![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 26, Issue 11, 1102-1112, November 1998
Department of Toxicology and Drug Disposition (C.H.J.V., S.F.M.K., R.M.E.V.) and Analytical Chemistry for Development (G.N.W., C.J.B.), NV Organon, and Department of Pharmacokinetics and Drug Delivery, University Centre for Pharmacy, Groningen Institute for Drug Studies (P.O., G.M.M.G.)
 |
Abstract |
|---|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
The metabolism of Org 30659 [(17
)-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 contraceptive and hormone replacement therapy,
was studied in vivo after oral administration to rats and
monkeys and in vitro using rat, rabbit, monkey, and human
liver microsomes and rat and human hepatocytes. After oral
administration of [7-3H]Org 30659 to rats and
monkeys, Org 30659 was extensively metabolized in both species. Fecal
excretion appeared to be the main route of elimination. In rats,
opening of the A-ring, resulting in a 2-OH,4-carboxylic acid, 5-H
metabolite of Org 30659, was the major metabolic route in
vivo. Other metabolic routes involved the introduction of an OH
group at C15
, followed by a shift of the
15-double bond to a 16/17-double bond with
subsequent removal of the OH group at C17 and reduction of the
3-keto,4 moiety followed by sulfate
conjugation of the 3-OH substituent. These metabolic routes observed
in vivo were also major routes in incubations with rat
hepatocytes. In rat liver microsomes, Org 30659 was metabolized by
reduction of the 3-keto,4 moiety. Rat
hepatocyte incubations with Org 30659 were more representative of the
in vivo metabolism of Org 30659, compared with rat
microsomal incubations. Both in vitro and in
vivo, the majority of the metabolites were 3-OH,4,5-dihydro
derivatives. In monkeys, Org 30659 was mainly metabolized at the C3-
and C17-positions in vivo. The 3-keto moiety was reduced to
both 3-OH and 3-OH substituents. In addition to phase I
metabolites, glucuronic acid conjugates were observed in
vivo. In monkey liver microsomes, the 6-OH metabolite of Org 30659 was the major metabolite present. Similar to the monkey liver
microsomes, rabbit and human liver microsomes converted Org 30659 to
the 6-OH metabolite. This metabolite was also the major metabolite
in incubations with human hepatocytes.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Org 30659 [(17)-17-hydroxy-11-methylene-19-norpregna-4,15-dien-20-yn-3-one]
(fig. 1) is a new potent progestagen
currently under clinical development by NV Organon for use in oral
contraceptive and hormone replacement therapy. In pharmacological
studies in rats and rabbits, the progestational activity of Org 30659 (as determined in the ovulation inhibition test) was shown to be of the
same order as the activity of etonogestrel and was much higher than the
activities of norethisterone and levonorgestrel. In addition, a lack of
androgenic activity (according to the Hershberger test) distinguishes
Org 30659 from other progestagens. Except for some weak estrogenic
activity, Org 30659 is devoid of other hormonal activities, such as
glucocorticoid and antiglucocorticoid activity (Deckers et
al., 1992
).
|
The present investigation was performed to study the metabolism of Org 30659 in vivo and in vitro in several species. The metabolism of Org 30659 was studied in vivo in female rats and female cynomolgus monkeys, the species used in the preclinical safety studies. To study the in vivo metabolism of Org 30659, the rats were dosed with 10 mg/kg [3H]Org 30659 and the monkeys with 3 mg/kg [3H]Org 30659. In vitro studies were performed with rat, rabbit, monkey, and human liver microsomes and rat and human hepatocytes. Metabolites were isolated from microsomal incubations and urine and feces samples by HPLC. Identification of the metabolites was performed by NMR, MS, and IR spectroscopy.
In vitro metabolic routes observed in incubations with human liver microsomes and human hepatocytes were used to predict the in vivo metabolic routes of Org 30659 in humans. The in vitro-in vivo correlation obtained for rats and monkeys in this study was taken into account to strengthen these extrapolations.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Chemicals.
[7-3H]Org 30569 (radiochemical purity, 
99%;
specific radioactivity, 888 GBq/mmol) was prepared by the Organic
Synthesis Section of the Department of Process Chemistry of NV Organon
(Oss, The Netherlands). Unlabeled Org 30659 was synthesized by the
Department of Process Chemistry, NV Organon. All other chemicals were
obtained from local commercial sources and were of analytical grade.
In Vivo Studies in Rats and Monkeys. Animals. Female Wistar rats (approximately 185 g, HSD/CPB:WU) were obtained from Harlan CPB (Zeist, The Netherlands). Each rat received standard pelleted food (diet RMH-B; Hope Farms BV, Woerden, The Netherlands) ad libitum.
Female cynomolgus monkeys (2-4 kg) were obtained from Inveresk Research (Tranent, Scotland). Each monkey was offered 0.2 kg/day of a complete dry diet of known formulation (SDS Mazuri Diet; Special Diet Services, Witham, Essex). Tap water was available ad libitum from drinking bottles and was refreshed each day. During sampling of urine and feces, the rats and monkeys were housed individually in stainless steel metabolism cages in a room under standard conditions (temperature range, 17-24°C; relative humidity range, 35-70%).Animal Treatment and Sampling. Six female rats were treated with a single oral dose of 10 mg/kg (12.6 MBq/kg) [3H]Org 30659. Four female cynomolgus monkeys received unlabeled Org 30659 once daily for 14 days, at a dose level of 3 mg/kg. On day 15, [3H]Org 30659 (834 kBq/kg) was administered at the same dose level.
Urine and feces samples were collected in fractions up to 168 hr after dosing. Urine samples were collected in chilled containers. Rat blood samples were taken from the tail vein at 0.5, 1, 2, 4, 7, and 24 hr after administration of the radioactive dose. Cynomolgus monkey blood samples were taken from the femoral vein at 0 (before the dose), 0.5, 1, 1.5, 2, 3, 4, 6, and 8 hr after radioactivity administration. All samples were stored at
20°C until required for analysis.
In Vitro Incubation Studies with Liver Microsomes and Hepatocytes. Liver Tissue. Liver tissue was obtained from female Wistar rats (HSD/Cpb:WU; Harlan CPB), female cynomolgus monkeys (Sanofi Recherche, Montpellier, France), and female New Zealand White rabbits (HSD/Cpb:NZW; Broekman Instituut, Someren, The Netherlands). Human liver microsomes and hepatocytes (female donors) were prepared from surgical waste liver tissue, after consent from legal authorities and patients had been obtained, in cooperation with the Groningen Human Liver Group, University of Groningen, The Netherlands. Human livers 1, 2, and 3 were used for the preparation of microsomes and human livers 4 and 5 for the isolation of hepatocytes.
Preparation of Liver Microsomes.
Liver samples were homogenized using a Potter-Elvehjem homogenizer at
0°C in 50 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose.
Microsomes were prepared by centrifugation (20 min at 100,000 N/kg;
supernatant, 2 × 75 min at 1,000,000 N/kg). The microsomal pellet
was resuspended in potassium phosphate buffer (100 mM, pH 7.4)
containing 20% (v/v) glycerol. The microsomal suspensions were stored
at 80°C until used. The microsomal suspensions were characterized
by the determination of cytochrome P450 and protein levels. The
concentration of cytochrome P450 was calculated from the carbon
monoxide-reduced difference spectrum, according to the method of Omura
and Sato (1964). The concentration of protein was measured by the
method of Lowry et al. (1951), using bovine serum
albumin as the standard.
Analytical Incubations.
Analytical microsomal incubations with [3H]Org
30659 were performed for the analysis of metabolite profiles.
Microsomes (cytochrome P450 concentration, 0.25 µM) were incubated in
2 ml of potassium phosphate buffer (100 mM, pH 7.4) containing 3 mM
MgCl2, 5 mM glucose-6-phosphate, 0.5 mM
NADP+, 1.25 units/ml glucose-6-phosphate
dehydrogenase, and 10 µM (37 kBq/ml) [3H]Org
30659, at 37°C. After a preincubation period of 2 min at 37°C,
incubations were started by the addition of the test compound. Incubations were carried out in a shaking water bath under a gentle stream of 95% O2/5% CO2.
Incubations were stopped after 30 min by freezing (solid
CO2/ethanol, approximately 80°C). In
addition, incubations with [14C]testosterone
were performed and served as positive controls for the enzymatic
activity of the microsomal batches. These incubations were performed
singularly, as described above. All the samples were stored at 20°C
until required for analysis.
Preparative Incubations.
A preparative incubation was performed with
[3H]Org 30659, using female rabbit hepatic
microsomes, for the isolation and identification of metabolites.
Microsomes (cytochrome P450 concentration, 4 µM) were incubated in
100 ml of potassium phosphate buffer (100 mM, pH 7.4) containing 3 mM
MgCl2, 20 mM glucose-6-phosphate, 1 mM NADP+, 1.5 units/ml glucose-6-phosphate
dehydrogenase, and 199 µM (29.3 kBq/ml)
[3H]Org 30569, at 37°C. After a preincubation
period of 5 min, the incubations were started by the addition of the
test compound. Incubations were carried out under a gentle stream of
95% O2/5% CO2.
Incubations were stopped after 2 hr of incubation by freezing (solid
CO2/ethanol, approximately 80°C). All samples
were stored at 20°C until required for analysis.
Hepatocyte Isolation.
A female Wistar rat was anesthetized with Nembutal, an abdominal
midline incision was made, and the bile duct, vena porta, and thoracic
vena cava inferior were cannulated. Hepatocytes were further isolated
by collagenase perfusion (Braakman et al., 1989). Human
hepatocytes were isolated at the Department of Pharmacokinetics and
Drug Delivery, Groningen Utrecht Institute for Drug Exploration, University of Groningen, by collagenase perfusion of a liver section with a single cut surface (Olinga et al., 1998b).
Hepatocyte Incubations.
Hepatocytes were suspended in Krebs-Henseleit buffer, supplemented with
bovine serum albumin (pH 7.4), at a cell density of 3-4 × 106 cells/ml (total volume, 1 ml). After
preincubation for 15 min at 37°C in a shaking water bath under a
gentle stream of 95% O2/5% CO2, [3H]Org 30659 was
added at a final concentration of 215-580 kBq/ml (equivalent to
approximately 75-201 ng/ml). Hepatocytes were incubated with the test
compound for a total time period of 3 hr at 37°C. The hepatocyte
suspension was then centrifuged for 1 min in an Eppendorf centrifuge,
and the pellet (cells) and supernatant (cell medium) were separated.
Cell and cell medium samples were frozen at approximately 80°C
until required for analysis.
Sample Analysis. Determination of Radioactivity Concentrations. The concentrations of radioactivity in plasma, urine, and incubation samples were determined by liquid scintillation counting (Tri-Carb 2500 TR/2; Canberra Packard, Groningen, The Netherlands). The concentrations of radioactivity in feces were determined by combustion in a Sample Oxidizer 306 or 387 (Canberra Packard), followed by liquid scintillation counting. Feces samples were homogenized with Milli-Q water before combustion.
Determination of Metabolite Profiles. Pooled rat plasma was analyzed by HPLC without pretreatment. Pooled rat urine was dried in a Speed Vac concentrator (Dumee, The Netherlands); the residue was dissolved in Milli-Q water and used for HPLC analysis.
Monkey pooled plasma and urine samples were applied to C18 solid-phase extraction columns. Columns were washed twice with Milli-Q water and eluted with methanol. The methanol extracts were evaporated to dryness in a Speed Vac concentrator. Residues were dissolved in methanol or Milli-Q water/methanol for HPLC analysis.
|
|
|
|
|
|
|
|
|
|
HPLC Analysis of Metabolite Profiles. HPLC analysis of the plasma, urine, feces, and incubation samples was performed using a µ-Bondapak C18 column (7.8 × 300 mm) and a gradient of ammonium acetate buffer (0.1 M, pH 4.2) (solvent A) and methanol (solvent B). Elution was performed with a linear gradient of 10-90% (v/v) solvent B in 35 min, at 50°C. The flow rate was 2.5 ml/min.
HPLC analysis was performed with an HP1090 liquid chromatograph equipped with an HP1040 diode-array detector (Hewlett Packard, Walbron, Germany) and a Flo-one [B]eta model A525 on-line radioactivity detector (Canberra Packard). Samples were spiked with unlabeled Org 30659 as a reference for retention time (UV signal at 254 nm). Metabolite numbers were assigned on the basis of retention times. Metabolites from rat plasma, urine, and feces (R), monkey plasma, urine, and feces (C), microsomal incubations for all species (M), and rat and human hepatocyte incubations (H) were numbered independently.Isolation of Metabolites from Urine, Feces, and In Vitro Incubation Samples. Urine and Feces. Rat urine was applied to C18 solid-phase extraction columns. Monkey urine was concentrated in a Speed Vac concentrator and then applied to C18 solid-phase extraction columns. The columns were washed with Milli-Q water and eluted with methanol. The methanol effluents were concentrated by vacuum centrifugation and subjected to HPLC.
Feces homogenates from rats and monkeys were extracted with acetonitrile. The extracts were concentrated in a Speed Vac concentrator, diluted with Milli-Q water, and subsequently applied to activated C18 solid-phase columns. The columns were washed with Milli-Q water and eluted with methanol. The methanol effluents were dried, dissolved in methanol, and subjected to HPLC analysis. The chromatographic conditions for the urine and feces samples were as described in HPLC Analysis of Metabolite Profiles. The monkey feces samples were eluted with a linear gradient of 10-90% solvent B in 30 min, instead of 10-90% solvent B in 35 min. The effluent was collected in fractions. Fractions constituting a peak of radioactivity were pooled and dried in a Speed Vac concentrator or under a gentle stream of nitrogen. The residues isolated from monkey urine were desalted and further processed, as appropriate, for NMR and MS analysis. Residues from rat urine and feces and monkey feces were dissolved in methanol and/or Milli-Q water and then subjected to a second HPLC analysis using a µ-Bondapak phenyl column (3.9 × 300 mm) and a gradient of ammonium acetate buffer (0.1 M, pH 4.2) (solvent A) and methanol (solvent B). Elution was performed with a linear gradient of 5-60% (v/v) solvent B in 30 min, at 50°C. The flow rate was 1.5 ml/min. Elution for the feces samples from monkeys was performed with 5-80% methanol in 35 min. The HPLC effluent was collected in fractions at the approximate retention times of the eluting peaks of radioactivity. Fractions constituting a peak of radioactivity were pooled and then dried. The residues were further processed, as appropriate, for NMR and MS analysis and, if necessary, for IR analysis.Preparative Incubation Samples. The incubation mixture from the preparative microsomal incubation was applied to 6-ml Bakerbond SPE C18 solid-phase extraction columns. The columns were washed with Milli-Q water and eluted with methanol. The methanol effluents were concentrated by vacuum centrifugation, diluted with Milli-Q water, and again subjected to C18 solid-phase extraction. The methanol effluents were concentrated to an appropriate volume and subjected to HPLC. The chromatographic conditions used for HPLC analysis were as described in HPLC Analysis of Metabolite Profiles. The HPLC effluent was collected in fractions. Fractions constituting a peak of radioactivity were pooled and dried. Residues were taken up in methanol and further purified by a second HPLC separation, using the same chromatographic conditions as described for the isolation of metabolites from urine and feces (second HPLC analysis). The HPLC effluent was collected in fractions at the approximate retention times of the eluting peaks of radioactivity. Fractions constituting a peak of radioactivity were pooled and dried. Residues were further processed, as appropriate, for NMR and MS analysis and, if necessary, for IR analysis.
Identification of Metabolites. NMR Spectroscopy. The 1H spectra were recorded at 400 and 600 MHz with Bruker DRX400 and DRX600 instruments (Bruker Spectrospin AG, Fällanden, Switzerland), respectively, under standard conditions. The chemical shifts are given in parts per million. The samples were dissolved in deuteromethanol. The CHD2OD was used as a reference and was set to 3.30 ppm.
MS. Electron ionization spectra were recorded at 70-eV electron energy with a HP-5989 mass spectrometer (Hewlett Packard, Palo Alto, CA), using an HP59980B particle-beam interface. Sample introduction was performed by HPLC, using a Prodigy ODS column (5 µm, 2 × 150 mm) and a gradient of Milli-Q water (solvent A) and acetonitrile (solvent B). Isocratic elution was performed with 40% solvent B for 5 min, followed by a linear gradient of 40-100% (v/v) solvent B in 10 min. The flow rate was 0.4 ml/min. The ion source temperature was 250°C, and the desolvation chamber was maintained at 60°C. Particle-beam chemical ionization spectra were recorded using methane as the reagent gas, at 200-eV electron energy.
The metabolites were also analyzed by HPLC/ion-spray MS, using a LC-200 Quad pump (Perkin-Elmer Sciex) and a Perkin-Elmer ISS-200 autosam-pler. Ion-spray mass spectra were recorded in positive- and negative-ion modes, using the ion-spray interface of the PE-Sciex API-100 mass spectrometer (Perkin-Elmer). The PE-Sciex API-100 mass spectrometer was operated at positive and negative ion-spray voltages of approximately 5800 and4500 V, respectively, with a nebulizer
Org gas flow of air at 10 bar
(prepressure, approximately 4 bar) and a curtain gas flow of nitrogen
at 8 bar (prepressure, approximately 3 bar). The orifice was held at 25 and 25 V, respectively, whereas the ring was held at 275 and 275 V,
respectively. Mass spectra were recorded from approximately 200 to 1000 amu, with a step size of 0.1 amu and a dwell time of 0.35 msec.
Sample introduction was performed by HPLC, using a Prodigy ODS column
(5 µm, 2 × 150 mm) and a gradient of 5 mM ammonium acetate solution (solvent A) and methanol (solvent B). Isocratic elution was
performed with solvent B (10%) for 1 min, followed by a linear gradient of 10-90% (v/v) solvent B in 10 min. Isocratic elution was
then performed with solvent B (90%) for 9 min. The flow rate was 0.4 ml/min.
IR Spectrometry.
IR spectra were recorded using a Bio-Rad FTS-60 spectrometer, which was
continuously purged with nitrogen. DRIFT1 spectra were
obtained using the Barnes DRIFT accessory. The samples were dissolved
in a small amount of methanol and were transferred, using a syringe,
onto a DRIFT mirror. The spectra were recorded in Kubelka-Munk mode,
with a resolution of 2 cm
1 and 128 scans.
| |
Results |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
Excretion of Radioactivity in Urine and Feces from Rats and Monkeys. Data on the excretion of radioactivity after an oral dose of [3H]Org 30659 to rats and monkeys are given in table 1. The total excretion of radioactivity in the 0-168-hr period after a single oral administration of 10 mg/kg [3H]Org 30659 to female Wistar rats was 93.8 ± 3.2% (mean ± SD of individual samples, N = 3). The 0-168-hr excretion in the urine was 43.0 ± 2.3% and that in the feces was 50.8 ± 0.9%.
The total excretion of radioactivity (0-168 hr) after 2 weeks of daily oral doses of 3 mg/kg Org 30659, followed by a single oral dose of 3 mg/kg [3H]Org 30659, to female cynomolgus monkeys was 71.2 ± 14.2%, of which 27.3 ± 4.7% was excreted in the urine and 43.9 ± 16.5% in the feces. The total recovery of radioactivity in urine and feces from the monkeys was not quantitative. During the study, the feces from the monkeys were of a liquid nature, resulting in high cage wash values (range, 2.4-29.4%) (results not shown). The liquid nature of the feces was presumably the result of the stress caused by the dosing and handling of the animals.Metabolites of Org 30659 Present in Plasma, Urine, and Feces from
Rats and Monkeys.
Rat plasma metabolite profiles after a single oral dose of 10 mg/kg
[3H]Org 30659 contained nine compounds,
i.e. R1, R6, R8,
R10, R13-R16, and R18. The
metabolites R13 and R15, followed by
R8, were the major metabolites present in the plasma samples
at 0.5, 1, and 2 hr. On the basis of the retention times of the
metabolites isolated from urine and feces, the putative structures of
R13 and R14 were the A-ring-opened
2-OH,4-carboxylic acid,5-H metabolite of Org 30659 and the
A-ring-opened 2-OH,4-carboxylic acid,5-H,15-OH,16,17-ethinyl metabolite
of Org 30659, respectively (see below). The putative structure of
compound R15 was the 3-OSO3H,5-H metabolite of Org 30659, based on comparison with the retention time of
a metabolite isolated from the liver perfusate after circulatory perfusion of Org 30659 in rat liver (results not shown). The structures of the other compounds present in plasma remained unidentified. The
HPLC metabolite profile of rat plasma (at 0.5 hr, the time of the
maximal plasma concentration) is given in fig. 2A
Identification of the Metabolites Present in Urine and Feces from Rats and Monkeys. Analysis. The major compounds present in urine and feces samples from rats and monkeys were isolated and identified by NMR and MS analysis. In addition, IR analysis was performed for the identification of compound R13. Minor metabolites remained unidentified because the isolated amounts of metabolites were insufficient or the mixtures were too complex for NMR and MS analysis. The NMR and MS (and IR, if available) data for the metabolites isolated from urine and feces from rats and monkeys are summarized in table 3.
A-Ring-Opened 2-OH,4-Carboxylic Acid,5-H Metabolite of Org 30659 (R13).
The presence of a
C10-CH2-CH2OH moiety, the
absence of the 3-keto,4 moiety, and the
chemical shifts of the 4-H (2H) protons were indications that the A
ring was opened and a carboxylic acid group at C4 and an OH substituent
at C2 were formed. In addition, the IR spectrum showed the presence of
a carboxylic acid substituent.
A-Ring-Opened 2-OH,4-Carboxylic
Acid,5-H,15-OH,16,17-Ethinyl Metabolite of Org
30659 (R14).
The shift of the H15 signal, the change in the multiplicity of the H16
signal, and the shifts of the 17-ethinyl-H (which is now probably under
the moisture peak) and H18 signals were indications for
17-ethinyl-16 and 15-OH substituents. The
presence of a C10-CH2-CH2OH
moiety, the absence of the proton signals at C3, and the shifts of the H4 proton signals indicated that the A-ring was opened and a carboxylic acid group at C4 and a OH substituent at C2 were formed.
17-O-Glucuronide of Org 30659 (C12A).
Metabolite C12 consisted of two metabolites, C12A
and C12B. The presence of the anomeric proton at 4.99 ppm
together with the glucuronide signals at 3.28-3.62 ppm suggested a
glucuronic acid conjugate. All of the signals, compared with Org 30659, were present, so the glucuronic acid is probably conjugated at the
17-OH group.
O-Glucuronide of the 2-OH,3-OH,5-H
Metabolite of Org 30659 (O-Glucuronide at Either C2 or
C3) (C12B).
The presence of a broad multiplet at 3.85 ppm indicated a 2-OH
substituent. This signal was coupled with the narrow multiplet at 4.16 ppm. No signal was present at 5.87 ppm, which indicated the absence of
the 4 moiety Together with the glucuronide
signals between 3.20 and 3.57 ppm and the anomeric proton at 4.37 ppm,
the presence of a 3-glucuronide was indicated. The position of the
O-glucuronide could be at either C2 or C3; the exact
position could not be assigned on the basis of the NMR spectrum but was
most likely at C3.
3-O-Glucuronide of the 5-H Metabolite of Org
30659 (C14A).
Metabolite C14 consisted of two metabolites, C14A
and C14B. The presence of a broad multiplet at 3.77 ppm,
glucuronide signals between 3.15 and 3.69 ppm, and the anomeric proton
signal at 4.43 ppm indicated the presence of a 3-glucuronide. No
signal was present at 5.87 ppm, which indicated the absence of the
4 moiety.
3-O-Glucuronide of the 5-H Metabolite of Org
30659 (C14B).
The presence of a narrow multiplet at 4.07 ppm, the absence of the C4
protons, the presence of the glucuronide signals between 3.28 and 3.62 ppm, and the anomeric proton signal at 4.36 ppm suggested a
3-glucuronide. No signal was present at 5.87 ppm, which indicated
the absence of the 4 moiety.
3-OH,5-H Metabolite of Org 30659 (C17).
A broad multiplet at 3.57 ppm and the absence of the C4 protons
indicated a 3-OH,5-H substituent.
3-OH,5-H (C18A) and 3-OH,5-H
(C18B) Metabolites of Org 30659.
Metabolite C18 consisted of two metabolites, C18A
and C18B. Metabolites C18A and C18B
were very similar but showed differences in the shifts of the A-ring protons. The 3-H of metabolite C18A was present at 4.03 ppm and the 3-H of C18B was present at 3.55. The
differences in the shifts of the A-ring protons indicated that 5-H of
metabolite C18A was in the -position and 5-H of
metabolite C18B was in the -position.
Metabolites Present in Microsomal Incubations. [3H]Org 30659 was well metabolized by rat, rabbit, and monkey hepatic microsomes; after 30 min of incubation, 0, 29.1, and 45.5%, respectively, was recovered as the unchanged drug for these species. The extent of metabolism of [3H]Org 30659 by three different batches of human hepatic microsomes was variable; after 30 min of incubation, 11.3-87.6% was recovered as the unchanged drug. Microsomes from all species used showed good conversion with [14C]testosterone (results not shown).
At least 10 compounds (M1-M10), in addition to Org 30659, were observed in the HPLC radioactivity profiles. The compounds present in HPLC metabolite profiles from incubations with rat, rabbit, monkey, and human liver microsomes are listed in table 4. The metabolite profiles after incubations of Org 30659 with rat, rabbit, monkey, and human liver 2 microsomes are given in fig. 3. The major metabolite of Org 30659 from incubations with rat hepatic microsomes was M10. Metabolite M4 was the major metabolite from incubations with rabbit, monkey, and human hepatic microsomes. For the identity of the metabolites, see below.Identification of the Metabolites Present in Microsomal Incubations. Analysis. The NMR, MS, and IR (only M7) data for the metabolites isolated from microsomal incubations are summarized in table 5. Minor metabolites remained unidentified because the isolated amounts of metabolites were insufficient or the mixtures were too complex for NMR and MS analysis.
6-OH Metabolite of Org 30659 (M4).
The 4-H multiplicity and the presence of 10-H at 3.07 ppm indicated a
substituent at C6. A triplet at 4.35 ppm pointed to the presence of
a 6-OH substituent.
15,16-Epoxide Metabolite of Org 30659 (M7).
The signals for 15-H and 16-H were observed at 3.48 ppm, which
indicated the presence of a 15,16-epoxy group. In the
1H-NMR NOE difference spectrum, a NOE contact was
found between 15-H and 14-H. No NOE contact was observed between
18-methyl and 15-H and/or 16-H. This indicated the presence of a
15,16-epoxide moiety.
-OH,5-H metabolite of Org 30659 and/or the 5-H metabolite of
Org 30659, whereas the putative structure of metabolite M10 was the 3-OH,5-H metabolite of Org 30659.
Metabolites Present in Hepatocyte Incubations. The cell medium contained the major fraction (>82%) of radioactivity after 3 hr of incubation of [3H]Org 30659 with rat and human hepatocytes. At least 26 compounds (H1-H26), in addition to Org 30659, were observed in the HPLC radioactivity profiles.
Org 30659 was extensively metabolized by rat hepatocytes; no Org 30659 was present in the cell medium, and <2% of the integrated radioactivity in the cell medium eluted at the retention time of Org 30659. In human hepatocyte incubations, Org 30659 was reasonably well metabolized; approximately 37% (human liver 4) and 53% (human liver 5) was accounted for by Org 30659 in cell media from human hepatocyte incubations. H11, H15, and H17 were the major metabolites present in cell media from rat samples, and H13 and Org 30659 were the major compounds present in cell media from human liver 4 and human liver 5 samples. The metabolites present in incubation samples were identified by comparison with the retention times of metabolites isolated from urine and feces from rats, from the preparative incubation samples with rabbit liver microsomes, or from the liver perfusate after a circulatory perfusion of Org 30659 in rat liver (results not shown). The compounds present in HPLC metabolite profiles of cell media from incubations with rat and human hepatocytes and the putative structures are given in table 6.| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
In Vivo Metabolism of Org 30659 in Rats and Monkeys. Org 30659 was extensively metabolized after oral administration to rats and monkeys; no unchanged Org 30659 was observed in urine and feces samples from rats and monkeys. The feces appeared to be the main route of excretion.
In rats, opening of the A-ring was the major metabolic route for Org 30659. The A-ring-opened 2-OH,4 carboxylic acid,5-H metabolite of
Org 30659 was a major metabolite present in plasma (0.5 hr), urine, and
feces. A possible mechanism for the opening of the A-ring is
via Baeyer-Villiger oxidation. Baeyer-Villiger oxidation is
an important reaction in organic synthesis for the oxidation of ketones
to esters or lactones. In Baeyer-Villiger oxidation, a carbonyl
compound is oxidized by a peroxy compound in the presence of an acid
catalyst, in a manner involving insertion of an oxygen atom into one of
the carbon-carbon bonds at the carbonyl group (Carey and Sundberg,
1990). In the literature, Baeyer-Villiger oxidation reactions of
several substrates, such as camphor and cyclohexanone, have been
reported to be catalyzed by bacterial FMOs (Walsh and Chen,
1988; Fang et al., 1995; Hrycko et al., 1989;
Ryerson et al., 1982; Alphand et al., 1990;
Levitt et al., 1990). These enzymatic Baeyer-Villiger
reactions require a bound flavin cofactor in the presence of NADPH and
oxygen. The flavin 4-hydroperoxide intermediate reacts as a
nucleophile with the carbonyl group of the substrate (Walsh and Chen,
1988; Fang et al., 1995; Ryerson et al., 1982).
The cleavage of the side chain of progesterone to form androstenedione
is also assumed to occur via a Baeyer-Villiger
rearrangement. Cytochrome P450 17, a microsomal steroidogenic
cytochrome P450 enzyme located in the adrenal cortex, ovary, and
testis, is proposed to catalyze this side chain cleavage of
progesterone (Swinney and Mak, 1994).
Chen et al. (1995) purified FMO1 from pig liver microsomes.
Pig liver FMO1 catalyzes the Baeyer-Villiger oxidation of
salicylaldehyde to pyrocatechol. The mechanism of this
C-oxidation of salicylaldehyde is probably the same as the
mechanism of the Baeyer-Villiger oxidation catalyzed by bacterial
enzymes. Demonstrating the involvement of pig liver FMO1 in the
C-oxidation of salicylaldehyde, Chen et al.
(1995) suggested that the oxidation of ketones or aldehydes by
Baeyer-Villiger reactions catalyzed by FMO isoforms should be
considered as a possibility. By analogy with the mechanism of
Baeyer-Villiger oxidation reactions catalyzed by bacterial monooxygenases, a possible mechanism for the A-ring opening of Org
30659 is proposed in fig. 4.
In rats, Org 30659 was also metabolized at the C3-position. At the
C3-position, 3-OH was observed in combination with the reduction of
the 4 double bond to 5-H. Metabolites of
etonogestrel (structurally related to Org 30659) were also mainly
3-OH,5-H derivatives, so it seems that reduction of the 3-keto
moiety to a 3-OH group is preferred in rats. The 3-OH,5-H
metabolite was further metabolized to a
3-OSO3H,5-H metabolite of Org 30659. An
additional major metabolite present in rat urine showed introduction of
an OH group at C15 (15-OH), a shift of the
15-double bond to a 16/17-double bond, and
subsequent removal of the OH group at C17 (the
15-shifted metabolite of Org 30659).
In monkeys, Org 30659 was mainly metabolized at the C3- and the
C17-positions. The 3-keto moiety was reduced to 3-OH and 3-OH
groups. No specific preference for reduction of the 3-keto moiety to
3-OH or 3-OH was observed. The 3-OH metabolites were present in
combination with a 5-H group in most cases, but 5-H derivatives
were also observed.
In monkey urine, conjugates of 3-OH metabolites with glucuronic acid
were present. The formation of a 2-O-glucuronide
derivative has not been proven but cannot be excluded as a possibility,
because the position of the glucuronic acid in metabolite
C12B could be at either C2 or C3. Conjugation with
glucuronic acid at the 17-OH group was also observed, yielding a major
metabolite excreted in the urine.
The majority of the metabolites isolated from monkey urine and feces
were glucuronides, so it seems that glucuronidation is the predominant
form of steroid conjugation in cynomolgus monkeys. Guillemette et
al. (1996) studied the levels of glucuronidated and sulfated
steroids, e.g. androstane-3,17-diol glucuronide, in
plasma of humans, cynomolgus monkeys, and rats. High levels of
circulating glucuronidated steroids were found in plasma of cynomolgus
monkeys, whereas no glucuronic acid conjugates could be detected in
plasma of rats. The levels of glucuronidated steroids in human plasma
samples were 10-fold lower than those in cynomolgus monkey samples.
With respect to the sulfated steroids circulating in plasma, very low
levels were detected in plasma of cynomolgus monkeys, compared with humans.
In Vitro Metabolism of Org 30659.
In rat liver microsomes, Org 30659 was metabolized very rapidly by
reduction of the 3-keto,4 moiety to yield a
3-OH,4,5-dihydro derivative and a 3-OH,4,5-dihydro and/or
4,5-dihydro derivative. The reduction of the 3-keto moiety was
mainly to the 3-OH group, as was observed in vivo. In rat hepatocyte incubations, these 3-OH,5-dihydro derivatives and the
sulfate conjugates of these derivatives were minor metabolites.
4 moiety
of Org 30659 by 5-reductase and 3-HSD, as a result of differences
in the affinities of these enzymes (compared with FMO) for Org 30659 under the conditions used and/or differences in the levels of
expression of these enzymes in female liver microsomes. Further study
is clearly required to establish both the mechanism and the enzyme(s)
involved in the formation of an A-ring-opened metabolite of Org 30659 in rats.
In contrast to the microsomal incubations, hepatocyte incubations
showed the presence of the A-ring-opened metabolites of Org 30659. This
metabolite was also observed as a major metabolite in urine from rats.
An additional major metabolite present in rat hepatocyte incubations
was the A-ring-opened and 15-shifted
metabolite of Org 30659. All of the metabolic routes for Org 30659 observed in vitro were also observed in vivo,
with the exception of reduction of the 3-keto moiety to a 3-OH group (yielding a minor metabolite), which was observed in vitro
but not in vivo. However, the metabolism of Org 30659 in
rat hepatocyte incubations was a better reflection of the in
vivo metabolism of Org 30659 in rats, compared with the metabolism
of Org 30659 in rat microsomal incubations. The metabolic routes of Org
30659 found in rats are given in fig. 5.
In monkey liver microsomes, the 6-OH metabolite of Org 30659 was the
major metabolite present, which was unexpected, because the 6-OH
metabolite of Org 30659 was not observed in vivo. As can be
seen from the in vivo data, glucuronidation is a major metabolic route for Org 30659 in monkeys. Glucuronidated conjugates formed in the liver can be easily excreted in the bile or urine. Probably the glucuronidation of Org 30659 in monkeys is so fast that
hydroxylation at C6 in vivo does not take place to a major extent. In general, the metabolism of Org 30659 in monkey liver microsomes was not representative of the in vivo metabolism
of Org 30659 in monkeys. The metabolic routes of Org 30659 observed in
monkeys are shown in fig. 5.
Similar to the monkey liver microsomes, rabbit and human liver
microsomes mainly converted Org 30659 to the 6-OH metabolite of Org
30659. The 6-OH metabolite of Org 30659 was also the major metabolite after incubations with human hepatocytes. The
6-hydroxylation is a known metabolic route for steroids,
e.g. testosterone, androstenedione, and progesterone, in
human liver microsomes and is primarily catalyzed by cytochrome P450
3A4 (Waxman et al., 1988). In monkey and human hepatocytes,
6-hydroxylation was also observed as a major metabolic route for
testosterone (Olinga et al., 1998a; Kanter et
al., 1998).
In addition to the 6-OH metabolite of Org 30659, a 15,16-epoxy
metabolite of Org 30659 (M7) was observed after incubation with rabbit liver microsomes. The metabolic routes of Org 30659 in
rabbit liver microsomes are shown in fig. 5.
In human liver microsomes, 3-keto,4 reduction
of Org 30659 was not observed. Early studies investigating the 3-HSD
enzyme in human liver (Iyer et al., 1992) showed no activity
with 3-ketosteroids containing a 4 double
bond. However, the 3-HSD enzyme reduced 5-H and 5-H dihydro
derivatives at similar rates. The 4-reductase
responsible for the reduction of the 4/5-double bond is predominantly
found in the cytosolic fractions of human liver (Ward and Back, 1993).
Hence, the 3-keto,4-reduced metabolites of Org
30659 were not present in microsomes.
In human hepatocyte incubations, 3-OH,5-H derivatives and
possibly 3-OH,5-H derivatives were present as minor metabolites. Other minor metabolites of Org 30659 in human hepatocyte incubations were glucuronic acid and sulfate conjugates of the 3-OH,5-H derivatives. Also, the A-ring-opened metabolite of Org 30659 (H15) and the 15,16-epoxide metabolite of Org 30659 (H18) were observed as minor metabolites in human hepatocyte
incubations. The metabolism of Org 30659 in human liver microsomes and
human hepatocytes is shown in fig. 5.
Considering the in vitro-in vivo correlation of Org 30659 in
rats, the rat hepatocyte incubations with Org 30659 were more representative of the in vivo metabolism of Org 30659 in
rats than were rat microsomal incubations. In addition, the metabolism of Org 30659 in monkey liver microsomes was not representative of the
in vivo metabolism of Org 30659 in monkeys. The major
metabolic routes for Org 30659 in human microsomal incubations
(6-hydroxylation) and human hepatocyte incubations were similar.
Hence, extrapolating the in vitro metabolic routes observed
in incubations with human liver microsomes and human hepatocytes, it is
likely that in vivo hydroxylation at the C6-position
(6-OH) would occur.
In addition, there is the possibility of finding D-homo metabolites, in
which the five-membered D-ring is expanded to a six-membered D-ring, in
humans; Abdel Aziz and Williams (1970) isolated a D-homo metabolite
of ethinylestradiol from human urine after oral administration of
ethinylestradiol to humans. Düsterberg et al. (1987)
demonstrated the formation of a D-homo metabolite of gestodene (a
steroid structurally related to Org 30659).
Also, 3-keto,4-reduced metabolites can be
expected to be formed in vivo, because the hepatocyte
incubations showed the presence of 3-OH,5-H derivatives and
possibly 3-OH,5-H derivatives. In addition, major urinary
metabolites of gestodene (a steroid structurally related to Org 30659)
in humans were the tetrahydro-reduced metabolites (Ward and Back,
1993). Phase II metabolism is also likely to occur in vivo,
because sulfate and glucuronide conjugates of Org 30659 were
observed in incubations with human hepatocytes.
Various phase I reactions, such as carbon hydroxylations, opening of
the A-ring, and reduction of the 3-keto,4
moiety, are observed in the metabolism of Org 30659 in several species.
Cytochrome P450 is known to catalyze a large number of phase I
reactions. 3-Keto,4 reduction of Org 30659 may
be catalyzed by 5- and/or 5-reductase and 3- and/or 3-HSDs.
The identification of the enzymes responsible for the phase I
metabolism of Org 30659 will be an interesting subject for further investigations.
| |
Footnotes |
|---|
Received April 21, 1998; accepted June 22, 1998.
Send reprint requests to: C. H. J. Verhoeven, NV Organon, Department of Toxicology and Drug Disposition, P.O. Box 20, 5340 BH, Oss, The Netherlands. e-mail: c.h.verhoeven{at}organon.sck.akzonobel.nl
| |
Abbreviations |
|---|
Abbreviations used are: DRIFT, diffuse reflectance; HSD, hydroxysteroid dehydrogenase; FMO, flavin-containing monooxygenase; NOE, nuclear Overhauser effect.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
-ethinyl oestradiol by women.
Steroids
15:
695-710[Medline].-reduced C19 steroid glucuronides in primates, rodents, and domestic animals.
Am J Physiol Endocrinol Metab
34:
348-353.-hydroxysteroid dehydrogenase.
J Steroid Biochem Mol Biol
43:
343-349[Medline].-hydroxylase cytochrome P450 enzyme.
Arch Biochem Biophys
263:
424-436[Medline].
This article has been cited by other articles:
|
|
 |
|
  M. J. Blom, M. G. Wassink, F. van Wijk, A. G. H. Ederveen, H. J. Kloosterboer, C. H. J. Verhoeven, J. G. D. Lambert, and H. J. Th. Goos Metabolism of Norethisterone and Norethisterone Derivatives in Rat Uterus, Vagina, and Aorta Drug Metab. Dispos., July 1, 2001; 29(7): 976 - 982. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
