Tibolone is not converted by human aromatase to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE): analyses with sensitive bioassays for estrogens and androgens and with LC-MSMS

To exclude that aromatization plays a role in the estrogenic activity of tibolone, we studied the effect tibolone and metabolites on the aromatization of androstenedione and the aromatization of tibolone and its metabolites to 7alpha-methyl-17alpha-ethynylestradiol (7alpha-MEE) by human recombinant aromatase. Testosterone (T), 17alpha-methyltestosterone (MT), 19-nortestosterone (Nan), 7alpha-methyl-19-nortestosterone (MENT) and norethisterone (NET) were used as reference compounds. Sensitive in vitro bioassays with steroid receptors were used to monitor the generation of product and the reduction of substrate. LC-MSMS without derivatization was used for structural confirmation. A 10 times excess of tibolone and its metabolites did not inhibit the conversion of androstenedione to estrone by human recombinant aromatase as determined by estradiol receptor assay whereas T, MT, Nan, and MENT inhibited the conversion for 75, 53, 85 and 67%, respectively. Tibolone, 3alpha- and 3beta-hydroxytibolone were not converted by human aromatase whereas the estrogenic activity formed with the Delta4-isomer suggests a conversion rate of 0.2% after 120 min incubation. In contrast T, MT, Nan, and MENT were completely converted to their A-ring aromates within 15 min while NET could not be aromatized. Aromatization of T, MT, Nan and MENT was confirmed with LC-MSMS. Structure/function analysis indicated that the 17alpha-ethynyl-group prevents aromatization of (19-nor)steroids while 7alpha-methyl substitution had no effect. Our results with the sensitive estradiol receptor assays show that in contrast to reference compounds tibolone and its metabolites are not aromatized.     

Synthesis and aromatization of 19-norandrogens in the stallion testis

The results of the measurement of 19-nortestosterone in the testiscular artery and vein of the stallion, the very low levels of this steroid in the peripheral blood of geldings and the similar patterns of increase in the peripheral levels of 19-nortestosterone and testosterone after hCG stimulation, show that 19-nortestosterone, like testosterone, is essentially synthesized in the testis. This testicular origin was confirmed by the ability of testicular tissue to synthesize 19-norandrogens from [4-14C]androgens in vitro. 19-Nortestosterone was 50% conjugated in the peripheral blood and almost entirely conjugated after biosynthesis in vitro. The sequence of appearance of steroids in the peripheral blood after a single injection of 10,000 IU hCG suggests that, in the equine testis, 19-norandrogens are produced by a specific C10-19 desmolase (estrene synthetase), stimulable by hCG. 19-Nortestosterone was aromatized into estradiol-17 beta by stallion testicular microsomes. The affinity of the aromatase for 19-nortestosterone was very low compared to that for testosterone. At low and presumably physiological levels, and at a high testosterone/19-nortestosterone ratio, testosterone did not inhibit 19-nortestosterone aromatization by more than 53%. Thus, 19-nortestosterone may be aromatized in vivo in the testis in spite of the endogenous concentrations of androgens. However, the low velocity of 19-nortestosterone aromatization by testicular microsomes at roughly physiological concentrations suggests that 19-norandrogen aromatization may only participate slightly in the testicular estrogen production. These results suggest that in the equine testis, two aromatizing enzyme systems may exist: one which aromatizes both androgens and 19-norandrogens, and a minority system more specific for 19-norandrogens.     

Aromatization of 16alpha-hydroxyandrostenedione by human placental microsomes: effect of preincubation with suicide substrates of androstenedione aromatization

Estrogen synthase (aromatase) catalyzes the aromatization of androstenedione (AD) as well as 16alpha-hydroxyandrostenedione (16alpha-OHAD) leading to estrone and estriol, respectively. We found that several steroid analogs including 4-hydroxyandrostenedione (1), 6-oxoandrostenedione (6-oxoAD, 2) and its 19-hydroxy analog (3), 10beta-acetoxyestr-5-ene-7,17-dione (4), androst-5-ene-4,7,17-trione (5), and 17alpha-ethynyl-19-norteststerone (6), which are known suicide inactivators of AD aromatization, are not effective in inactivating 16alpha-OHAD aromatization in a time-dependent manner. The compounds were tested with the use of human placental microsomes and 1beta-tritiated-16alpha-OHAD as the substrate. The results of the tritium water method of 16alpha-OHAD aromatization was confirmed by the gas chromatography-mass spectrometry (GC-MS) method of estriol formation. The 1beta-tritiated-AD was used to measure AD aromatization as a positive control for these experiments. The compounds were tested at concentrations up to 40-fold higher than the K(i)'s determined for inhibition of AD aromatization. These studies suggest that differences exist in the binding site structures responsible for aromatization of 16alpha-OHAD and AD.     

Aromatization of testosterone and 19-nortestosterone by a single enzyme from equine testicular microsomes. Differences from human placental aromatase

A single enzyme in the stallion testis was able to aromatize both testosterone and nortestosterone. This enzyme had a much lower affinity for nortestosterone than for testosterone. In contrast to human placental estrogen synthetase, this enzyme aromatized testosterone and 19-nortestosterone with similar efficiency. The differences observed (effects of monovalent cations, inhibition of androstenedione aromatization by testosterone and 19-nortestosterone and, above all, rate of norandrogen aromatization) suggest that the aromatase in the horse testis is not the same as that in the human placenta.     

Equine testicular aromatase: substrates specificity and kinetic characteristics.

1. In the stallion, estrogens were synthesized and sulfated in vivo by the testis. 2. The equine testicular enzyme aromatized androgens and 19-norandrogens with similar velocity, but not 16 alpha-hydroxytestosterone or epitestosterone in contrast to the human placental aromatase. 3. One single enzyme was implicated in the aromatization of androstenedione, testosterone, 19-norandrostenedione and 19-nortestosterone by ETMES. 4. During the process of androstenedione aromatization by ETMES, 19-hydroxyandrostenedione and 19-oxoandrostenedione were released and 4-hydroxyandrostenedione was a competitive inhibitor causing an additional irreversible enzyme inactivation which is what occurs with HPMES. 5. Dihydrotestosterone was a potent competitive inhibitor of aromatase activity.     

7alpha-methyl-19-nortestosterone, a synthetic androgen with high potency: structure-activity comparisons with other androgens

CNNT. There was a good correlation between bioactivity and binding affinity to AR for the 7alpha-substituted androgens compared to T. In contrast, relative to their binding affinity to AR, the androgenic potency of DHT and 19-NT was lower compared to T. The reason for the lower in vivo androgenic activity of 19-NT is attributable to its enzymatic conversion to 5alpha-reduced-19-NT in the prostate. In the case of DHT, the lower bioactivity could be attributed to its faster metabolic clearance rate relative to T. The correlation was further investigated in vitro by co-transfection of rat ARcDNA expression plasmid and a reporter plasmid encoding the chloramphenicol acetyl transferase (CAT) gene driven by an androgen inducible promoter into CV-1 cells. All the androgens led to a dose-dependent increase in the CAT activity. MENT was found to be the most potent followed by DHT, 19-NT, T, and CNNT. The specificity of the androgenic response was confirmed by its inhibition with hydroxyflutamide, an antiandrogen. Thus, there was a good correlation between binding affinity and in vitro bioactivity in the transient transfection assay for the androgens. This suggests that the in vivo bioactivity of androgens could be influenced not only by binding affinity to receptors but also by factors such as absorption, binding to serum proteins and metabolism. However, the high potency of MENT is primarily related to its higher affinity to AR.     

Different patterns of metabolism determine the relative anabolic activity of 19-norandrogens

Testosterone, the principal androgen secreted by Leydig cells, exerts a wide range of actions including growth of the male reproductive tract (androgenic effects) and growth of non-reproductive tissues such as muscle, kidney, liver, and salivary gland (anabolic effects). As androgenic steroids were discovered some were found to have relatively more anabolic than androgenic activity. The results reviewed in this report suggest that these differences result, in part, from the differential metabolism of the steroids in individual tissues and the varied activities of the individual metabolites. In the accessory sex organs (e.g. the prostate) testosterone is 5-reduced to dihydrotestosterone (DHT) which, due to its higher affinity for androgen receptors (AR), amplifies the action of testosterone. In contrast, when 19-nortestosterone (NT) is 5-reduced, its affinity for AR decreases, resulting in a decrease in its androgenic potency. However, their anabolic potency remains unchanged since significant 5-reduction of the steroids does not occur in the muscle. 7-methyl-19-nortestosterone (MENT) does not get 5-reduced due to steric hindrance from the 7-methyl group. Therefore, the androgenic potency of MENT is not amplified as happens with testosterone. These metabolic differences are responsible for the increased anabolic activity of NT and MENT compared to testosterone. Part of the biological effects of testosterone are mediated by its aromatization to estrogens. The fact that MENT is also aromatized to 7-methyl estradiol, a potent estrogen, in vitro by human placental and rat ovarian aromatase suggests that some of the anabolic actions of MENT may be mediated by this estrogen.     

Hydroxylation of 19-norandrogens by porcine Leydig cells.

The metabolism of 19-norandrogens by porcine Leydig cells was investigated. Non-radioactive 19-norandrostenedione (19-Nor A) and [3H]19-nortestosterone (19-Nor T) were used as substrates in incubations with cell preparations from mature male pigs. Steroid products were separated by reversed-phase HPLC and material in selected peaks was rechromatographed before attempts to identify them by GC-MS. Both 11 beta- and 6 beta-hydroxylated derivatives of 19-Nor A were found and a third product (11-oxo-19-Nor A) was tentatively identified. The profile of radioactive metabolites from [3H]19-Nor T also favours the view of a capacity for hydroxylation of 19-norandrogens by porcine Leydig cells. The significance of these findings together with our earlier report of direct 11 beta-hydroxylation of C19 steroids by such cells remains to be examined.     

Androgen and 19-norandrogen aromatization by equine and human placental microsomes

The ability of equine and human placental microsomes to aromatize testosterone and 19-nortestosterone was studied. When 3 microM [1 beta,2 beta-3H]testosterone was used as substrate, the specific activity of equine placental microsomal aromatase was 2.5 times higher than that of the human microsomal enzyme. Although 19-nortestosterone was aromatized 67 times more rapidly by equine than by human aromatase, we found that equine aromatase exhibited a markedly weaker affinity for this substrate than did the human enzyme. Competitive inhibition of testosterone aromatization by 19-nortestosterone occurred with both equine and human aromatases. While having no effect on mare placental microsomes, Na+ and K+ (500 mM) stimulated testosterone aromatization by human placental microsomes by 73 and 52% respectively. If indeed a single enzyme is responsible for the aromatization of testosterone and 19-nortestosterone, which seems to be the case in both equine and human placental aromatase, our results show that differences in the structure of the active sites exist between equine and human aromatases.     

Synthesis of nitrile derivatives of estrogens

The first time synthesis of 7alpha- and 11beta-nitrile estradiol is described. Reaction of 7alpha-cyano-19-nortestosterone with copper(II)bromide in acetonitrile at room temperature results in aromatization of the A-ring. Treatment of 11beta-cyano-19-nortestosterone-17-one under similar condition does not induce A-ring aromatization but rather results in bromination at the 2beta-position. However A-ring aromatized products are obtained when the latter compound is treated with Ac2O-Py-AcOCl, NBS and HCl.     

Molecular and crystallographic structure of D-11-aza-19-nortestosterone

Crystal and molecular structure of D-11-aza-19-nortestosterone monohydrate C17H25NO2.H2O (a 8,288(2), b 12,433(2), c 7,570(2) A, beta 90,25(1) degrees; space group P2(1), R 8.3%) has been determined by X-ray analysis. Its comparison with the molecular structure of D-19-nortestosterone showed that the decrease in the hormonal activity upon 11-aza-substitution may be due to difference in chemical properties of imino and methylene groups.     

Radioimmunoassay of 7 alpha-methyl-19-nortestosterone and investigation of its pharmacokinetics in animals

A method for the measurement of 7 alpha-methyl-19-nortestosterone (7MENT) in serum/plasma by radioimmunoassay (RIA) is described. The antiserum, raised against 7 alpha-methyl-19-nortestosterone-3-O-oxime-bovine serum albumin, had a low titer (final dilution = 1:4500) and low affinity (Ka = 1.17 x 10(9) l/mol) but showed little or no cross-reactivity with several of the steroids tested. The sensitivity of the RIA was 28.2 pg/ml and the mean recovery of added cold steroid was 86 to 100%. Intra- and inter-assay coefficients of variation ranged from 4.3 to 7.3% and 7.3 to 8.4%, respectively. This RIA was used to follow plasma 7MENT levels after a single i.v. injection of the steroid in rats and rabbits. The metabolic clearance rates (MCR) of 7MENT as determined from the plasma disappearance curve for rats and rabbits were 50 l/day and 336 l/day, respectively. The MCR of 7MENT in rats and rabbits lies in the same range as for testosterone. When compared to other nortestosterone derivatives such as norethisterone, 7MENT is metabolized relatively faster.     

Distribution, metabolism and excretion of a synthetic androgen 7alpha-methyl-19-nortestosterone, a potential male-contraceptive

A synthetic androgen 7α-Methyl-19-nortestosterone (MENT) has a potential for therapeutic use in ‘androgen replacement therapy’ for hypogonadal men or as a hormonal male-contraceptive in normal men. Its tissue distribution, excretion and metabolic enzyme(s) have not been reported. Therefore, the present study tested the distribution and excretion of MENT in Sprague-Dawley rats castrated 24 h prior to the injection of tritium-labeled MENT (³H-MENT). Rats were euthanized at different time intervals after dosing, and the amount of radioactivity in various tissues/organs was measured following combustion in a Packard oxidizer. The radioactivity (% injected dose) was highest in the duodenal contents in the first 30 min of injection. Specific uptake of the steroid was observed in target tissues such as ventral prostate and seminal vesicles at 6 h, while in other tissues radioactivity equilibrated with blood. Liver and duodenum maintained high radioactivity throughout, as these organs were actively involved in the metabolism and excretion of most drugs. The excretion of ³H-MENT was investigated after subcutaneous injection of ³H-MENT into male rats housed in metabolic cages. Urine and feces were collected at different time intervals (up to 72 h) following injection. Results showed that the radioactivity was excreted via feces and urine in equal amounts by 30 h.Aiming to identify enzyme(s) involved in the MENT metabolism, we performed in vitro metabolism of ³H-MENT using rat and human liver microsomes, cytosol and recombinant cytochrome P₄₅₀ (CYP) isozymes. The metabolites were separated by thin-layer chromatography (TLC). Three putative metabolites (in accordance with the report of Agarwal and Monder [Agarwal AK, Monder C. In vitro metabolism of 7α-methyl-19-nortestosterone by rat liver, prostate, and epididymis. Endocrinology 1988;123:2187-93]), [i] 3-hydroxylated MENT by both rat and human liver cytosol; [ii] 16α-hydroxylated MENT (a polar metabolite) by both rat and human hepatic microsomes; and [iii] 7α-methyl-19-norandrostenedione (a non-polar metabolite) by human hepatic microsomes, were obtained. By employing chemical inhibitors and specific anti-CYP antibodies, ³H-MENT was found to be metabolized specifically by rat CYP 2C11 and 3-hydroxysteroid dehydrogenase (3-HSD) enzymes whereas in humans it was accomplished by CYP 3A4, 17β-hydroxysteroid dehydrogenase (17β-HSD) and 3-HSD enzymes.     

Development of a competitive enzyme immunoassay for 17α-19-nortestosterone

Because 17β-19-nortestosterone and its esters are frequently used anabolic steroids in cattle in Europe, there is a need for an assay that can also detect certain metabolites. The enzyme immunoassay described here involves the detection and quantitation of the major metabolite 17α-19-nortestosterone in urine. The assay is based on the coating of an antibody raised in a rabbit against 17α-19-nortestosterone-3-carboxy-methyloxime—bovine serum albumin (17α-19-NT-3-CMO-BSA), the competitive incubation of 17α-19-NT and the 17α-19-nortestosterone-3-CMO—horseradish peroxidase label, followed by the detection of the blue colour developed by the action of the enzyme on tetramethylbenzidine. The 3-CMO conjugate of 17α-19-nortestosterone was used to produce an antibody with selective affinity for the 17α-epimer. For the optimization of the assay, different coatings and incubation conditions were tested. The standard curve ranged between 0.98 and 4000 pg per well, with a B/B₀ 50% of ± 65 pg per well. Colour was measured with a microtitre plate reader. The method was validated by means of certified blank and spiked cattle urine samples.     

Influence of 19-nortestosterone and androgens on progesterone biosynthetic enzymes in early human placental explants

We recently showed that the production of progesterone (P4) in human placental explant culture from early gestation is enhanced by treatment with 19-nortestosterone (19-NT) or with certain androgens, namely androsen, namely androstenedione (A-dione), 5-androstane-3, 17β diol (3-diol) and 5-androstane-3β, 17β diol (3β-diol). This stimulation of P4 was explored further in this study. There was little metabolism of radioactive P4 when incubated for 24 h in the presence or absene of these steroids. The role of different steroids in the regulation of P450 cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD) was evaluated by measuring the conversion of P4 derived from unlabelled 25-hydroxycholesterol and from labelled pregnenolone, respectively. The results showed that 19-NT, A-dione and 3-diol stimulated (P450scc) activity; however, 3β-diol was ineffective. While 19-NT and 3β-diol enhanced the bioconversion of pregnenoloe to P4, A-dione and 3-diol were without effect.

The initial rapid stimulation of P4 by 19-NT within 2 h of incubation was not blocked by concurrent treatment with cycloheximide (CH). However, after incubation for 24 h, 70% of the 19-NT-stimulated P4 was abolished by CH. During the same incubation period,] P4 stimulation by A-dione, 3- and 3β-diol were completely blocked by treatment with CH. Thus our observations suggest that 19-NT-stimulated P4 accumulation is due to the combined effects on P450scc adn 3β-HSD enzyme activities. A-dioneand 3-diol increase biosynthesis of P4 by acting selectively on P450scc enzyme. However, the stimulatory action of 3β-diol on P4 is only at the level of 3β-HSD. Since CH blocks the stimulatory actions, the mechanism(s) by which androgens (A-dione, 3-diol and 3β-diol) and norandrogen (19-NT) augment the biosynthetic enzyme activities appears to be mediated by a process inhibited by CH. Since CH interference was absent during the initial rapid P4-stimulation by 19-NT, there may be a direct action of this steroid at the cellular level which is not dependent on new protein synthesis.     

Time-dependent aromatase inactivation by 4 beta,5 beta-epoxides of the natural substrate androstenedione and its 19-oxygenated analogs

Aromatase catalyzes the conversion of androgens to estrogens through three sequential oxygenations. To gain insight into the catalytic function of aromatase and its aromatization mechanism, we studied the inhibition of human placental aromatase by 4 beta,5 beta-epoxyandrostenedione (5) as well as its 19-hydroxy and 19-oxo derivatives (6 and 7, respectively), and we also examined the biochemical aromatization of these steroids. All of the epoxides were weak competitive inhibitors of aromatase with apparent K(i) values ranging from 5.0 microM to 30 microM. The 19-methyl and 19-oxo compounds 5 and 7 inactivated aromatase in a time-dependent manner with k(inact) of 0.048 and 0.110 min(-1), respectively, in the presence of NADPH. In the absence of NADPH, only the former inhibited aromatase with a k(inact) of 0.091 min(-1). However, 19-hydroxy steroid 6 did not cause irreversible inactivation either in the presence or absence of NADPH. Gas chromatography-mass spectrometric analysis of the metabolite produced by a 5-min incubation of the three epoxides with human placental microsomes in the presence of NADPH under air revealed that all three compounds were aromatized to produce estradiol with rates of 8.82, 0.51, and 1.62 pmol/min/mg protein for 5, 6, and 7, respectively. In each case, the aromatization was efficiently prevented by 19-hydroxyandrost-4-en-17-one, a potent aromatase inhibitor. On the basis of the aromatization and inactivation results, it seems likely that the two pathways, aromatization and inactivation, may proceed, in part, through a common intermediate, 19-oxo compound 7, although they may be principally different.     

Structure-activity relationships of 2alpha-substituted androstenedione analogs as aromatase inhibitors and their aromatization reactions

Aromatase catalyzes the conversion of androstenedione (1a, AD) to estrone through three sequential oxygenations of the 19-methyl group. To gain insight into the spatial nature of the AD binding (active) site of aromatase in relation to the catalytic function of the enzyme, we tested for the ability of 2alpha-substituted (halogeno, alkyl, hydroxy, and alkoxy) ADs (1b-1i) to inhibit aromatase in human placental microsomes as well as their ability to serve as a substrate for the enzyme. All of the steroids inhibited the enzyme in a competitive manner with the apparent K(i)'s ranging from 45 to 1150 nM. 2alpha-Halogeno (F, Cl, and Br) and 2alpha-alkyl (CH3 and CH2CH3) steroids 1b-1f were powerful to good inhibitors (Ki=45-171 nM) whereas steroids 1g-1i, having an oxygen function (hydroxy or alkoxy) at C-2alpha, were poor inhibitors (Ki=670-1150 nM). Aromatization of some of the steroids with placental microsomes was analyzed by gas chromatography-mass spectrometry, indicating that the aromatization rate of the bromide 1d was about two-fold that of the natural substrate AD and that of 2alpha-methoxide 1h was similar to that of AD. Kinetic analysis of the aromatization of androgens revealed that a good substrate was not essentially a good inhibitor for aromatase.     

Pharmacokinetics of 19-nortestosterone esters in normal men

A reliable method for the isolation of 19-nortestosterone (NT), testosterone (T) and dihydrotestosterone (DHT) by high-performance liquid chromatography (HPLC) and quantitation of the individual steroids by radioimmunoassays is described. The method was used to measure serum concentrations of NT, T and DHT in a pharmacokinetic study and in a clinical trial for male fertility control. Following intramuscular injection of either 50 mg 19-nortestosterone-3-(p-hexoxyphenyl)-propionate (NP) or 50 mg 19-nortestosterone-decanoate (ND) serum NT increased rapidly to maximal concentrations of 4.6 +/- 3.2 and 2.0 +/- 1.3 nmol/l (+/-SD), respectively, in the 6 volunteers. The half-life time was 8 days for ND and 21 days for NP. Based on these findings a clinical trial with NP was performed. NP was given to 5 healthy men in doses of 100 mg/week for the first 3 weeks followed by 200 mg/week for 10 further weeks. Serum NT levels increased gradually and maximal concentrations were reached in the 13th treatment week (20.2 +/- 3.4 nmol/l). Measurable amounts of NT were detectable for 19 weeks after the last injection. The study shows that NT accumulates under this treatment regime and wider spacing of the injection intervals may be possible in future trials.     

Biochemical aromatization of 2-methyleneandrostenedione: stereochemistry of hydrogen removal at the C-1 position

To explore a stereochemistry of hydrogen removal at C-1 of the powerful aromatase inhibitor 2-methyleneandrostenedione (1), of which the A-ring conformation is markedly different from that of the natural substrate androstenedione (AD), in the course of the aromatase-catalyzed A-ring aromatization producing 2-methylestrone (2), we synthesized [1-²H]labeled steroid 1 and its [1β-²H]stereoisomer, and the metabolic fate of the C-1 deuterium in aromatization was analyzed by gas chromatography–mass spectrometry (GC–MS) in each. Parallel experiments with the natural substrates [1-²H] and [1β-²H]ADs were also carried out. The GC–MS analysis indicated that 2-methyl estrogen 2 produced from [1-²H]labeled substrate 1 retained completely the 1-deuterium (1β-H elimination), while product 2 obtained from [1β-²H]isomer 1 lost completely the 1β-deuterium. Stereospecific 1β-hydrogen elimination was also observed in the parallel experiments with the labeled ADs as established previously. The results indicate that biochemical aromatization of the 2-methylene steroid 1 proceeds through the 1β-hydrogen removal concomitant with cleavage of the C₁₀–C₁₉ bond, yielding 1(10),4-dienone 9, in a similar manner to that involved in AD aromatization. This would give additional evidence for the stereomechanisms for the last step of aromatization of AD, requiring the stereospecific 1β-hydrogen abstraction and cleavage of the C₁₀–C₁₉ bond, and for the enolization of a carbonyl group at C-3 in the A-ring aromatization.