogen receptor of the cells in vitro nor induce levels of progesterone receptors in intact cells. In vivo studies of 7 alpha-APTA in the DMBA-induced rat mammary carcinoma model resulted in 80% of the tumors responding completely or partially at doses of 25 and 50 mg/kg body wt/day. Thus, these 7 alpha-substituted steroidal aromatase inhibitors are effective medicinal agents and may be useful for the treatment of estrogen-dependent breast cancer.

Estrogen and androgen radioimmunoactive substances were measured throughout development in whole body homogenates of the flesh fly Sarcophaga crassipalpis. Estradiol-equivalent substances (EES) (range of 1–9 pg/mg protein) and testosterone-equivalent substances (TES) (1–17 pg/mg protein) were present throughout larval and pupal development, during pupal diapause, and in both male and female adults. Males and females of each age contained nearly the same amounts of EES and TES, thus suggesting that these steroids are unlikely to contribute to sexual differences. A major increase in EES and TES was detected in both males and females on day 3, a point coinciding with the onset of mating. High levels present in these ovoviviparous females during the last half of the reproductive cycle may be due partially to the embryos within her uterus since very high amounts of both steroids were detected in first instar larvae dissected from the uterus.