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Medroxyprogesterone acetate and estradiol cypionate injectable suspension (Cyclofem) monthly contraceptive injection: steady-state pharmacokinetics
Contraception 87 (2013) 738 – 743
Original research article
Medroxyprogesterone acetate and estradiol cypionate injectable suspension (Cyclofem) monthly contraceptive injection: steady-state pharmacokinetics☆ Andrea Thurman a,⁎, Thomas Kimble a , Peter Hall b , Jill L. Schwartz c , David F. Archer a a
CONRAD Clinical Research Center, Eastern Virginia Medical School, Norfolk, VA 23507, USA b Concept Foundation, Pathumthani, Thailand 12120 c CONRAD, Eastern Virginia Medical School, Rosslyn, VA 22209, USA Received 26 September 2012; revised 19 October 2012; accepted 8 November 2012
Abstract Background: Cyclofem is a combined injectable contraceptive, containing medroxyprogesterone acetate (MPA) and estradiol cypionate. The objective was to characterize the steady-state pharmacokinetics (PK) using tandem liquid chromatography/mass spectrometry and compare these data to a previous PK study of this formulation in US women. Study Design: Fifteen ovulatory, surgically sterile women received three Cyclofem injections, once every 28 days, with serum PK measurements on 23 separate days. Trough levels of estradiol and MPA were obtained on Days 1, 29 and 57, prior to each of the three injections. Steady-state concentrations of MPA and estradiol were assessed during the third treatment month on Days 58, 60, 62, 64, 67, 69, 71, 75, 78 and 85. MPA and serum progesterone levels were measured during the follow-up phase to assess MPA clearance (Days 92, 99, 106, 113, 120, 127, 134 and 141) and return of ovulation (Days 103, 106, 131 and 134). Results: In the steady state, mean serum MPA concentrations peaked at 1.31 ng/mL at 4.1 days. Mean estradiol levels peaked at 254 pg/mL by 3.3 days. Ovulation was suppressed for at least 77 days post third injection in all but one woman. Conclusions: Once monthly injections of Cyclofem resulted in contraceptive levels of MPA without accumulation of hormones, consistent with a previous US study. © 2013 Elsevier Inc. All rights reserved. Keywords: Cyclofem; Injectable contraceptive; Pharmacokinetics; Combined contraceptive; Lunelle
1. Introduction Unintended or mistimed pregnancy is a worldwide problem with far reaching implications. Accordingly, there has been an emphasis on developing alternative contraceptive options with acceptable side effect profiles to improve compliance and decrease the incidence of unintended
☆
Funding Info: This study was funded by CONRAD (with funds from a Cooperative Agreement with the US Agency for International Development) and by the Concept Foundation and Sun Pharmaceuticals. The views expressed by the authors do not necessarily represent those of the funding organizations. ⁎ Corresponding author. CONRAD Clinical Research Center, Eastern Virginia Medical School, Norfolk, VA 23507, USA. Tel.: + 1 757 446 7444; fax: + 1 757 446 8998. E-mail address: [email protected] (A. Thurman). 0010-7824/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.contraception.2012.11.010
pregnancies. The majority of long-acting reversible contraceptive methods are progestin (P) based regimens, and despite excellent contraceptive efficacy, breakthrough bleeding and menstrual irregularities are the most prevalent side effects and a common reason for their discontinuation [1,2]. Despite the existence of several effective contraceptives, incorrect use, inconsistent use and discontinuation of the methods contribute to the estimated 3.0 million unintended pregnancies in the United States (US) each year [3,4]. New contraceptive options are needed to meet the goals of Healthy People 2010 to increase the percentage of intended pregnancies from 49% to 71% [5]. Cyclofem is a monthly injectable contraceptive containing 25 mg of medroxyprogesterone acetate (MPA) and 5 mg of estradiol cypionate (E2C), a long-acting ester of estradiol (E2), as a microcrystalline suspension in 0.5-mL aqueous solution. This combined injectable contraceptive (CIC) is
A. Thurman et al. / Contraception 87 (2013) 738–743
administered at 28-day intervals and results in predictable, cyclic bleeding in most users [6,7]. Due to the presence of an estrogen component, bleeding patterns with Cyclofem are more predictable than with MPA alone [7]. Studies of Cyclofem were previously undertaken in populations throughout the world, with approximately 46,000 womenmonths of experience generated in clinical trials and N 100,000 women-months of experience in the introductory studies [8]. Cyclofem has been shown to be highly effective with a reported overall cumulative life table failure rate of 0.1 per 100 women-years [9]. This formulation became available in the US, under the brand name of Lunelle (Pfizer, New York, NY, USA) in 2000. Twelve-month pregnancy rates were low (b 0.2%) in clinical trials with Lunelle [10]. It was estimated that approximately 350,000 US women had used Lunelle when it was withdrawn from the US market because of production problems. The goal of the current study was to replicate the study design and findings of a previous pharmacokinetic (PK) and pharmacodynamic (PD) study performed in the US [11] using a recently manufactured Cyclofem (Sun Pharmaceuticals, India) with the most current method of PK and PD analysis, namely, tandem liquid chromatography/mass spectrometry. This PK study of Cyclofem is the first step in the process of obtaining US Food and Drug Administration (FDA) approval with the ultimate goal to expand the access and range of methods available to women in resource poor areas worldwide through the introduction of a highly effective family planning method. Approval of Cyclofem by the US FDA would also allow the reintroduction of a CIC to the US market. The current study is designed to provide critical information to determine similar bioavailability of Cyclofem to Lunelle in women residing in the US.
2. Materials and methods This study was approved by the Chesapeake Institutional Review Board (#Pro00004759) and registered with ClinicalTrials.gov (#NCT01699022). It was an open-label Phase I study conducted at the CONRAD Clinical Research Center located in the Jones Institute for Reproductive Medicine at the Eastern Virginia Medical School in Norfolk, Virginia. Each potential participant was counseled regarding the rationale for the study and the risks and benefits of the test product and was asked to sign an informed consent before enrollment into the protocol. Participants had to be between 18 and 45 years of age, inclusive, in good health, not at risk of pregnancy due to surgical tubal occlusion, have had regular menstrual cycles (every 21–35 days) for the past two cycles and have a body mass index (BMI) between 18 and 30 kg/m 2 inclusive. To confirm that participants were ovulatory, serum P measurements in the control menstrual cycle, on Days 18 and 21, had to be both at least 3 ng/mL. Potential participants were
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excluded if they had category 3 or 4 contraindications to estrogen-containing hormonal contraception, based on the World Health Organization medical eligibility criteria for contraception [12]. Twenty-four women were screened for the study and 17 were enrolled. After the control menstrual cycle, which documented normal ovulatory function, women subsequently underwent 23 blood draws for PK analyses. Seventeen women received the first intramuscular injection of Cyclofem, starting on the first day of menstruation in the first treatment menstrual cycle (Day 1). Of these 17 women, 2 discontinued prior to the second Cyclofem injection, leaving 15 women, who received a total of three injections, once every 28 days, with evaluable data. MPA and E2 serum levels were measured on Days 1, 29 and 57 just before each Cyclofem injection, in order to obtain MPA and E2 trough levels. Peripheral blood samples for measurement of E2 and MPA were obtained by venipuncture three times a week to determine steady-state PK assessments during the third treatment month (Days 58, 60, 62, 64, 67, 69, 71, 75, 78 and 85). MPA levels were also measured on Days 92, 99, 106, 113, 120, 127, 134 and 141 after the first injection to assess clearance of MPA after three treatment cycles. Serum P levels were measured on Days 103, 106, 131 and 134 during the follow-up phase to determine time to return of ovulation after the third injection. Study end points were (1) trough MPA and E2 levels just prior to the first, second and third Cyclofem injections; (2) steady-state PK parameters of MPA and E2 after the third Cyclofem injection including mean maximum serum concentration (Cmax), time (days) of Cmax (Tmax) and the area under the curve (AUC) of 28 days for E2 and 84 days for MPA and (3) return of ovulation as measured by changes in serum P concentration. Blood was collected in serum separator tubes and centrifuged in a refrigerated centrifuge at 3500 rpm for 25 min. Samples were stored at − 20°C prior to analysis. AUC was calculated over the dosing interval using the trapezoidal rule. MPA and E2 concentrations were determined by tandem liquid chromatography/mass spectrometry at PPD Laboratories (Richmond, VA, USA), which is a GLP (good laboratory practice) certified laboratory. All samples from the same subject were run in the same assay in order to reduce intra assay variability. The assay sensitivity (lower limit of quantitation) for E2 was 1 pg/mL, and the assay was validated over a range of 1–100 pg/mL. For MPA, the assay sensitivity was to 0.02 ng/mL, and the assay was validated over a range of 0.02–5 ng/mL. Serum P concentrations were determined using an enzyme-amplified chemiluminescence assay by the Reproductive Endocrinology laboratory at the Jones Institute (Norfolk, VA, USA). Statistical analysis was performed with SAS version 9.3 (Cary, NC), and p values of less than .05 were considered significant. PK analysis of data was performed by Sun Pharmaceuticals, India. Data from this study were compared to a previous study of Lunelle in US women, which included 14 women [11].
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A. Thurman et al. / Contraception 87 (2013) 738–743 Screened 24 Participants
7 Screen Fails: 3 patients with control cycle luteal phase serum p<3 ng/mL. 3 patients withdrew consent 1 patient screened for study but study closed prior to enrollment
17 Enrolled Participants
Two patients discontinued: 1 due to rash (possibly related to product) 1 due to ankle edema (unlikely related to product)
15 women with evaluable follow up data 1 patient missed day 67 blood draw 1 patient missed last 5 blood draws
Fig. 1. Screening, enrollment and loss to follow-up of participants.
3. Results Fig. 1 documents study enrollment, screen fails and loss to follow-up. Twenty-four women were screened for the study, 17 received the first injection. Of these 17 women, 2 were discontinued early due to a rash, which was possibly related to product use and ankle edema, which was unlikely related to product use. Therefore, 15 women who received all three injections have evaluable data. The mean BMI of participants enrolled in our study (24.26 kg/m 2) was not statistically different from the mean BMI of participants enrolled in the previous study of Lunelle in US women by Rahimy et al. (22.92 kg/m 2) (p=.25) [11]. There was no statistical difference between the average age of women enrolled in our study (mean=34.5 years old) versus those enrolled in the previous US study (mean=35.3 years old) (p=.66) [11]. Our study enrolled significantly more nonHispanic Black women (6/15, 40%) than the previous study (1/14, 7.1%) (p=.05, Fisher's Exact Test). For E2, the inter-assay precision ranged from 3.29% to 6.87%. For MPA, the inter-assay precision ranged from 3.84% to 11.5%. The mean trough (Cmin) levels of MPA
and E2 are summarized in Table 1. For MPA, the serum levels at these four sampling periods were significantly different (ANOVA pb.001) reflecting differences from baseline. Post hoc analysis showed no accumulation of MPA once steady state was reached (concentrations at Day 57 versus Day 85, Tukey p value=.99), and steady-state concentrations were achieved by the end of the second injection (Day 29 versus Day 57, Tukey p=.007). The mean trough levels of E2 on Days 1, 29, 57 and 85 were not significantly different from each other (ANOVA p=.57). These data indicate no accumulation of exogenous hormones in the steady state. In addition, mean trough (Cmin, Day 28) concentrations of MPA for the three consecutive monthly injections ranged from 0.27 (± 0.09) to 0.44 (± 0.19) ng/mL (Table 2), indicating that the Cmin concentrations 28 days after each injection were all greater than the threshold MPA concentrations expected to exert a contraceptive effect (0.10–0.20 ng/mL) [13,14]. The mean serum concentration-time profile for MPA post third injection is shown in Fig. 2, and a summary of the steady-state PK parameters of MPA is shown in Table 2. Mean serum MPA concentrations peaked at 4.1 days (range
Table 1 Trough concentrations (Cmin) of MPA and estradiol (E2) after three monthly injections of MPA/E2C Day of study
1 29 57 85
MPA (ng/mL)
Baseline 28 days post 1st dose 28 days post 2nd dose 28 days post 3rd dose
E2 (pg/mL)
Mean (S.D.)
Range
Mean (S.D.)
Range
0 0.27 (0.09) 0.43 (0.13) 0.44 (0.19)
0 0.14–0.48 0.28–0.65 0.24–1.02
45.55 (17.78) 43.92 (18.73) 51.55 (21.94) 41.65 (16.69)
18.40–70.90 7.66–69.50 29.90–119.00 16.80–71.30
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Table 2 Comparison of PK data from this study to previous US study 11 Variable MPA (ng/mL) Cmax (ng/mL) Tmax (days) AUC 0–28 days (ng a day/mL) AUC 0–84 days (ng a day/mL) AUC 0–∞ days (ng a day/mL) T1/2 (days) Cmin prior to third injection (Day 57) (ng/mL) Estradiol (pg/mL) Cmax (pg/mL) Tmax (days) AUC 0–28 (pg a day/mL) AUC 0–∞ (pg a day/mL) T1/2 (days) Cmin prior to third injection (Day 57) (pg/mL) a
Mean±S.D. from this study
Mean±S.D. from the study of Rahimy et al.
p value
1.31±0.44 4.1±5.0 20.58±6.58 31.53±7.48 33.51±8.17 17.1±7.5 0.43±0.13
1.25±0.33 3.5±2.9 21.51±3.98 32.13±6.26 33.65±7.59 14.7±7.8 0.44–0.47
.58 .67 .59 .76 .95 .24 .21
254±94 3.3±2.5 3290±889 3922±1019 10.1±4.9 51.55±21.94
247±97 2.1±1.9 2741±557 2992±592 8.4±4.3 40–55
.75 .09 .03 .003 .20 .49
Rahimy et al. [11].
1–21 days) after the third monthly administration of Cyclofem. The average steady-state Cmax for MPA was 1.31 ng/mL. Serum MPA concentrations gradually declined thereafter with a mean half-life (T1/2) of 17.05 days, indicating that absorption of MPA from the injection site is prolonged after intramuscular administration. The time for MPA concentrations to fall below the lower limit of quantification (b 0.02 ng/mL) after the third injection ranged from 56 to 84 days after the third injection (the last PK sampling time point). The mean serum concentration-time profile for E2 indicates that absorption of E2 from the injection site, like MPA, was prolonged after the Cyclofem injection (Fig. 3). Mean serum concentrations of E2 peaked by 3.3 days (range
1–7 days) after the third monthly injection, and the average Cmax was 254.73 pg/mL (Table 2). Serum E2 levels declined faster than MPA with a T1/2 of approximately 10.09 days; E2 concentrations were at baseline levels (approximately 50 pg/mL) by Day 24 post-injection. One patient had a serum P level N 3 ng/mL by Day 106 (49 days after the third Cyclofem injection), while none of the other 14 patients had a serum P level of more than 3 ng/ mL, indicating ovulation by Day 134 (77 days after the third Cyclofem injection), the final day of serum sampling. Table 2 compares the PK data obtained in this study to the previous data obtained in US women receiving Lunelle [11], as assessed by gas chromatography tandem mass spectrometry. Our sampling intervals were exactly the same as that
Fig. 2. Mean (S.D.) serum concentration-time profile of MPA after the third monthly intramuscular injection of MPA/E2C to surgically sterile women.
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A. Thurman et al. / Contraception 87 (2013) 738–743
Fig. 3. Mean (S.D.) serum concentration-time profile of estradiol after the third monthly im injection of MPA/E2C to surgically sterile females.
done in the previous US study [11]. The previous study of US women reported on 14 women [11], while we have evaluable data on 15 women.
4. Discussion We have duplicated the results of the previous US PK study of this highly effective CIC containing E2C and MPA [11] using current tandem liquid chromatography/mass spectrometry methodology. The previous US based study of Lunelle was designed to address both potential differences in US women and the variations in E2 and MPA values of prior studies. Our participants were similar in age and BMI as the previous US study [11]. We enrolled significantly more non-Hispanic Black women than the previous PK study of Rahimy et al. For the contraceptive component of Cyclofem, MPA, our data were statistically not different from that obtained with Lunelle by Rahimy et al. [11]. Our findings are also in parallel to previous PK studies that evaluated steady-state E2, P and MPA levels after Lunelle administration to women in Latin America, Europe and Asia. Our data are also in accordance with a small study that evaluated PK profiles after prolonged treatment of Chinese women (1-year duration) with Lunelle [17]. Previous studies of Lunelle, like ours, found that mean E2 levels peaked 2–4 days in the steady state, reflecting the exogenous contribution of E2C [7,16]. This peak simulated the pre-ovulatory peak in a normal menstrual cycle and returned to baseline levels approximately 14–24 days postinjection, without a subsequent ovulatory peak in serum P [16]. MPA levels peaked in the first week after injection and became undetectable 28–90 days after the third injection [6,7,16]. We did find higher AUC values for E2 than Rahimy et al. [11]. However, trough data of E2 from our study and
that of Rahimy et al. [11] are reassuring in that they demonstrate there was no accumulation of E2 above pretreatment levels. It is not clear why the AUC for E2 was higher in our patient population than those described by Rahimy et al. [11]. Our participants were of similar age and BMI but did differ significantly in race and ethnicity. We assessed return to ovulation by weekly P measurements after the third and final injection, until Day 134 (77 days after the final Cyclofem injection). One subject had a serum P level N 3 ng/mL by Day 106 (49 days after the third Cyclofem injection), while none of the other 14 subjects had a serum P level over 3 ng/mL prior to Day 134. These data may suggest that ovulation is suppressed for an extended period after the final Cyclofem injection, as is seen with other MPA based methods [13], but we cannot rule out the possibility that weekly P sampling may have missed increases in P levels, indicating earlier return to ovulation. Previous studies of Lunelle found that serum P levels returned to normal luteal phase values in most subjects by 60–90 days after the third injection [6,7,16,17]. Previous studies of Lunelle used radioimmunoassay or gas chromatography tandem mass spectrometry to analyze plasma and serum concentration profiles of MPA and E2 [6,15–17]. In order to obtain FDA approval of Cyclofem, PK analysis must be done by GLP accredited laboratories, as was done in this study. Most current PK analyses are now performed by tandem liquid chromatography/gas spectrometry due to the superior ability of this technique to assess a variety of metabolic and chemical compounds. However, steroid hormones may be accurately assessed by either tandem liquid chromatography/gas spectrometry or gas chromatography tandem mass spectrometry techniques [18]. In general, we found that release of MPA and E2 from the injection site was prolonged, with no accumulation of either hormone. Contraceptive levels of MPA were reached within
A. Thurman et al. / Contraception 87 (2013) 738–743
the first day after the Cyclofem injection. For the contraceptive component of Cyclofem, MPA, our PK findings were not statistically different from those of previous investigators [11]. Accumulation of MPA and E2 were followed due to safety concerns, and our data demonstrated appropriate clearance of both components. The Cmin of E2 and MPA in the steady state were not statistically different from the Rahimy study of Lunelle [11]. These data will be used in support of a new drug application with the United States FDA for Cyclofem produced by Sun Pharmaceuticals, India. In summary, access to highly effective, reversible contraception offers one way to potentially decrease the incidence of unintended and mistimed pregnancies. CICs have more predictable bleeding patterns than MPA alone and are highly effective, discreet and controlled by the woman. Offering various options, in varying dosage forms may provide women with contraceptive alternatives which fit their lifestyle and family planning goals. The goal of this study was to duplicate previous PK data of this CIC in US women, in order to forward the reintroduction of this effective contraceptive into the US and worldwide markets. Acknowledgments The authors thank Bela Oza, MS who served as the clinical study coordinator. References [1] Westhoff C. Depot-medroxyprogesterone acetate injection (DepoProvera): a highly effective contraceptive option with proven longterm safety. Contraception 2003;68(2):75–87. [2] Kaunitz AM. Injectable depot medroxyprogesterone acetate contraception: an update for U.S. clinicians. Int J Fertil Womens Med 1998;43:73–83. [3] Moreau C, Cleland K, Trussell J. Contraceptive discontinuation attributed to method dissatisfaction in the United States. Contraception 2007;76:267–72. [4] Vaughan B, Trussell J, Kost K, Singh S, Jones R. Discontinuation and resumption of contraceptive use: results from the 2002 National Survey of Family Growth. Contraception 2008;78(4):271–83.
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[5] healthy pople.gov. 2010 [cited 19 January 2010]; Available from: http://www.healthypeople.gov/document/html/objectives/09-01.htm. [6] Fotherby K, Benagiano G, Toppozada HK, et al. A preliminary pharmacological trial of the monthly injectable contraceptive cycloprovera. Contraception 1982;25:261–72. [7] Garza-Flores J, Rodriguez V, Perez-Palacios G, et al. A multicentered pharmacokinetic, pharmacodynamic study of once-a-month injectable contraceptives. I. Different doses of HRP112 and of DepoProvera. World Health Organization Task Force on Long-acting Systemic Agents for Fertility Regulation. Contraception 1987;36:441–57. [8] Hall PE. New once-a-month injectable contraceptives, with particular reference to Cyclofem/Cyclo-Provera. Int J Gynaecol Obstet 1998; 62(Suppl 1):S43–56. [9] Said S, Sadek W, Knoieif A, et al. A multicentred phase III comparative study of two hormonal contraceptive preparations given once-a-month by intramuscular injection: I. Contraceptive efficacy and side effects. World Health Organization. Task Force on Long-Acting Systemic Agents for Fertility Regulation. Contraception 1988;37:1–20. [10] Kaunitz AM, Garceau RJ, Cromie MA. Comparative safety, efficacy, and cycle control of Lunelle monthly contraceptive injection (medroxyprogesterone acetate and estradiol cypionate injectable suspension) and Ortho-Novum 7/7/7 oral contraceptive (norethindrone/ethinyl estradiol triphasic). Lunelle Study Group. Contraception 1999;60:179–87. [11] Rahimy MH, Ryan KK, Hopkins NK. Lunelle monthly contraceptive injection (medroxyprogesterone acetate and estradiol cypionate injectable suspension): steady-state pharmacokinetics of MPA and E2 in surgically sterile women. Contraception 1999;60:209–14. [12] World Health Organization. Medical Eligibility Criteria for Contraceptive Use. Update 2008. http://whqlibdoc.who.int/hq/2008/WHO_ RHR_08.19_eng.pdf. [13] Mishell Jr DR. Pharmacokinetics of depot medroxyprogesterone acetate contraception. J Reprod Med 1996;41(5 Suppl):381–90. [14] Rahimy MH, Ryan KK. Lunelle monthly contraceptive injection (medroxyprogesterone acetate and estradiol cypionate injectable suspension): assessment of return of ovulation after three monthly injections in surgically sterile women. Contraception 1999;60:189–200. [15] Rahimy MH, Cromie MA, Hopkins NK, Tong DM. Lunelle monthly contraceptive injection (medroxyprogesterone acetate and estradiol cypionate injectable suspension): effects of body weight and injection sites on pharmacokinetics. Contraception 1999;60:201–8. [16] Aedo AR, Landgren BM, Johannisson E, Diczfalusy E. Pharmacokinetic and pharmacodynamic investigations with monthly injectable contraceptive preparations. Contraception 1985;31:453–69. [17] Zhou XF, Shao QX, Han XJ, Weng LJ, Sang GW. Pharmacokinetics of medroxyprogesterone acetate after single and multiple injection of Cyclofem in Chinese women. Contraception 1998;57:405–11. [18] Diaz-Cruz MS, Lopez de Alda MJ, Lopez R, Barcelo D. Determination of estrogens and progestogens by mass spectrometric techniques (GC/ MS, LC/MS and LC/MS/MS). J Mass Spectrom 2003;38:917–23.
Original research article
Medroxyprogesterone acetate and estradiol cypionate injectable suspension (Cyclofem) monthly contraceptive injection: steady-state pharmacokinetics☆ Andrea Thurman a,⁎, Thomas Kimble a , Peter Hall b , Jill L. Schwartz c , David F. Archer a a
CONRAD Clinical Research Center, Eastern Virginia Medical School, Norfolk, VA 23507, USA b Concept Foundation, Pathumthani, Thailand 12120 c CONRAD, Eastern Virginia Medical School, Rosslyn, VA 22209, USA Received 26 September 2012; revised 19 October 2012; accepted 8 November 2012
Abstract Background: Cyclofem is a combined injectable contraceptive, containing medroxyprogesterone acetate (MPA) and estradiol cypionate. The objective was to characterize the steady-state pharmacokinetics (PK) using tandem liquid chromatography/mass spectrometry and compare these data to a previous PK study of this formulation in US women. Study Design: Fifteen ovulatory, surgically sterile women received three Cyclofem injections, once every 28 days, with serum PK measurements on 23 separate days. Trough levels of estradiol and MPA were obtained on Days 1, 29 and 57, prior to each of the three injections. Steady-state concentrations of MPA and estradiol were assessed during the third treatment month on Days 58, 60, 62, 64, 67, 69, 71, 75, 78 and 85. MPA and serum progesterone levels were measured during the follow-up phase to assess MPA clearance (Days 92, 99, 106, 113, 120, 127, 134 and 141) and return of ovulation (Days 103, 106, 131 and 134). Results: In the steady state, mean serum MPA concentrations peaked at 1.31 ng/mL at 4.1 days. Mean estradiol levels peaked at 254 pg/mL by 3.3 days. Ovulation was suppressed for at least 77 days post third injection in all but one woman. Conclusions: Once monthly injections of Cyclofem resulted in contraceptive levels of MPA without accumulation of hormones, consistent with a previous US study. © 2013 Elsevier Inc. All rights reserved. Keywords: Cyclofem; Injectable contraceptive; Pharmacokinetics; Combined contraceptive; Lunelle
1. Introduction Unintended or mistimed pregnancy is a worldwide problem with far reaching implications. Accordingly, there has been an emphasis on developing alternative contraceptive options with acceptable side effect profiles to improve compliance and decrease the incidence of unintended
☆
Funding Info: This study was funded by CONRAD (with funds from a Cooperative Agreement with the US Agency for International Development) and by the Concept Foundation and Sun Pharmaceuticals. The views expressed by the authors do not necessarily represent those of the funding organizations. ⁎ Corresponding author. CONRAD Clinical Research Center, Eastern Virginia Medical School, Norfolk, VA 23507, USA. Tel.: + 1 757 446 7444; fax: + 1 757 446 8998. E-mail address: [email protected] (A. Thurman). 0010-7824/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.contraception.2012.11.010
pregnancies. The majority of long-acting reversible contraceptive methods are progestin (P) based regimens, and despite excellent contraceptive efficacy, breakthrough bleeding and menstrual irregularities are the most prevalent side effects and a common reason for their discontinuation [1,2]. Despite the existence of several effective contraceptives, incorrect use, inconsistent use and discontinuation of the methods contribute to the estimated 3.0 million unintended pregnancies in the United States (US) each year [3,4]. New contraceptive options are needed to meet the goals of Healthy People 2010 to increase the percentage of intended pregnancies from 49% to 71% [5]. Cyclofem is a monthly injectable contraceptive containing 25 mg of medroxyprogesterone acetate (MPA) and 5 mg of estradiol cypionate (E2C), a long-acting ester of estradiol (E2), as a microcrystalline suspension in 0.5-mL aqueous solution. This combined injectable contraceptive (CIC) is
A. Thurman et al. / Contraception 87 (2013) 738–743
administered at 28-day intervals and results in predictable, cyclic bleeding in most users [6,7]. Due to the presence of an estrogen component, bleeding patterns with Cyclofem are more predictable than with MPA alone [7]. Studies of Cyclofem were previously undertaken in populations throughout the world, with approximately 46,000 womenmonths of experience generated in clinical trials and N 100,000 women-months of experience in the introductory studies [8]. Cyclofem has been shown to be highly effective with a reported overall cumulative life table failure rate of 0.1 per 100 women-years [9]. This formulation became available in the US, under the brand name of Lunelle (Pfizer, New York, NY, USA) in 2000. Twelve-month pregnancy rates were low (b 0.2%) in clinical trials with Lunelle [10]. It was estimated that approximately 350,000 US women had used Lunelle when it was withdrawn from the US market because of production problems. The goal of the current study was to replicate the study design and findings of a previous pharmacokinetic (PK) and pharmacodynamic (PD) study performed in the US [11] using a recently manufactured Cyclofem (Sun Pharmaceuticals, India) with the most current method of PK and PD analysis, namely, tandem liquid chromatography/mass spectrometry. This PK study of Cyclofem is the first step in the process of obtaining US Food and Drug Administration (FDA) approval with the ultimate goal to expand the access and range of methods available to women in resource poor areas worldwide through the introduction of a highly effective family planning method. Approval of Cyclofem by the US FDA would also allow the reintroduction of a CIC to the US market. The current study is designed to provide critical information to determine similar bioavailability of Cyclofem to Lunelle in women residing in the US.
2. Materials and methods This study was approved by the Chesapeake Institutional Review Board (#Pro00004759) and registered with ClinicalTrials.gov (#NCT01699022). It was an open-label Phase I study conducted at the CONRAD Clinical Research Center located in the Jones Institute for Reproductive Medicine at the Eastern Virginia Medical School in Norfolk, Virginia. Each potential participant was counseled regarding the rationale for the study and the risks and benefits of the test product and was asked to sign an informed consent before enrollment into the protocol. Participants had to be between 18 and 45 years of age, inclusive, in good health, not at risk of pregnancy due to surgical tubal occlusion, have had regular menstrual cycles (every 21–35 days) for the past two cycles and have a body mass index (BMI) between 18 and 30 kg/m 2 inclusive. To confirm that participants were ovulatory, serum P measurements in the control menstrual cycle, on Days 18 and 21, had to be both at least 3 ng/mL. Potential participants were
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excluded if they had category 3 or 4 contraindications to estrogen-containing hormonal contraception, based on the World Health Organization medical eligibility criteria for contraception [12]. Twenty-four women were screened for the study and 17 were enrolled. After the control menstrual cycle, which documented normal ovulatory function, women subsequently underwent 23 blood draws for PK analyses. Seventeen women received the first intramuscular injection of Cyclofem, starting on the first day of menstruation in the first treatment menstrual cycle (Day 1). Of these 17 women, 2 discontinued prior to the second Cyclofem injection, leaving 15 women, who received a total of three injections, once every 28 days, with evaluable data. MPA and E2 serum levels were measured on Days 1, 29 and 57 just before each Cyclofem injection, in order to obtain MPA and E2 trough levels. Peripheral blood samples for measurement of E2 and MPA were obtained by venipuncture three times a week to determine steady-state PK assessments during the third treatment month (Days 58, 60, 62, 64, 67, 69, 71, 75, 78 and 85). MPA levels were also measured on Days 92, 99, 106, 113, 120, 127, 134 and 141 after the first injection to assess clearance of MPA after three treatment cycles. Serum P levels were measured on Days 103, 106, 131 and 134 during the follow-up phase to determine time to return of ovulation after the third injection. Study end points were (1) trough MPA and E2 levels just prior to the first, second and third Cyclofem injections; (2) steady-state PK parameters of MPA and E2 after the third Cyclofem injection including mean maximum serum concentration (Cmax), time (days) of Cmax (Tmax) and the area under the curve (AUC) of 28 days for E2 and 84 days for MPA and (3) return of ovulation as measured by changes in serum P concentration. Blood was collected in serum separator tubes and centrifuged in a refrigerated centrifuge at 3500 rpm for 25 min. Samples were stored at − 20°C prior to analysis. AUC was calculated over the dosing interval using the trapezoidal rule. MPA and E2 concentrations were determined by tandem liquid chromatography/mass spectrometry at PPD Laboratories (Richmond, VA, USA), which is a GLP (good laboratory practice) certified laboratory. All samples from the same subject were run in the same assay in order to reduce intra assay variability. The assay sensitivity (lower limit of quantitation) for E2 was 1 pg/mL, and the assay was validated over a range of 1–100 pg/mL. For MPA, the assay sensitivity was to 0.02 ng/mL, and the assay was validated over a range of 0.02–5 ng/mL. Serum P concentrations were determined using an enzyme-amplified chemiluminescence assay by the Reproductive Endocrinology laboratory at the Jones Institute (Norfolk, VA, USA). Statistical analysis was performed with SAS version 9.3 (Cary, NC), and p values of less than .05 were considered significant. PK analysis of data was performed by Sun Pharmaceuticals, India. Data from this study were compared to a previous study of Lunelle in US women, which included 14 women [11].
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A. Thurman et al. / Contraception 87 (2013) 738–743 Screened 24 Participants
7 Screen Fails: 3 patients with control cycle luteal phase serum p<3 ng/mL. 3 patients withdrew consent 1 patient screened for study but study closed prior to enrollment
17 Enrolled Participants
Two patients discontinued: 1 due to rash (possibly related to product) 1 due to ankle edema (unlikely related to product)
15 women with evaluable follow up data 1 patient missed day 67 blood draw 1 patient missed last 5 blood draws
Fig. 1. Screening, enrollment and loss to follow-up of participants.
3. Results Fig. 1 documents study enrollment, screen fails and loss to follow-up. Twenty-four women were screened for the study, 17 received the first injection. Of these 17 women, 2 were discontinued early due to a rash, which was possibly related to product use and ankle edema, which was unlikely related to product use. Therefore, 15 women who received all three injections have evaluable data. The mean BMI of participants enrolled in our study (24.26 kg/m 2) was not statistically different from the mean BMI of participants enrolled in the previous study of Lunelle in US women by Rahimy et al. (22.92 kg/m 2) (p=.25) [11]. There was no statistical difference between the average age of women enrolled in our study (mean=34.5 years old) versus those enrolled in the previous US study (mean=35.3 years old) (p=.66) [11]. Our study enrolled significantly more nonHispanic Black women (6/15, 40%) than the previous study (1/14, 7.1%) (p=.05, Fisher's Exact Test). For E2, the inter-assay precision ranged from 3.29% to 6.87%. For MPA, the inter-assay precision ranged from 3.84% to 11.5%. The mean trough (Cmin) levels of MPA
and E2 are summarized in Table 1. For MPA, the serum levels at these four sampling periods were significantly different (ANOVA pb.001) reflecting differences from baseline. Post hoc analysis showed no accumulation of MPA once steady state was reached (concentrations at Day 57 versus Day 85, Tukey p value=.99), and steady-state concentrations were achieved by the end of the second injection (Day 29 versus Day 57, Tukey p=.007). The mean trough levels of E2 on Days 1, 29, 57 and 85 were not significantly different from each other (ANOVA p=.57). These data indicate no accumulation of exogenous hormones in the steady state. In addition, mean trough (Cmin, Day 28) concentrations of MPA for the three consecutive monthly injections ranged from 0.27 (± 0.09) to 0.44 (± 0.19) ng/mL (Table 2), indicating that the Cmin concentrations 28 days after each injection were all greater than the threshold MPA concentrations expected to exert a contraceptive effect (0.10–0.20 ng/mL) [13,14]. The mean serum concentration-time profile for MPA post third injection is shown in Fig. 2, and a summary of the steady-state PK parameters of MPA is shown in Table 2. Mean serum MPA concentrations peaked at 4.1 days (range
Table 1 Trough concentrations (Cmin) of MPA and estradiol (E2) after three monthly injections of MPA/E2C Day of study
1 29 57 85
MPA (ng/mL)
Baseline 28 days post 1st dose 28 days post 2nd dose 28 days post 3rd dose
E2 (pg/mL)
Mean (S.D.)
Range
Mean (S.D.)
Range
0 0.27 (0.09) 0.43 (0.13) 0.44 (0.19)
0 0.14–0.48 0.28–0.65 0.24–1.02
45.55 (17.78) 43.92 (18.73) 51.55 (21.94) 41.65 (16.69)
18.40–70.90 7.66–69.50 29.90–119.00 16.80–71.30
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Table 2 Comparison of PK data from this study to previous US study 11 Variable MPA (ng/mL) Cmax (ng/mL) Tmax (days) AUC 0–28 days (ng a day/mL) AUC 0–84 days (ng a day/mL) AUC 0–∞ days (ng a day/mL) T1/2 (days) Cmin prior to third injection (Day 57) (ng/mL) Estradiol (pg/mL) Cmax (pg/mL) Tmax (days) AUC 0–28 (pg a day/mL) AUC 0–∞ (pg a day/mL) T1/2 (days) Cmin prior to third injection (Day 57) (pg/mL) a
Mean±S.D. from this study
Mean±S.D. from the study of Rahimy et al.
p value
1.31±0.44 4.1±5.0 20.58±6.58 31.53±7.48 33.51±8.17 17.1±7.5 0.43±0.13
1.25±0.33 3.5±2.9 21.51±3.98 32.13±6.26 33.65±7.59 14.7±7.8 0.44–0.47
.58 .67 .59 .76 .95 .24 .21
254±94 3.3±2.5 3290±889 3922±1019 10.1±4.9 51.55±21.94
247±97 2.1±1.9 2741±557 2992±592 8.4±4.3 40–55
.75 .09 .03 .003 .20 .49
Rahimy et al. [11].
1–21 days) after the third monthly administration of Cyclofem. The average steady-state Cmax for MPA was 1.31 ng/mL. Serum MPA concentrations gradually declined thereafter with a mean half-life (T1/2) of 17.05 days, indicating that absorption of MPA from the injection site is prolonged after intramuscular administration. The time for MPA concentrations to fall below the lower limit of quantification (b 0.02 ng/mL) after the third injection ranged from 56 to 84 days after the third injection (the last PK sampling time point). The mean serum concentration-time profile for E2 indicates that absorption of E2 from the injection site, like MPA, was prolonged after the Cyclofem injection (Fig. 3). Mean serum concentrations of E2 peaked by 3.3 days (range
1–7 days) after the third monthly injection, and the average Cmax was 254.73 pg/mL (Table 2). Serum E2 levels declined faster than MPA with a T1/2 of approximately 10.09 days; E2 concentrations were at baseline levels (approximately 50 pg/mL) by Day 24 post-injection. One patient had a serum P level N 3 ng/mL by Day 106 (49 days after the third Cyclofem injection), while none of the other 14 patients had a serum P level of more than 3 ng/ mL, indicating ovulation by Day 134 (77 days after the third Cyclofem injection), the final day of serum sampling. Table 2 compares the PK data obtained in this study to the previous data obtained in US women receiving Lunelle [11], as assessed by gas chromatography tandem mass spectrometry. Our sampling intervals were exactly the same as that
Fig. 2. Mean (S.D.) serum concentration-time profile of MPA after the third monthly intramuscular injection of MPA/E2C to surgically sterile women.
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Fig. 3. Mean (S.D.) serum concentration-time profile of estradiol after the third monthly im injection of MPA/E2C to surgically sterile females.
done in the previous US study [11]. The previous study of US women reported on 14 women [11], while we have evaluable data on 15 women.
4. Discussion We have duplicated the results of the previous US PK study of this highly effective CIC containing E2C and MPA [11] using current tandem liquid chromatography/mass spectrometry methodology. The previous US based study of Lunelle was designed to address both potential differences in US women and the variations in E2 and MPA values of prior studies. Our participants were similar in age and BMI as the previous US study [11]. We enrolled significantly more non-Hispanic Black women than the previous PK study of Rahimy et al. For the contraceptive component of Cyclofem, MPA, our data were statistically not different from that obtained with Lunelle by Rahimy et al. [11]. Our findings are also in parallel to previous PK studies that evaluated steady-state E2, P and MPA levels after Lunelle administration to women in Latin America, Europe and Asia. Our data are also in accordance with a small study that evaluated PK profiles after prolonged treatment of Chinese women (1-year duration) with Lunelle [17]. Previous studies of Lunelle, like ours, found that mean E2 levels peaked 2–4 days in the steady state, reflecting the exogenous contribution of E2C [7,16]. This peak simulated the pre-ovulatory peak in a normal menstrual cycle and returned to baseline levels approximately 14–24 days postinjection, without a subsequent ovulatory peak in serum P [16]. MPA levels peaked in the first week after injection and became undetectable 28–90 days after the third injection [6,7,16]. We did find higher AUC values for E2 than Rahimy et al. [11]. However, trough data of E2 from our study and
that of Rahimy et al. [11] are reassuring in that they demonstrate there was no accumulation of E2 above pretreatment levels. It is not clear why the AUC for E2 was higher in our patient population than those described by Rahimy et al. [11]. Our participants were of similar age and BMI but did differ significantly in race and ethnicity. We assessed return to ovulation by weekly P measurements after the third and final injection, until Day 134 (77 days after the final Cyclofem injection). One subject had a serum P level N 3 ng/mL by Day 106 (49 days after the third Cyclofem injection), while none of the other 14 subjects had a serum P level over 3 ng/mL prior to Day 134. These data may suggest that ovulation is suppressed for an extended period after the final Cyclofem injection, as is seen with other MPA based methods [13], but we cannot rule out the possibility that weekly P sampling may have missed increases in P levels, indicating earlier return to ovulation. Previous studies of Lunelle found that serum P levels returned to normal luteal phase values in most subjects by 60–90 days after the third injection [6,7,16,17]. Previous studies of Lunelle used radioimmunoassay or gas chromatography tandem mass spectrometry to analyze plasma and serum concentration profiles of MPA and E2 [6,15–17]. In order to obtain FDA approval of Cyclofem, PK analysis must be done by GLP accredited laboratories, as was done in this study. Most current PK analyses are now performed by tandem liquid chromatography/gas spectrometry due to the superior ability of this technique to assess a variety of metabolic and chemical compounds. However, steroid hormones may be accurately assessed by either tandem liquid chromatography/gas spectrometry or gas chromatography tandem mass spectrometry techniques [18]. In general, we found that release of MPA and E2 from the injection site was prolonged, with no accumulation of either hormone. Contraceptive levels of MPA were reached within
A. Thurman et al. / Contraception 87 (2013) 738–743
the first day after the Cyclofem injection. For the contraceptive component of Cyclofem, MPA, our PK findings were not statistically different from those of previous investigators [11]. Accumulation of MPA and E2 were followed due to safety concerns, and our data demonstrated appropriate clearance of both components. The Cmin of E2 and MPA in the steady state were not statistically different from the Rahimy study of Lunelle [11]. These data will be used in support of a new drug application with the United States FDA for Cyclofem produced by Sun Pharmaceuticals, India. In summary, access to highly effective, reversible contraception offers one way to potentially decrease the incidence of unintended and mistimed pregnancies. CICs have more predictable bleeding patterns than MPA alone and are highly effective, discreet and controlled by the woman. Offering various options, in varying dosage forms may provide women with contraceptive alternatives which fit their lifestyle and family planning goals. The goal of this study was to duplicate previous PK data of this CIC in US women, in order to forward the reintroduction of this effective contraceptive into the US and worldwide markets. Acknowledgments The authors thank Bela Oza, MS who served as the clinical study coordinator. References [1] Westhoff C. Depot-medroxyprogesterone acetate injection (DepoProvera): a highly effective contraceptive option with proven longterm safety. Contraception 2003;68(2):75–87. [2] Kaunitz AM. Injectable depot medroxyprogesterone acetate contraception: an update for U.S. clinicians. Int J Fertil Womens Med 1998;43:73–83. [3] Moreau C, Cleland K, Trussell J. Contraceptive discontinuation attributed to method dissatisfaction in the United States. Contraception 2007;76:267–72. [4] Vaughan B, Trussell J, Kost K, Singh S, Jones R. Discontinuation and resumption of contraceptive use: results from the 2002 National Survey of Family Growth. Contraception 2008;78(4):271–83.
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[5] healthy pople.gov. 2010 [cited 19 January 2010]; Available from: http://www.healthypeople.gov/document/html/objectives/09-01.htm. [6] Fotherby K, Benagiano G, Toppozada HK, et al. A preliminary pharmacological trial of the monthly injectable contraceptive cycloprovera. Contraception 1982;25:261–72. [7] Garza-Flores J, Rodriguez V, Perez-Palacios G, et al. A multicentered pharmacokinetic, pharmacodynamic study of once-a-month injectable contraceptives. I. Different doses of HRP112 and of DepoProvera. World Health Organization Task Force on Long-acting Systemic Agents for Fertility Regulation. Contraception 1987;36:441–57. [8] Hall PE. New once-a-month injectable contraceptives, with particular reference to Cyclofem/Cyclo-Provera. Int J Gynaecol Obstet 1998; 62(Suppl 1):S43–56. [9] Said S, Sadek W, Knoieif A, et al. A multicentred phase III comparative study of two hormonal contraceptive preparations given once-a-month by intramuscular injection: I. Contraceptive efficacy and side effects. World Health Organization. Task Force on Long-Acting Systemic Agents for Fertility Regulation. Contraception 1988;37:1–20. [10] Kaunitz AM, Garceau RJ, Cromie MA. Comparative safety, efficacy, and cycle control of Lunelle monthly contraceptive injection (medroxyprogesterone acetate and estradiol cypionate injectable suspension) and Ortho-Novum 7/7/7 oral contraceptive (norethindrone/ethinyl estradiol triphasic). Lunelle Study Group. Contraception 1999;60:179–87. [11] Rahimy MH, Ryan KK, Hopkins NK. Lunelle monthly contraceptive injection (medroxyprogesterone acetate and estradiol cypionate injectable suspension): steady-state pharmacokinetics of MPA and E2 in surgically sterile women. Contraception 1999;60:209–14. [12] World Health Organization. Medical Eligibility Criteria for Contraceptive Use. Update 2008. http://whqlibdoc.who.int/hq/2008/WHO_ RHR_08.19_eng.pdf. [13] Mishell Jr DR. Pharmacokinetics of depot medroxyprogesterone acetate contraception. J Reprod Med 1996;41(5 Suppl):381–90. [14] Rahimy MH, Ryan KK. Lunelle monthly contraceptive injection (medroxyprogesterone acetate and estradiol cypionate injectable suspension): assessment of return of ovulation after three monthly injections in surgically sterile women. Contraception 1999;60:189–200. [15] Rahimy MH, Cromie MA, Hopkins NK, Tong DM. Lunelle monthly contraceptive injection (medroxyprogesterone acetate and estradiol cypionate injectable suspension): effects of body weight and injection sites on pharmacokinetics. Contraception 1999;60:201–8. [16] Aedo AR, Landgren BM, Johannisson E, Diczfalusy E. Pharmacokinetic and pharmacodynamic investigations with monthly injectable contraceptive preparations. Contraception 1985;31:453–69. [17] Zhou XF, Shao QX, Han XJ, Weng LJ, Sang GW. Pharmacokinetics of medroxyprogesterone acetate after single and multiple injection of Cyclofem in Chinese women. Contraception 1998;57:405–11. [18] Diaz-Cruz MS, Lopez de Alda MJ, Lopez R, Barcelo D. Determination of estrogens and progestogens by mass spectrometric techniques (GC/ MS, LC/MS and LC/MS/MS). J Mass Spectrom 2003;38:917–23.