J. O'Neal Johnston

Biological characterization of 10-(2-propynyl) estr-4-ene-3,17-dione (MDL 18,962), an enzyme-activated inhibitor of aromatase

MDL 18,962 was shown to be a highly specific, potent (Ki = 3-4 nM), enzyme-activated inhibitor of aromatase with minimal intrinsic endocrine properties. The affinity of MDL 18,962 was higher for human and baboon placental aromatase than for rhesus placental or rodent ovarian aromatase. These species differences necessitated the development of a novel model of peripheral aromatase utilizing human enzyme. Human choriocarcinoma trophoblast xenografts in athymic nude mice were used for pharmacologic and pharmacokinetic evaluation of MDL 18,962. The ED50 for inhibition of aromatase activity in these trophoblast tumors at 6 h post-treatment was 1.4 mg/kg, s.c. and 3.0 mg/kg, oral. Preliminary results indicated that the ED50 for inhibition of peripheral aromatization of androgen by MDL 18,962 in female baboons was 0.01 mg/kg, i.v. and 4 mg/kg, oral.

Novel silylated steroids as aromatase inhibitors

Androst-4-en-3-one analogs incorporating a trimethylsilyl or a trimethylsilylmethyl group at C-1, C-2 or C-19 were prepared and evaluated as inhibitors of aromatase. Only 10-[1-hydroxy-2-(trimethylsilyl)ethyl]estr-4-ene-3,17-dione inhibited human placental aromatase. Enzyme kinetic analysis revealed competitive inhibition [apparent dissociation constant (Ki) of 562 +/- 12 nM] associated with marginal time-dependent inhibition.

Stereoselective inhibition of aromatase by novel epoxysteroids

The diastereoisomeric 10-(epoxyethyl)estr-4-ene-3,17-diones (2) and (3) have been synthesized, their conformations and configurations have been established by X-ray crystallographic analysis, and they have been shown to be powerful inhibitors of human placental aromatase.

Human trophoblast xenografts in athymic mice: a model for peripheral aromatization.

A novel procedure was developed for evaluating aromatase inhibitors using human enzyme in a rodent model. Human choriocarcinoma trophoblast (JAr line) cells injected subcutaneously into athymic nude mice develop into tumor xenografts in 7-14 days which represent sites for peripheral aromatization of androgens. The rapid growth of these trophoblast tumors is estrogen independent. The tumors provide a source of nonovarian human tissue which has relatively high levels of enzyme activity (248 +/- 12 pmol estrogen/g/h) for biochemical determination of in vivo aromatase inhibition. These are major advantages for pharmacological evaluations in comparison to the slow tumor growth response of most carcinogen-induced rodent mammary cancers, which are usually devoid of aromatase activity. In addition, the hormonal dependent components of rodent mammary tumors require several weeks to regress as a result of the indirect effects of estrogen deprivation on tumor growth via inhibition of prolactin dependency, a minor component relative to the role estrogen occupies in hormonally-dependent breast cancer in humans. This model of peripheral aromatization was utilized to evaluate in vivo pharmacological parameters of MDL 18,962 (10-(2-propynyl)estr-4-ene-3,17-dione) such as bioavailability of several formulations, time course and dose responses following different routes of drug administration, pharmacokinetics and tissue distribution of [14C]MDL 18,962. Tumor aromatase activities of trophoblast xenografts were significantly (P less than or equal to 0.05) inhibited when MDL 18,962 was administered intravenously, orally, subcutaneously, or via subcutaneous silastic implants. The ED50 of MDL 18,962 for tumor aromatase inhibition at 6 h after a single treatment was 1.4 mg/kg, s.c. and 3.0 mg/kg, orally. MDL 18,962 blocked aromatase activity more effectively in human trophoblast than in mouse ovarian tissue. Human trophoblast aromatase activity was inhibited by 70% following a single oral dose of 100 mg/kg of MDL 18,962, while the hosts ovarian aromatase activity exhibited only marginal inhibition. In vitro, the addition of 10 microM MDL 18,962 to trophoblast tumor cytosol or mouse ovarian cytosol resulted in 99.6 and 91.4% inhibition of aromatase activity, respectively. Tissue distribution of [14C]MDL 18,962 was predominantly associated with endocrine tissues with aromatase activity and organ systems involved in steroid metabolism and excretion. These in vivo data show that MDL 18,962 an enzyme-activated aromatase inhibitor, causes prolonged aromatase inhibition in the absence of saturating levels of inhibitor.

Degeneration of the Rat and Canine Adrenal Cortex Caused by α-(1,4-Dioxido-3-methylquinoxalin-2-yl)-N-methylnitrone (DMNM)

The antibacterial drug alpha-(1,4-dioxido-3-methylquinoxalin-2-yl) N-methylnitrone (DMNM) given at a dose of 22.5 mg/kg bid to four dogs for 14 days caused diminished adrenal cortical reserves as determined by decreased plasma cortisol (three dogs) and lower aldosterone levels (four dogs) following the intravenous infusion of ACTH. A dose of 100 mg/kg/day of DMNM administered to rats for 31 or 35 days resulted in significant decreases in blood glucose. Histologically, the adrenal glands of both species treated with DMNM for a maximum period of 21 days (dogs) and 35 days (rats) had widespread granular and vacuolar degeneration of the cortex. This degeneration in treated rats began in the zona reticularis and inner regions of the zona fasciculata and eventually involved the entire cortex including the zona glomerulosa. As a result of treatment, significant ultrastructural alterations within cells of the rat and canine adrenal cortex consisted of degeneration of the mitochondria and an increase in the numbers and lipolysis of lipid droplets. The ultrastructure of the zona reticularis and fasciculata was most severely affected.

Inhibition of hamster adrenal 11β19-hydroxylase activity

Abstract Increased mineralocorticoid activity has been associated with elevated urinary levels of 19-nordeoxycorticosterone in several forms of experimental and human hypertension. Biosynthesis of 19-norsteroids involves hydroxylation of the C-19 methyl group. We synthesized the 4-hydroxy analogs of deoxycorticosterone, deoxycorticosterone acetate, progesterone, and androstenedione and evaluated them as inhibitors of deoxycorticosterone llβll9-hydroxylase using hamster adrenal mitochondrial preparations. These 4-hydroxy analogs were inhibitors of this P 450 hydroxylase, with approximately 10 times weaker affinity than their respective natural substrates. 4-Hydroxydeoxycorticosterone was the most potent inhibitor evaluated in this study. The half-maximal inhibitory concentration of deoxycorticosterone hydroxylation was 5 μm, 15 μm, more than 50 μm, and 14 μM, respectively, for the above compounds. (Steroids 55 :378–382, 1990)

Comparison of the mineralocorticoid activity of 19-oxygenated and 19-nor derivatives of deoxycorticosterone.

19-Nordeoxycorticosterone (19-nor-DOC) is a mineralocorticoid with several unresolved physiologic questions. First, is 19-nor-DOC synthesized in the kidney from a circulating adrenocortical precursor (19-oicdeoxycorticosterone [19-oic-DOC] or 19-oxodeoxycorticosterone [19-oxo-DOC])? Second, does 19-nor-DOC, synthesized in the kidney, have mineralocorticoid activity or is it excreted in the urine without biologic activity? To answer this question, we administered two of the putative 19-nor-DOC precursors (19-oxo-DOC and 19-oic-DOC) to adrenalectomized rats and measured the formation of 19-nor-DOC and bioactivity as the urinary Na+ to K+ ratio. Each of the 10-microgram steroid treatments produced an elevation of urinary-free 19-nor-DOC (0 to 2 hours), whereas at the 1-micrograms dose only 19-oic-DOCA produced an increased UF 19-nor-DOC. None of the treatments led to an increase of conjugated 19-nor-DOC except 10 microgram 19-oic-DOCA. Increased mineralocorticoid activity (decreased urinary Na+ to K+ ratio) was produced by aldosterone, 1 and 10 micrograms 19-nor-DOC, and 10 micrograms 19-oic-DOCA over the same time period. An anti-mineralocorticoid effect (increased urinary Na+ to K+ ratio) was produced by 1 microgram 19-oxo-DOC. Urinary-free 19-nor-DOC, but not conjugated 19-nor-DOC, correlated with the urinary mineralocorticoid effect (decreased Na+ to K+ ratio). These data support the contention that 19-oic-DOC is the circulating 19-nor-DOC precursor and that, at least at the higher dose, it has a mineralocorticoid action on the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)