Endocrinological R&D Labs, Organon International . V., The Netherlands
METABOLITES OF LYNESTRENOL ACETATE IN THE BILE OF RATS AFTER INTRAVENOUS ADMINISTRATION; A COMPARISON WITH LYNESTRENOL
By A. Coert,
J. Geelen
and
J.
van
der Vies
ABSTRACT Some aspects of the metabolism of lynestrenol acetate, an orally active contraceptive compound, were studied in female rats. Lynestrenol acetate is stable in gastric and intestinal juice in vitro. After intravenous administration of lynestrenol acetate and lynestrenol with a 14C label in the nucleus approximately 40 % of the administered radioactivity was excreted in the bile within 90 min. After administration of lynestrenol acetate labelled in the ester group, 6% of the radioactivity was found in the bile. This means that the greater part of the lynestrenol acetate had lost its acetate group during the process of metabolism. There was an important difference between the autoradiograms of the thin layer patterns of post-hydrolysis extracts after administration of [4-14C]lynestrenol acetate and those after administration of [1\m='\-14C]lynestrenol acetate and [4-14C]lynestrenol: the major metabolite of [4-14C]\x=req-\ lynestrenol acetate did not appear on the autoradiograms of [1\m='\-14C]lynestrenol acetate and [4-14C] lynestrenol. This indicates that lynestrenol acetate was altered in the nucleus in the presence of the acetate group. The acetate group itself was removed, either when the alteration took place, or after it had been completed. The results of IR, NMR and mass spectrometry analysis indicate the introduction of a 15\g=a\hydroxyl group. Results of gas-liquid chromatography and thin layer chromatography indicate that a second important metabolite is 19-nor-17\g=a\-pregn-20-yne-3\g=a\, 17\g=b\-diol. The main conclusions are: 1. A part of the lynestrenol acetate is metabolized and excreted in the bile, the acetate group still being present. 2. Lynestrenol acetate is to some extent metabolized via another pathway than lynestrenol. This indicates that esterification of a steroid can lead to deviation from the metabolic pathway of the free original steroid.
Pharmacological investigations of lynestrenol acetate1) have shown that this compound has biological activities similar to those of lynestrenol2) which is used as a progestational component in oral contraceptives3) (Overbeek et al. 1962). Because it seemed very likely that the acetyl moiety of lynestrenol acetate would be readily split off in the body fluids and tissues to yield free lynestre¬ nol, the metabolic fate of the
ester in rats
was
studied.
MATERIALS AND METHODS Female Wistar rats,
body weight
supplied by TNO4). Thin layer Silicagel HF^^gg plates (Merck, Darm¬
180-200 g
were
chromatography (TLC) performed stadt, West-Germany). Silver florisil chromatography was performed according to the method of Ercoli et al. (1964), but with one modification: 0.1 ml of NH4OH (25 °/o) was used instead of the original 1 ml. A one g column was made in diethyl ether. AgN03-silica-gel plates containing 1 *Vo AgNOg (w/v) were prepared according to the normal procedure. Radioactivity was measured with a Packard Tricarb (3314) liquid scintillation spectro¬ meter. A toluene scintillator, containing 4 g PPO (2,5-diphenyloxazole) and 0.1 g dimethyl-POPOP (2,2'-p-phenyl-ene-bis(4-methyl-5-phenyloxazole)) per litre was used for counting organic solutions. Aqueous solutions were counted in a Bray scintillator (Bray 1960). Autoradiography was performed with Kodak Medical X-Ray film NS-2. Identifica¬ tion of metabolites was performed with the aid of IR-spectrometry using a Perkin Elmer spectrophotometer type 21, NMR analysis with a Varían A 60 D spectrometer MS 902 equipped with a spectrosystem 100 computer, mass spectrometry with an Mass Spectrometer and all the gas-liquid Chromatographie (GLC) analysis with a Hewlett Packard gas Chromatograph model F Se M 402. The following radiochemicals was
were
on
used5):
[4-14C] lynestrenol acetate, specific activity 48.1 /¿Ci/mg abbreviated to [4-14C]LA; [l'-14C] lynestrenol acetate, specific activity 42.4 pCi/mg, abbreviated to [l'-14C]LA; [4-14C] lynestrenol, specific activity 27.8 /
METABOLITES OF LYNESTRENOL ACETATE IN THE BILE OF RATS AFTER INTRAVENOUS ADMINISTRATION; A COMPARISON WITH LYNESTRENOL
By A. Coert,
J. Geelen
and
J.
van
der Vies
ABSTRACT Some aspects of the metabolism of lynestrenol acetate, an orally active contraceptive compound, were studied in female rats. Lynestrenol acetate is stable in gastric and intestinal juice in vitro. After intravenous administration of lynestrenol acetate and lynestrenol with a 14C label in the nucleus approximately 40 % of the administered radioactivity was excreted in the bile within 90 min. After administration of lynestrenol acetate labelled in the ester group, 6% of the radioactivity was found in the bile. This means that the greater part of the lynestrenol acetate had lost its acetate group during the process of metabolism. There was an important difference between the autoradiograms of the thin layer patterns of post-hydrolysis extracts after administration of [4-14C]lynestrenol acetate and those after administration of [1\m='\-14C]lynestrenol acetate and [4-14C]lynestrenol: the major metabolite of [4-14C]\x=req-\ lynestrenol acetate did not appear on the autoradiograms of [1\m='\-14C]lynestrenol acetate and [4-14C] lynestrenol. This indicates that lynestrenol acetate was altered in the nucleus in the presence of the acetate group. The acetate group itself was removed, either when the alteration took place, or after it had been completed. The results of IR, NMR and mass spectrometry analysis indicate the introduction of a 15\g=a\hydroxyl group. Results of gas-liquid chromatography and thin layer chromatography indicate that a second important metabolite is 19-nor-17\g=a\-pregn-20-yne-3\g=a\, 17\g=b\-diol. The main conclusions are: 1. A part of the lynestrenol acetate is metabolized and excreted in the bile, the acetate group still being present. 2. Lynestrenol acetate is to some extent metabolized via another pathway than lynestrenol. This indicates that esterification of a steroid can lead to deviation from the metabolic pathway of the free original steroid.
Pharmacological investigations of lynestrenol acetate1) have shown that this compound has biological activities similar to those of lynestrenol2) which is used as a progestational component in oral contraceptives3) (Overbeek et al. 1962). Because it seemed very likely that the acetyl moiety of lynestrenol acetate would be readily split off in the body fluids and tissues to yield free lynestre¬ nol, the metabolic fate of the
ester in rats
was
studied.
MATERIALS AND METHODS Female Wistar rats,
body weight
supplied by TNO4). Thin layer Silicagel HF^^gg plates (Merck, Darm¬
180-200 g
were
chromatography (TLC) performed stadt, West-Germany). Silver florisil chromatography was performed according to the method of Ercoli et al. (1964), but with one modification: 0.1 ml of NH4OH (25 °/o) was used instead of the original 1 ml. A one g column was made in diethyl ether. AgN03-silica-gel plates containing 1 *Vo AgNOg (w/v) were prepared according to the normal procedure. Radioactivity was measured with a Packard Tricarb (3314) liquid scintillation spectro¬ meter. A toluene scintillator, containing 4 g PPO (2,5-diphenyloxazole) and 0.1 g dimethyl-POPOP (2,2'-p-phenyl-ene-bis(4-methyl-5-phenyloxazole)) per litre was used for counting organic solutions. Aqueous solutions were counted in a Bray scintillator (Bray 1960). Autoradiography was performed with Kodak Medical X-Ray film NS-2. Identifica¬ tion of metabolites was performed with the aid of IR-spectrometry using a Perkin Elmer spectrophotometer type 21, NMR analysis with a Varían A 60 D spectrometer MS 902 equipped with a spectrosystem 100 computer, mass spectrometry with an Mass Spectrometer and all the gas-liquid Chromatographie (GLC) analysis with a Hewlett Packard gas Chromatograph model F Se M 402. The following radiochemicals was
were
on
used5):
[4-14C] lynestrenol acetate, specific activity 48.1 /¿Ci/mg abbreviated to [4-14C]LA; [l'-14C] lynestrenol acetate, specific activity 42.4 pCi/mg, abbreviated to [l'-14C]LA; [4-14C] lynestrenol, specific activity 27.8 /
