Heme-Containing Oxygenases

The pharmacology and clinical uses of tamoxifen

Much of what I will discuss here today relates to brain differentiation in rodents. This subject was the interest of two outstanding British scientists, Geoffrey Harris and Peter MacDonald. Harris spent his scientific life and considerable intellectual powers observing and enlarging upon this very interesting phenomenon, which was originally described so brilliantly by Carroll Pfeiffer in 1936. Peter MacDonald was an outstanding young man who had already shown the key role of estrogens in central steroid actions by experiments such as the use of antiestrogens to block the action of androgens on brain differentiation, as described below. Geoffrey Harris’ life ended at the pinnacle and fruition of a career that included the elucidation of the neurovascular and chemical links between the brain and pituitary and the isolation of the LH releasing factor. Peter MacDonald's life and continued work were prematurely lost in the recent Paris air disaster; we can only guess what he might have contributed in years to come. However, both of these men left important scientific and intellectual legacies, part of which you have already heard about. If a man's work is what others judge him by, these two will stand up well to any test, any time. We owe them our gratitude and continued remembrance.

The formation of estrogens by central neuroendocrine tissues

In the past decade there has been considerable progress in the characterization of individual human P450 enzymes. These advances were initiated by early work on the purification of individual enzymes from human liver and other sources. The early work in the area was guided by a focus on the most abundant and easily purified enzymes.1–4It is now apparent, in retrospect, that these proteins were in the 2C and 3A families. Efforts were shifted to attempts to purify individual P450s on the basis of catalytic activities with the evidence that in some cases a single P450 could be identified in this way; e.g., the P450 now known as P450 2D6 was found to be under monogenic control. 5 The approach is technically demanding because of the need to do separations in the presence of detergents and then remove them before analysis of catalytic activity. Nevertheless, human P450s 1A1,6 1A2,7 2A6,8 2C8,9 2C9,9,10 2D67,11,12 2E1 13,14 3A4,15 3A5,16 4A11,17 and lanosterol 14α-demethylase18 were isolated in this general manner. Another approach that has been used, sometimes in a mode complementary to catalytic specificity, is purification on the basis of immunochemical similarity to animal P450s. 7,11,19,20

Human Cytochrome P450 Enzymes

Utilization of oxygen and reduced nicotinamide adenine dinucleotide phosphate by human placental microsomes during aromatization of androstenedione.

4-Hydroxy-f-androstene-3,17-dione (4-OH-A) when tested at various concentrations was found to inhibit markedly the conversion of 4-andorstene-3,17-dione to estrogens inhuman placental and rat ovarian microsomes. To obtain evidence that estrogen biosynthesis could also be reduced in vivo with 4-OH-A, rats were treated sc at a dose level of 50 mg/kg body weight. After 3 h the ovarian veins were cannulated and blood collected. Estradiol concentrations in the plasma were reduced by 80% compared to control values during the proestrous surge and on Day 4 of pregnancy. 4-OH-A was also found to be effective in controlling estrogen-dependent reproductive and neoplastic processes. In rats treated from Day 2-7 of pregnancy, implantation of fertilized ova was completely prevented in some rats, while in others either implantation was delayed or the development of implants was retarded. 4-OH-A treatment of rats having estrogen-dependent breast tumors induced by 7,12-dimethylbenz(a)anthracene caused 80% of the tumors to regress significantly in 4 weeks of treatment; 42% of these regressed completely.

The effect of an aromatase inhibitor 4-hydroxy-4-androstene-3 17-dione on estrogen-dependent processes in reproduction and breast cancer.

Structural features of over 100 steroids and some non—steroids were evaluated systematically as inhibitors of estrogen biosynthesis in human placental microsomes. The microsomes were preincubated with and without potential inhibitors, and then were incubated with [4–14C] androst–4–ene–3,17–dione or [4–14C]3P—hydroxyandrost— S—en—17–one (DHA) and an NADPH generating system. After adding tritiated estrogens as recovery markers, the estrogens were isolated by thin—layer chromatography and were assayed using dual—label scintillation counting to determine percent conversion and inhibition produced. C10 A4–3–keto compounds were better inhibitors than the corresponding ring A reduced (5<x or 5P) compounds or A4–3–keto 19–nor compounds. Further conjugation with the A4—3–keto system was also beneficial so that A1.4'6–3–one, A4–6–3–one and A4– 3,6–dione compounds were effective. The 17–keto was superior to the 17P—hydroxy group in providing inhibitory properties in all cases, while the 17P—acetate and formate group...

Studies on the mechanism of estrogen biosynthesis. 8. The development of inhibitors of the enzyme system in human placenta.

Studies on the mechanism of estrogen biosynthesis in the rat ovary--I.

Studies on the mechanism of estrogen biosynthesis. 3. The stereochemistry of aromatization of C19 and C18 steroids

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.

Harry J. Brodie

Papers

The effect of an aromatase inhibitor 4-hydroxy-4-androstene-3 17-dione on estrogen-dependent processes in reproduction and breast cancer.

Studies on the mechanism of estrogen biosynthesis. 8. The development of inhibitors of the enzyme system in human placenta.

Studies on the mechanism of estrogen biosynthesis in the rat ovary--I.

Studies on the mechanism of estrogen biosynthesis. 3. The stereochemistry of aromatization of C19 and C18 steroids

Aromatase inhibitors. Synthesis and biological activity of androstenedione derivatives