Alternative markers for Methylnortestosterone misuse in human urine.

Methylnortestosterone is a progestin and synthetic androgenic anabolic steroid, prohibited by WADA. Methylnortestosterone misuse is commonly detected by monitoring the parent compound and its main metabolites, 17α-methyl-5α-estrane-3α, 17ß-diol (M1) and 17α-methyl-5ß-estrane-3α, 17ß-diol (M2), in the glucuronide fraction. In the current study, a direct detection of methylnortestosterone sulfo-conjugated metabolites after ethyl acetate extraction and analysis by LC/Q/TOF-MS in negative ionization mode was performed, detecting two main sulfate metabolites (S1, S2). For the characterization of metabolites, samples from the excretion study, were additionally analyzed by GC-MS, after solvolysis and per TMS derivatization. RT and MS data collected, were compared with RT and MS data from metabolites of 17z-methyl-5α/ß-estrane-3α/ß, 17z-diols structures with prefixed stereochemistry at 3 and 5 positions, synthesized through Grignard reaction from 19-noretiocholanolone, 19-norandrosterone and 19-norepiandrosterone. Confirmed sulfate metabolites were S1, 17α-methyl-5α-estrane-3α, 17ß-diol 3α sulfate (detected up to 72 h) and S2, 17α-methyl-5ß-estrane-3α, 17ß-diol 3α sulfate (detected up to 192 h). Furthermore, applying targeted analysis based on RT and MS data of the synthesized metabolites two additional metabolites M3, 17ß-methyl-5ß-estrane-3α, 17α-diol and M4, 17ß-methyl-5α-estrane-3α, 17α-diol were detected in the glucuronide fraction and one more metabolite (S3) 17ß-methyl-5ß-estrane-3α, 17α-diol was detected in the sulfate fraction in lower abundance until the end of the excretion study (192 h). Interestingly, S2 could also be detected after the direct analysis of non-hydrolyzed steroid by GC-MS/MS as artifact, following normal ProcIV anabolic steroid procedure and using diethylether as extraction solvent.

Pharmacokinetics of altrenogest in horses.

The Federation Equestre Internationale has permitted the use of altrenogest in mares for the control of oestrus. However, altrenogest is also suspicious to misuse in competition horses for its potential anabolic effects and suppression of typical male behaviour, and thus is a controlled drug. To investigate the pharmacokinetics of altrenogest in horses we conducted an elimination study. Five oral doses of 44 mug/kg altrenogest were administered to 10 horses at a dose interval of 24 h. Following administration blood and urine samples were collected at appropriate intervals. Altrenogest concentrations were measured by liquid chromatography-tandem mass spectrometry. The plasma levels of altrenogest reached maximal concentrations of 23-75 ng/mL. Baseline values were achieved within 3 days after the final administration. Urine peak concentrations of total altrenogest ranged from 823 to 3895 ng/mL. Twelve days after the final administration concentrations were below the limit of detection (ca 2 ng/mL).

Excretion balance and metabolism of the progestagen Org 30659 in healthy postmenopausal women.

Metabolism of Org 30659 ((17alpha)-17-hydroxy-11-methylene-19-norpregna-4, 15-dien-20-yn-3-one), a new potent progestagen currently under clinical development by NV Organon for use in oral contraception and hormone replacement therapy, was studied in vivo after oral administration to healthy postmenopausal women. After oral administration of [14C]-Org 30659 to postmenopausal women, the compound was extensively metabolized. The dosed radioactivity was predominantly excreted via urine. Org 30659 was to a large extent metabolized at the C3- and the C17-positions. Phase II metabolism, and in particular conjugation with glucuronic acid at the 17beta-hydroxy group, is the major metabolic route for Org 30659 in vivo. Not only phase II metabolism was observed for Org 30659 after oral administration to postmenopausal volunteers, but also metabolism in the A-ring occurred, especially reduction of the 3-keto-Delta(4) moiety to give 3alpha-hydroxy, 5alpha(beta)-dihydro and 3beta-hydroxy, 5alpha-dihydro derivatives. Oxidative metabolism (6beta-hydroxylation) observed in human liver preparations in vitro, was not observed to a significant extent in vivo. So, in vitro human metabolism is different from the in vivo metabolism, indicating that the in vitro-in vivo extrapolation is far from straightforward, at least when only liver preparations are used. The proper choice of the in vitro system (e.g., microsomes, hepatocytes, slices or individually expressed enzymes) and the substrate concentration can be very important determinative factors for the predictability of the in vitro system for the in vivo situation. Species comparison of the metabolic routes of Org 30659 after oral administration indicated that the monkey seems to be a better representative species than the rat for the metabolism of Org 30659 in humans.

Pharmacokinetics of the progestin dienogest (STS 557) in rabbits.

Disposition and excretion of the progestin Dienogest (17 alpha-cyanomethyl-17 beta-hydroxy-estra-4,9-dien-3-one, STS 557) were investigated in female rabbits. Following single and repeated administration of the tritium-labelled compound the plasma concentration courses of total radioactivity (Dienogest + metabolites) and of the parent drug alone were estimated and also the urinary and fecal excretion of total radioactivity. From these data basic pharmacokinetic parameters were calculated. Additionally, the enterohepatic recirculation of biliary excreted metabolites was studied using bile of donors for oral administration to recipients. Following oral administration the high bioavailability of Dienogest which was already found in other animal species and in man could also be confirmed with rabbits. The parameters of Dienogest disposition do not differ significantly from those of the progestin levonorgestrel. Thus, the different effects of the both progestins in the McPhail-Clauberg assay in the rabbit cannot be attributed to differences in pharmacokinetics.

Studies on the biotransformation of the progestagen dienogest in the rabbit.

Following administration of 14 alpha, 15 alpha-3H-Dienogest (STS 557, 17 alpha-cyanomethyl-17 beta-hydroxy-4, 9-estradien-3-one) to female rabbits, extracts from urine, bile and plasma were separated by means of HPLC. Urinary and biliary metabolites are characterized by patterns of high complexity. From the mass spectra and UV absorption data of the urinary Dienogest metabolites a variety of biotransformation reactions has been derived like: hydroxylation in different positions of the Dienogest molecule, among these the 11-position; reduction of the 3-oxo group to 3-hydroxy; introduction of 2 and 4 hydrogen atoms; aromatization of ring A; transformation of 17 alpha-CH2CN to CH2OH, and formation of compounds with a 5(10), 9(11)-diene structure. Some of these reactions occur simultaneously resulting in a very complicated metabolite spectrum. Possibly the multiple effects of Dienogest established in animals are partially caused by metabolites.

Urinary metabolites of the new progestagen STS 557 (17 alpha-cyanomethyl-17-hydroxy-4,9-estradien-3-one) in the dog and rat.

Urinary metabolites of the new progestagen STS 557 (17 alpha-cyanomethyl-17-hydroxy-4,9-estradien-3-one) were isolated and characterized following oral administration of the 14 alpha, 15 alpha-tritium labelled compound to dogs and rats. 17 alpha-Cyanomethyl-11 beta, 17-dihydroxy-4,9-estradien-3-one (11 beta-OH-STS 557) and 17 alpha-cyanomethyl-1,3,5(10),9(11)-estratetraene-3,17-diol were identified by comparison with synthesized reference compounds. Mass spectra data indicate the following other pathways of STS 557 biotransformation: Hydroxylation in other positions than 11; hydrogenation; hydroxylation + hydrogenation; alteration of the 17 alpha-side chain.

Metabolism of a new synthetic progestagen, Org 2969, in female volunteers. The distribution and excretion of radioactivity after an oral dose of the labelled drug.

The metabolism of a new synthetic progestagen, Org 2969 was studied in 4 healthy female volunteers. During the first part of the study (Phase I), the volunteers ingested 50 microgram (about 0.1 mCi) of [16-3H5Org 2969 together with 50 microgram of ethinyloestradiol as a single dose. During the second part of the study (Phase II), a 10-day pre-treatment with the same dosage of non-radioactive compound preceded the administration of the radioactive steroid. A peak level of total radioactivity, representing 3.16-5.02% of the dose given/l of serum, was achieved within 2-3 h in Phase I. During Phase II, the corresponding figures were 4.54-5.13% after 1.5-3 h. The difference was mainly due to an increase of freely-extractable steroids during Phase II. The difference can at least partly be explained by assuming a change in the kinetics of the metabolism of Org 2969 by pre-treatment with Org 2969 and ethinyloestradiol. The mean recovery of radio activity in urine and faeces was 83.0%/48.1%/34.9% (total/urine/faeces) of the total dose in Phase I and 76.1%/45.2%/30.9% during Phase II. The differences in the total excretion and in the radioactivity excreted in the faeces were significant.

PIP: Org 2969, a newly synthesized progestin from the laboratory of Organon Int B.V., Oss, the Netherlands, was ingested by women volunteers in an oral, radioactive dose to learn more about the drug's metabolism in humans. 4 female volunteeers were studied. The study was conducted in 2 phases, the first had volunteers ingesting about .1 mCi (50 mcg) of tritiated Org 2969 along with 50 mg of ethinylestradiol (as a single dose), and the second phase was a 10-day treatment with the same (50 mcg) dose of Org 2969 without radiolabel followed by administration of tritiated Org 2969. In Phase 1, peak level of total radioactivity occurred within 2-3 hours; this peak represented 3.16-5.02% of the dose administered per liter of serum. The corresponding figures for Phase 2 were 1.5-3 hours and 4.54-5.13%. Phase 2 samples had increased levels of freely-extractable steroids compared with Phase 1, which explains the difference in measurements. Another aspect of the difference in metabolism of the 2 doses must result from a change in kinetics of the metabolism of Org 2969 by pretreatment with Org 2969 (Phase 2) or simultaneous administration with ethinylestradiol (Phase 1). 1 level was significantly different between Phase 1 and 2 and that was amount of drug excreted in feces. In Phase 1, mean recoveries of radioactivity in urine and feces were 83%/48.1%/34.9% (total/urine/feces) and for Phase 2 the same recoveries were 76.1%/45.2%/30.9%.