Dimethandrolone (7alpha,11beta-dimethyl-19-nortestosterone) and 11beta-methyl-19-nortestosterone are not converted to aromatic A-ring products in the presence of recombinant human aromatase

Abstract

Dimethandrolone undecanoate (DMAU: 7alpha,11beta-dimethyl-19-nortestosterone 17beta-undecanoate) is a potent orally active androgen in development for hormonal therapy in men. Cleavage of the 17beta-ester bond by esterases in vivo leads to liberation of the biologically active androgen, dimethandrolone (DMA), a 19-norandrogen. For hormone replacement in men, administration of C19 androgens such as testosterone (T) may lead to elevations in circulating levels of estrogens due to aromatization. As several reports have suggested that certain 19-norandrogens may serve as substrates for the aromatase enzyme and are converted to the corresponding aromatic A-ring products, it was important to investigate whether DMA, the related compound, 11beta-methyl-19-nortestosterone (11beta-MNT), also being tested for hormonal therapy in men, and other 19-norandrogens can be converted to aromatic A-ring products by human aromatase. The hypothetical aromatic A-ring product corresponding to each substrate was obtained by chemical synthesis. These estrogens bound with high affinity to purified recombinant human estrogen receptors (ER) alpha and beta in competitive binding assays (IC50's: 5-12 x 10(-9) M) and stimulated transcription of 3XERE-luciferase in T47Dco human breast cancer cells with a potency equal to or greater than that of estradiol (E2) (EC50's: 10(-12) to 10(-11) M). C19 androgens (T, 17alpha-methyltestosterone (17alpha-MT), androstenedione (AD), and 16alpha-hydroxyandrostenedione (16alpha-OHAD)), 19-norandrogens (DMA, 11beta-MNT, 19-nortestosterone (19-NT), and 7alpha-methyl-19-nortestosterone (MENT)) or the structurally similar 19-norprogestin, norethindrone (NET) were incubated at 50 microM with recombinant human aromatase for 10-180 min at 37 degrees C. The reactions were terminated by extraction with acetonitrile and centrifugation, and substrate and potential product were separated by HPLC. Retention times were monitored by UV absorption, and UV peaks were quantified using standard curves. Aromatization of the positive controls, T, AD, and 16alpha-OHAD was linear for 40-60 min, and conversion of T or AD was complete by 120 min. The nonsteroidal aromatase inhibitor, letrozole, demonstrated concentration-dependent suppression of T aromatization. Under the same conditions, there was no detectable conversion of DMA, 11beta-MNT, or NET to their respective hypothetical aromatic A-ring products during incubation times up to 180 min. Aromatization of MENT and 19-NT proceeded slowly and was limited. Collectively, these data support the notion that in the absence of the C19-methyl group, which is the site of attack by oxygen, aromatization of androgenic substrates proceeds slowly or not at all and that this reaction is impeded by the presence of a methyl group at the 11beta position.

Fig. 1

Structures of the C19 androgens and 19-norsteroids used in this study.

Fig. 2

Standard curves for quantification of (A) testosterone, (B) estradiol, (C) dimethandrolone, and (D) 7α,11β-dimethylestradiol by HPLC. Various volumes (10−50 μl) of solutions containing a mixture of T and E₂ standards (A and B) or DMA and 7α,11β-DME standards (C and D), each at 2 or 20 μg/ml, were injected (final amount injected 0.02−0.80 μg) into a Luna 5 μm C18(2) column and eluted isocratically (Table 1). AU's at 245 nm for the peaks of T at ∼35 min or DMA at ∼27 min (Table 1) and at 216 nm for the peaks of E₂ and 7α,11β-DME at 31−32 min were determined by the Waters Empower™ software and plotted vs. the amount of steroid applied to the column. Amounts (μg) of substrate and product present following incubation of T or DMA with SUPERSOMES™ for various times were calculated from the standard curves. Similar methodology was used to prepare standard curves for all other substrates and hypothetical aromatic A-ring products.

Fig. 3

Time course of conversion of testosterone to estradiol (A) or androstenedione to estrone (B) after incubation with GENTEST Human CYP19 + P450 SUPERSOMES™ and analysis by HPLC under the conditions specified in Table 1. The limit of detection was 20 ng.

Fig. 4

Effect of various concentrations of letrozole on conversion of testosterone to estradiol following incubation with SUPERSOMES™ for 60 min. The limit of detection was 20 ng.

Fig. 5

Amount of substrate (μg) in reaction mixtures after various times of incubation of dimethandrolone (A) or 11β-methyl-19-nortestosterone (B) with SUPERSOMES™ and analysis by HPLC under the conditions specified in Table 1. The limit of detection was 20 ng.

Fig. 6

Time course of conversion of MENT to 7α-methylestradiol (A) and of 19-nortestosterone to estradiol (B) after incubation with SUPERSOMES™ and analysis by HPLC under the conditions specified in Table 1. In (B) estradiol was undetectable (<20 ng) at 0, 10, 20, 40, and 60 min and equivalent to 20 ng at 120 and 180 min.