Harry J. Brodie

Studies on the mechanism of estrogen biosynthesis in the rat ovary–I☆

Studies on the mechanism of estrogen biosynthesis in the rat ovary are presented. Short-term treatment with luteinizing hormone and follicle stimulating hormone in various combinations did not produce appreciable stimulation whereas 8-16 day treatment (long-term) with pregnant mares serum gonadotropin increased activity in homogenates up to 9 fold per mg wet weight of tissue. A similar increase per mg protein was noted in the 105000 g microsomal fractions where the bulk of the activity was seen. The potencies of 6 steroids as aromatase inhibitors were compared in the rat ovaries and human placental microsomal systems and were found to be comparable in all cases except 1.

Aromatase inhibitors. Synthesis and biological activity of androstenedione derivatives

The synthesis and biological evaluation of androstenedione derivatives as inhibitors of estrogen biosynthesis are described. The results show that 4-hydroxy analogues are among the most potent in vitro inhibitors of the series. Esterification of the 4-hydroxy steroids generally reduced activity. Further conjugation of the 3-keto 4-ene system to give 4-hydroxy-4,6-androstadiene-3,17-dione caused more rapid inactivation of aromatase in rat ovarian microsomes than 4-hydroxyandrostenedione. Some compounds exhibited differences in activity when tested for inhibition of human placental microsomes vs. rat ovarian microsomes. The 4-hydroxyandrostenedione derivatives and their nonbulky esters were generally more potent in vitro and in vivo inhibitors than other substituted steroids in the series. Several of the synthesized compounds markedly reduce (50-81%) estrogen levels in rats on proestrus and/or had antifertility action. To date, none of the compounds surpassed the in vivo inhibitory action of 4-hydroxy-4-androstene-3,17-dione or its 4-acetate derivative.

Aromatase inhibitors—iv. Regression of hormone-dependent, mammary tumors in the rat with 4-acetoxy-4-androstene-3,17-dione

Abstract An aromatase (estrogen synthetase) inhibitor, 4-acetoxy-4-androstene-3,17-dione (4-acetoxy-A)‡, was effective in causing regression of DMBA (7,12-dimethylbenz(a)anthracene)-induced, hormone-dependent, mammary tumors in the rat. At a similar dose (15 mg/day), treatment was more effective when 4-acetoxy-A was administered by silastic wafer supplemented with injections than by injections alone. When 4-acetoxy-A was administered by wafers to 7 rats for 4 weeks, 64% of the tumors completely regressed. The mean ovarian estradiol secretion rate measured at the end of treatment with 4-acetoxy-A was reduced to 28% of the control value. Estrone and progesterone secretion rates were reduced but not significantly. Gonadotropin levels were unchanged. Tumor regression with 4-acetoxy-A was counteracted when estradiol-17β was administered concomitantly. The above finclings indicate that 4-acetoxy-A probably acts to cause tumor regression by reducing estrogen production via aromatase inhibition.

Aromatase inhibitors and their use in controlling oestrogen-dependent processes

Abstract The goal of this work is to develop potent in vivo inhibitors to the aromatase (oestrogen synthetase) enzyme system and to evaluate them for controlling oestrogen-dependent processes. To date, the most potent compounds in vitro are 4-hydroxy-4-androstene-3,17-dione (4-OH-A), 4-acetoxy-4-androstene-3, 17-dione (4-acetoxy-A) and 1,4,6-androstatriene-3,17-dione (ATD). In antifertility studies, rats were treated from dioestrus 1 of the cycle with aromatase inhibitors. The oestrogen surge and subsequent LH surge normally occurring during pro-oestrus were inhibited and mating and ovulation prevented. These effects were counteracted when oestradiol was administered concomitantly with aromatase inhibitors. The compounds also retarded implantation, an oestrogen-dependent process in the rat, and ovarian oestradiol secretion was reduced on day 4 of pregnancy. In contrast, hamsters do not require oestrogen for implantation and aromatase inhibitors were without effect. The compounds were also highly effective in causing regression of dimethylbenzanthracene-induced, hormone-dependent, mammary tumours of the rat. Ovarian oestrogen secretion measured at the end of treatment with 4-acetoxy-A was reduced compared with controls, and when supplemental oestradiol was given, together with aromatase inhibitors, tumour regression was prevented. Our studies indicate that aromatase inhibitors act in vivo to reduce oestrogen production by aromatase inhibition. They thus have potential for treating oestrogen related disorders, for fertility control and as tools for investigating the role of oestrogen in physiological and pathological processes.

Synthesis of deuterium- and tritium-labeled 4-hydroxyandrostene-3,17-dione, an aromatase inhibitor, and its metabolism in vitro and in vivo in the rat

The metabolism of the aromatase inhibitor-4-hydroxyandrostenedione (4-OHA) was studied in vitro and in vivo in the rat. To accomplish this, deuterium- and tritium-labeled 4-OHA were prepared from 4-hydroxyandrosta-4, 6-dione-3,17-dione. The latter was synthesized from 4-androstene-3,17-dione. Using deuterated 4-OHA in in vitro incubations of rat ovarian microsomes, 4-hydroxytesterone (4-OHT) was identified by gas chromatography/mass spectroscopy as the major metabolite. 4-OHT constituted approximately 20% of the total radioactivity from [6,7-3H]-4-OHA in the ovarian microsomal incubations. Conversion of [6,7-3H]-4-OHA to 4-hydroxyesterone was approximately 0.1%. The major metabolite of [6, 7-3H]-4-OHA in vivo identified in the free, neutral fraction of rat blood was 3 beta-hydroxyandrostane-4,17-dione. The metabolite accounted for approximately 5% of the total radio-activity in the blood, Whereas 4-OHT accounted for only 0.1%, 4-OHT inhibited in vitro ovarian aromatization by 59%, but 3 beta-hydroxyandrostane-4-17-dione had little effect. It was concluded that the in vivo effects of 4-OHA previously reported are largely due to its own activity although additional effects of its metabolic products cannot be excluded.

Studies on the mechanism of estrogen biosynthesis. IV. Ovarian metabolism of estr-4-ene-3,17-dione☆

Abstract 1. 1. The role of estr-4-ene-3,17-dione (1) as an ovarian estrogen precursor has been examined. 2. 2. Radiochemically pure 3-hydroxyestra-1,3,5(10)-trien-17-one (11) and 1β-hydroxyestr-4-ene-3,17-dione (III) were isolated from an incubation of minced human ovary with [4-14C]-1 in the presence of a NADPH-generating system. 3. 3. The formation of II confirms Shorts suggestion that I, which he had isolated from equine follicular fluid, may serve as an estrogen precursor in the ovary. 4. 4. The simultaneous formation of II and III parallels the situation in preparations of human-term placenta and supports our previous suggestion that the two products may be related biosynthetically. 5. 5. The demonstration of 1β-hydroxylase activity in human ovary provides an explanation of the formation of urinary estrogen artifacts from labile 1β-hydroxy-19-nor steroids during studies of ig-nor steroid metabolism in non-pregnant women.

Estrogen biosynthesis and 10-hydroxylation using C19 and 19-nor steroid precursors

(1) In order to study the relationship between aromatization (estrogen biosynthesis) and 1beta-hydroxylation, the effects of a variety of factors on these processes were evaluated. (2) Using the C18 substrate, 4-estrene-3,17-dione, it was found that carbon monoxide, SU-4885, amphenone B, potassium cyanide, 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione inhibited the above transformations significantly and to varying degrees. However, within a given experiment the inhibition of each process was similar. (3) SKF-525A did not inhibit either transformation. In addition, phosphate, Tris and barbital buffers, as well as pH changes from 6.9 to 7.7, had no stimulatory or inhibitory effect on the production of estrogen and 1beta-hydroxy compounds. (4) In contrast, several inhibitors affected the aromatization of C19 and C18 steroids differently. These include carbon monoxide, SU-4885 and amphenone B. (5) When a mixture of 4-[7beta-3Hi1estrene-3,17-dione and 19-[4-14C]nortestosterone were incubated together the former was preferentially converted to estrogen. This preference for the 17-keto steroidal form mimics results observed for C19 substrates. (6) We conclude that while estrogen biosynthesis and 1beta-hydroxylation appear to be mediated by the same enzyme system, the same conclusion cannot be drawn for the aromatization of C19 and C18 substrates.

Studies on the mechanism of estrogen biosynthesis, V. stereochemical comparison of aromatization in placenta! and microbiological systems☆

1. 1. To provide definitive evidence as to whether 19-hydroxyandrost-4-ene-3,17-dione (I) may aromatize by similar mechanisms in human placental and in microbiological systems, the stereochemistry of hydrogen removal at C-1 in the conversion of I to estrogen was investigated. 2. 2. Incubations of 19-hydroxy-[1-3H(83%-β)]androst-4-ene-3,17-dione with a placental microsomal preparation and a NADPH-generating system gave estrone in which 84% of the tritium was lost, showing that the 1β-hydrogen was eliminated. When the NADPH-generating system was replaced by an artificial electron acceptor active in converting 19-hydroxyandrostenedione to estrone in microorganisms, no conversion to estrone was noted. 3. 3. Incubations of the same steroid substrate with respiring cultures of Pseudomonas sp. (ATCC 13262) and Nocardia restrictus showed that 22 and 28% of the tritium, respectively, was lost in the transformation to estrone, thus implicating removal of the α-hydrogen in the transformation. A similar result for N. restrictus was obtained with a 6000 × g supernate preparation and the artificial electron acceptor, phenazine methosulfate. 4. 4. The results show that the transformation of I to estrone in the two types of systems occurs by different stereochemical mechanisms, and that probably different electronic factors are involved also.

The catalytic reduction of 3β-hydroxyandrost-5-en-17-one with tritium. The distribution and orientation of label in the product ☆

Abstract 3β-Hydroxyandrost-5-en-17-one (I) was reduced catalytically with carrier-free tritium. The product was mainly 3β-hydroxy-5α-androstan-17-one (II), which was converted to androst-4-ene-3, 17-dione (VI) with a loss of 43% of the label. 6β-Bromination and 6β-methoxylation was carried out on VI with little loss of label, indicating that the tritium at C-6 was alpha orientated. Chemical and kinetic experiments established that the additional label was at C-7α. The distribution of tritium in the reduced product II was 43% 5α, 35% 6α and 22% 7α.

Quantification of urinary 3α,21-dihydroxy-5β-pregnan-20-one and 5-pregnene-3β, 20α-diol by mass fragmentography

Abstract A sensitive and accurate method is described for measuring urinary corticosteroids by gas chromatography-mass spectroscopy (GC-MS). Using single peak monitoring (mass fragmentography) and electron impact ionization, the acetates of 3α,21-dihydroxy-5β-pregnan-20-one (tetrahydrodeoxycorticoster-one) and 5-pregnene-3β,20α-diol were estimated with deuterio-acetate carriers as recovery markers. With this technique, the coefficient of variation did not exceed 3% for GC-MS analyses of the urinary corticosteroid samples by single peak monitoring. An evaluation of the trimethylsilyl ether derivatives of the two steroids by chemical ionization was also made. Secretion rates determined for deoxycorticos-terone derived from specific activities of urinary tetrahydrodeoxycorticosterone and excretion levels of 5-pregnene-3β,20α-diol were slightly lower than those obtained by other methods.