Steroids 75 (2010) 252–264
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Single-dose Pharmacokinetics of Nestorone® , a potential female-contraceptive Pramod Vishwanath Prasad a,∗ , Mohammad Bashir b , Regine Sitruk-Ware a , Narender Kumar a,∗ a b
Center for Biomedical Research, The Population Council, The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA Covance Laboratories Inc., 3301 Kinsman Boulevard, Madison, WI 53704, USA
a r t i c l e
i n f o
Article history: Received 17 June 2009 Received in revised form 26 December 2009 Accepted 29 December 2009 Available online 11 January 2010 Keywords: Tissue distribution of Nestorone Metabolism of Nestorone 17␣-Deacetyl-Nestorone 4,5-Dihydro-17␣-deacetyl-Nestorone Excretion of Nestorone
a b s t r a c t A synthetic progestin Nestorone® is being developed for female-contraception. This study was conducted to determine the distribution, metabolism, and excretion of tritium-labeled Nestorone (3 H Nestorone) in adult female rats. Rats were injected subcutaneously (S.C.) with a single dose of 400 Ci 3 H Nestorone/kg BW. Its distribution and concentrations in blood, plasma and other tissues were determined at defined times. The excreta were examined for elimination of 3 H Nestorone. Radioactivity in all samples was analyzed by liquid scintillation counter. Metabolite profiling was performed by HPLC and LC/MS analysis of the plasma, urine, and feces samples. Following subcutaneous injection of 3 H Nestorone, the mean peak concentrations of radioactivity (Cmax ) in the blood and plasma were 58.1 and 95.5 ng equiv. 3 H Nestorone/g, respectively, at 2-h postdose (Tmax ). Thereafter, the concentration of drug steadily declined through 96-h postdose with a terminal elimination half-life (t1/2 ) of 15.6 h. 3 H Nestorone-derived radioactivity was widely distributed in most tissues by 0.5 h and attained a mean maximal concentration by 2-h postdose. Approximately, 81.4% and 7.62% of the administered dose was excreted via feces and urine, respectively. In vivo metabolism of 3 H Nestorone resulted into a total of 19 metabolites. Among them, two metabolites viz., 17␣-deacetyl-Nestorone (M9) and 4,5-dihydro-17␣deacetyl-Nestorone (M19) were identified by HPLC and LC/MS analysis. Metabolite profiling of plasma samples showed that most of the circulating radioactivity was associated with unchanged parent drug, and M19. The M19 was a major metabolite in the profiled urine and feces samples. Presence of large proportion of drug/drug-related material in feces suggested that the biliary excretion is a main elimination route of 3 H Nestorone. The distribution, metabolism, and excretion profiles of 3 H Nestorone obtained in this study provide a fairly good insight about its fate in women. © 2010 Elsevier Inc. All rights reserved.
1. Introduction Nestorone® (16-methylene-17∝-acetoxy 19-norpregn-4-ene3, 20-dione) is a 19-norprogesterone derivative. It is highly selective and potent progestin [1]. It is being investigated for its potential use as a female contraceptive and hormone replacement therapy (HRT) [2–5]. Several formulations such as silastic implants, vaginal ring and transdermal gels are in various phases of clinical trials. Vaginal rings delivering 150 g/day of Nestorone and 15 g/day of ethinyl estradiol is in the most advanced phase of development [6,7]. Earlier studies have shown that Nestorone has maximal progestational activity (as measured by McPhail index) and ovulation inhibition ability, when compared to most of the commonly used progestins. In addition, Nestorone has a unique pharmacological profile as it has neither androgenic nor estrogenic activity [1,8]. It does not affect serum lipid levels or carbohydrate
metabolism in women [9,10]. One of the interesting characteristics of Nestorone is its low bioavailability when given orally. Upon oral ingestion, it is inactivated rapidly during passage through gastrointestinal tract and liver. This property of Nestorone renders it suitable for contraception in lactating women. This is because the infant liver will rapidly metabolize any trace amounts of the Nestorone present in the milk ingested from nursing mother [11]. Although Nestorone has been extensively studied for its use as a female contraceptive, its absorption, distribution, metabolism and excretion (ADME) has not yet been reported. Therefore, in the present study, we investigated the distribution, metabolism and excretion of Nestorone in the adult female rats using 3 H labeled Nestorone. 2. Experimental 2.1. Materials and reagents
∗ Corresponding authors. Tel.: +1 212 327 8743; fax: +1 212 327 7678. E-mail addresses:
[email protected] (P.V. Prasad),
[email protected] (N. Kumar). 0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.steroids.2009.12.011
Nestorone was custom synthesized by Gedeon Richter Ltd., Budapest, Hungary. The tritium-labeled Nestorone (3 H Nestorone) was custom prepared by New England Nuclear (NEN) Corp., Boston,
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MA, USA. The specific activity was reported to be 837 Ci/mg of Nestorone (99.6% radiochemical purity). Absolute ethanol and acetic acid were purchased from Sigma–Aldrich, Inc., St. Louis, MO, USA. Acetonitrile was purchased from ICN Biomedicals, Inc., Costa Mesa, CA, USA. All other organic solvents and reagents were of HPLC and analytical grade, respectively. 2.2. Animal model Twenty-seven adult, female, Sprague–Dawley rats (body weight: 200–225 g; age: 8 weeks) were purchased from Harlan Sprague Dawley, Inc., Indianapolis, IN, USA, and housed in a temperature-controlled vivarium with a 12-h/12-h light/dark cycle, at the Covance Laboratories Inc., Wisconsin, USA, in accordance with the National Institutes of Health (NIH) guidelines outlined in the Public Health Service Policy on Humane Care and Use of Laboratory Animals. The rats were provided certified rodent diet #8728CM (from Harlan Sprague Dawley, Inc.) and fresh water ad libitum. 2.3. Procedures 2.3.1. Study design Following acclimatization to the standard housing conditions for a week, the rats were randomly assigned to the following three groups and were housed as appropriate for the sample collection. (i) Group 1 consisted of only three animals designated for collection of excreta. They were housed in the metabolic cages [Nalge Nunc International (Nalgene), Rochester, NY, USA] designed to quantitatively collect and separate urine and feces. (ii) Group 2 included nine animals designated for collection of blood; and (iii) Group 3 was comprised of 15 animals designated for tissues collection. Rats in both Groups 2 and 3 were housed in individual, suspended, stainless steel wire-mesh cages (Nalge Nunc International). At different time intervals after dosing, blood, urine, feces, and selected tissues were collected from rats. 2.3.2. Dose formulation and administration On the day of injection, dose was prepared as follows: 0.919 mL of 3 H Nestorone solution in absolute ethanol and 0.327 mL of cold Nestorone solution (10.05 mg/mL ethanol) were mixed in a serum vial and the solvent was evaporated under a stream of nitrogen gas. After drying, the residue was dissolved by adding 33 mL of 30% Cavitron (2-hydroxypropyl -cyclodextrin) [Cargill, Wayzata, MN, USA] solution in reverse osmosis (RO) water. The dose formulation was sonicated for 5 min and mixed well by stirring to achieve a homogeneous solution. All animals of Groups 1–3 were individually administered 5 mL of above mixture containing 500 g of 3 H Nestorone/kg BW (400 Ci/kg BW) by subcutaneous injection. The dose was administered in the mid-scapular region. 2.3.3. Collection of samples The urine and feces were collected from the Group 1 animals for the determination of elimination of total radioactivity by housing them in metabolic cages. The sample collection was performed in the plastic containers surrounded by dry ice at 24-h intervals through 120-h postdose. The weight of each sample was recorded. After the last collection, animals were sacrificed with an overdose of isoflurane anesthesia and carcasses discarded. Blood samples were collected for serial pharmacokinetic analyses (approx. 1 mL each) from the jugular vein using hypodermic syringe and transferred into tubes containing sodium-heparin at 0.5-, 1-, 2-, 4-, 8-, and 24-h postdose from three animals/time point in Group 2. Each rat was bled twice. After the collection of second sample of blood, three animals were sacrificed by exsanguination
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(cardiac puncture) under isofluorane anesthesia at 48-h postdose and blood (as much as possible) was collected into tubes containing sodium-heparin. Blood was placed on wet ice until centrifuged and the plasma collected. Three animals/time point in Group 3 were sacrificed by exsanguination (cardiac puncture) under isofluorane anesthesia at 0.5-, 2-, 4-, 24-, and 72-h postdose. Blood (as much as possible) was collected into tubes containing sodium-heparin from all animals at the time of sacrifice. The weight of each sample was recorded. Samples were maintained on wet ice until centrifuged to obtain plasma. The tissues viz., adrenal glands, bone (both femurs), bone marrow (from both femurs), brain, breasts, eyes (both), fat (reproductive), heart, kidneys, large-intestine (including cecum), large-intestinal and cecal contents/wash, liver, lungs, lymph nodes (mesenteric), muscle (thigh), ovaries, pancreas, salivary glands, skin (dorsal, shaved), small-intestine, small-intestinal contents/wash, spleen, stomach, stomach contents/wash, thymus, thyroid, uterus and urinary bladder were collected from each animal. Tissues were excised, rinsed using saline and blotted dry, as appropriate, weighed, and placed on wet ice. All samples, except blood, were stored at −20 ◦ C. Blood was stored at 4 ◦ C until centrifuged. The plasma was harvested, aliquoted, and stored at −20 ◦ C. 2.3.4. Determination of radioactivity in samples Plasma and urine samples were subjected to radioanalyses by measuring total radioactivity by direct liquid scintillation counting in Packard Tri-Carb Scintillation Spectrometer Model 2900 TR. Radioanalyses in all other tissue and excreta samples were accomplished by combustion in a Packard Sample Oxidizer, Model 307 (Packard Instrument Co., Downers Grove, IL, USA) and the resulting tritium was recovered in 10 mL Monophase-S scintillation fluid (PerkinElmer, Inc., Waltham, MA, USA). An Ultima GoldTM XR scintillation cocktail was used for samples analyzed directly for radioactivity in Model 2900 TR liquid scintillation counter (Packard Instrument Co.) for at least 5 min. 2.3.5. Metabolite profiling by HPLC The pooled plasma, urine, and fecal homogenate samples were subjected to 3 H Nestorone metabolite profiling by HPLC using Vydac C18 column. The mobile phases used were 0.1% acetic acid in water (phase A) and acetonitrile (phase B) in a gradient manner. The HPLC flow rate was adjusted to 1 mL/min. The LSC cocktail used was Ultima-FloTM M (PerkinElmer, USA) with a flow rate of 3 mL/min. The radioactivity detection was performed at 254 nm using Packard 500 Series, 2300TR liquid scintillation counter detector (Packard Instrument Co., Meriden, Conn., USA). The total eluent for each sample was collected and analyzed by LSC to determine the column recovery. For samples with low radioactivity levels, 0.5 min fractions were collected using a Foxy fraction collector throughout the run and analyzed by LSC to determine the radioactive profile and column recovery. 2.3.6. Liquid chromatography tandem mass spectrometry (LC–MS/MS) analysis For LC–MS/MS analysis, the HPLC column effluent of each sample was split with approximately 50% of the flow diverted to the mass spectrometer and remaining 50% towards the radiometric detector. To minimize contamination of the mass spectrometer source, the first 10 min of each run was diverted to waste using the switching valve. 2.3.7. Statistical analyses The analyses of data were limited to simple expressions of variation, such as mean and standard deviation. Dose tables were compiled with mean and standard deviation values calculated
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using Excel, Version 8.0e (Microsoft Corporation). Radioanalyses data tables were generated by Debra, Version 5.2a (LabLogic Systems Ltd., Sheffield, UK). Debra is an automated and validated data capture and management system for data collection in absorption, distribution, and excretion studies using radiolabeled test article. Debra captures data from balances and scintillation counters. The maximum concentration (Cmax ) in plasma and the time to reach maximum concentration (Tmax ) were obtained by visual inspection of the raw data. Pharmacokinetic parameters calculated included half-life (t1/2 ), area under the concentration–time curve from time 0 to the last measurable time point (AUC0–t ), and area under the concentration–time curve from 0 to infinity (AUC0–∞ ). Pharmacokinetic parameters were calculated using WinNonlin Professional Edition, Version 4.1 (Pharsight Corporation). 3. Results 3.1. Distribution of 3 H Nestorone in blood and other tissues The mean concentration of radioactivity (ng equiv. 3 H Nestorone/g) in blood and plasma at specified time intervals postdose is graphically presented in Fig. 1. The results clearly indicated that the peak mean concentrations of radioactivity in the blood and plasma were 58.1 and 95.5 ng equiv. 3 H Nestorone/g at 2-h postdose, respectively. Thereafter, the concentrations of 3 H Nestorone-derived radioactivity in both samples steadily declined through 96-h postdose. The overall distribution of radioactivity (ng equiv. 3 H Nestorone/g) in the blood, plasma, and other tissues as a function of time is shown by histogram (Fig. 2). Following S.C. administration of 3 H Nestorone to the female rats, most of the tissues achieved mean maximum concentrations of radioactivities at 2-h postdose. Tissues with the highest mean concentration of radioactivity, besides the GI tract, were: adrenal glands, liver, skin, fat (reproductive), ovaries, breasts, pancreas, salivary glands, and uterus with values of 458, 453, 348, 287, 216, 198, 149, 124, and 119 ng equiv. 3 H Nestorone/g, respectively. Low concentration of
Table 1 Pharmacokinetic parameters for non-volatile radioactivitya in the plasma collected from female rats after a single S.C. injection of 3 H Nestorone (Group 2, 400 Ci/kg BW). Cmax (ng equiv./g)
Tmax (h)
t1/2 (h)
AUC0–t (ng equiv. h/g)
AUC0–∞ (ng equiv. h/g)
95.4
2
15.6
1122
1135
Pharmacokinetic parameters were calculated using single animal data for each time point, except 48 (mean of three animals), 72 (average of 2 animals) and 96 (average of 2 animals)-h postdose. Cmax , maximum concentration; Tmax , time of maximum concentration; t1/2 , elimination half-life; AUC, total area under the curve; equiv., equivalents. Non-volatile radioactivity (remainder radioactivity in dried tissue sample) = (total radioactivity in tissue sample) − (radioactivity lost during evaporation as tritiated-water). a The radioactivity associated with tritiated-water is termed as ‘volatile radioactivity’; whereas, the radioactivity associated with completely dried tissue samples is called as ‘non-volatile radioactivity’. Measurement of non-volatile radioactivity facilitates to quantify the radioactivity lost during evaporation as tritiated-water, as well, the radioactivity remained in completely dried tissue samples. In an ideal condition, it is justified to precise “non-volatile” for the radioactivity measured in completely dried samples.
radioactivity was found in the bone marrow (from both femurs), bone (both femurs), and eyes (both) with mean values 49.6, 31.3, and 31.3 ng equiv. 3 H Nestorone/g, respectively. The distribution of radioactivity steadily declined over time with low concentrations of 3 H Nestorone-derived radioactivity detected in all the tissues at 72-h postdose. The liver, kidneys, skin (dorsal, shaved), and fat (reproductive) contained the highest levels of radioactivity at 72-h postdose, with mean concentrations of 21.0, 7.67, 7.05 and 6.99 ng equiv. 3 H Nestorone/g, respectively. 3.2. Pharmacokinetic parameters The PK parameters of non-volatile radioactivity in the plasma achieved a peak value at 2-h (Tmax ) postdose with a Cmax value 95.4 ng equiv. 3 H Nestorone/g. The Cmax value was found to decline steadily with a terminal elimination half-life (t1/2 ) of 15.6 h (Table 1). 3.3. Excretion of 3 H Nestorone The percent amount (%) of 3 H Nestorone eliminated in urine and feces during the consecutive intervals of 24-h postdose over the period of 120 h, from each animal, is shown in Table 2; whereas, Table 2 Percentage radioactive doses in the urine and feces at specified time intervals after a single S.C. administration of 3 H Nestorone to the female rats (Group 1, 400 Ci/kg BW). Collection interval (h)
Percent radioactive dose Mean ± SD
Animal number 1
Fig. 1. Mean concentrations of radioactivity in the blood and plasma versus time profiles. Following an S.C. injection of 3 H Nestorone (400 Ci/kg BW) to the adult female rats of Group 2, the peak concentrations of drug in the blood and plasma were achieved at 2-h postdose and were found to decline steadily through 96 h, the period of study.
2
3
Urine 0–24 24–48 48–72 72–96 96–120 Subtotal
5.68 0.64 0.21 0.08 0.04 6.65
6.63 1.17 0.47 0.20 0.06 8.53
6.14 1.02 0.34 0.13 0.05 7.68
6.15 ± 0.48 0.94 ± 0.27 0.34 ± 0.13 0.14 ± 0.06 0.05 ± 0.01 7.62 ± 0.94
Feces 0–24 24–48 48–72 72–96 96–120 Subtotal
49.1 26.8 5.40 1.74 0.39 83.4
31.5 34.6 9.65 2.70 0.73 79.2
37.5 32.7 8.66 2.03 0.62 81.5
39.4 ± 9.0 31.4 ± 4.1 7.90 ± 2.22 2.16 ± 0.49 0.58 ± 0.17 81.4 ± 2.1
SD, standard deviation.
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Fig. 2. Histogram showing mean concentrations of radioactivity in the blood, plasma, and other tissues at different time intervals of a single S.C. dose of 3 H Nestorone to adult female rats (Group 3, 400 Ci/kg BW). In both figures (a) and (b), the histograms obtained at 72-h (red color) postdose are not visible in most cases because the distribution of 3 H Nestorone in majority of tissues were not well pronounced on the scale used on the Y-axis. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
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Table 3 Cumulative amount (%) of radioactive dose in the urine and feces at specified time intervals after a single S.C. administration of 3 H Nestorone to the female rats (Group 1, 400 Ci/kg BW). Collection interval (h)
Percent radioactive dose Mean ± SD
Animal number 1 Urine 0–24 0–48 0–72 0–96 0–120 Feces 0–24 0–48 0–72 0–96 0–120
2 5.68 6.32 6.53 6.61 6.65
49.1 75.9 81.3 83.0 83.4
3 6.63 7.80 8.27 8.47 8.53
31.5 66.1 75.8 78.5 79.2
6.15 ± 0.48 7.09 ± 0.74 7.43 ± 0.87 7.57 ± 0.93 7.62 ± 0.94
6.14 7.16 7.50 7.63 7.68 37.5 70.2 78.9 80.9 81.5
39.4 ± 9.0 70.7 ± 4.9 78.7 ± 2.8 80.8 ± 2.3 81.4 ± 2.1
SD, standard deviation.
Fig. 4. Representative HPLC chromatogram of Nestorone.
the feces and 7.62% was excreted in the urine by 120-h postdose. Approximately 78% of the radioactivity was recovered in the feces and urine within 48 h of S.C. injection. The overall mean recovery of radioactivity was 90.4%, after subcutaneous dosing. The analyses of urine samples for radioactivity before and after drying indicated that the presence of tritiated-water generally increased over time. 3.4. 3 H Nestorone metabolite profiling in plasma, urine and feces by HPLC
Fig. 3. Mean cumulative percent of radioactive dose in the urine and feces at specified time intervals after a single S.C. dose of 3 H Nestorone to the adult female rats (Group 1, 400 Ci/kg BW).
both Table 3 and Fig. 3 present a trend of the cumulative amounts (%) of excreted radioactivity via urine and feces until 120-h postdose. After S.C. administration of 3 H Nestorone to the female rats, a mean of 81.4% of the administered radioactivity was excreted in
3.4.1. Plasma The profile of 3 H Nestorone and its metabolites in pooled plasma samples at 1-, 2-, and 8-h postdose was determined. The percentage and concentrations (ng equiv. 3 H Nestorone/g) of parent drug and metabolites in the plasma are presented in Table 4. A representative HPLC chromatogram of Nestorone is presented in Fig. 4. Representative radiochromatograms of plasma samples are shown in Fig. 5. The profiles of plasma samples collected after S.C. administration of the drug showed 10 metabolites in addition to unchanged parent drug. An unchanged parent drug was major component in the plasma, with a maximum concentration of 27.1 ng equiv. 3 H Nestorone/g, at 2-h postdose, corresponding to 23.5% of the sample radioactivity. The concentration of parent drug was then declined to 3.49 ng equiv. 3 H Nestorone/g, by 8-h postdose. A metabolite M19 (4,5-dihydro-17␣-deacetyl-Nestorone) was the major circulating metabolite in the plasma samples at all evaluated time points, excluding void volume radioactivity (M1). A Cmax value of 36.8 ng equiv./g of 4,5-dihydro-17␣-deacetyl-Nestorone was
Table 4 The concentration and percentage of parent drug and metabolites in the pooled plasma samples collected at 1-, 2-, and 8-h postdose from female rats after a single S.C. administration of 3 H Nestorone (Group 2, 400 Ci/kg BW). Peak identification
Retention time (min)
Collection time (h) 1
2
8
3
ng equiv. H Nesterone/g (% total radioactivity in the sample) M1 2.80–2.90 M2 13.40–13.70 M3 14.20 M4 15.00 M5 15.60–15.80 M6 16.40–16.90 M9 26.80–27.00 M12 30.80 M14 33.30–33.80 M19 45.70–46.30 Nestorone 56.60–57.00 Total radioactivity concentration (ng equiv. 3 H Nesterone/g) Pool radioactivity concentration (ng equiv. 3 H Nesterone/g) in sample (% total radioactivity in the sample) ND, peak not detected; limit of quantitation 1.0% of run or greater.
49.4 (55.5) 1.39 (1.56) ND (ND) 2.27 (2.55) 1.64 (1.84) 1.01 (1.13) ND (ND) ND (ND) ND (ND) 11.5 (12.9) 14.4 (16.2) 81.5 76.5 (91.68)
48.4 (41.9) ND (ND) 1.17 (1.01) ND (ND) ND (ND) ND (ND) ND (ND) ND (ND) ND (ND) 36.8 (31.8) 27.1 (23.5) 114 95.5 (98.2)
22.5 (41.0) ND (ND) ND (ND) ND (ND) ND (ND) 0.63 (1.14) 0.81 (1.47) 0.89 (1.63) 0.89 (1.63) 21.1 (38.4) 3.49 (6.37) 50.3 49.2 (91.7)
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Fig. 5. Metabolic profile in the plasma sample at 8 h following a single S.C. dose of 3 H Nestorone to the adult female rats (Group 2, 400 Ci/kg BW). The metabolic profiles of Nestorone were studied in the plasma samples collected at 1, 2 and 8 h following a single S.C. dose of 3 H Nestorone to the adult female rats. The HPLC profiles appeared to be almost similar in the said samples. Therefore, only one chromatogram obtained from 8-h plasma sample is shown because it exhibits the maximum conversion of tritiated Nestorone into the metabolites M1 and M19.
detected in the 2-h sample, corresponding to 31.8% of the sample radioactivity. The minor metabolite, 17␣-deacetyl-Nestorone (M9), was only detected at the 8-h time point. The concentration of unidentified M1 ranged from 22.5 to 49.4 ng equiv. 3 H Nestorone/g. The remaining unidentified M2 though M6 and M12 and M14 metabolites were present at various time points, where concentrations of any single metabolite never exceeded 2.5 ng equiv. 3 H Nestorone/g. 3.4.2. Urine The profile of 3 H Nestorone metabolites in the urine was determined in a pooled (0–48 h) sample. The parent drug and metabolites in the urine, calculated as percentage radioactivity in the sample and percent of dose, are presented in Table 5. The metabolic profile of the urine sample showed 12 metabolites in addition to unchanged parent drug [Fig. 6(a)]. The unchanged parent drug excreted in the urine through 48 h accounted for 0.47% of the dose. The 4,5-dihydro-17␣-deacetylNestorone was a major metabolite in urine, accounting for 2.61% of the dose. The void volume radioactivity (M1) accounted for 1.51% of the dose. The remaining unidentified metabolites each accounted for
