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Formation of Ethinyl Estradiol in Women during Treatment with Norethindrone Acetate

Columbia University College of Physicians and Surgeons (M.C.C., R.A.L.), New York, New York 10032; and University of Southern California School of Medicine, Women’s & Children’s Hospital (X.Z., E.G., F.Z.S.), Los Angeles, California 90033

Address all correspondence and requests for reprints to: Rogerio A. Lobo, M.D., 622 West 168th Street, PH 16-69, New York, New York 10032. E-mail: ral35{at}columbia.edu.

	Abstract

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Context: The significance of the conversion of norethindrone acetate (NET-A), a commonly prescribed, synthetic, oral progestogen, to ethinyl estradiol (EE₂) is controversial.

Objective: We sought to determine accurately the extent to which NET-A may be converted to EE₂, and if so, whether circulating levels of EE₂ would be of clinical significance.

Design, Subjects, and Interventions: We administered NET-A 10, 20, or 40 mg once daily for 7 d to 20 regularly menstruating premenopausal women and measured NET-A and EE₂ levels before drug intake; at 1, 2, 4, 8, and 24 h after the first dose; and 2 h after the seventh dose.

Results: The mean EE₂ maximum serum concentration obtained from the 10-, 20-, and 40-mg doses of NET-A in this study is 58, 178, and 231 pg/ml, respectively. The conversion ratio of NET-A to EE₂ ranged from 0.20 to 0.33% for the different doses.

Conclusions: NET-A is converted to EE₂, and although the conversion rate is relatively small, higher doses of NET-A, as used clinically, give rise to substantial levels of EE₂.

	Introduction

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NORETHINDRONE ACETATE (NET-A) is a synthetic, orally active progestogen used in premenopausal women for the treatment of secondary amenorrhea, endometriosis, and abnormal uterine bleeding. It is also given in combination with estrogen in oral contraceptive formulations and hormone therapy. Previous studies with postmenopausal women receiving oral doses of NET-A-containing preparations have shown that a small fraction of the dose may be converted to ethinyl estradiol (EE₂), although the extent to which this occurs is controversial (1, 2, 3). It has been suggested that every 1 mg of NET-A ingested equates with the ingestion of an oral dose of 6 µg of EE₂ (4). Although this may appear to be a large amount of conversion, it remains to be confirmed whether EE₂ is authentically produced from NET-A and whether the levels produced would have clinical significance (3, 4). In premenopausal women, doses of up to 40 mg are routinely used to treat severe bleeding, endometriosis, and endometrial hyperplasia. If high doses of EE₂ are produced from NET-A, this may be of clinical importance in that EE₂ may have deleterious effects on hemostasis and hepatic metabolism.

Therefore, in this study we sought to determine accurately the extent to which NET-A may be converted to EE₂, and if so, whether circulating levels of EE₂ would be of clinical significance.

	Subjects and Methods

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Study population

We recruited 20 regularly menstruating premenopausal women (ages, 28 ± 1 yr; weight, 62 ± 2 kg; height, 1.65 ± 0.01 m). All women had not received any oral or topical sex hormones for at least 7 d before this study. The subjects underwent a laboratory screening and a thorough medical and gynecological examination before entering the study. Excluded from participation were subjects who had any contraindication to the use of contraceptive steroids or a concomitant medication that might interfere with the pharmacokinetics of NET-A or EE₂. Smoking more than 10 cigarettes per day was prohibited. The study was approved by the Columbia University Institutional Review Board, and all patients gave written informed consent for participation in the study.

Protocol

Subjects were randomized to receive 10, 20, or 40 mg of NET-A daily for 7 d in the early follicular phase of the menstrual cycle. The sequence generation was determined using a computer program that randomly assigned the subjects to one of the three treatment doses. Five-milligram tablets of NET-A were obtained commercially (Barr Laboratories, Inc., Pomona, NY). Blood samples were collected before drug intake; at 1, 2, 4, 8, and 24 h after the first dose; and 2 h after the seventh dose.

Assays

EE₂ and NET were quantified in serum by highly specific and sensitive assays (5). EE₂ was measured by RIA after extraction with ethyl acetate:hexane (3:2) and subsequent Celite column partition chromatography. Separation of antibody-bound steroid from unbound steroid was achieved by the second antibody method. NET was also measured by RIA after extraction with ethyl acetate:hexane (3:2) and Celite column partition chromatography, in a manner similar to that described for the EE₂ assay. The two RIA methods had been previously validated with respect to sensitivity, accuracy, precision, and specificity. Low-, medium-, and high-level quality control samples were used in duplicate in each assay to assess assay reliability. Also, in both RIA methods, the appropriate internal standard ³H-EE₂ or ³H-NET was used to follow procedural losses. The assay sensitivities were 15 and 50 pg/ml for the EE₂ and NET RIAs, respectively. Intraassay and interassay coefficients of variation were in the range of 5–10% and 10–15%, respectively, for both RIAs.

To cross-validate the specificity of the EE₂ RIA, EE₂ was also measured by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (n = 7). The assay was performed at Pharmaceutical Products Development, Inc. (Richmond, VA) as follows: an aliquot (0.05 ml) of each sample was combined with 0.05 ml of internal standard, and the analyte was extracted with organic solvent. After evaporating the solvent, the residue was derivatized, and the derivative was extracted with hexane. The hexane was evaporated, and the residue was reconstituted in 0.3 ml of acetonitrile:water. This extract was then subjected to analysis by HPLC with detection by tandem mass spectrometry. The lower limit of quantification was 2 pg/ml. The intraassay and interassay coefficients of variation were less than 9% and less than 9%, respectively. Identification of authentic EE₂ by LC-MS/MS and its correlation with values by RIA was considered to be confirmatory evidence of conversion between NET-A and EE₂.

Pharmacokinetic evaluation

The area under the serum level-time curve, AUC_{(0–24 h)}, was calculated according to the linear trapezoidal rule. Maximum serum concentrations (C_max) and the times to reach C_max were obtained directly from the data.

The conversion ratio (CR) of NET-A to EE₂ was calculated according to the formula: CR^NET-A/EE2 = AUC (0–24 h)_EE2/AUC (0–24 h)_NET-A.

	Results

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Age and BMI were not statistically different among the three treatment groups.

At baseline, levels of EE₂ were essentially undetectable. Among the eight women who received the 40-mg dose, EE₂ levels peaked at 4 h and were 231 ± 38 pg/ml. At 24 h after the first dose, EE₂ was still significantly detectable (63 ± 8 pg/ml). After 7 d of NET-A administration, levels of EE₂ at 2 h after treatment were 358 ± 73 pg/ml. Women who received the 20-mg dose (n = 6) had a peak EE₂ level of 178 ± 35 pg/ml at 1 h, and the 2-h EE₂ level after 7 d was 134 ± 16 pg/ml. At the 10-mg dose (n = 6), the maximum EE₂ level was 58 ± 7 pg/ml at 2 h, and EE₂ was as high as 77 ± 13 pg/ml at 2 h after 7 d (Fig. 1A and Table 1).

View larger version (24K): [in this window] [in a new window]   FIG. 1. Time courses of mean EE2 levels (A) and mean NET levels (B) with varying doses of NET-A (10 mg, n = 6; 20 mg, n = 6; 40 mg, n = 8).

Seven samples in which EE₂ was detected (range, 30 to 76 pg/ml) were further analyzed by LC-MS/MS. EE₂ was 54.7 ± 5.6 pg/ml by RIA and 37.6 ± 5.9 pg/ml by LC-MS/MS. Higher levels of EE₂ were measured by RIA, but this difference was not statistically significant. The Pearson product moment correlation coefficient r for the RIA and LC-MS/MS values of these seven samples was 0.87.

After single doses of NET-A, peak NET levels were 58 ± 7 ng/ml at 2 h with the 10-mg dose, 122 ± 11 ng/ml at 1 h with 20 mg, and 181 ± 40 ng/ml with 40 mg (Fig. 1B). The conversion ratios of NET-A to EE₂ ranged from 0.20 to 0.33% for the different doses. Because gas chromatography/mass spectrometry detects lower EE₂ levels by approximately 30%, we might expect the conversion to be at least 0.14%.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
The mean EE₂ C_max obtained from the 10-, 20-, and 40-mg doses of NET-A in this study is 58, 178, and 231 pg/ml, respectively. After reviewing some of our EE₂ data and those in the literature, it appears that the EE₂ C_max is in the range of 100–135 pg/ml after doses with 30–40 µg of EE₂ (6, 7, 8, 9). This C_max range is substantially lower than that obtained with 20 mg of NET-A but considerably higher than that obtained with 10 mg of NET-A. Therefore, we can safely say that 20 mg of NET-A may be equivalent to taking a pill containing 30 µg of EE₂.

For the conversion of the calculated AUC_{0–24 h} values of EE₂ into oral dose equivalents (µg EE₂), a mean AUC_{0–24 h} value of 806 ± 222 pg*h/ml was considered to be equivalent to an oral dose of 30 µg EE₂. This represents the mean AUC_{0–24 h} value observed in six independent clinical studies in which women received single doses of different combination oral contraceptives, each containing a dose of 30 µg EE₂. In the present study, the mean AUC_{0–24 h} of EE₂ was 837 pg*h/ml for the 10-mg dose of NET-A. Thus, this finding is also consistent with the equivalence of a 20-mg dose of NET-A to a 30-µg pill.

U.S. Food and Drug Administration-approved doses of NET-A are from 5–15 mg (Aygestin) for bleeding and endometriosis, but often doses as high as 40 mg may be used in clinical practice for various indications. The 40-mg dose of NET-A had an EE₂ C_max of 231 ± 38 pg/ml. This translates into an oral dose of EE₂ in excess of 50 µg. Oral contraceptive pills containing a 50-µg dose of EE₂ have been associated with an increased risk of thrombosis, particularly in older women and in smokers. Our data would suggest that even doses between 10 and 20 mg, if taken chronically, may equate with the estrogenicity of oral contraceptive formulations in the 20–30 µg EE₂ range; which in certain women may be associated with risk.

These data demonstrate that NET-A is converted to EE₂, and although the conversion rate is relatively small, higher doses of NET-A, which may be used clinically, give rise to substantial levels of EE₂. This exposure may be of concern for those women who are more sensitive to high concentrations of estrogen, particularly if taken on a longer term basis.

	Footnotes

 
Disclosure Statement: The authors have nothing to disclose.

First Published Online March 6, 2007

Abbreviations: AUC_{(0–24 h)}, Area under the serum level-time curve; C_max, maximum serum concentration; EE₂, ethinyl estradiol; LC-MS/MS, liquid chromatography-tandem mass spectrometry; NET-A, norethindrone acetate.

Received January 9, 2007.

Accepted February 28, 2007.

	References

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7. Kuhnz W, al-Yacoub G, Fuhrmeister A 1992 Pharmacokinetics of levonorgestrel and ethinylestradiol in 9 women who received a low-dose oral contraceptive over a treatment period of 3 months and, after a wash-out phase, a single oral administration of the same contraceptive formulation. Contraception 46:455–469[CrossRef][Medline]
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