Identification of the human cytochrome P450 enzymes involved in the in vitro biotransformation of lynestrenol and norethindrone

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Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 56–66

Identification of the human cytochrome P450 enzymes involved in the in vitro biotransformation of lynestrenol and norethindrone夽 Tuomas Korhonen a,b,c,∗ , Miia Turpeinen d,e,2 , Ari Tolonen d,e,f,3 , Kari Laine b,c,1 , Olavi Pelkonen d,2 a

Department of Pharmacology, Drug Development and Therapeutics, University of Turku, It¨ainen Pitk¨akatu 4B, FIN-20520 Turku, Finland b Clinical Pharmacology, TYKSLAB, Health Care District of Southwest Finland, Finland c Medbase Ltd., Turku, Finland d Department of Pharmacology and Toxicology, University of Oulu, PO Box 5000, FIN-90014 Oulu, Finland e Novamass Analytical Ltd., Oulu, Finland f Department of Chemistry, University of Oulu, Oulu, Finland Received 2 November 2006; accepted 13 September 2007

Abstract This study examined the cytochrome P450 (CYP) enzyme selectivity of in vitro bioactivation of lynestrenol to norethindrone and the further metabolism of norethindrone. Screening with well-established chemical inhibitors showed that the formation of norethindrone was potently inhibited by CYP3A4 inhibitor ketoconazole (IC50 = 0.02 ␮M) and with CYP2C9 inhibitor sulphaphenazole (IC50 = 2.13 ␮M); the further biotransformation of norethindrone was strongly inhibited by ketoconazole (IC50 = 0.09 ␮M). Fluconazole modestly inhibited both lynestrenol bioactivation and norethindrone biotransformation. Lynestrenol bioactivation was mainly catalysed by recombinant human CYP2C9, CYP2C19 and CYP3A4; rCYP3A4 was responsible for the hydroxylation of norethindrone. A significant correlation was observed between norethindrone formation and tolbutamide hydroxylation, a CYP2C9-selective activity (r = 0.63; p = 0.01). Norethindrone hydroxylation correlated significantly with model reactions of CYP2C19 and CYP3A4. The greatest immunoinhibition of lynestrenol bioactivation was seen in incubations with CYP2C-Ab. The CYP3A4-Ab reduced norethindrone hydroxylation by 96%. Both lynestrenol and norethindrone were weak inhibitors of CYP2C9 (IC50 of 32 ␮M and 46 ␮M for tolbutamide hydroxylation, respectively). In conclusion, CYP2C9, CYP2C19 and CYP3A4 are the primary cytochromes in the bioactivation of lynestrenol in vitro, while CYP3A4 catalyses the further metabolism of norethindrone. © 2008 Elsevier Ltd. All rights reserved. Keywords: Lynestrenol; Norethindrone; Cytochrome P450; Metabolism; Prodrug

1. Introduction

Abbreviations: AUC, area under the plasma concentration–time curve; CYP, cytochrome P450. 夽 Finnish Technological Research Agency (TEKES) through DRUG2000 programme; Turku University Hospital Grant EVO13990. ∗ Corresponding author at: Department of Pharmacology, Drug Development and Therapeutics, University of Turku, It¨ainen Pitk¨akatu 4B, FIN-20520 Turku, Finland. Tel.: +358 2 3337513; fax: +358 2 3337516. E-mail addresses: [email protected] (T. Korhonen), [email protected] (M. Turpeinen), [email protected] (A. Tolonen), [email protected] (K. Laine), [email protected] (O. Pelkonen). 1 Tel.: +358 10 3976300. 2 Tel.: +358 8 5375231. 3 Tel.: +358 20 7639666. 0960-0760/$ – see front matter © 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jsbmb.2007.09.025

Lynestrenol (19-norpregn-4-en-20-yn-17-ol,[17␣]-; C20 H28 O) (Fig. 1) and norethindrone (19-norpregn-4-en-20-yn-3one,17-hydroxy-,[17␣-]-; C20 H26 O2 ) (Fig. 1) are progestins classified as 19-nortestosterone derivates [1]. They have been used widely for decades in oral contraceptives (OC) particularly in combination preparations with ethinylestradiol and in progestin-only-pills. In addition, both lynestrenol and norethindrone monotherapy have been indicated for treatment of endometriosis and menstrual disturbances. Norethindrone has also been used for treatment of hormone replacement therapy (HRT) in post-menopausal women with intact uterus in combination with estrogen. Lynestrenol is a prodrug, which is converted to norethindrone both in vitro [2] and in vivo [3,4]. Parent drug lacks progesto-

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Fig. 1. The proposed metabolic pathway of lynestrenol bioactivation to norethindrone and further norethindrone metabolism in vitro. The percentage of norethindrone production from lynestrenol is derived from the incubations in human liver microsomes performed in the identification phase of lynestrenol metabolites. Norethindrone metabolite (M1–M3) production represents the results of identification phase of norethindrone metabolites. In the identification phase both substrates at the concentration of 100 ␮M were incubated for 20 min with human liver microsomal protein at 37 ◦ C. Product formations were linear with respect to incubation time, protein content and substrate concentration under assay conditions described in the text.

genic activity thus requiring bioactivation to norethindrone in order to exert therapeutic effect [5]. Several pharmacokinetic studies have confirmed the biotransformation of lynestrenol to norethindrone in vivo and showed that this reaction is rapid and almost complete after oral administration of lynestrenol [4,6,7]. Although the pharmacokinetics of both lynestrenol and norethindrone have been intensively studied in humans [8–11], the involvement of cytochrome P450 (CYP) enzymes in the metabolism of lynestrenol and norethindrone have never been formally characterized. However, there is indirect evidence that CYP isoforms may contribute in the disposition of norethindrone. Back et al. [12] showed that the potent CYP enzyme inducer rifampicin caused a significant, approximately 42% reduction in the AUC of norethindrone. These results were more recently confirmed by Barditch-Crovo et al. [13]. In addition, both carbamazepine and St. John’s wort were found to cause an increase in the norethindrone clearance, which suggests the involvement of CYP3A isoforms in the norethindrone disposition [14,15]. Furthermore, recently a known CYP2C9 and CYP3A4 inducer bosentan has been shown to decrease norethindrone exposure by 23%, while an individual subject showed a 56% decrease in norethindrone AUC [16]. Given the wide-spread use of lynestrenol and norethindrone, an understanding of the bioactivation of lynestrenol and metabolism of norethindrone is imperative to anticipate and avoid drug interactions, which may impair the therapeutic response to lynestrenol or norethindrone or to cause increased risk for concentration-dependent adverse effects due to norethindrone accumulation.

The purpose of the present investigation was to evaluate the role of specific cytochrome enzymes in the bioactivation of lynestrenol to norethindrone and to determine the metabolic pathways of further norethindrone metabolism in vitro using human liver microsomes with chemical inhibitors as well as baculovirus–insect cells expressed human CYP isoforms, inhibitory antibodies raised against specific CYP isoforms and correlation with probe substrate activities. 2. Experimental procedures 2.1. Materials Lynestrenol and norethindrone were generous donations from N.V. Organon (Oss, The Netherlands) and Schering AG (Berlin, Germany); bupropion and hydroxybupropion from Glaxo SmithKline (Research Triangle, NC); midazolam and 1 -hydroxymidazolam from F. Hoffmann-La Roche (Basel, Switzerland) and omeparazole, omeprazole sulphone and 5-hydroxyomeprazole from Astra Zeneca (M¨olndal, Sweden). The metabolite standards dextrometorphan, 6-hydroxy-chlorzoxazone, hydroxytolbutamide and 6␤-hydroxytestosterone were purchased from Ultrafine Chemical Company (Manchester, UK). Formic acid, Lichro-Solv GG methanol and acetonitrile were obtained from Merck KGaA (Darmstadt, Germany). All other chemicals used were from Sigma Chemical Company (St. Louis, MO) and were of the highest purity available. Water was in-house freshly prepared with Simplicity 185 (Millipore S.A., Molsheim, France)

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water purification system and was UP grade (ultra pure, 18.2 M).

2.2. Human liver microsomes, cDNA expressed human P450s, and anti-CYP antibodies Human liver samples used in this study were obtained from the University Hospital of Oulu as a surplus from kidney transplantation donors. The collection of surplus tissue was approved by the Ethics Committee of the Medical Faculty of the University of Oulu, Finland. The demographic data of the kidney transplantation doners from whom the liver tissue was obtained is given in Table 1. The livers were transferred to ice immediately after the surgical excision, cut into pieces, snap-frozen in liquid nitrogen and stored at −80 ◦ C until the microsomes were prepared by standard differential ultracentrifugation [17]. A weight-balanced microsomal pool of 15 liver microsomal preparations which have been extensively characterized (sufficient model activities, no CYP2D6 or CYP2C19 poor metabolizers as measured by appropriate probe activities, expected effects of model inhibitors, and quantification of the amount of CYP protein by Western blotting) was employed for the primary screening. The final microsomal pellet was suspended in 0.1 M phosphate buffer pH 7.4. Accurate protein content was determined by the method of Bradford [18]. Baculovirus-expressed human CYPs (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5) co-expressing human P450 reductase (SUPERSOMESTM ) and anti-CYP antibodies for immunoinhibition (anti-CYP1A2, 2A6, 2B6, 2C, 2D6, 2E1 and 3A4) were purchased from BD Biosciences Discovery Labware (Bedford, MA). Anti-CYP2C-Ab show strong inhibitory effect on CYP2C8, CYP2C9 and CYP2C19. Anti-CYP3A4-Ab strongly inhibits CYP3A5 in addition to CYP3A4. Other monoclonal antibodies inhibitory to CYP isoforms are highly specific for target enzyme isoform only, and show no inhibitory effect on other CYP isoforms according to manufacturer.

2.3. Incubations using human liver microsomes Incubation mixtures contained 0.5 mg microsomal protein/ml, 0.1 M phosphate buffer (pH 7.4), 1 mM NADPH, a model inhibitor (in five different concentrations) and a substrate. Final substrate concentration in the incubations was 30 ␮M for the both substrates lynestrenol and norethindrone. In metabolite identification phase the final substrate concentration was 100 ␮M for both substrates and no model inhibitors were present. The first dissolution of substrates was done in methanol. The final amount of primary solvents in the incubation mixture was under 0.5% (v/v), which is expected to a have minimal effect on the studied CYP activities. Reaction mixture, in a final volume of 200 ␮l, was preincubated for 2 min at 37 ◦ C in a shaking incubator block (Eppendorf Thermomixer 5436, Hamburg, Germany) before reaction was initiated by addition of NADPH. Each reaction was determined after 20 min by adding 100 ␮l of ice-cold acetonitrile. Samples were cooled in an ice bath to precipitate the proteins. Cooled samples were stored at −20 ◦ C until analysed. Product formations were linear with respect to incubation time, protein content and substrate concentration under assay conditions described above. Experiments with anti-CYP antibodies for immunoinhibition were carried out according to manufacturer’s instructions. Shortly, 10 ␮l of antibody was added to microsomes (0.5 mg protein/ml), mixed gently and incubated for 15 min (20 min for anti-CYP1A2) on ice. After that, 0.1 M phosphate buffer (pH 7.4), lynestrenol or norethindrone and 1 mM NADPH were added and incubations were carried out as described above. As a control, comparable incubations with 25 mM Tris buffer, pH 7.5 (goat nonimmune serum for anti-CYP2C) instead of antibody were carried out. 2.4. Incubations using cDNA expressed human P450s For measuring the metabolites formation of lynestrenol and norethindrone in human recombinant expressed CYP enzymes

Table 1 The demographic data of the kidney transplantation doners from whom the liver tissue for microsomal preparations were obtained as surplus tissue Liver

Age

Sex

Cause of death

Drug history

Liver pathology

HL15 HL16 HL20 HL21 HL22 HL23 HL24 HL25 HL26 HL27 HL28 HL29 HL30 HL31 HL32

NA NA 54 44 40 43 47 33 70 52 21 39 53 44 62

M F M M F M M M F M M F F F M

Gun shot ICH ICH ICH ICH ICH ICH Astrocytoma Melanoma NA Stroke ICH, SAH ICH, SAH ICH, SAH ICH, SDH

No medication No medication Diazepama Phenytoina , alcohol abuse Dexamethasonea , nizatidinea , phenytoina Diazepama , smoker No medication, smoker Not known Not known Not known Dexamethasonea , smoker Dexamethasonea No medication No medication Metformin, alcohol abuse, smoker

None None None Cirrothic None None None None Steatosis Steatosis None None Steatosis Steatosis None

a Drugs were administrated only during the last 24 h before death; M, male; F, female; ICH, intracerebral haemorrhage; SAH, subarachnoidal haemorrhage; SDH, subdural haematoma; NA, not available.

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the standard incubation mixture (200 ␮l) contained 0.1 M phosphate buffer (pH 7.4), 1 mM NADPH, 30 ␮M substrate and recombinant expressed CYP enzymes (50 pmol CYP/ml). Incubations were carried out according to manufacturer’s instructions. Shortly, the reaction was started by adding recombinant enzymes into the preincubated reaction mixture (2 min at +37 ◦ C), mixed gently and incubated for 30 min at +37 ◦ C in incubator block without agitating the reaction. Otherwise the incubation protocol was similar to microsomal incubations. 2.5. In vitro inhibition of lynestrenol and norethindrone metabolism and reference inhibitors For determination of the IC50 values, the CYP-selective reference inhibitors were added into the incubation mixture in five different concentrations (0.01, 0.1, 1, 10, 100 ␮M) in a small volume of an appropriate solvent. Reference inhibitors used were fluvoxamine (CYP1A2), tranylcypromine (CYP2A6), ticlopidine (CYP2B6), quercetin (CYP2C8), sulphaphenazole (CYP2C9), fluconazole (CYP2C19), quinidine (CYP2D6), pyridine (CYP2E1) and ketoconazole (CYP3A4) [19–21]. The solvents used were water (fluvoxamine, tranylcypromine, ticlopidine, pyridine and quinidine), ethanol (quercetin), and acetonitrile (sulphaphenazole, fluconazole, ketoconazole). The IC50 values for inhibitors were determined graphically by a linear regression analysis of the logarithmic plot of inhibitor concentration versus percentage of activity remaining after inhibition using Microcal Origin, version 6.0 (Microcal Software Inc., Northapton, MA). All data points represent the mean of two incubations. The methods have been earlier validated in the laboratory: intraassay coefficient of variation has been <5% (depends on a specific assay) and day-to-day variation <10%. The enzyme activities in the presence of inhibitors were compared with control incubations (incubations with the solvent but without an inhibitor). 2.6. Inhibition of CYP enzymes by lynestrenol and norethindrone Conduction of the n-in-one assay has been previously described in detail [17]. Shortly, each incubation mixture contained 0.5 mg microsomal protein/ml, 0.1 M phosphate buffer (pH 7.4), 1 mM NADPH and all the ten probe substrates. Substrates and their concentrations for the incubations were: melatonin (4 ␮M), coumarin (2 ␮M), bupropion (1 ␮M), amodiaquine (2 ␮M), tolbutamide (4 ␮M), omeprazole (2 ␮M), dextromethorphan (0.2 ␮M), chlorzoxazone (6 ␮M), midazolam (0.4 ␮M) and testosterone (1 ␮M). Lynestrenol and norethindrone (separately) were added in five different concentrations (0.01, 0.1, 1, 10, 100 ␮M). Reaction mixture, in a final volume of 200 ␮l, was preincubated for 2 min at +37 ◦ C in a shaking incubator block (Eppendorf Thermomixer 5436, Hamburg, Germany) before the reaction was initiated by addition of NADPH. Each reaction was terminated after 30 min by adding 100 ␮l of ice-cold acetonitrile. Analysis of the assay was carried out using a LC/MS/MS method optimized for Micromass Quattro II triple

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quadrupole mass spectrometer (Micromass Corp., Altrincham, UK) from the method described by Turpeinen et al. [21]. 2.7. Correlation with probe substrate activities A bank of 15 livers was used to assess the metabolism of lynestrenol and norethindrone. The CYP activities of microsomes from these livers have been well characterized. A correlation was performed between metabolite formation of both 30 ␮M lynestrenol and 30 ␮M norethindrone and each CYP activity across the human liver bank. Lynestrenol and norethindrone were incubated separately with human microsomes. Probe substrate reactions used for correlations were ethoxyresorufin O-de-ethylation (CYP1A2), coumarin 7hydroxylation (CYP2A6), bupropion hydroxylation (CYP2B6), amodiaquine de-ethylation (CYP2C8), tolbutamide hydroxylation (CYP2C9), omeprazole 5-hydroxylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzoxazone 6-hydrolylation (CYP2E1), midazolam 1 -hydroxylation (CYP3A4/5), and omeprazole sulfoxidation (CYP3A4) [22–25]. Correlation with statistical analysis was carried out with GraphPad Software Prism 3.03 (GraphPad Software, Inc.). For all data points the mean of duplicate incubations were used. Bivariate linear Pearson’s correlation coefficients (r) were calculated between metabolite formations and model activities in livers. The limit of statistical significance was set at p < 0.05. 2.8. Analytical methods 2.8.1. Chemicals All chemicals and solvents were HPLC grade. Glacial acetic acid and LichroSolv GG acetonitrile were obtained from Merck KGaA (Darmstadt, Germany). 2.8.2. Sample preparation All incubation samples were thawed at room temperature (RT), shaken and centrifuged for 10 min at 13,400 × g (Eppendorf Mini Spin, Eppendorf AG, Hamburg, Germany) and transferred to Total Recovery vials (Waters Corporation, Milford, Massachusetts, USA) to wait for an autosampler run. 2.8.3. LC/MS conditions A Waters Alliance 2695 high-performance liquid chromatographic (HPLC) system (Waters Corporation, Milford, Massachusetts, USA) was used in all analyses. Waters XTerra RP18 column (2.1 mm × 50 mm, 3.5 ␮m particle size) together with Phenomenex Luna C18 precolumn (4.0 mm × 2.0 mm, 3.0 ␮m, Phenomenex, Torrance, California, USA) were used. The eluents were (A) 0.1% acetic acid in water (pH 3.2) and (B) acetonitrile. A linear gradient elution from 20% B to 55% B in 8 min and to 60% B in next 2 min was applied, following a fast wash of the column with 80% B and equilibration with initial conditions. The eluent flow rate was 0.3 ml/min and the column oven temperature was 30 ◦ C. The flow was split postcolumn with acurate post-column stream splitter (LC Packings, Amsterdam, The Netherlands) with ratio 1:3 to MS and waste, respectively. The LC/MS data was recorded with a Micromass

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LCT time-of-flight (TOF) high-resolution mass spectrometer (Micromass Ltd., Manchester, England) equipped with LockSpray electrospray ionization (ESI) source. LockSpray feature was used in metabolite identification with leucine enkephalin as a lock mass reference compound (M+H+ at m/z 556.2771). The ion count for leucine enkephalin was adjusted approximately to 160 ions/count. The ESI cone voltage 35 V and capillary voltage 3300 V were used in positive ion mode electrospray for both lynestrenol and norethindrone samples. Injection volume used was 10 ␮l. The mass spectrometer and HPLC system were operated under Micromass MassLynx 4.0 software. The formation of metabolites was determined semiquantitatively from LC/MS runs by comparing the peak areas in the samples, assuming their mass spectrometric response to be directly comparable. The lynestrenol was detected as its fragment ion due to loss of water [M+H–H2 O]+ at m/z 267, while norethindrone and all metabolites of both substances were detected as their protonated molecules [M+H]+ . The LC/MSMS experiments were performed with the same chromatographic method, but the Finnigan Surveyour HPLC equipped with MS-pump and Finnigan TSQ Quantum Discovery MAX (Thermo-Finnigan, San Jose, CA, USA) triple quadrupole mass spectrometer was used. Argon was used as a collision gas with 1.5 mTorr pressure, and collision energy of 20 eV was used for all compounds studied. Capillary voltage of 4500 V and capillary temperature of 350 ◦ C was used. 3. Results 3.1. Identification of lynestrenol metabolites Lynestrenol itself was detected with a retention time 12.5 min, not showing the molecular ion but only a fragment ion due to loss of water [M+H–H2 O]+ at m/z 267. Exact mass measured for the ion was m/z 267.2139, while the calculated value is m/z 267.2113 [C20 H27 ]+ . The main metabolite norethindrone (M1, 3-keto-lynestrenol) was detected as a [M+H]+ ion at m/z 299, having a retention time 7.8 min. Measured exact mass for norethindrone was m/z 299.2005, while the calculated value is m/z 299.2011 [C20 H27 O2 ]+ . Identification was also confirmed using authentic norethindrone standard in the same LC/MS system. The metabolite M2 was identified as a hydroxylation product of norethindrone due to its [M+H]+ ion at m/z 315, having exact mass value of m/z 315.1924, while the calculated value is m/z 315.1960 [C20 H27 O3 ]+ . Also, the retention time of 4.2 min for M2 supports the hydroxylated norethindrone structure. In addition, the same compound was detected also in incubation of norethindrone, confirming its formation from norethindrone. The very minor metabolite M3 at retention time 7.5 min was also identified as a further metabolite of norethindrone, formed via formation of double bond (or alternatively via cleavage of water from hydroxymetabolite M2). Also for this compound the formation from norethindrone was confirmed by norethindrone incubation. The exact mass measured for M3 was m/z 297.1849, while the calculated value is 297.1854 [C20 H25 O2 ]+ . In the lynestrenol incubations without any CYP-inhibitors, the norethindrone (M1) constituted about 90% of all metabolite peak

areas, being clearly the main metabolite, while M2 and M3 had relative peak areas of about 8% and 2% of all metabolites. 3.2. Identification of norethindrone metabolites As in lynestrenol incubations, norethindrone was detected as a [M+H]+ ion at m/z 299, having a retention time 7.8 min. Exact mass obtained for it was m/z 299.2010 (calculated m/z 299.2011). Two hydroxymetabolites were detected with retention times 4.2 min (norethindrone M1) and 5.8 min (norethindrone M2), the former being the same compound that was formed also in lynestrenol incubations. The former was clearly a main metabolite, while the latter had only a low abundance. The exact masses measured for these compounds were m/z 315.1939 and 315.1924, while the calculated value is m/z 315.1960. Also a very minor norethindrone metabolite M3 due to two hydroxylations was detected with retention time 2.6 min, having exact mass of m/z 331.1924 (calculated m/z 331.1909). The fourth detected norethindrone metabolite M4 was found also from lynestrenol incubations, having a retention time 7.5 min and formed via formation of double bond (or alternatively via cleavage of water from hydroxymetabolite). The exact mass measured for [M+H]+ ion was 297.1863 (calculated m/z 297.1854). The main norethindrone metabolite M1 consisted of 94% LC/MS peak area of the total metabolite peak area, while norethindrone M2 had about 4%, and both norethindrone M3 and M4 only about 1% share of total metabolite peak area. For more accurate identification of the main hydroxymetabolite M1 of norethindrone (present also in lynestrenol incubations), tandem mass spectrometric experiments (MS/MS) with triple quadrupole mass spectrometer were carried out, colliding the [M+H]+ ions at m/z 299 and 315 for norethindrone and M1, respectively. The abundance of the minor metabolites was too low for the MS/MS experiments. For norethindrone, the main detected fragment ions were at m/z 281, 263, 231, 213, 185, 171, 145 and 109. The first two of these are due to the losses of one and two water molecules from the [M+H]+ ion, and the corresponding fragments were detected also for the M1 at m/z 297 and 279. The ions at m/z 109 and 145 were detected also for norethindrone M1, while the m/z values for the other norethindrone M1 fragments were increased by 16 u due to hydroxylation, to m/z 247, 229, 201 and 187. These fragment ions can be identified by comparing the MS/MS data with corresponding results for closely related lynestrenol and norethandrolone [26,27]. The norethindrone fragment ion at m/z 231 is due to a [M+H–H2 O–C4 H2 ]+ ion including the loss of C2-side chain, and the ion at m/z 213 due to the consecutive loss of another water molecule. The ion at m/z 171 is probably due to the simultaneous loss of water and B-ring cleavage from the b2-position (between carbons 6–7 and 9–10), and the ion at m/z 185 due to the loss of water and B-ring cleavage from the b1-position (between carbons 5–6 and 9–10). Ion at m/z 145 is then due to the loss of ethyl side chain as neutral C2 H2 -molecule from the m/z 171, and the ion at m/z 109 is due to the well known A-ring fragment ion [C7 H9 O]+ . According to these results, the hydroxylation site can be pointed to the any of the C-ring carbons, C18-methyl group or the D-ring carbon C-15 of the substrate. According to the Williams et al. [28], the

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ion at m/z 109 contains also both the B-ring carbons 6 and 7, excluding also the hydroxylation to the B-ring. Due to the very complicated nature of steroid structure MS/MS behaviour, the spectra contain numerous minor fragment ions for which the identification is not possible, as the ions with the same m/z values may be formed via number of different fragmentation routes. Therefore, more accurate identification of the metabolites with LC/MSMS methods cannot be achieved, and for unambiguous identification of the structure the analytes should be isolated for NMR studies. The suggested bioactivation of lynestrenol to norethindrone and further metabolism of norethindrone to hydroxylated metabolites are shown in Fig. 1. 3.3. Inhibition of lynestrenol metabolism in human liver microsomes When lynestrenol was incubated with selective CYP inhibitors the formation of active metabolite norethindrone was potently inhibited by CYP3A4 inhibitor ketoconazole (IC50 = 0.02 ␮M) (Fig. 2a). Also the CYP2C9-selective inhibitor sulphaphenazole (IC50 = 2.13 ␮M) reduced the bioactivation of lynestrenol to norethindrone (Fig. 2b). A moderate inhibition of norethindrone formation was observed in the presence of fluconazole (IC50 = 10.10 ␮M). Other selective inhibitors were without prominent effect even at the concentration of 100 ␮M. 3.4. Inhibition of norethindrone metabolism in human liver microsomes When norethindrone was incubated with specific CYP inhibitors, the hydroxylation of norethindrone to main metabolite M1 was found to be markedly reduced in the presence of ketoconazole (IC50 = 0.09 ␮M) suggesting a CYP3A4-catalysed reaction (Fig. 2c). Furthermore, the formation of less abundant hydroxylated metabolite M2 and dihydroxylated metabolite M3 in addition to M4 (formed via formation of double bond or alternatively via cleavage of water from hydroxymetabolite) was strongly decreased by ketoconazole (IC50 values <0.01, 0.10,

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Table 2 The correlation analysis of CYP-specific activities against the formation of norethindrone from lynestrenol Isoform

Probe reaction

Correlation coefficient (r)

CYP1A2 CYP2A6 CYP2B6 CYP2C8 CYP2C9 CYP2C19 CYP2D6 CYP2E1 CYP3A4

Ethoxyresorufin O-de-ethylation 0.13 Coumarin 7-hydroxylation 0.23 Bupropion hydroxylation 0.33 Amodiaquine de-ethylation 0.49 Tolbutamide hydroxylation 0.63* Omeprazole 5-hydroxylation 0.17 Dextromethorphan O-demethylation −0.17 Chlorzoxazone 6-hydrolylation 0.14 Midazolam 1 -hydroxylation 0.34 Omeprazole sulfoxidation 0.22

Number of human livers used in the correlation analysis was 15. * p < 0.05.

and 0.07 ␮M, respectively). Fluconazole (CYP2C19 inhibitor) inhibited the formation of norethindrone M1, M2, and M4 characterized by IC50 values of 23.20, 20.0 and 23.20 ␮M, respectively. Fluvoxamine (CYP1A2 inhibitor) and tranylcypromine (CYP2A6 inhibitor) showed inhibitory effect on the formation of dihydroxylated norethindrone metabolite M3 (IC50 values of 1.0 and 4.0 ␮M, respectively). 3.5. Characterisation of lynestrenol bioactivation and norethindrone hydroxylation in human liver bank Microsome preparations from a bank of 15 individual human livers were used to assess the rate of norethindrone formation from lynestrenol and further metabolism of norethindrone. The correlation data for norethindrone formation and probe reactions for specific CYP enzymes are shown in Table 2. The highest and only statistically significant correlation was observed with tolbutamide hydroxylation (r = 0.63; p = 0.01) (Fig. 3) suggesting the participation of CYP2C9, which is in accordance with the results from incubations carried out with “diagnostic” inhibitors. However, bioactivation of lynestrenol did not correlate with CYP3A4-specific model reaction. Modest but statistically insignificant correlation was observed with

Fig. 2. The inhibition of lynestrenol biotransformation to norethindrone in human liver microsomes by ketoconazole (a) and sulphaphenazole (b); and the effect of ketoconazole on the further metabolism of norethindrone (metabolites M1–M4) in human liver microsomes (c). All data points are means of duplicate measurements. Incubation mixtures contained 0.5 mg microsomal protein/ml, 0.1 M phosphate buffer (pH 7.4), 1 mM NADPH, a model inhibitor (in five different concentrations) and a substrate at a concentration of 30 ␮M for the both substrates lynestrenol and norethindrone. After 2 min preincubation NADPH was added, and reaction mixture was further incubated for 20 min before determination.

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3.6. The bioactivation of lynestrenol to norethindrone and further metabolism of norethindrone in expressed recombinant CYP enzymes

Fig. 3. A correlation between tolbutamide hydroxylation (probe substrate reaction for CYP2C9 activity) and lynetrenol bioactivation, e.g. norethindrone formation. A bank of 15 livers was used to assess the metabolism of lynestrenol. Lynestrenol at concentration of 30 ␮M was incubated for 20 min with microsomal protein derived from individual liver and a correlation was performed between norethindrone formation and each CYP activity across the human liver bank. For all data points the mean of duplicate incubations were used. Bivariate linear Pearson’s correlation coefficients (r) were calculated between metabolite formations and model activities in livers.

CYP2C8 probe reaction amodiaquine de-ethylation (r = 0.49; p = 0.06). Formation of all hydroxylated norethindrone metabolites significantly correlated with CYP2C19 and CYP3A4 probe reactions as seen in Table 3. Norethindrone hydroxylation to metabolite M1 modestly correlated also with coumarin 7hydroxylation (r = 0.40) and the formation of metabolite M2 correlated with amodiaquine de-ethylation (r = 0.45).

Microsomes from baculovirus-infected insect cells expressing human CYP isoforms were proven to be functionally competent, i.e. shown to catalyse appropriate model reactions (Table 2), prior they were used in the incubations with lynestrenol and norethindrone. A screen of nine human recombinant CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) showed that all tested CYPs except 2A6 and 2C8 had detectable activity toward lynestrenol bioactivation to norethindrone. Taking into account the average human hepatic microsomal protein amounts of CYPs [(1A2, 52); (2A6, 36); (2B6, 11); (2C8, 24); (2C9, 73); (2C19, 14); (2D6, 8); (2E1, 61); (3A4, 111 pmol/mg microsomal protein)] [29] and the actual intrinsic activity values for various CYPs, relative contributions of each CYP enzymes values were calculated and shown in Table 4. Biotransformation of norethindrone to main hydroxylated metabolite M1 was catalysed by only rCYP3A4, while the predominant metabolite was not detected in the incubations with other rCYP isoforms. The formation of minor norethindrone metabolite M2 was determined only by rCYP2D6. 3.7. Incubations with inhibitory antibodies in human liver microsomes

Norethidrone metabolite

Probe reaction (CYP)

Correlation coefficient (r)

Several inhibitory CYP antibodies used in this study showed ability to reduce the biotransformation of lynestrenol to norethindrone when compared to control. The greatest inhibition was seen in incubations with antibodies to CYP2C, CYP2B6, CYP2E1 and CYP2A6 as seen in Fig. 4a. Also, antibodies to CYP1A2 and CYP2D6 had a moderate inhibitive effect on norethindrone formation. Contrary to incubations with diagnostic inhibitors, specific inhibitory antibody against CYP3A4 had only a minor effect on the norethindrone formation. All the inhibitory CYP specific antibodies prevented the formation of the minor norethindrone metabolites M2, M3, and M4; only norethindrone metabolite M1 could be detected. Rea-

M1

Coumarin 7-hydroxylation (CYP2A6) Omeprazole 5-hydroxylation (CYP2C19) Midazolam 1 -hydroxylation (CYP3A4) Omeprazole sulfoxidation (CYP3A4)

0.40 0.92*** 0.96*** 0.96***

Table 4 Relative contribution of human recombinant CYP enzymes on the lynestrenol biotransformation to norethindrone

Amodiaquine de-ethylation (CYP2C8) Omeprazole 5-hydroxylation (CYP2C19) Midazolam 1 -hydroxylation (CYP3A4) Omeprazole sulfoxidation (CYP3A4)

0.45 0.82*** 0.90*** 0.91***

M3

Coumarin 7-hydroxylation (CYP2A6) Omeprazole 5-hydroxylation (CYP2C19) Midazolam 1 -hydroxylation (CYP3A4) Omeprazole sulfoxidation (CYP3A4)

0.33 0.81*** 0.75*** 0.79***

M4

Coumarin 7-hydroxylation (CYP2A6) Omeprazole 5-hydroxylation (CYP2C19) Midazolam 1 -hydroxylation (CYP3A4) Omeprazole sulfoxidation (CYP3A4)

0.41 0.90*** 0.96*** 0.95***

Table 3 The correlation analysis of CYP-specific activities against the metabolite formation from norethindrone

M2

Number of human livers used in the correlation analysis was 15. *** p < 0.001.

Cytochrome P450 isoforms

Intrinsic activitya

Relative contribution (%)b

1A2 2A6 2B6 2C8 2C9 2C19 2D6 3A4

6 0 22 0 125 1160 6 60

1.0 0 0.70 0 28.0 49.8 0.10 20.4

a Intrinsic activity of rCYP isoforms toward lynestrenol biotransformation is a mean of duplicate incubations. b Average microsomal amounts of CYP enzymes used in calculations are taken from Ref. [29].

T. Korhonen et al. / Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 56–66

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bodies had only a minor effect on the norethindrone metabolite M1 formation. 3.8. In vitro inhibition of CYP enzymes by lynestrenol and norethindrone Inhibition of CYP activities was investigated at progestin concentrations up to 100 ␮M. Inhibition profiles of lynestrenol and norethindrone are shown in Table 5. Both lynestrenol and norethindrone were found to be weak inhibitors of CYP2C9 characterized by IC50 values of 32 and 46 ␮M, respectively, for tolbutamide hydroxylation. 4. Discussion

Fig. 4. The inhibitory effect of CYP isoenzyme specific antibodies on the formation of norethindrone from lynestrenol (a) and the inhibitory effect of anti-CYP-Ab on the formation of norethindrone metabolite M1 (b). These experiments were performed in human liver microsomes. Antibody was added to microsomes, mixed gently and incubated for 15 min (20 min for anti-CYP1A2) on ice. After that lynestrenol or norethindrone at concentration of 30 ␮M were added and incubations were carried out as described in the text.

son for non-appearance of other metabolites is not known. CYP3A4 antibody was observed to inhibit the formation of M1 by approximately 96% indicating that CYP3A4 catalysed hydroxylation of norethindrone (Fig. 4b). CYP2C and CYP1A2 antibodies moderately inhibited the formation of M1 (49% and 39%, respectively) compared to control, while other CYP anti-

The aim of the present study was to identify CYP isoforms involved in the bioactivation of lynestrenol to form norethindrone and to elucidate metabolic routes of norethindrone metabolism in vitro. In the metabolite identification phase it was found that lynestrenol undergoes rather rapid metabolism to form norethindrone, which was the principal metabolite observed in the incubations with human liver microsomes. This finding is in accordance with earlier observations [2,3]. No other metabolites were formed directly from lynestrenol except norethindrone, which was further biotransformed via hydroxylation and double bond formation (or alternatively via cleavage of water from hydroxymetabolite) to four metabolites. A number of well-documented chemical inhibitors were used to assess their inhibitory potential on lynestrenol and norethindrone biotransformation. Bioactivation of lynestrenol to norethindrone was strongly inhibited by sulphaphenazole at low inhibitor concentrations (IC50 = 2.13 ␮M) in human liver microsomes. Taking into account the high selectivity of sulphaphenazole to inhibit CYP2C9 at micromolar concentrations [30,31], this observation strongly implicates involvement of CYP2C9 isoform in the catalysis of lynestrenol bioactivation. The incubations with recombinant human CYP isoforms showed that the relative contribution of CYP2C9 to the formation of norethindrone from lynestrenol was 28% of the total. The important role of CYP2C9 in lynestrenol bioactivation was further supported by statistically significant positive correlation (r = 0.63) between norethindrone formation and tolbutamide hydroxylation, used as a model reaction of CYP2C9 activity in correlation studies. To further substantiate the relative contribution of CYP enzyme isoforms in the lynestrenol bioactivation, the effect of inhibitory CYP enzyme antibodies was evaluated. In the immunoinhibition studies anti-CYP2C antibody possessed the strongest ability to reduce lynestrenol bioactivation. However, the particular contribution of CYP2C9 isoform cannot be directly estimated from this finding since anti-CYP2C antibodies suppress the activity of all CYP2C isoforms, not only the CYP2C9. Inhibition studies with diagnostic inhibitors in human liver microsomes suggested that in addition to CYP2C9 also CYP2C19 is involved in the lynestrenol bioactivation. Fluconazole reduced lynestrenol biotransformation to norethindrone with an IC50 of 10.06 ␮M. Fluconazole has been suggested

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Table 5 The effect of lynestrenol and norethindrone on the CYP-specific model reactions CYP isoform

1A2 2A6 2B6 2C8 2C9 2C19 2C19 2D6 2E1 3A4 3A4 3A4 3A4

Substrate

Melatonin Coumarin Bupropion Amodiaquine Tolbutamide Omeprazole Omeprazole Dextromethorphan Chloroxazone Midazolam Testosterone Omaprazole Omeprazole

Metabolite

6-OH-MEL 7-OH-COU OH-BUP deEt-AMO OH-TOL 5-OH-OME dem-OME O-dem-DEX 6-OH-CLZ 1 -OH-MDZ 6␤-OH-TES SO2 -OME 3-OH-OME

as a selective inhibitor for CYP2C19 [32], although it actually displays widely variable inhibition potencies on several CYP isoforms depending on the test configuration [21]. It has been demonstrated that the Ki values of fluconazole against the CYP2C19 model reaction, (S)-mephenytoin 4 -hydroxylation, may range from 2.0 to 12.3 ␮M [32,33]. Moreover, fluconazole has also ability to inhibit CYP2C9; Ki values of 7.0–8.0 ␮M against (S)-warfarin hydroxylation [34] and 30.3 ␮M against tolbutamide hydroxylation have been reported [33]. In addition, fluconazole is a moderate CYP3A4 inhibitor [35]. Accordingly, the reduction of norethindrone formation from lynestrenol in the presence of fluconazole can be regarded as a net result of CYP2C9, CYP2C19 and CYP3A4 inhibition produced by fluconazole. Unfortunately, no established and reliably selective inhibitor for CYP2C19 has been presented hitherto, which would obviate the use of fluconazole. Nevertheless, incubations with recombinant CYP isoforms further confirmed the important role of CYP2C19 in the lynestrenol bioactivation. Significant appearance of norethindrone was detected in the lynestrenol incubations with rCYP2C19 and the relative contribution of the CYP2C19 to the formation of norethindrone was approximately 49.8% of the total despite the low abundance of CYP2C19 in human liver microsomes [29]. In addition, anti-CYP2C antibody performed most prominent inhibition on the lynestrenol bioactivation in immunoinhibition phase. However, on the contrary to other findings, correlation analysis did not support participation of CYP2C19 in the lynestrenol bioactivation. Altogether, it is still prudent to state that the involvement of CYP2C19 in lynestrenol disposition is highly probable. Incubations with diagnostic inhibitors showed that ketoconazole strongly inhibited lynestrenol bioactivation to norethindrone at low inhibitor concentrations. Taking into account the low but variable expression of CYP3A5 in human liver [36], particularly the inhibition of CYP3A4 may explain the reduced norethindrone formation in the presence of ketoconazole. However, in immunoinhibition studies the norethindrone formation was reduced only by 22% in the presence of antiCYP3A4 antibodies when compared to control. The relative contribution of the CYP3A4 to the lynestrenol bioactivation

IC50 values (␮M) of Lynestrenol

Norethindrone

>100 >100 >100 >100 31.6 63.5 78.1 >100 >100 >100 >100 >100 80.9

>100 >100 >100 >100 46.4 >100 >100 >100 >100 >100 >100 >100 >100

was 20.4% of the total according to human rCYP incubations which suggests relatively important role of CYP3A4 isoform in lynestrenol bioactivation although, norethindrone formation from lynestrenol did not correlate with any of the CYP3A4 model reactions used in the correlation studies with human liver bank. The further hydroxylation of norethindrone was catalyzed mainly by CYP3A4. IC50 values of ketoconazole against the norethindrone hydroxylation to major metabolites were 0.01–0.097 ␮M. Moreover, the formation of predominant norethindrone metabolite M1 was determined only by recombinant CYP3A4 when norethindrone was incubated with the baculovirus expression system. The involvement of CYP3A4 in norethindrone disposition was further confirmed by the results obtained from norethindrone incubations with inhibitory antibodies and from the correlations studies. Also fluconazole showed a weak inhibition on the formation of major norethindrone metabolites in the inhibition studies. In addition, correlation and immunoinhibition studies referred that CYP2C19 may have a minor role in the norethindrone hydroxylation. However, recombinant CYP2C19 did not catalyse norethindrone biotransformation. Inhibition studies with diagnostic inhibitors suggested the participation of CYP1A2 and CYP2A6 in the formation of dihydroxylated norethindrone metabolite (M3), but taking into account the low relative abundancy of this metabolite (approximately 1% of the total share of metabolites), these metabolic routes are not likely to be of importance. The effect of lynestrenol on CYP enzyme activities has not been examined earlier, while norethindrone has been reported to be a weak inhibitor of losartan oxidation by CYP2C9 and Romeprazole 5-hydroxylation by CYP2C19 according an in vitro study [37]. In the present study, lynestrenol weakly reduced the activities of CYP2C9, CYP2C19 and CYP3A4. Also norethindrone displayed a weak inhibition of CYP2C9, which is in agreement with the earlier findings [37]. However, the activities of other CYP isoenzymes were unaffected by norethindrone. According to the findings of our study, the liability of lynestrenol

T. Korhonen et al. / Journal of Steroid Biochemistry & Molecular Biology 110 (2008) 56–66

and norethindrone to inhibit CYP enzymes seems to be very distinctive and it is prudent to say that the clinical relevance of these interactions is probably negligible. The metabolic fate of a prodrug like lynestrenol may be adversely affected by genetic factors. Both CYP2C9 and CYP2C19, the isoforms probably responsible for lynestrenol bioactivation, are known to exhibit genetic polymorphism with large interethnic differences in the frequency of mutated alleles [38,39]. Approximately 1% and 3% of the western Caucasian population lack the active CYP2C9 or CYP2C19 enzyme, respectively [40–43]. It has been previously described that the individuals hetero- or homozygous for the CYP2C9*3, or homozygous for the CYP2C9*2, have significantly impaired CYP2C9 enzyme mediated drug metabolism compared to individuals with wild-type alleles of CYP2C9 [44,45]. Accordingly, lynestrenol bioactivation may be affected by the polymorphism of CYP2C9 and CYP2C19, which may result in reduction of the therapeutic efficacy of lynestrenol in individuals carrying mutated alleles. This may have particular clinical relevance when lynestrenol is used for contraception. As lynestrenol is a prodrug, it is vulnerable to pharmacokinetic drug–drug interactions affecting its bioactivation. Both CYP2C isoforms and CYP3A4 are expressed in the gastrointestinal tract in addition to liver [46], and the biotransformation of lynestrenol may be readily affected during first-pass metabolism and later during hepatic elimination. Concurrent use of lynestrenol with potent CYP2C9 and CYP2C19 inhibitors may decrease the amount of norethindrone converted from lynestrenol and thereby predispose to reduced therapeutic efficacy of lynestrenol. On the other hand, CYP3A4 inhibitors may increase the exposure to norethindrone by decreasing the elimination of norethindrone. Accordingly, risk for concentration-dependent adverse effects of norethindrone is increased in concomitant use of CYP3A4 inhibitors. In contrast, drugs that induce CYP3A4 enzyme may impair the biological effects of lynestrenol or norethindrone by decreasing the plasma levels of norethindrone as previously described [12–15] and enhance the risk for therapeutic failure. While extrapolating the in vitro results into in vivo, the restrictions of present test system have to be taken into account. Human liver microsomes derived from endoplasmic reticulum contain only phase I enzymes (CYPs) and UDP-glucuronosyltransferase isoforms (UGTs) [47]. Therefore, the effect of cytoplasmic enzymes on lynestrenol and norethindrone metabolism cannot be evaluated by approaches used in the present study. The lack of presence of phase II drug metabolising enzymes in the test system may explain the differences in the metabolite spectra obtained in the present study compared to those observed in previous human studies in which norethindrone metabolites were present predominatly as tetrahydrometabolites, mainly sulfate conjugates [48]. Furthermore, the in vivo predictive value of the data obtained from heterologously expressed enzyme systems has been debated, because the enzymes are studied in isolation from other hepatic enzymes [47]. However, ascertifying role of recombinant enzymes in vitro setting has been widely accepted. Nevertheless, it is prudent to underline that clinical studies are needed to confirm the role of CYP isoforms in the lynestrenol

65

biotransformation and norethindrone disposition. Well-designed drug–drug interaction studies are needed to elucidate the importance of CYP enzyme catalysed bioactivation for progestagenic response to lynestrenol. In conclusion, several approaches were adopted to identify the CYP enzymes involved in the lynestrenol and norethindrone metabolism in the present study. The data strongly suggests an important role of the polymorphic CYP2C9 and CYP2C19 in lynestrenol bioactivation to norethindrone. In addition, CYP3A4 was tentatively found to be involved in the lynestrenol bioactivation. The further hydroxylation of norethindrone was catalyzed by CYP3A4 while no other isoforms seemed to be that important in norethindrone disposition. Neither lynestrenol nor norethindrone showed distinctive inhibitory effect on the CYP isoforms. Genetic polymorphism of CYP2C9 and 2C19 as well as concurrent use of lynestrenol with drugs, which possess inhibitory or inducing effect on CYP2C and CYP3A isoforms, may modify the therapeutic response for lynestrenol, while metabolic fate of norethindrone may be affected particularly by CYP3A4 inhibitors or inducers. In vivo studies, however, are required to ascertain the clinical relevance of these drug interactions. Acknowledgements Mr. Amir Snapir, PhD, is thanked for his skilful assistance in computing. Miia Turpeinen is a postgraduate student of the Graduate School of Clinical Drug Research. References [1] A.E. Schindler, C. Campagnoli, R. Druckmann, J. Huber, J.R. Pasqualini, K.W. Schweppe, J.H. Thijssen, Classification and pharmacology of progestins, Maturitas 46 (Suppl. 1) (2003) S7–S16. [2] A. Mazaheri, K. Fotherby, J.R. Chapman, Metabolism of lynestrenol to norethisterone by liver homogenate, J. Endocrinol. 47 (2) (1970) 251–252. [3] S. Kamyab, K. Fotherby, A.I. Klopper, Metabolism of [4-14 C]lynestrenol in man, J. Endocrinol. 42 (2) (1968) 337–343. [4] H. Kuhl, H.J. Bremser, H.D. Taubert, Serum levels and pharmacokinetics of norethisterone after ingestion of lynestrenol: its relation to dose and stage of the menstrual cycle, Contraception 26 (3) (1982) 303–315. [5] K. Kontula, O. Janne, R. Vihko, E. de Jager, J. de Visser, F. Zeelen, Progesterone-binding proteins: in vitro binding and biological activity of different steroidal ligands, Acta Endocrinol. (Copenh.) 78 (3) (1975) 574–592. [6] M. H¨umpel, H. Wendt, G. Dogs, C. Weiss, S. Rietz, U. Speck, Intraindividual comparison of pharmacokinetics parameters of d-norgestrel, lynestrenol, and cyproterone acetate in 6 women, Contraception 16 (2) (1977) 199–215. [7] K. Shrimanker, J. Akpoviroro, K. Fotherby, J. Watson, Bioavailability of lynestrenol, Arzneimittelforschung 30 (3) (1980) 500–502. [8] D.J. Back, A.M. Breckenridge, F.E. Crawford, M. Mciver, M.L. Orme, B.K. Park, P.H. Rowe, E. Smith, Kinetics of norethindrone in women. I. Radioimmunoassay and concentrations during multiple dosing, Clin. Pharmacol. Ther. 24 (4) (1978) 439–447. [9] D.J. Back, A.M. Breckenridge, F.E. Crawford, M. Mciver, M.L. Orme, P.H. Rowe, E. Smith, Kinetics of norethindrone in women. II. Single-dose kinetics, Clin. Pharmacol. Ther. 24 (4) (1978) 448–453. [10] H. Singh, J.P. Uniyal, P. Jha, K. Murugesan, D. Takkar, V. Hingorani, K.R. Laumas, Pharmacokinetics of norethindrone acetate in women, Am. J. Obstet. Gynecol. 135 (3) (1979) 409–414. [11] S. Zalanyi, B.M. Landgren, E. Johannisson, Pharmacokinetics, pharmacodynamic and endometrial effects of a single dose of 200 mg norethisterone enanthate, Contraception 30 (3) (1984) 225–237.

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