Eur J Drug Metab Pharmacokinet DOI 10.1007/s13318-014-0224-7
ORIGINAL PAPER
Pharmacokinetics, tissue distribution, and excretion of nomegestrol acetate in female rats Qingbiao Huang • Xiaoke Chen • Yan Zhu Lin Cao • Jim E. Riviere
•
Received: 16 May 2014 / Accepted: 20 August 2014 Springer International Publishing Switzerland 2014
Abstract Nomegestrol acetate (NOMAC), a synthetic progestogen derived from 19-norprogesterone, is an orally active drug with a strong affinity for the progesterone receptor. NOMAC inhibits ovulation and is devoid of undesirable androgenic and estrogenic activities. The aim of this study was to evaluate the pharmacokinetics, tissue distribution, and excretion of NOMAC in female rats. Sprague–Dawley female rats were orally administered a single dose of NOMAC (10, 20 or 40 mg/kg) and drug plasma concentrations at different times were determined by RP-HPLC. Tissue distribution at 1, 2, and 4 h and excretion of NOMAC into bile, urine, and feces after dosing were investigated. The results showed that NOMAC was rapidly absorbed after oral administration, with tmax of Q. Huang and X. Chen contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s13318-014-0224-7) contains supplementary material, which is available to authorized users. Q. Huang (&) State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China e-mail: [email protected] X. Chen Department of Research and Development, Pharmaceutics International, Inc., Hunt Valley, MD 21031, USA Y. Zhu L. Cao (&) Department of Reproductive Pharmacology, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China e-mail: [email protected] J. E. Riviere Institute of Computational Comparative Medicine, Kansas State University, Manhattan, KS 66506, USA
1–2 h. The plasma concentration–time curves were fitted in a two-compartment model. The exposure to NOMAC (Cmax and AUC) increased dose proportionally from 10 to 40 mg/ kg. The average CL and t1=2b were 5.58 L/(hkg) and 10.8 h, respectively. The highest concentrations of NOMAC in ovary, liver, kidney, lung, heart, brain, spleen, muscle, and uterus were observed at 2 h, whereas the highest concentrations in stomach, pituitary, and hypothalamus appeared at 1 h. The total cumulative excretion of NOMAC in feces (0–72 h), urine (0–72 h), and bile (0–48 h) was *1.06, 0.03, and 0.08 % of the oral administered dose, respectively. This study indicated that NOMAC had a widespread distribution in tissues, including ovary, pituitary, and hypothalamus, which are main target tissues where NOMAC inhibits ovulation. NOMAC was excreted via both feces and urine with few unchanged NOMAC excreted. Enterohepatic circulation was found in the drug elimination; however, it did not significantly affect tmax . Keywords Nomegestrol acetate NOMAC Pharmacokinetics Tissue distribution Excretion HPLC Abbreviations NOMAC Nomegestrol acetate HPLC High-pressure liquid chromatography AUC Area under the plasma concentration–time curve CL Clearance Cmax Maximum plasma concentration V/F Apparent distribution volume t1=2b Terminal half-life Time to maximum plasma concentration tmax Ka Absorption rate constant K10 Elimination rate constant
Eur J Drug Metab Pharmacokinet
K12 K21 a b
Distribution rate constant from the central compartment to the peripheral compartment Distribution rate constant from the peripheral compartment to the central compartment Rate constant associated with the distribution phase of the concentration–time curve Rate constant associated with the terminal phase of the concentration–time curve
1 Introduction Nomegestrol acetate (NOMAC) is a 19-norprogesterone derivative with a high progestational activity, first reported by Miyake and Rooks (1966). It is an orally active progestogen with a favorable tolerability profile and neutral metabolic characteristics (Lello 2010). NOMAC is designed to bind selectively for the progesterone receptor and lacks significant affinity with other steroid receptors, showing strong antiestrogenic and antigonadotropic activity, but without androgenic or glucocorticoid properties (Lello 2010; van Diepen 2012; Ruan et al. 2012; Yang and Plosker 2012). Unlike some other progestogens, the antigonadotropic effect of NOMAC is mediated at the hypothalamic and pituitary level (Couzinet et al. 1999). In in vitro functional assay, nanomolar affinity of NOMAC was demonstrated in radioligand binding with cytosolic progesterone receptor in human endometrium (Botella et al. 1988) and breast tissue (Duc et al. 1990), and the potency of NOMAC was greater than progesterone. NOMAC had no agonist or antagonist activity at a or b estrogen or mineralocorticoid receptors in Hela cells (a human cervical carcinoma cell line) or CHO cells transfected with human steroid receptors (Merk Sharp and Dohme (Australia) Pty Limited 2011). In addition, NOMAC inhibited the estrogen-induced stimulation of progesterone receptor expression in T47-D human breast cancer cells in vitro (van Diepen 2012). NOMAC has been approved in Europe and Australia and widely used for the treatment of gynecological disorders (menstrual disturbances, dysmenorrhoea, and premenstrual syndrome) (Alsina 2010) and for hormone replacement therapy (HRT) in combination with estradiol (E2) for the relief of post-menopausal symptoms (Shields-Botella et al. 2003). At a dosage of 1.25 mg/day, NOMAC inhibited ovulation while follicle growth was not affected; at a dosage of 2.5 or 5 mg/day, both ovulation and follicle development were significantly suppressed (Bazin et al. 1987). The studies on NOMAC/E2 as a combined oral contraceptive (COC) showed that NOMAC preserved the beneficial hemostatic effects of estrogen and had a neutral or beneficial effect on lipid profiles, while not changing body weight and having no adverse effects on glucose metabolism. In addition, NOMAC
showed a lack of proliferative activity in normal and cancerous breast tissues and did not have a deleterious effect on bone remodeling (Lello 2010; van Diepen 2012; Yang and Plosker 2012). Despite NOMAC has been used in humans in some developed countries, limited information on pharmacokinetics, tissue distribution (especially for targeted organs including ovary, uterus, hypothalamic, and pituitary), and excretion of NOMAC following a single oral administration in animals and humans was available (or disclosed) in the literature due to confidential reasons. In this study, RP-HPLC method was adopted to determine NOMAC concentrations in rat biological matrices, including plasma, tissues, urine, feces, and bile. The pharmacokinetic profiles of NOMAC in female rats were investigated, including (1) the plasma pharmacokinetics of NOMAC; (2) the tissue distribution of NOMAC; (3) the excretion of NOMAC in bile, urine, and feces after oral administration. The results were also useful for new formulation development in the future.
2 Materials and methods 2.1 Chemicals and reagents NOMAC (lot no. 980616, purity [99.2 %, Fig. 1) was gifted from School of Pharmacy, Fudan University Medical Center (Shanghai, China). Two internal standards (IS) included flutamide (lot no. 981217, purity [99.2 %), purchased from Fudan Forward Pharmaceutical Co., Ltd (Shanghai, China) and mifepristone (lot no. 980607, purity [99.8 %), obtained from Zhejiang Xianju Junye Pharmaceutical Co., Ltd (Zhejiang, China). Methanol (HPLC grade) was purchased from Shanghai Chemical Reagent Research Institute Co., Ltd (Shanghai, China). All other chemicals and solvents were of the highest grade of commercially available materials. Purified water obtained via a Milli-Q system (Millipore, Bedford, MA, USA) was used throughout the experiments. 2.2 In vivo animal experiment Healthy Sprague–Dawley female rats weighing 200–320 g (certificate no. 02-49-2) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Upon arrival in the laboratory, each animal was evaluated by a laboratory veterinarian. The selected healthy female rats were allowed to acclimate for at least 1 week before the experiments. The animal room was maintained at 25 ± 2 C and 50–70 % relative humidity with 12 h light/ dark cycles. Feed and municipal water were provided ad libitum, except when feed was withdrawn *12 h before dosing. The experiments were carried out in compliance
Eur J Drug Metab Pharmacokinet
Fig. 1 Chemical structure of NOMAC
with Chinese Regulations for the Care and Use of Experimental Animals. At the end of the experiment, pentobarbital sodium was used for the euthanasia of the animals. 2.3 Plasma collection and NOMAC extraction Female rats were assigned randomly into three groups (five rats per group) and received 10, 20, and 40 mg/kg NOMAC in a solution of saline (0.5 % Tween-80), respectively, through oral administration. By cutting the tails, blood samples (800 lL) were collected into a clean test tube containing sodium heparin before and 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24 h after drug administration (group 1 in Table 1). Plasma was prepared by centrifugation at 3,000 rpm for 5 min after the blood samples were placed for 30 min, and then stored at -80 C until further analysis (within 4 weeks). Extraction of NOMAC from plasma involved the addition of 10 lL (containing total 1 lg) flutamide (IS) and 3.0 mL diethyl ether into 500 lL of plasma sample (6:1, v/v) in 5 mL centrifuging glass tube and vortex mixing for 1 min. The mixed samples were equilibrated at room temperature for 5 min, extracted twice with diethyl ether (3.0 mL) with vortex mixing for 5 min each time, and then centrifuged at 3,000 rpm for 5 min. The organic and aqueous layers were separated by allowing the mixture to stand in room temperature for 10 min. The top (organic) layer (2.5 mL) was transferred to glass tube and evaporated to dryness under a stream of nitrogen at 40 C. The dried residue was then reconstituted with 30 lL of mobile phase (methanol: water = 70:30, v/v) and centrifuged at 10,000 rpm for 5 min. After vortex mixing for 1 min, a 20-lL aliquot was injected into HPLC system for analysis. 2.4 Tissue distribution studies Fifteen female rats were divided randomly into three groups (five rats per group) and orally administered NOMAC in a solution of saline (0.5 % Tween-80) at a dose of
20 mg/kg. One additional rat was killed pre-dose to provide blank control tissues. The animals were killed at 1, 2, or 4 h after the dosing (group 2 in Table 1). The tissues, including liver, kidney, stomach, brain, heart, lung, muscle, ovary, pituitary, hypothalamus, spleen, and uterus, were promptly removed and washed with saline solution to remove any residual blood. Each tissue sample (*0.5 g) was homogenized with 1.5 mL saline using Polytron PTMR 3000 homogenizer (Kinematica AG, Switzerland) and the leftover on the homogenizer was washed with 0.5 mL saline and transferred to the same tube. Each sample was added with 10 lL flutamide (IS). NOMAC was isolated from the homogenate as described previously for the plasma samples and stored at -80 C until further analysis (within 4 weeks). 2.5 Metabolism and excretion studies Three female rats were orally administered a single dose of NOMAC at 20 mg/kg in a solution of saline (0.5 % Tween-80). The rats (n = 3) were then placed into separate metabolic cages designed for the separation and collection of urine and feces. Urine and feces were collected 3 h before the dosing and 0–6, 6–12, (or 0–12), 12–24, 24–48, and 48–74 h after the dosing (group 3 and 4 in Table 1). Both urine and feces were collected in separate containers surrounded by ice and then frozen at -80 C at the end of each collection interval for further analysis (within 4 weeks). Polyethylene tubes were surgically cannulated into the bile duct of female rats (n = 5). A 20 mg/kg dose of NOMAC in a solution of saline (0.5 % Tween-80) was orally administered to the rats. Bile was collected into successive vials on ice at 3 h before the dosing as a blank control and at 0–2, 2–4, 4–8, 8–12, 12–24, 24–36, and 36–48 h after the dosing (group 5 in Table 1). The bile samples were stored at -80 C for further analysis (within 4 weeks). The volumes of urine and bile, and dry weight of feces during each collection period were measured before being stored in the refrigerator (Table 1). Before HPLC analysis, the urine sample was added with 20 lL (containing 2 lg) mifepristone as IS. After liquid– liquid extraction with diethyl ether for three times, the organic layers were evaporated to dryness under a stream of nitrogen at 40 C. The residue was reconstituted in 30 lL mobile phase and then centrifuged at 10,000 rpm for 5 min after vortex mixing for 1 min. Twenty microliter aliquot of the supernatant was injected into the HPLC system. Fecal samples (50 mg) were dried at 80 C for 2 h, and then soaked in 1 mL methanol at 4 C for 24 h. Five hundred microliters of supernatant was transferred and 20 lL mifepristone (containing 2 lg) was added as IS. The mixed samples were vertically blended for 2 min and then
Eur J Drug Metab Pharmacokinet Table 1 Protocol for the pharmacokinetic study of NOMAC in rats Group
Tissues
1
Plasma
2
Tissues
3
n
Route
Dose (mg/kg)
15
Oral
10, 20, 40
0, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h
15
Oral
20
1, 2, and 4 h
Urine
3
Oral
20
–3–0, 0–6, 6–12, 12–24, 24–48 and 48–72 h
4
Feces
3
Oral
20
–3–0, 0–12, 12–24, 24–48 and 48–72 h
5
Bile
5
Oral
20
–3–0, 0–2, 2–4, 4–8, 8–12, 12–24, 24–36, and 36–48 h
extracted as described previously for urine. Bile sample was subjected to the same procedure as described for urine. 2.6 HPLC analysis Samples were analyzed using Waters system equipped with binary pump, on-line vacuum degasser, autosampler, column compartment, UV detector, and Waters Millennium32 software, as described previously (Huang et al. 2000, 2014). Chromatographic separation was achieved on a lBondapak-C18 column (300 9 3.9 mm, 5 lm; Waters Instruments, Marlborough, MA) and a lBondapak-C18 guard column. An isocratic mobile phase consisted of a mixture of methanol and water (70:30, v/v, %) with a flow rate of 1.2 mL/min, and the column temperature was maintained at 25 ± 2 C throughout the analysis. The eluent was detected by UV detector with the wavelength set at 293 nm. The HPLC chromatograms of the extracted rat plasma, bile, and urine samples with the presence of IS were shown in Fig. 2. 2.7 Pharmacokinetic and statistical analysis The data of plasma concentration versus time for NOMAC were analyzed using PK-GRAPH package (Yi 1992). The pharmacokinetic parameters were estimated by appropriate compartmental methods. The goodness of fit and the most appropriate model were determined by accessing the randomness of the scatter of actual data points around the fitted function. The Student’s t test was used to analyze differences between two groups. The difference in two groups of data with p-value of \0.05 or 0.01 was considered significant. The data were presented as mean ± SD.
Collection times
in female rats was shown in Fig. 3 and the pharmacokinetic parameters were calculated and summarized in Table 2. There were no significant differences in pharmacokinetic parameters compared with groups of 10, 20, and 40 mg/kg, except AUC and Cmax . Both AUC and Cmax exhibited linear increase with the dose administered (r2 [ 0.98, p \ 0.01). The Cmax of NOMAC was obtained at 1–2 h (tmax ) after dosing, and the drug concentration decreased slowly after Cmax . The t1=2b values for NOMAC were 13.14 ± 3.70, 9.33 ± 4.82, and 9.93 ± 3.71 h for dosing groups of 10, 20, and 40 mg/kg, respectively. The values of V/F and CL, as well as K10, were relatively constant compared with three groups. The K12 and K21 values were very similar in the same dosing group, and no significant difference was observed among different groups (p [ 0.05). 3.2 Tissue distribution Tissue distribution studies in Sprague–Dawley female rats after oral dose of 20 mg/kg revealed wide tissue distribution (Fig. 4). In three sampling times (1, 2, and 4 h), the highest tissue concentrations of NOMAC were observed at 2 h after oral dosing, excluding stomach, pituitary, and hypothalamus, whose highest concentrations appeared at 1 h. The stomach samples exhibited the highest drug exposure. The mean Cmax values of ovary, liver, pituitary, hypothalamus, and kidney were 6.4, 4.8, 3.9, 2.5, and 2.2 lg/g, respectively. Other tissues that displayed relative high exposure were lung, heart, brain, and spleen. NOMAC concentrations in muscle and uterus were very low (1.1 and 1.0 lg/g, respectively). 3.3 Urinary, fecal, and biliary excretion
3 Results 3.1 Plasma pharmacokinetics Plasma concentration versus time was modeled by PKGRAPH package and the best fit was achieved by a twocompartment model. A plot of mean plasma drug concentration versus time for oral administration of NOMAC
The NOMAC excretion-time profiles for the urine and feces within 72 h and the bile within 48 h following 20 mg/kg oral administration were described in Fig. 5. The urinary and fecal excretions of NOMAC were completed before 24 h, when the cumulative excretion curve reached the maximum. However, biliary excretion seemed to continue after 48 h according to the ascending curve. The cumulative percentages of intact NOMAC excreted
Eur J Drug Metab Pharmacokinet Fig. 2 Representative HPLC chromatograms of the extracted plasma (a) biliary (b) urinary (c) and fecal (d) samples obtained from rats following a single oral dose of 20 mg/kg of NOMAC. Internal standards (IS) in plasma and biliary samples were flutamide; IS in urinary and fecal samples were mifepristone
Fig. 3 Mean plasma concentration–time profile of NOMAC after oral administration of 10, 20, and 40 mg/kg of NOMAC to rats. At 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h following NOMAC oral administration, plasma samples were collected and processed for HPLC determination. Each data point represents mean ? SD of five rats
Eur J Drug Metab Pharmacokinet Table 2 Pharmacokinetic parameters following NOMAC administration to rats Parameter
Units
Mean ± SD at different doses (mg/kg) 10
20
40
3.87 ± 2.63
1.06 ± 1.06
1.09 ± 0.53•
a
h
-1
1.33 ± 1.62
1.01 ± 0.82
0.37 ± 0.14•
b
h-1
0.06 ± 0.02
0.09 ± 0.03
0.07 ± 0.02•
0.25 ± 0.15
0.74 ± 0.29•
1.63 ± 1.89
1.65 ± 1.95
2.09 ± 0.90•
13.14 ± 3.70
9.33 ± 4.82
9.93 ± 3.71•
0.12 ± 0.05
0.16 ± 0.10•
0.10 ± 0.08•
Ka
t1=2ðKaÞ
h-1
h
t1=2a
h
t1=2b
h
K10 K12 K21
h
-1
h
-1
h
-1
V=F
L/kg
CL
L/h/kg -1
tmax
h
Cmax
ng/mL
AUC
ng 9 h/mL
1.49 ± 1.09
0.17 ± 0.11
0.73 ± 1.06
0.55 ± 0.68
0.54 ± 0.54
0.18 ± 0.01•
0.35 ± 0.29
68.46 ± 53.58
58.95 ± 33.22
52.60 ± 20.59•
5.42 ± 1.09
5.87 ± 0.34
5.44 ± 2.24•
0.99 ± 0.34
2.68 ± 1.56
2.50 ± 0.60**•
143 ± 28
280 ± 91*
589 ± 65**#
1,910 ± 404
3,417 ± 188**
8,698 ± 4,421**#
n = 5 per group; differences of pharmacokinetic parameters between groups 20 mg/kg (or 40 mg/kg) and 10 mg/kg: p [ 0.05; * p \ 0.05; ** p \ 0.01; differences of pharmacokinetic parameters between groups 40 and 20 mg/kg: • p [ 0.05; # p \ 0.01
through the urine and feces within 72 h were 0.03 ± 0.01 and 1.06 ± 0.55 %, respectively. The cumulative percentage excreted to bile was 0.08 ± 0.01 % within 48 h.
4 Discussion
Fig. 4 Tissue distribution of NOMAC in rats (n = 5) after a single oral dose of 20 mg/kg. At 1, 2, and 4 h following oral administration, tissue samples were collected and processed for HPLC determination. Values are presented as mean ? SD
This study showed that NOMAC is rapidly absorbed into the bloodstream, reaching maximum blood concentrations at 1–2 h after dosing, which was similar to that observed in mice, cynomogus monkeys (Merk Sharp and Dohme (Australia) Pty Limited 2011), and humans(1.5–2 h) (Gerrits et al. 2013). NOMAC was distributed fast in tissues and excreted through feces and urine. The hepatoenteral circulation occurred during drug elimination. By comparing pharmacokinetic parameters of NOMAC at 10, 20, and 40 mg/kg using the Student’s t test, no significant difference was observed except for Cmax and AUC, which were dose-proportional. This suggested that NOMAC exhibited linear first-order pharmacokinetic characteristics and no saturation of metabolism occurred in the dose range of 10–40 mg/kg. The value of V/F in rats was 60 L/kg, 2.5-fold larger than in humans (27 L/kg) (Gerrits et al. 2013), which follows allometric principle. The t1=2b of NOMAC was about 10 h, shorter than human t1=2b (42 h) (Gerrits et al. 2013) due to the faster and more extensive metabolism of NOMAC in animals compared with humans (Merk Sharp and Dohme (Australia) Pty Limited 2011).
Eur J Drug Metab Pharmacokinet Table 3 The structures of possible metabolites of NOMAC observed in rat plasma Metabolites no#
Metabolite structure
Metabolite #1
Metabolite #2
Fig. 5 Cumulative excretion of NOMAC in fences, urine, and bile following a single oral dose of 20 mg/kg. Feces and urine samples were collected at 6, 12, 24, 48, and 72 h and bile samples were collected at 2, 4, 8, 12, 24, 36, and 48 h. Values are presented as mean ? SD (urine and bile) or mean ± SD (feces)
The distribution of NOMAC in tissues was rapid, with the highest concentration observed at 2 h post-administration in most tissues. NOMAC had widespread tissue distribution in different tissues, including stomach, ovary, liver, pituitary, kidney and hypothalamus, lung, heart, brain, spleen, muscle, and uterus. The high NOMAC concentration appeared in hypothalamus, pituitary, and brain at 2 h, which indicated that NOMAC can transfer across the blood–brain barrier (BBB) easily. In addition, high levels of NOMAC were observed in the organs including ovary, hypothalamus, and pituitary, which is consistent with the distribution to receptors in target tissues (Bazin et al. 1987; Botella et al. 1986, 1988; Couzinet et al. 1999; Duc et al. 1990). The concentrations of NOMAC in the urine were analyzed till their concentrations decreased below HPLC detection limit. About 0.08 % cumulative amount of NOMAC was eliminated via biliary excretion within 48 h; the excretion was projected to keep active after 48 h according to the slope of the cumulative excretion curve at 48 h, whereas little amount of NOMAC was found in feces after 48 h, and negligible NOMAC excretion was observed in urine after 24 h. The polar nature of NOMAC with good liposolubility prevents drug excretion through the kidney. These findings are consistent with the previous report that compounds whose molecular weight ranged between 150 and 700 demonstrated an increase in the proportion of compounds excreted in the bile versus urine when the molecular weight increased (Calabrese 1983). However,
Metabolite #3
Metabolite #4
Metabolite #5
Nomegestrol (minor)
the enterohepatic circulation of NOMAC did not significantly change tmax because (1) only small portion (*15 %) of NOMAC in the bloodstream undergoes enterohepatic
Eur J Drug Metab Pharmacokinet
circulation; (2) the excretion rates of NOMAC in the bile decreased after 2 h, which can be observed by the slopes of the biliary cumulative excretion curve in Fig. 5. The excretion of intact NOMAC in rat was detected only at low concentrations in feces, urine, and bile, similar to that observed in monkeys and humans (Merk Sharp and Dohme (Australia) Pty Limited 2011; Gerrits et al. 2013). NOMAC is metabolized primarily by hepatic CYP3A4 and CYP3A5, and a possible contributory role by CYP2C19 and CYP2C8 (Yang and Plosker 2012). NOMAC is metabolized into several hydroxylated metabolites and subsequently conjugated with glucuronide or sulfate (Lello 2010), which accounts for only 1.08 or\1 % of intact NOMAC being detected in feces, urine or bile samples of rats. All metabolites of NOMAC had little or no effects on progesterone receptor activity (Lello 2010). The possible structures of NOMAC metabolites in rats were shown in Table 3 (Merk Sharp and Dohme (Australia) Pty Limited 2011).
5 Conclusions NOMAC was fast absorbed, widely distributed throughout tissues, eliminated in female rats via both fecal and renal routes, and has a long terminal half-life. The results can be useful for drug formulation development and pharmacokinetic studies in humans. Acknowledgments The authors would like to thank Gengdi You and Rongfa Lu for excellent technical support. This work was financially supported by Shanghai Modern Biology and Drug Industry Development Foundation (No. 955419004). Conflict of interest The authors report no conflict of interest. The authors are responsible for the content and writing of the paper.
References Alsina JC (2010) After 50 years of ethinylestradiol, another oestrogen in combined oral contraceptives. Eur J Contracept Reprod Health Care 15:1–3 Bazin B, Thevenot R, Bursaux C, Paris J (1987) Effect of nomegestrol acetate, a new 19-nor-progesterone derivative, on pituitaryovarian function in women. Br J Obstet Gynaecol 94:1199–1204 Botella J, Porthe-Nibelle J, Paris J, Lahlou B (1986) Interaction of new 19-nor progesterone derivatives with progestagen,
mineralocorticoid and glucocorticoid cytosolic receptors. J Pharmacol 17:699–706 Botella J, Paris J, Duc I, Lahlou B (1988) Nomegestrol acetate binding to cytosolic progesterone receptor in human endometrium. Med Sci Res 16:299–300 Calabrese EJ (1983) Principles of Animal Extrapolation. Wiley, New York Couzinet B, Young J, Kujas M, Meduri G, Brailly S, Thomas JL, Chanson P, Schaison G (1999) Theantigonadotropic activity of a 19-nor-progesterone derivative is exerted both at the hypothalamic and pituitary levels in women. J Clin Endocrinol Metab 84:4191–4196 Duc I, Botella J, Gillet JY, Duforestel T, Paris J (1990) Nomegestrol acetate binding to human breast tissue. Med Sci Res 18:57–58 Ezan E, Benech H, Bucourt R, Ardouin T, Tchernatinsky C, Thomas JL, Paris J, Grognet JM (1993) Enzyme immunoassay for nomegestrol acetate in human plasma. J Steroid Biochem Mol Biol 46:507–514 Gerrits MG, Schnabel PG, Post TM, Peeters PA (2013) Pharmacokinetic profile of nomegestrol acetate and 17b-estradiol after multiple and single dosing in healthy women. Contraception 87:193–200 Gibaldi M, Perrier D (1982) Pharmacokinetics. Marcel Dekker, New York Huang Q, Cao L, Gu Z (2000) RP-HPLC determination of nomegestrol acetate in plasma of rabbit. Chin J Pharm Anal 20:379–380 Huang Q, Chen X, Zhu Y, Cao L (2014) Development and validation of an HPLC method for the quantitation of nomegestrol acetate in biological matrices of rats. Biomed Chromatogr (Submitted) Jamin C (1992) Female contraception by a normal dose progestogen after 40 years of age. Possible association of nomegestrol-17beta-estradiol acetate by percutaneous route. Rev Fr Gynecol Obstet 87:370–376 Lello S (2010) Nomegestrol acetate: pharmacology, safety profile and therapeutic efficacy. Drugs 70:541–559 Merk Sharp and Dohme (Australia) Pty Limited (2011) Australian public assessment report for nomegestrol acetate/oestradiol. Health safety regulation http://www.tga.gov.au/pdf/auspar/ auspar-zoely.pdf. Accessed 13 April 2014 Miyake T, Rooks WHII (1966) The relation between the structure and physiological activity of progestational steroids. In: Dorfman RI (ed) Methods in hormone research, 5th edn. Academic, New York, pp 59–145 Ruan X, Seeger H, Mueck AO (2012) The pharmacology of nomegestrol acetate. Maturitas 71:345–353 Shields-Botella J, Duc I, Duranti E, Puccio F, Bonnet P, Delansorne R, Paris J (2003) An overview of nomegestrol acetate selective receptor binding and lack of estrogenic action on hormonedependent cancer cells. J Steroid Biochem Mol Biol 87:111–122 Thevenot R (1984) Nomegestrol acetate. Drugs Future 9:657–659 van Diepen HA (2012) Preclinical pharmacological profile of nomegestrol acetate, a synthetic 19-nor-progesterone derivative. Reprod Biol Endocrinol 8:10–85 Yang LP, Plosker GL (2012) Nomegestrol acetate/estradiol: in oral contraception. Drugs 72:1917–1928 Yi Z (1992) PK-GRAPH software to calculate pharmacokinetic parameters. Acta Univ Med Second Shanghai 12:272–274
ORIGINAL PAPER
Pharmacokinetics, tissue distribution, and excretion of nomegestrol acetate in female rats Qingbiao Huang • Xiaoke Chen • Yan Zhu Lin Cao • Jim E. Riviere
•
Received: 16 May 2014 / Accepted: 20 August 2014 Springer International Publishing Switzerland 2014
Abstract Nomegestrol acetate (NOMAC), a synthetic progestogen derived from 19-norprogesterone, is an orally active drug with a strong affinity for the progesterone receptor. NOMAC inhibits ovulation and is devoid of undesirable androgenic and estrogenic activities. The aim of this study was to evaluate the pharmacokinetics, tissue distribution, and excretion of NOMAC in female rats. Sprague–Dawley female rats were orally administered a single dose of NOMAC (10, 20 or 40 mg/kg) and drug plasma concentrations at different times were determined by RP-HPLC. Tissue distribution at 1, 2, and 4 h and excretion of NOMAC into bile, urine, and feces after dosing were investigated. The results showed that NOMAC was rapidly absorbed after oral administration, with tmax of Q. Huang and X. Chen contributed equally to this work.
Electronic supplementary material The online version of this article (doi:10.1007/s13318-014-0224-7) contains supplementary material, which is available to authorized users. Q. Huang (&) State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China e-mail: [email protected] X. Chen Department of Research and Development, Pharmaceutics International, Inc., Hunt Valley, MD 21031, USA Y. Zhu L. Cao (&) Department of Reproductive Pharmacology, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China e-mail: [email protected] J. E. Riviere Institute of Computational Comparative Medicine, Kansas State University, Manhattan, KS 66506, USA
1–2 h. The plasma concentration–time curves were fitted in a two-compartment model. The exposure to NOMAC (Cmax and AUC) increased dose proportionally from 10 to 40 mg/ kg. The average CL and t1=2b were 5.58 L/(hkg) and 10.8 h, respectively. The highest concentrations of NOMAC in ovary, liver, kidney, lung, heart, brain, spleen, muscle, and uterus were observed at 2 h, whereas the highest concentrations in stomach, pituitary, and hypothalamus appeared at 1 h. The total cumulative excretion of NOMAC in feces (0–72 h), urine (0–72 h), and bile (0–48 h) was *1.06, 0.03, and 0.08 % of the oral administered dose, respectively. This study indicated that NOMAC had a widespread distribution in tissues, including ovary, pituitary, and hypothalamus, which are main target tissues where NOMAC inhibits ovulation. NOMAC was excreted via both feces and urine with few unchanged NOMAC excreted. Enterohepatic circulation was found in the drug elimination; however, it did not significantly affect tmax . Keywords Nomegestrol acetate NOMAC Pharmacokinetics Tissue distribution Excretion HPLC Abbreviations NOMAC Nomegestrol acetate HPLC High-pressure liquid chromatography AUC Area under the plasma concentration–time curve CL Clearance Cmax Maximum plasma concentration V/F Apparent distribution volume t1=2b Terminal half-life Time to maximum plasma concentration tmax Ka Absorption rate constant K10 Elimination rate constant
Eur J Drug Metab Pharmacokinet
K12 K21 a b
Distribution rate constant from the central compartment to the peripheral compartment Distribution rate constant from the peripheral compartment to the central compartment Rate constant associated with the distribution phase of the concentration–time curve Rate constant associated with the terminal phase of the concentration–time curve
1 Introduction Nomegestrol acetate (NOMAC) is a 19-norprogesterone derivative with a high progestational activity, first reported by Miyake and Rooks (1966). It is an orally active progestogen with a favorable tolerability profile and neutral metabolic characteristics (Lello 2010). NOMAC is designed to bind selectively for the progesterone receptor and lacks significant affinity with other steroid receptors, showing strong antiestrogenic and antigonadotropic activity, but without androgenic or glucocorticoid properties (Lello 2010; van Diepen 2012; Ruan et al. 2012; Yang and Plosker 2012). Unlike some other progestogens, the antigonadotropic effect of NOMAC is mediated at the hypothalamic and pituitary level (Couzinet et al. 1999). In in vitro functional assay, nanomolar affinity of NOMAC was demonstrated in radioligand binding with cytosolic progesterone receptor in human endometrium (Botella et al. 1988) and breast tissue (Duc et al. 1990), and the potency of NOMAC was greater than progesterone. NOMAC had no agonist or antagonist activity at a or b estrogen or mineralocorticoid receptors in Hela cells (a human cervical carcinoma cell line) or CHO cells transfected with human steroid receptors (Merk Sharp and Dohme (Australia) Pty Limited 2011). In addition, NOMAC inhibited the estrogen-induced stimulation of progesterone receptor expression in T47-D human breast cancer cells in vitro (van Diepen 2012). NOMAC has been approved in Europe and Australia and widely used for the treatment of gynecological disorders (menstrual disturbances, dysmenorrhoea, and premenstrual syndrome) (Alsina 2010) and for hormone replacement therapy (HRT) in combination with estradiol (E2) for the relief of post-menopausal symptoms (Shields-Botella et al. 2003). At a dosage of 1.25 mg/day, NOMAC inhibited ovulation while follicle growth was not affected; at a dosage of 2.5 or 5 mg/day, both ovulation and follicle development were significantly suppressed (Bazin et al. 1987). The studies on NOMAC/E2 as a combined oral contraceptive (COC) showed that NOMAC preserved the beneficial hemostatic effects of estrogen and had a neutral or beneficial effect on lipid profiles, while not changing body weight and having no adverse effects on glucose metabolism. In addition, NOMAC
showed a lack of proliferative activity in normal and cancerous breast tissues and did not have a deleterious effect on bone remodeling (Lello 2010; van Diepen 2012; Yang and Plosker 2012). Despite NOMAC has been used in humans in some developed countries, limited information on pharmacokinetics, tissue distribution (especially for targeted organs including ovary, uterus, hypothalamic, and pituitary), and excretion of NOMAC following a single oral administration in animals and humans was available (or disclosed) in the literature due to confidential reasons. In this study, RP-HPLC method was adopted to determine NOMAC concentrations in rat biological matrices, including plasma, tissues, urine, feces, and bile. The pharmacokinetic profiles of NOMAC in female rats were investigated, including (1) the plasma pharmacokinetics of NOMAC; (2) the tissue distribution of NOMAC; (3) the excretion of NOMAC in bile, urine, and feces after oral administration. The results were also useful for new formulation development in the future.
2 Materials and methods 2.1 Chemicals and reagents NOMAC (lot no. 980616, purity [99.2 %, Fig. 1) was gifted from School of Pharmacy, Fudan University Medical Center (Shanghai, China). Two internal standards (IS) included flutamide (lot no. 981217, purity [99.2 %), purchased from Fudan Forward Pharmaceutical Co., Ltd (Shanghai, China) and mifepristone (lot no. 980607, purity [99.8 %), obtained from Zhejiang Xianju Junye Pharmaceutical Co., Ltd (Zhejiang, China). Methanol (HPLC grade) was purchased from Shanghai Chemical Reagent Research Institute Co., Ltd (Shanghai, China). All other chemicals and solvents were of the highest grade of commercially available materials. Purified water obtained via a Milli-Q system (Millipore, Bedford, MA, USA) was used throughout the experiments. 2.2 In vivo animal experiment Healthy Sprague–Dawley female rats weighing 200–320 g (certificate no. 02-49-2) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). Upon arrival in the laboratory, each animal was evaluated by a laboratory veterinarian. The selected healthy female rats were allowed to acclimate for at least 1 week before the experiments. The animal room was maintained at 25 ± 2 C and 50–70 % relative humidity with 12 h light/ dark cycles. Feed and municipal water were provided ad libitum, except when feed was withdrawn *12 h before dosing. The experiments were carried out in compliance
Eur J Drug Metab Pharmacokinet
Fig. 1 Chemical structure of NOMAC
with Chinese Regulations for the Care and Use of Experimental Animals. At the end of the experiment, pentobarbital sodium was used for the euthanasia of the animals. 2.3 Plasma collection and NOMAC extraction Female rats were assigned randomly into three groups (five rats per group) and received 10, 20, and 40 mg/kg NOMAC in a solution of saline (0.5 % Tween-80), respectively, through oral administration. By cutting the tails, blood samples (800 lL) were collected into a clean test tube containing sodium heparin before and 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24 h after drug administration (group 1 in Table 1). Plasma was prepared by centrifugation at 3,000 rpm for 5 min after the blood samples were placed for 30 min, and then stored at -80 C until further analysis (within 4 weeks). Extraction of NOMAC from plasma involved the addition of 10 lL (containing total 1 lg) flutamide (IS) and 3.0 mL diethyl ether into 500 lL of plasma sample (6:1, v/v) in 5 mL centrifuging glass tube and vortex mixing for 1 min. The mixed samples were equilibrated at room temperature for 5 min, extracted twice with diethyl ether (3.0 mL) with vortex mixing for 5 min each time, and then centrifuged at 3,000 rpm for 5 min. The organic and aqueous layers were separated by allowing the mixture to stand in room temperature for 10 min. The top (organic) layer (2.5 mL) was transferred to glass tube and evaporated to dryness under a stream of nitrogen at 40 C. The dried residue was then reconstituted with 30 lL of mobile phase (methanol: water = 70:30, v/v) and centrifuged at 10,000 rpm for 5 min. After vortex mixing for 1 min, a 20-lL aliquot was injected into HPLC system for analysis. 2.4 Tissue distribution studies Fifteen female rats were divided randomly into three groups (five rats per group) and orally administered NOMAC in a solution of saline (0.5 % Tween-80) at a dose of
20 mg/kg. One additional rat was killed pre-dose to provide blank control tissues. The animals were killed at 1, 2, or 4 h after the dosing (group 2 in Table 1). The tissues, including liver, kidney, stomach, brain, heart, lung, muscle, ovary, pituitary, hypothalamus, spleen, and uterus, were promptly removed and washed with saline solution to remove any residual blood. Each tissue sample (*0.5 g) was homogenized with 1.5 mL saline using Polytron PTMR 3000 homogenizer (Kinematica AG, Switzerland) and the leftover on the homogenizer was washed with 0.5 mL saline and transferred to the same tube. Each sample was added with 10 lL flutamide (IS). NOMAC was isolated from the homogenate as described previously for the plasma samples and stored at -80 C until further analysis (within 4 weeks). 2.5 Metabolism and excretion studies Three female rats were orally administered a single dose of NOMAC at 20 mg/kg in a solution of saline (0.5 % Tween-80). The rats (n = 3) were then placed into separate metabolic cages designed for the separation and collection of urine and feces. Urine and feces were collected 3 h before the dosing and 0–6, 6–12, (or 0–12), 12–24, 24–48, and 48–74 h after the dosing (group 3 and 4 in Table 1). Both urine and feces were collected in separate containers surrounded by ice and then frozen at -80 C at the end of each collection interval for further analysis (within 4 weeks). Polyethylene tubes were surgically cannulated into the bile duct of female rats (n = 5). A 20 mg/kg dose of NOMAC in a solution of saline (0.5 % Tween-80) was orally administered to the rats. Bile was collected into successive vials on ice at 3 h before the dosing as a blank control and at 0–2, 2–4, 4–8, 8–12, 12–24, 24–36, and 36–48 h after the dosing (group 5 in Table 1). The bile samples were stored at -80 C for further analysis (within 4 weeks). The volumes of urine and bile, and dry weight of feces during each collection period were measured before being stored in the refrigerator (Table 1). Before HPLC analysis, the urine sample was added with 20 lL (containing 2 lg) mifepristone as IS. After liquid– liquid extraction with diethyl ether for three times, the organic layers were evaporated to dryness under a stream of nitrogen at 40 C. The residue was reconstituted in 30 lL mobile phase and then centrifuged at 10,000 rpm for 5 min after vortex mixing for 1 min. Twenty microliter aliquot of the supernatant was injected into the HPLC system. Fecal samples (50 mg) were dried at 80 C for 2 h, and then soaked in 1 mL methanol at 4 C for 24 h. Five hundred microliters of supernatant was transferred and 20 lL mifepristone (containing 2 lg) was added as IS. The mixed samples were vertically blended for 2 min and then
Eur J Drug Metab Pharmacokinet Table 1 Protocol for the pharmacokinetic study of NOMAC in rats Group
Tissues
1
Plasma
2
Tissues
3
n
Route
Dose (mg/kg)
15
Oral
10, 20, 40
0, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h
15
Oral
20
1, 2, and 4 h
Urine
3
Oral
20
–3–0, 0–6, 6–12, 12–24, 24–48 and 48–72 h
4
Feces
3
Oral
20
–3–0, 0–12, 12–24, 24–48 and 48–72 h
5
Bile
5
Oral
20
–3–0, 0–2, 2–4, 4–8, 8–12, 12–24, 24–36, and 36–48 h
extracted as described previously for urine. Bile sample was subjected to the same procedure as described for urine. 2.6 HPLC analysis Samples were analyzed using Waters system equipped with binary pump, on-line vacuum degasser, autosampler, column compartment, UV detector, and Waters Millennium32 software, as described previously (Huang et al. 2000, 2014). Chromatographic separation was achieved on a lBondapak-C18 column (300 9 3.9 mm, 5 lm; Waters Instruments, Marlborough, MA) and a lBondapak-C18 guard column. An isocratic mobile phase consisted of a mixture of methanol and water (70:30, v/v, %) with a flow rate of 1.2 mL/min, and the column temperature was maintained at 25 ± 2 C throughout the analysis. The eluent was detected by UV detector with the wavelength set at 293 nm. The HPLC chromatograms of the extracted rat plasma, bile, and urine samples with the presence of IS were shown in Fig. 2. 2.7 Pharmacokinetic and statistical analysis The data of plasma concentration versus time for NOMAC were analyzed using PK-GRAPH package (Yi 1992). The pharmacokinetic parameters were estimated by appropriate compartmental methods. The goodness of fit and the most appropriate model were determined by accessing the randomness of the scatter of actual data points around the fitted function. The Student’s t test was used to analyze differences between two groups. The difference in two groups of data with p-value of \0.05 or 0.01 was considered significant. The data were presented as mean ± SD.
Collection times
in female rats was shown in Fig. 3 and the pharmacokinetic parameters were calculated and summarized in Table 2. There were no significant differences in pharmacokinetic parameters compared with groups of 10, 20, and 40 mg/kg, except AUC and Cmax . Both AUC and Cmax exhibited linear increase with the dose administered (r2 [ 0.98, p \ 0.01). The Cmax of NOMAC was obtained at 1–2 h (tmax ) after dosing, and the drug concentration decreased slowly after Cmax . The t1=2b values for NOMAC were 13.14 ± 3.70, 9.33 ± 4.82, and 9.93 ± 3.71 h for dosing groups of 10, 20, and 40 mg/kg, respectively. The values of V/F and CL, as well as K10, were relatively constant compared with three groups. The K12 and K21 values were very similar in the same dosing group, and no significant difference was observed among different groups (p [ 0.05). 3.2 Tissue distribution Tissue distribution studies in Sprague–Dawley female rats after oral dose of 20 mg/kg revealed wide tissue distribution (Fig. 4). In three sampling times (1, 2, and 4 h), the highest tissue concentrations of NOMAC were observed at 2 h after oral dosing, excluding stomach, pituitary, and hypothalamus, whose highest concentrations appeared at 1 h. The stomach samples exhibited the highest drug exposure. The mean Cmax values of ovary, liver, pituitary, hypothalamus, and kidney were 6.4, 4.8, 3.9, 2.5, and 2.2 lg/g, respectively. Other tissues that displayed relative high exposure were lung, heart, brain, and spleen. NOMAC concentrations in muscle and uterus were very low (1.1 and 1.0 lg/g, respectively). 3.3 Urinary, fecal, and biliary excretion
3 Results 3.1 Plasma pharmacokinetics Plasma concentration versus time was modeled by PKGRAPH package and the best fit was achieved by a twocompartment model. A plot of mean plasma drug concentration versus time for oral administration of NOMAC
The NOMAC excretion-time profiles for the urine and feces within 72 h and the bile within 48 h following 20 mg/kg oral administration were described in Fig. 5. The urinary and fecal excretions of NOMAC were completed before 24 h, when the cumulative excretion curve reached the maximum. However, biliary excretion seemed to continue after 48 h according to the ascending curve. The cumulative percentages of intact NOMAC excreted
Eur J Drug Metab Pharmacokinet Fig. 2 Representative HPLC chromatograms of the extracted plasma (a) biliary (b) urinary (c) and fecal (d) samples obtained from rats following a single oral dose of 20 mg/kg of NOMAC. Internal standards (IS) in plasma and biliary samples were flutamide; IS in urinary and fecal samples were mifepristone
Fig. 3 Mean plasma concentration–time profile of NOMAC after oral administration of 10, 20, and 40 mg/kg of NOMAC to rats. At 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 24 h following NOMAC oral administration, plasma samples were collected and processed for HPLC determination. Each data point represents mean ? SD of five rats
Eur J Drug Metab Pharmacokinet Table 2 Pharmacokinetic parameters following NOMAC administration to rats Parameter
Units
Mean ± SD at different doses (mg/kg) 10
20
40
3.87 ± 2.63
1.06 ± 1.06
1.09 ± 0.53•
a
h
-1
1.33 ± 1.62
1.01 ± 0.82
0.37 ± 0.14•
b
h-1
0.06 ± 0.02
0.09 ± 0.03
0.07 ± 0.02•
0.25 ± 0.15
0.74 ± 0.29•
1.63 ± 1.89
1.65 ± 1.95
2.09 ± 0.90•
13.14 ± 3.70
9.33 ± 4.82
9.93 ± 3.71•
0.12 ± 0.05
0.16 ± 0.10•
0.10 ± 0.08•
Ka
t1=2ðKaÞ
h-1
h
t1=2a
h
t1=2b
h
K10 K12 K21
h
-1
h
-1
h
-1
V=F
L/kg
CL
L/h/kg -1
tmax
h
Cmax
ng/mL
AUC
ng 9 h/mL
1.49 ± 1.09
0.17 ± 0.11
0.73 ± 1.06
0.55 ± 0.68
0.54 ± 0.54
0.18 ± 0.01•
0.35 ± 0.29
68.46 ± 53.58
58.95 ± 33.22
52.60 ± 20.59•
5.42 ± 1.09
5.87 ± 0.34
5.44 ± 2.24•
0.99 ± 0.34
2.68 ± 1.56
2.50 ± 0.60**•
143 ± 28
280 ± 91*
589 ± 65**#
1,910 ± 404
3,417 ± 188**
8,698 ± 4,421**#
n = 5 per group; differences of pharmacokinetic parameters between groups 20 mg/kg (or 40 mg/kg) and 10 mg/kg: p [ 0.05; * p \ 0.05; ** p \ 0.01; differences of pharmacokinetic parameters between groups 40 and 20 mg/kg: • p [ 0.05; # p \ 0.01
through the urine and feces within 72 h were 0.03 ± 0.01 and 1.06 ± 0.55 %, respectively. The cumulative percentage excreted to bile was 0.08 ± 0.01 % within 48 h.
4 Discussion
Fig. 4 Tissue distribution of NOMAC in rats (n = 5) after a single oral dose of 20 mg/kg. At 1, 2, and 4 h following oral administration, tissue samples were collected and processed for HPLC determination. Values are presented as mean ? SD
This study showed that NOMAC is rapidly absorbed into the bloodstream, reaching maximum blood concentrations at 1–2 h after dosing, which was similar to that observed in mice, cynomogus monkeys (Merk Sharp and Dohme (Australia) Pty Limited 2011), and humans(1.5–2 h) (Gerrits et al. 2013). NOMAC was distributed fast in tissues and excreted through feces and urine. The hepatoenteral circulation occurred during drug elimination. By comparing pharmacokinetic parameters of NOMAC at 10, 20, and 40 mg/kg using the Student’s t test, no significant difference was observed except for Cmax and AUC, which were dose-proportional. This suggested that NOMAC exhibited linear first-order pharmacokinetic characteristics and no saturation of metabolism occurred in the dose range of 10–40 mg/kg. The value of V/F in rats was 60 L/kg, 2.5-fold larger than in humans (27 L/kg) (Gerrits et al. 2013), which follows allometric principle. The t1=2b of NOMAC was about 10 h, shorter than human t1=2b (42 h) (Gerrits et al. 2013) due to the faster and more extensive metabolism of NOMAC in animals compared with humans (Merk Sharp and Dohme (Australia) Pty Limited 2011).
Eur J Drug Metab Pharmacokinet Table 3 The structures of possible metabolites of NOMAC observed in rat plasma Metabolites no#
Metabolite structure
Metabolite #1
Metabolite #2
Fig. 5 Cumulative excretion of NOMAC in fences, urine, and bile following a single oral dose of 20 mg/kg. Feces and urine samples were collected at 6, 12, 24, 48, and 72 h and bile samples were collected at 2, 4, 8, 12, 24, 36, and 48 h. Values are presented as mean ? SD (urine and bile) or mean ± SD (feces)
The distribution of NOMAC in tissues was rapid, with the highest concentration observed at 2 h post-administration in most tissues. NOMAC had widespread tissue distribution in different tissues, including stomach, ovary, liver, pituitary, kidney and hypothalamus, lung, heart, brain, spleen, muscle, and uterus. The high NOMAC concentration appeared in hypothalamus, pituitary, and brain at 2 h, which indicated that NOMAC can transfer across the blood–brain barrier (BBB) easily. In addition, high levels of NOMAC were observed in the organs including ovary, hypothalamus, and pituitary, which is consistent with the distribution to receptors in target tissues (Bazin et al. 1987; Botella et al. 1986, 1988; Couzinet et al. 1999; Duc et al. 1990). The concentrations of NOMAC in the urine were analyzed till their concentrations decreased below HPLC detection limit. About 0.08 % cumulative amount of NOMAC was eliminated via biliary excretion within 48 h; the excretion was projected to keep active after 48 h according to the slope of the cumulative excretion curve at 48 h, whereas little amount of NOMAC was found in feces after 48 h, and negligible NOMAC excretion was observed in urine after 24 h. The polar nature of NOMAC with good liposolubility prevents drug excretion through the kidney. These findings are consistent with the previous report that compounds whose molecular weight ranged between 150 and 700 demonstrated an increase in the proportion of compounds excreted in the bile versus urine when the molecular weight increased (Calabrese 1983). However,
Metabolite #3
Metabolite #4
Metabolite #5
Nomegestrol (minor)
the enterohepatic circulation of NOMAC did not significantly change tmax because (1) only small portion (*15 %) of NOMAC in the bloodstream undergoes enterohepatic
Eur J Drug Metab Pharmacokinet
circulation; (2) the excretion rates of NOMAC in the bile decreased after 2 h, which can be observed by the slopes of the biliary cumulative excretion curve in Fig. 5. The excretion of intact NOMAC in rat was detected only at low concentrations in feces, urine, and bile, similar to that observed in monkeys and humans (Merk Sharp and Dohme (Australia) Pty Limited 2011; Gerrits et al. 2013). NOMAC is metabolized primarily by hepatic CYP3A4 and CYP3A5, and a possible contributory role by CYP2C19 and CYP2C8 (Yang and Plosker 2012). NOMAC is metabolized into several hydroxylated metabolites and subsequently conjugated with glucuronide or sulfate (Lello 2010), which accounts for only 1.08 or\1 % of intact NOMAC being detected in feces, urine or bile samples of rats. All metabolites of NOMAC had little or no effects on progesterone receptor activity (Lello 2010). The possible structures of NOMAC metabolites in rats were shown in Table 3 (Merk Sharp and Dohme (Australia) Pty Limited 2011).
5 Conclusions NOMAC was fast absorbed, widely distributed throughout tissues, eliminated in female rats via both fecal and renal routes, and has a long terminal half-life. The results can be useful for drug formulation development and pharmacokinetic studies in humans. Acknowledgments The authors would like to thank Gengdi You and Rongfa Lu for excellent technical support. This work was financially supported by Shanghai Modern Biology and Drug Industry Development Foundation (No. 955419004). Conflict of interest The authors report no conflict of interest. The authors are responsible for the content and writing of the paper.
References Alsina JC (2010) After 50 years of ethinylestradiol, another oestrogen in combined oral contraceptives. Eur J Contracept Reprod Health Care 15:1–3 Bazin B, Thevenot R, Bursaux C, Paris J (1987) Effect of nomegestrol acetate, a new 19-nor-progesterone derivative, on pituitaryovarian function in women. Br J Obstet Gynaecol 94:1199–1204 Botella J, Porthe-Nibelle J, Paris J, Lahlou B (1986) Interaction of new 19-nor progesterone derivatives with progestagen,
mineralocorticoid and glucocorticoid cytosolic receptors. J Pharmacol 17:699–706 Botella J, Paris J, Duc I, Lahlou B (1988) Nomegestrol acetate binding to cytosolic progesterone receptor in human endometrium. Med Sci Res 16:299–300 Calabrese EJ (1983) Principles of Animal Extrapolation. Wiley, New York Couzinet B, Young J, Kujas M, Meduri G, Brailly S, Thomas JL, Chanson P, Schaison G (1999) Theantigonadotropic activity of a 19-nor-progesterone derivative is exerted both at the hypothalamic and pituitary levels in women. J Clin Endocrinol Metab 84:4191–4196 Duc I, Botella J, Gillet JY, Duforestel T, Paris J (1990) Nomegestrol acetate binding to human breast tissue. Med Sci Res 18:57–58 Ezan E, Benech H, Bucourt R, Ardouin T, Tchernatinsky C, Thomas JL, Paris J, Grognet JM (1993) Enzyme immunoassay for nomegestrol acetate in human plasma. J Steroid Biochem Mol Biol 46:507–514 Gerrits MG, Schnabel PG, Post TM, Peeters PA (2013) Pharmacokinetic profile of nomegestrol acetate and 17b-estradiol after multiple and single dosing in healthy women. Contraception 87:193–200 Gibaldi M, Perrier D (1982) Pharmacokinetics. Marcel Dekker, New York Huang Q, Cao L, Gu Z (2000) RP-HPLC determination of nomegestrol acetate in plasma of rabbit. Chin J Pharm Anal 20:379–380 Huang Q, Chen X, Zhu Y, Cao L (2014) Development and validation of an HPLC method for the quantitation of nomegestrol acetate in biological matrices of rats. Biomed Chromatogr (Submitted) Jamin C (1992) Female contraception by a normal dose progestogen after 40 years of age. Possible association of nomegestrol-17beta-estradiol acetate by percutaneous route. Rev Fr Gynecol Obstet 87:370–376 Lello S (2010) Nomegestrol acetate: pharmacology, safety profile and therapeutic efficacy. Drugs 70:541–559 Merk Sharp and Dohme (Australia) Pty Limited (2011) Australian public assessment report for nomegestrol acetate/oestradiol. Health safety regulation http://www.tga.gov.au/pdf/auspar/ auspar-zoely.pdf. Accessed 13 April 2014 Miyake T, Rooks WHII (1966) The relation between the structure and physiological activity of progestational steroids. In: Dorfman RI (ed) Methods in hormone research, 5th edn. Academic, New York, pp 59–145 Ruan X, Seeger H, Mueck AO (2012) The pharmacology of nomegestrol acetate. Maturitas 71:345–353 Shields-Botella J, Duc I, Duranti E, Puccio F, Bonnet P, Delansorne R, Paris J (2003) An overview of nomegestrol acetate selective receptor binding and lack of estrogenic action on hormonedependent cancer cells. J Steroid Biochem Mol Biol 87:111–122 Thevenot R (1984) Nomegestrol acetate. Drugs Future 9:657–659 van Diepen HA (2012) Preclinical pharmacological profile of nomegestrol acetate, a synthetic 19-nor-progesterone derivative. Reprod Biol Endocrinol 8:10–85 Yang LP, Plosker GL (2012) Nomegestrol acetate/estradiol: in oral contraception. Drugs 72:1917–1928 Yi Z (1992) PK-GRAPH software to calculate pharmacokinetic parameters. Acta Univ Med Second Shanghai 12:272–274
