0021-972x/92/7.506-1268503.00/0 rJournal of’Climca1 Endocrinology and Metabolism Copyright B 1992 by The Rndocrlne Sow&y
Vol. 75, No. 5 Printed in U.S A
Existence of Multiple Peaks in Plasma Ethinyl and Norethindrone after Oral Administration Contraceptive Pill* HOWARD A. ZACUR, PETER KWITEROVICH,
RONALD AND
T. BURKMAN, ALLYN WILLIAM R. BELL
Estradiol of a
W. KIMBALL,
Departments of Gynecology and Obstetrics, Biostatistics, Pediatrics, and Medicine, Johns Hopkins Medical Institutions (H.A.Z., A. W.K., P.K., W.R.B.), Baltimore, Maryland 21205; and the Department of Gynecology and Obstetrics, Henry Ford Hospital (R.T.B.), Detroit, Michigan 48202 ABSTRACT
of multiple hormone peaks. Two peaks of EE2 were identified in 44.8% of women during the first pill cycle and in 75.9%, 55.2%, and 67.2% of women after 3, 6, and 9 months of pill use. Two hormone peaks of NE were observed in 29.3% of women during the first cycle and in 36.2%, 50%, and 44.8% at 3, 6, and 9 months, respectively. Existence of these multiple peaks at the frequency observed has not previously been reported. Further quantification of the frequency and magnitude of these peaks could be helpful in explaining differences in biological responses associated with pill use. (J Clin Endocrinol Metab 75: 12681272,1992)
Previous measurements of plasma ethinyl estradiol (EE,) and norethindrone (NE) over 24 h after oral administration of a contraceptive pill have demonstrated a single steroid peak occurring 1-2 h after pill ingestion, with a gradual decline over the next 22 h. In the present study plasma concentrations of EE2 and NE were measured 0,0.5,0.75, 1,2,4, 12, and 24 h after oral ingestion of a contraceptive pill containing 35 fig EE, and 1 mg NE at 0, 3, 6, and 9 months of use in 58 normal healthy women. Contrary to previous reports, analysis of the 464 steroid curves (58 subjects x 4 time periods X 2 steroids) revealed the presence
P
Materials
HARMACOLOGICAL studies have previously been conducted in normal women to determine the absorption, peak plasma level, and half-life of steroid hormones contained within the pill. These studies have almost uniformly found that a single peak in plasma steroid hormone concentrations occurs within l-2 h after oral administration of the pill, with a subsequent decline over the next 22 h (1, 2). Using these pharmacokinetic data, 24-h contraceptive steroid hormone curves have been generated, and the area under these curves calculated. Tremendous variations within and between individuals have been reported using this method (3-5). Since a correlation between the dose of contraceptive steroid and adverse effects exists (6), concern has been raised that for certain individuals, even low dose pills could produce elevated serum levels of contraceptive steroids. An explanation for this variability has remained elusive, and it has not been found to be correlated with age, height, weight, blood pressure, cigarette smoking, alcohol use, total lifetime oral contraceptive use, or the type of estrogen used (3). The present report confirms the previously reported variability in steroid hormone levels in oral contraceptive pill users and demonstrates that the pharmacokinetics of the oral contraceptive steroids may be even more complex than previously realized.
and Methods
Healthy young women (n = 63) between 18-35 yr of age were recruited from the Baltimore area for a clinical study approved by the Johns Hopkins Medical Institutions, Joint Committee on Clinical Investigation. After obtaining written consent, all subjects were evaluated for full eligibility. Criteria for study included regular menstrual cycles between 25-35 days; no contraindications to oral contraceptive use; absence of significant endocrinological, hematological, or metabolic disorders; body weight within 115% of idea1 body weight; no pregnancy or breast feeding within 6 months; and no oral contraceptive or sex steroid use within 12 months. A standardized medical and obstetric/gynecological history was obtained from all participants, which included questions regarding demographic characteristics, tobacco and alcoholic beverage use, diet, and onset of physical exercise. All subjects underwent physical and pelvic examinations. After an overnight fast, serial venous blood samples were obtained following oral ingestion of a combination-type oral contraceptive pill containing 35 pg ethinyl estradiol (EE,) and 1 mg norethindrone (NE). Each participant was started on the pill during the first 5 days of her cycle, after which time she returned for repeat blood sampling during the third, sixth, and ninth months of pill use. With few exceptions, subjects were sampled during the 19th to 21st day of pill use. All subjects received a combination pill provided from the same source (Syntex Laboratories, Palo Alto, CA). While 63 women initially enrolled in this study, only 58 women completed 9 months of evaluation, and only their hormone data are included in the present report. Venous blood samples (15 mL) were obtained before taking the morning pill (generally between 0700-0800 h) and 0.5, 0.75, 1, 2, 4, 12, and 24 h after pill ingestion. In almost all instances, a heparin lock was used to facilitate blood drawing. Blood samples were taken at the time intervals listed to conform with previously reported pharmacokinetic data (l-5). Although they had fasted before the first blood sample was taken, subjects were able to eat thereafter and participate in their normal daily activities, EEI and NE concentrations were measured in plasma samples using a RIA procedure modified from that of Stanczyk et al. (7-9) to permit more rapid preassay chromatographic separation of EE? and NE. Antiserum for EEI was supplied by Dr. C. Edgar Cook of the Research Triangle Institute (Research Triangle Park, NC), unlabeled EE, was ob-
Received February 11, 1992. Address requests for reprints to: Howard A. Zacur, M.D., Ph.D., Johns Hopkins Hospital, Park 82, Room 202A, 600 North Wolfe Street, Baltimore, Maryland 21205. *This work was supported by Contract NOl-HD-32816 from the Contraceptive Evaluation Branch, NICHHD (Bethesda, MD). 1268
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PEAKS
IN CONTRACEPTIVE
STEROID
LEVELS
1269
120-
loo-
$
60-
x I2 z f W = .f 5 w
60-
40-
200
0’
0
I
2I
81
4
1
6I
!
Time FIG. 1. Plasma concentrations ingestion of 35 Kg EE, and cycle of pill use.
8I1
IO!
1
12./
v
l.i5
of EE1 measured over 24 h after 1 mg NE in one subject during the
2
4
Time
Hours oral first
tained from Steraloids, Inc. (Welton, NH), and tritiated steroid was purchased from New England Nuclear Corp. (Boston, MA). Antiserum for NE and NE for the standard were supplied by Dr. Ing Nieuweboer of Schering AG (West Berlin, Germany) and Syntex Laboratories, Inc. (Palo Alto, CA), respectively. Tritiated steroid was purchased from New England Nuclear Corp. Plasma samples were twice extracted with ether and chromatographed on Sephadex LH-20 before assay. All samples were assayed in duplicate. The cross-reactivity of the EEI antiserum to NE was l.l%, and there was less than 0.08% cross-reactivity of the NE antiserum to 17P-estradiol. The EEI assay has a sensitivity of 12 pg/mL, with a within-assay variation of 10.6% and a between-assay variation of 18.7%. The sensitivity of the NE assay was 15 pg/mL; within-assay variation was 11.6%, and between-assay variation was 12.5%.
Analysis Curve fitting for 24-h levels of EE2 and NE was attempted using both smooth compartment models and cubic spline regression models. The statistical methods used included t tests, logistic regression, multiple linear regression, and trichotomous regression, as previously described (10, 11).
Results Over the 9-month course of this study, 464 individual 24h steroid hormone curves were generated (58 subjects X 2 steroids X 4 time periods). Despite all efforts, attempts to apply smooth compartment modeling and cubic spline regression techniques were unsuccessful in fitting the observed RIA data with the modeled curves. Upon close inspection, it became apparent that the reason for this was that the individual curves contained more than one peak, a fea-
Hours
FIG. 2. Plasma concentrations of NE over 24 h after oral administration of 35 pg EE, and 1 mg NE after 6 months of pill use in a single individual.
ture that prevented the application of standard curve-fitting procedures. Figures 1 and 2 are examples of the multiple peaks observed for EE? during the first cycle of pill use in one individual and for NE after 6 months of pill use in another individual. The existence of these multiple peaks is less apparent when mean profiles of steroid concentrations are used, as shown in Figs. 3 and 4. Individuals could exhibit a double peak of EE2, NE, or both. Furthermore, the occurrence of a double peak during the first cycle of pill use did not mean that this would occur subsequently (Table 1). The first steroid hormone peak occurred between l-l.5 h, and the second steroid peak occurred between 3.9-6.7 h (Table 2). A peak was initially defined as a steroid hormone value that exceeded those values preceding and following it. To be certain that peaks were occurring and were not artifacts of the assay, a peak was also identified as such if its value was greater than 10% of the values immediately preceding or following it. When this criterion was used, 75% of all of the double peaks of EE2 and 64% of all of the double peaks of NE originally identified as peaks met this more stringent criteria. Discussion An understanding of the relationship between plasma drug levels and dosage is important to correlate the amount of drug ingested with the biological response (12, 13). For the oral contraceptive pill this has particular relevance, as associations have been sought between steroid dose and
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ZACUR
1270
A
0 MONTHS
3 MONTHS
B
7
160
100. w-
80 EE,
JCE & M. 1992 Vol75.No5
ET AL.
60
EE,
60.
m/ml
Pg/ml
-1
40
40.
20
20.
6
12
16
. . . . . .. . . . . . .. . . . . . .. .. .
+ 0
24
6
t (hrs)
6 MONTHS
C
12
16
24
16
24
t (hrs)
D
9 MONTHS w-
EE,
60.
w/ml
. 40.
0
12
6
16
“rn 0
24
6
t (hrs) FIG. 3. Mean concentrations of EE, in 58 subjects during oral contraceptive pill containing 35 pg EE, and 1 mg NE. TABLE 1. Percent frequency of multiple steroid and months of follow-up in 58 women volunteers
peaks
the initial
pill
by hormone
Month HORPXX
EE, NE
0
3
6
9
44.8
75.9 36.2
55.2 50.0
67.2 44.8
29.3
12
t (hrs)
adverse effects (14). Studies have demonstrated large differences in plasma hormone concentrations between individuals ingesting the same type of pill (15). The reasons for these differences have remained unexplained. After oral administration, steroids in the oral contraceptive pill are absorbed as free steroid or conjugated within the intestinal wall. Absorbed free steroid may then be conjugated by the liver or hydroxylated by mixed function oxidases within the liver. Conjugated steroids may be stored in the gall bladder, then released into the intestine, hydrolyzed, and reabsorbed as free or conjugated steroid or excreted in the feces (16). Previous studies analyzing plasma levels of the steroid components of the oral contraceptive pill have reported only a single early peak response at l-2 h, with a rapid decline in concentration over the next 22 h (1, 2, 17). Secondary peaks of EE2 and NE have occasionally been reported, but no attempt has been made to assess their frequency (18-23). Secondary peaks are presumed to be due to partial degradation associated with enterohepatic cycling. Some researchers have even suggested that these secondary peaks could reflect gall bladder contractions occurring after
cycle
(0 months)
and after
3, 6, and 9 months
of use of a combination
food ingestion (24). Our data confirm that significant differences in blood levels of EE2 and NE occur between individuals after ingestion of a pill containing a standard hormone dose. Our attempts to fit 24-h steroid curves to these values were unsuccessful because the curves frequently exhibited more than a single peak, a finding that had not been anticipated. Two peaks in EE2 levels were observed in 44.8% of women during the very first cycle of pill use. This percentage rose to 75.9% at 3 months and gradually declined to 55.2% at 6 months, only to rise again to 67.2% at 9 months. Subjects also experienced two or more peaks in NE, with 29.3% of the women exhibiting such patterns during the first month, and 36.2%, 50.0%, and 44.8% of the women showing these patterns at 3, 6, and 9 months, respectively. Initial peaks of both EE2 and NE were observed approximately 1.2 h after oral administration, and secondary peaks of both hormones were observed after 3.9-6.7 h. The steroid concentrations of secondary peaks frequently exceeded those of initial peaks, although the mean steroid concentrations between peaks were similar. Individuals could have two peaks during the first cycle of pill use, one peak during the third cycle, and two peaks during the sixth or ninth cycle. Therefore, no predictable pattern could be identified. Overall, however, multiple peaks increased in frequency with the duration of pill use. Previous studies may have failed to observe the steroid responses seen in our study due to the small numbers of subjects sampled, the frequency of blood sampling, or the
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PEAKS IN CONTRACEPTIVE
STEROID
LEVELS
o MONTHS
1271
3 MONTHS
A 8 rig/ml
NE
FIG. 4. Mean contraceptive TABLE month
ib---
~.,........‘,‘,.,.““‘,’ 6 0
concentrations pill containing
2, Mean of follow-up
1 . . . . >, , , , , . , . , . . , , , , , , , * 0 6 12 18 24
24
18
12
t (hrs)
t (hrs)
6 MONTHS
9 MONTHS
of NE in 58 subjects during 35 pg EE2 and 1 mg NE.
maximum hormone in 58 subjects
concentrations
the initial
(+SEM)
pill cycle
and mean
(0 months)
times
and after
of occurrence
Months
(n=o 26, First peak Time (h) Minimum interpeak Second peak Time (h)
99 1.3 65.4 98 6.7
cont.
f + f + +-
9 0.1 7 6 1.2
(n = 17)
(n=3 44) 117 1.2 80.0 109 5.0
+ + I!I + f
7 0.1 5 6 0.7
(n = 21)
3,6,
(+SEM)
and 9 months
for subjects
of use of a combination
with
multiple
steroid
peaks
oral
by
of follow-up
(n=6 32) 94 1.2 67.0 89 5.6
+ * + f t
6 0.1 4 5 0.9
(II = 29
(n,939) 93 1.2 65.3 91 5.1
+6 z!I + * +
0.05 4 5 0.5
(n = 24)
NE h&L) First peak Time (h) Minimum interpeak Second peak Time (h) Minimum
interpeak
concentrations
4.7 1.5 4.2 5.3 5.5
cont.
are provided
+ + + + +
0.6 0.2 0.6 0.6 1.4
9.8 1.3 8.5 10.1 4.0
f + + + f
0.6 0.1 0.5 0.6 0.4
8.8 1.2 7.3 8.9 3.9
+ + + + ii
0.6 0.05 0.4 0.4 0.3
7.3 1.1 6.5 8.7 4.3
+ + + + +
0.6 0.04 0.6 0.4 0.4
for comparison.
use of mean steroid profiles, which smooths the data, as shown in Figs. 3 and 4. Certainly, the frequency of multiple peaks in our own study may have even been greater had we performed more frequent blood sampling between 4-24 h after pill ingestion. In one previous study, when subject data did not fit the two-compartment pharmacokinetic model, these subjects (4 of 24) were not included in the final data analysis (25). In another study, when pronounced secondary peaks in EE2 levels were observed in the terminal phase, a zero weighing factor was introduced for data analysis (26). We also ob-
served variations in the pharmacokinetic curve of the oral contraceptive pill, but we believe that these variations are real and should be included in the data analysis. An explanation for the multiple peaks observed in the present study remains unknown, although changes in enterohepatic recycling, organ retention and rerelease, and gastrointestinal absorption occurring alone or in combination may be causative. Neither food ingestion nor activity after baseline fasting blood sampling were controlled in this study, and these variables may affect steroid production and clearance rates, which could have affected our results (27-30).
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ZACUR
1272
Additional studies designed specifically to study the pharmacokinetics of oral contraceptive pill use which control for dietary and activity factors may be required to determine their effect on the multiple peaks we observed. Since samples for individual subjects were analyzed together in a single assay, intraassay variation was not a factor in determining our results. Nevertheless, the possibility of technical errors influencing our results cannot be totally excluded. This is, however, unlikely, since errors would be expected to be random and not systematic, and would not explain the multiple peaks seen in other studies or the confirmation of double peaks in NE measured in our samples sent to another laboratory (Dr. Nieuweboer, Schering AG, Berlin, Germany). In conclusion, we have reported that the pharmacokinetics of oral contraceptive pill administration may be more complicated than previously realized. This phenomenon could explain in part the large variability in steroid concentrations between individuals when hormone measurements are taken at preset points in time. Methods other than single time point hormone measurements or area under the curve may be required to correlate physical and biochemical responses to oral contraceptive pill dose. Use of the initial slope of the steroid hormone rise serves as one example of an alternative method that we have suggested should be used when comparing pill dose with lipid response (11). Acknowledgments We are indebted to Ms. Kathy Zulty and Ms. Lynn Wilkins for manuscript preparation; to Ms. Beverly Smith, Ms. Sue Linkins, Ms. Janice Huth, and Ms. Victoria Chang for technical assistance; and to Drs. Edward E. Wallach, J. Donald Woodruff, and Giraud V. Foster for their critical and helpful editorial comments.
References 1. de la Pena A, Chenault CB, Goldzieher JW. 1975 Radioimmunoassay of unconjugated plasma ethinylestradiol in women given a single dose of ethylestrahiol or mestranol. Steroids. 25:773-780. 2. Warren RI. Fotherbv K. 1973 Plasma levels of ethvlestradiol or mestranol IO human subjects. J Endocrinol. 89:369-37b. 3. Stadel BV, Sternthal PM, Schlesselman JJ, et al. 1980 Variation of ethinyl estradiol blood levels among healthy women using oral contraceptives. Fertil Steril. 33:257-260. 4. Fotherby K, Akpoviroro J, Abdel-Rahman HA, et al. 1981 Pharmacokinetics of ethinyl estradiol in women from different populations. Contraception. 23:487-496. 5. Goldzieher JW, Dozier TS, de la Pena A. 1980 Plasma levels and pharmacokinetics of ethinyl estrogens in various populations. I. Ethinyl estradiol. Contraception. 21:1-16. 6. Mishell DR. 1989 Correcting misconceptions about oral contraceptives. Am J Obstet Gynecol. 161:1385-1389. 7. Zacur HA, Linkins S, Chang V, Smith B, Kimball AW, Burkman RT. 1991 Ethinyl estradiol and norethindrone radioimmunoassay following sephadex LH-20 column chromatography. Clin Chim Acta. 204:209-215. 8. Stanczyk FZ, Gale JA, Goebelsmann U, Nerenberg C, Matin S. 1980 Radioimmunoassay of plasma ethinyl estradiol in the presence
ET AL.
JCE & M. 1992 Vol75.No5
of circulatory norethindrone. Contraception. 22:457-470. 9. Stanczyk F, Brenner P, Mishell DR, Ortiz A, Gentzschein EKE, Goebelsmann V. 1978 A radioimmunoassay for norethindrone (NET): measurement of serum NET concentrations following ingestion of NET containing oral contraceptive steroids. Contraception. 18:615-632. RT, Zacur HA, Kimball AW, Kwiterovich I’, Bell WR. 10. Burkman 1989 Oral contraceptives, lipids, and lipoproteins, I. Variations in mean levels by oral contraceptive type. Contraception. 40:553-561. 11. Burkman RT, Zacur HA, Kimball AW, Kwiterovich P, Bell WR. 1989 Oral contraceptives, lipids, and lipoproteins. II. Relationship to plasma steroid levels and other status, Contraception. 40:675689. 12. Hvidberg EF. 1990 Why do we need pharmacokinetic studies. Am J Obstet Gynecol. 163:316-318. 13. Goldzieher JW. 1990 Selected aspects of the pharmacokinetics and metabolism of ethinyl estradiol and their clinical implications. Am J Obstet Gynecol. 163:318-322. 14. Back DJ, Breckenridge AM, Crawford FE, MacIver M, Orme MLE, Rowe PH. 1981 Interindividual variation, drug interactions with normal steroid contraceptives. Drugs. 21:46-61. 15. Kuhl H. 1990 Pharmacokinetics of oestrogens, progestogens. Maturitas. 12:171-197. 16. Orme MLE, Back DJ. 1990 Factors affecting the enterohepatic circulation of oral contraceptive steroids, Am J Obstet Gynecol. 163:2146-2152. 17. Orme MLE, Back DJ, Breckenridge AM. 1983 Clinical pharmacokinetics of oral contraceptive steroids. Clin Pharmacokinet. 8:95136. 18. Kaufman JM, Thiery M, Vermeulen A. 1981 Plasma levels of ethinyl estradiol (EE) during cyclic treatment with combined oral contraceptives, Contraception. 24:589-602. 19. Back DJ, Bates M, Breckenridge AM, et al. 1981 The pharmacokinetics of levonorgestrel and ethinyl estradiol in women-studies with ovran and ovranette. Contraception. 23:229-239. M, Nieuweboer B, Wendt H, Speck U. 1979 Investigations 20. Humpel of pharmacokinetics of ethinyl estradiol to specific consideration of a possible first pass effect in women. Contraception, 19:421-432. 21. Pasqualini JR, Castellet R, Portois MC, Hill JL, Kinel FA. 1977 Plasma concentrations of ethinyl oestradiol and norethindrone after oral administration to women. J Reprod Fertil. 49:189-193. AM, Crawford FE, et al. 1978 Kinetics of 22. Back DJ, Breckenridge norethindrone in women. II. Single dose kinetics, Clin Pharmacol Ther. 24:448-453. 23. Kiriwat 0, Fotherby K. 1983 Pharmacokinetics of oral contraceptive steroids after morning or evening administration, Contraception. 27:53-160. 24. Newburger J, Goldzieher JW. 1985 Pharmacokinetics of ethinyl estradiol: a current view. Contraception. 32:33-44. 25. Stanczyk FZ, Mroszczak EJ, Ling T, et al. 1983 Plasma levels and pharmacokinetics of norethindrone and ethinyl estradiol administered in solution and as tablets to women. Contraception. 28:241251. 26. Stanczyk F, Lobo RA, Chiang ST, Woutersz TB. 1990 Pharmacokinetic comparison of two triphasic oral contraceptive formulations containing levonorgestrel and ethinyl estradiol. Contraception. 41:39-53. 27. Balikian HM, Brodie AH, Dale SL, Melby JC, Tait JF. 1968 Effect of posture on the metabolic clearance rate, plasma concentration, and blood production rate of aldosterone in man. J Clin Endocrinol Metab. 28:1630-1640. 28. Baird DT, Horton R, Longcope C, Tait JF. 1969 Steroid dynamics under steady-state conditions. Recent Prog Horm Res. 25:611-664. 29. Flood C, Hunter SA, Lloyd CA, Longcope C. 1973 The effects of posture on the metabolism of androstenedione and estrone in males, J Clin Endocrinol Metab. 36:1180-1188. ST, Gibson M. 1989 The effect of a meal on circulating 30. Nakajima steady-state progesterone levels. J Clin Endocrinol Metab. 69:917919.
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Vol. 75, No. 5 Printed in U.S A
Existence of Multiple Peaks in Plasma Ethinyl and Norethindrone after Oral Administration Contraceptive Pill* HOWARD A. ZACUR, PETER KWITEROVICH,
RONALD AND
T. BURKMAN, ALLYN WILLIAM R. BELL
Estradiol of a
W. KIMBALL,
Departments of Gynecology and Obstetrics, Biostatistics, Pediatrics, and Medicine, Johns Hopkins Medical Institutions (H.A.Z., A. W.K., P.K., W.R.B.), Baltimore, Maryland 21205; and the Department of Gynecology and Obstetrics, Henry Ford Hospital (R.T.B.), Detroit, Michigan 48202 ABSTRACT
of multiple hormone peaks. Two peaks of EE2 were identified in 44.8% of women during the first pill cycle and in 75.9%, 55.2%, and 67.2% of women after 3, 6, and 9 months of pill use. Two hormone peaks of NE were observed in 29.3% of women during the first cycle and in 36.2%, 50%, and 44.8% at 3, 6, and 9 months, respectively. Existence of these multiple peaks at the frequency observed has not previously been reported. Further quantification of the frequency and magnitude of these peaks could be helpful in explaining differences in biological responses associated with pill use. (J Clin Endocrinol Metab 75: 12681272,1992)
Previous measurements of plasma ethinyl estradiol (EE,) and norethindrone (NE) over 24 h after oral administration of a contraceptive pill have demonstrated a single steroid peak occurring 1-2 h after pill ingestion, with a gradual decline over the next 22 h. In the present study plasma concentrations of EE2 and NE were measured 0,0.5,0.75, 1,2,4, 12, and 24 h after oral ingestion of a contraceptive pill containing 35 fig EE, and 1 mg NE at 0, 3, 6, and 9 months of use in 58 normal healthy women. Contrary to previous reports, analysis of the 464 steroid curves (58 subjects x 4 time periods X 2 steroids) revealed the presence
P
Materials
HARMACOLOGICAL studies have previously been conducted in normal women to determine the absorption, peak plasma level, and half-life of steroid hormones contained within the pill. These studies have almost uniformly found that a single peak in plasma steroid hormone concentrations occurs within l-2 h after oral administration of the pill, with a subsequent decline over the next 22 h (1, 2). Using these pharmacokinetic data, 24-h contraceptive steroid hormone curves have been generated, and the area under these curves calculated. Tremendous variations within and between individuals have been reported using this method (3-5). Since a correlation between the dose of contraceptive steroid and adverse effects exists (6), concern has been raised that for certain individuals, even low dose pills could produce elevated serum levels of contraceptive steroids. An explanation for this variability has remained elusive, and it has not been found to be correlated with age, height, weight, blood pressure, cigarette smoking, alcohol use, total lifetime oral contraceptive use, or the type of estrogen used (3). The present report confirms the previously reported variability in steroid hormone levels in oral contraceptive pill users and demonstrates that the pharmacokinetics of the oral contraceptive steroids may be even more complex than previously realized.
and Methods
Healthy young women (n = 63) between 18-35 yr of age were recruited from the Baltimore area for a clinical study approved by the Johns Hopkins Medical Institutions, Joint Committee on Clinical Investigation. After obtaining written consent, all subjects were evaluated for full eligibility. Criteria for study included regular menstrual cycles between 25-35 days; no contraindications to oral contraceptive use; absence of significant endocrinological, hematological, or metabolic disorders; body weight within 115% of idea1 body weight; no pregnancy or breast feeding within 6 months; and no oral contraceptive or sex steroid use within 12 months. A standardized medical and obstetric/gynecological history was obtained from all participants, which included questions regarding demographic characteristics, tobacco and alcoholic beverage use, diet, and onset of physical exercise. All subjects underwent physical and pelvic examinations. After an overnight fast, serial venous blood samples were obtained following oral ingestion of a combination-type oral contraceptive pill containing 35 pg ethinyl estradiol (EE,) and 1 mg norethindrone (NE). Each participant was started on the pill during the first 5 days of her cycle, after which time she returned for repeat blood sampling during the third, sixth, and ninth months of pill use. With few exceptions, subjects were sampled during the 19th to 21st day of pill use. All subjects received a combination pill provided from the same source (Syntex Laboratories, Palo Alto, CA). While 63 women initially enrolled in this study, only 58 women completed 9 months of evaluation, and only their hormone data are included in the present report. Venous blood samples (15 mL) were obtained before taking the morning pill (generally between 0700-0800 h) and 0.5, 0.75, 1, 2, 4, 12, and 24 h after pill ingestion. In almost all instances, a heparin lock was used to facilitate blood drawing. Blood samples were taken at the time intervals listed to conform with previously reported pharmacokinetic data (l-5). Although they had fasted before the first blood sample was taken, subjects were able to eat thereafter and participate in their normal daily activities, EEI and NE concentrations were measured in plasma samples using a RIA procedure modified from that of Stanczyk et al. (7-9) to permit more rapid preassay chromatographic separation of EE? and NE. Antiserum for EEI was supplied by Dr. C. Edgar Cook of the Research Triangle Institute (Research Triangle Park, NC), unlabeled EE, was ob-
Received February 11, 1992. Address requests for reprints to: Howard A. Zacur, M.D., Ph.D., Johns Hopkins Hospital, Park 82, Room 202A, 600 North Wolfe Street, Baltimore, Maryland 21205. *This work was supported by Contract NOl-HD-32816 from the Contraceptive Evaluation Branch, NICHHD (Bethesda, MD). 1268
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 01 April 2015. at 14:21 For personal use only. No other uses without permission. . All rights reserved.
PEAKS
IN CONTRACEPTIVE
STEROID
LEVELS
1269
120-
loo-
$
60-
x I2 z f W = .f 5 w
60-
40-
200
0’
0
I
2I
81
4
1
6I
!
Time FIG. 1. Plasma concentrations ingestion of 35 Kg EE, and cycle of pill use.
8I1
IO!
1
12./
v
l.i5
of EE1 measured over 24 h after 1 mg NE in one subject during the
2
4
Time
Hours oral first
tained from Steraloids, Inc. (Welton, NH), and tritiated steroid was purchased from New England Nuclear Corp. (Boston, MA). Antiserum for NE and NE for the standard were supplied by Dr. Ing Nieuweboer of Schering AG (West Berlin, Germany) and Syntex Laboratories, Inc. (Palo Alto, CA), respectively. Tritiated steroid was purchased from New England Nuclear Corp. Plasma samples were twice extracted with ether and chromatographed on Sephadex LH-20 before assay. All samples were assayed in duplicate. The cross-reactivity of the EEI antiserum to NE was l.l%, and there was less than 0.08% cross-reactivity of the NE antiserum to 17P-estradiol. The EEI assay has a sensitivity of 12 pg/mL, with a within-assay variation of 10.6% and a between-assay variation of 18.7%. The sensitivity of the NE assay was 15 pg/mL; within-assay variation was 11.6%, and between-assay variation was 12.5%.
Analysis Curve fitting for 24-h levels of EE2 and NE was attempted using both smooth compartment models and cubic spline regression models. The statistical methods used included t tests, logistic regression, multiple linear regression, and trichotomous regression, as previously described (10, 11).
Results Over the 9-month course of this study, 464 individual 24h steroid hormone curves were generated (58 subjects X 2 steroids X 4 time periods). Despite all efforts, attempts to apply smooth compartment modeling and cubic spline regression techniques were unsuccessful in fitting the observed RIA data with the modeled curves. Upon close inspection, it became apparent that the reason for this was that the individual curves contained more than one peak, a fea-
Hours
FIG. 2. Plasma concentrations of NE over 24 h after oral administration of 35 pg EE, and 1 mg NE after 6 months of pill use in a single individual.
ture that prevented the application of standard curve-fitting procedures. Figures 1 and 2 are examples of the multiple peaks observed for EE? during the first cycle of pill use in one individual and for NE after 6 months of pill use in another individual. The existence of these multiple peaks is less apparent when mean profiles of steroid concentrations are used, as shown in Figs. 3 and 4. Individuals could exhibit a double peak of EE2, NE, or both. Furthermore, the occurrence of a double peak during the first cycle of pill use did not mean that this would occur subsequently (Table 1). The first steroid hormone peak occurred between l-l.5 h, and the second steroid peak occurred between 3.9-6.7 h (Table 2). A peak was initially defined as a steroid hormone value that exceeded those values preceding and following it. To be certain that peaks were occurring and were not artifacts of the assay, a peak was also identified as such if its value was greater than 10% of the values immediately preceding or following it. When this criterion was used, 75% of all of the double peaks of EE2 and 64% of all of the double peaks of NE originally identified as peaks met this more stringent criteria. Discussion An understanding of the relationship between plasma drug levels and dosage is important to correlate the amount of drug ingested with the biological response (12, 13). For the oral contraceptive pill this has particular relevance, as associations have been sought between steroid dose and
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ZACUR
1270
A
0 MONTHS
3 MONTHS
B
7
160
100. w-
80 EE,
JCE & M. 1992 Vol75.No5
ET AL.
60
EE,
60.
m/ml
Pg/ml
-1
40
40.
20
20.
6
12
16
. . . . . .. . . . . . .. . . . . . .. .. .
+ 0
24
6
t (hrs)
6 MONTHS
C
12
16
24
16
24
t (hrs)
D
9 MONTHS w-
EE,
60.
w/ml
. 40.
0
12
6
16
“rn 0
24
6
t (hrs) FIG. 3. Mean concentrations of EE, in 58 subjects during oral contraceptive pill containing 35 pg EE, and 1 mg NE. TABLE 1. Percent frequency of multiple steroid and months of follow-up in 58 women volunteers
peaks
the initial
pill
by hormone
Month HORPXX
EE, NE
0
3
6
9
44.8
75.9 36.2
55.2 50.0
67.2 44.8
29.3
12
t (hrs)
adverse effects (14). Studies have demonstrated large differences in plasma hormone concentrations between individuals ingesting the same type of pill (15). The reasons for these differences have remained unexplained. After oral administration, steroids in the oral contraceptive pill are absorbed as free steroid or conjugated within the intestinal wall. Absorbed free steroid may then be conjugated by the liver or hydroxylated by mixed function oxidases within the liver. Conjugated steroids may be stored in the gall bladder, then released into the intestine, hydrolyzed, and reabsorbed as free or conjugated steroid or excreted in the feces (16). Previous studies analyzing plasma levels of the steroid components of the oral contraceptive pill have reported only a single early peak response at l-2 h, with a rapid decline in concentration over the next 22 h (1, 2, 17). Secondary peaks of EE2 and NE have occasionally been reported, but no attempt has been made to assess their frequency (18-23). Secondary peaks are presumed to be due to partial degradation associated with enterohepatic cycling. Some researchers have even suggested that these secondary peaks could reflect gall bladder contractions occurring after
cycle
(0 months)
and after
3, 6, and 9 months
of use of a combination
food ingestion (24). Our data confirm that significant differences in blood levels of EE2 and NE occur between individuals after ingestion of a pill containing a standard hormone dose. Our attempts to fit 24-h steroid curves to these values were unsuccessful because the curves frequently exhibited more than a single peak, a finding that had not been anticipated. Two peaks in EE2 levels were observed in 44.8% of women during the very first cycle of pill use. This percentage rose to 75.9% at 3 months and gradually declined to 55.2% at 6 months, only to rise again to 67.2% at 9 months. Subjects also experienced two or more peaks in NE, with 29.3% of the women exhibiting such patterns during the first month, and 36.2%, 50.0%, and 44.8% of the women showing these patterns at 3, 6, and 9 months, respectively. Initial peaks of both EE2 and NE were observed approximately 1.2 h after oral administration, and secondary peaks of both hormones were observed after 3.9-6.7 h. The steroid concentrations of secondary peaks frequently exceeded those of initial peaks, although the mean steroid concentrations between peaks were similar. Individuals could have two peaks during the first cycle of pill use, one peak during the third cycle, and two peaks during the sixth or ninth cycle. Therefore, no predictable pattern could be identified. Overall, however, multiple peaks increased in frequency with the duration of pill use. Previous studies may have failed to observe the steroid responses seen in our study due to the small numbers of subjects sampled, the frequency of blood sampling, or the
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PEAKS IN CONTRACEPTIVE
STEROID
LEVELS
o MONTHS
1271
3 MONTHS
A 8 rig/ml
NE
FIG. 4. Mean contraceptive TABLE month
ib---
~.,........‘,‘,.,.““‘,’ 6 0
concentrations pill containing
2, Mean of follow-up
1 . . . . >, , , , , . , . , . . , , , , , , , * 0 6 12 18 24
24
18
12
t (hrs)
t (hrs)
6 MONTHS
9 MONTHS
of NE in 58 subjects during 35 pg EE2 and 1 mg NE.
maximum hormone in 58 subjects
concentrations
the initial
(+SEM)
pill cycle
and mean
(0 months)
times
and after
of occurrence
Months
(n=o 26, First peak Time (h) Minimum interpeak Second peak Time (h)
99 1.3 65.4 98 6.7
cont.
f + f + +-
9 0.1 7 6 1.2
(n = 17)
(n=3 44) 117 1.2 80.0 109 5.0
+ + I!I + f
7 0.1 5 6 0.7
(n = 21)
3,6,
(+SEM)
and 9 months
for subjects
of use of a combination
with
multiple
steroid
peaks
oral
by
of follow-up
(n=6 32) 94 1.2 67.0 89 5.6
+ * + f t
6 0.1 4 5 0.9
(II = 29
(n,939) 93 1.2 65.3 91 5.1
+6 z!I + * +
0.05 4 5 0.5
(n = 24)
NE h&L) First peak Time (h) Minimum interpeak Second peak Time (h) Minimum
interpeak
concentrations
4.7 1.5 4.2 5.3 5.5
cont.
are provided
+ + + + +
0.6 0.2 0.6 0.6 1.4
9.8 1.3 8.5 10.1 4.0
f + + + f
0.6 0.1 0.5 0.6 0.4
8.8 1.2 7.3 8.9 3.9
+ + + + ii
0.6 0.05 0.4 0.4 0.3
7.3 1.1 6.5 8.7 4.3
+ + + + +
0.6 0.04 0.6 0.4 0.4
for comparison.
use of mean steroid profiles, which smooths the data, as shown in Figs. 3 and 4. Certainly, the frequency of multiple peaks in our own study may have even been greater had we performed more frequent blood sampling between 4-24 h after pill ingestion. In one previous study, when subject data did not fit the two-compartment pharmacokinetic model, these subjects (4 of 24) were not included in the final data analysis (25). In another study, when pronounced secondary peaks in EE2 levels were observed in the terminal phase, a zero weighing factor was introduced for data analysis (26). We also ob-
served variations in the pharmacokinetic curve of the oral contraceptive pill, but we believe that these variations are real and should be included in the data analysis. An explanation for the multiple peaks observed in the present study remains unknown, although changes in enterohepatic recycling, organ retention and rerelease, and gastrointestinal absorption occurring alone or in combination may be causative. Neither food ingestion nor activity after baseline fasting blood sampling were controlled in this study, and these variables may affect steroid production and clearance rates, which could have affected our results (27-30).
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ZACUR
1272
Additional studies designed specifically to study the pharmacokinetics of oral contraceptive pill use which control for dietary and activity factors may be required to determine their effect on the multiple peaks we observed. Since samples for individual subjects were analyzed together in a single assay, intraassay variation was not a factor in determining our results. Nevertheless, the possibility of technical errors influencing our results cannot be totally excluded. This is, however, unlikely, since errors would be expected to be random and not systematic, and would not explain the multiple peaks seen in other studies or the confirmation of double peaks in NE measured in our samples sent to another laboratory (Dr. Nieuweboer, Schering AG, Berlin, Germany). In conclusion, we have reported that the pharmacokinetics of oral contraceptive pill administration may be more complicated than previously realized. This phenomenon could explain in part the large variability in steroid concentrations between individuals when hormone measurements are taken at preset points in time. Methods other than single time point hormone measurements or area under the curve may be required to correlate physical and biochemical responses to oral contraceptive pill dose. Use of the initial slope of the steroid hormone rise serves as one example of an alternative method that we have suggested should be used when comparing pill dose with lipid response (11). Acknowledgments We are indebted to Ms. Kathy Zulty and Ms. Lynn Wilkins for manuscript preparation; to Ms. Beverly Smith, Ms. Sue Linkins, Ms. Janice Huth, and Ms. Victoria Chang for technical assistance; and to Drs. Edward E. Wallach, J. Donald Woodruff, and Giraud V. Foster for their critical and helpful editorial comments.
References 1. de la Pena A, Chenault CB, Goldzieher JW. 1975 Radioimmunoassay of unconjugated plasma ethinylestradiol in women given a single dose of ethylestrahiol or mestranol. Steroids. 25:773-780. 2. Warren RI. Fotherbv K. 1973 Plasma levels of ethvlestradiol or mestranol IO human subjects. J Endocrinol. 89:369-37b. 3. Stadel BV, Sternthal PM, Schlesselman JJ, et al. 1980 Variation of ethinyl estradiol blood levels among healthy women using oral contraceptives. Fertil Steril. 33:257-260. 4. Fotherby K, Akpoviroro J, Abdel-Rahman HA, et al. 1981 Pharmacokinetics of ethinyl estradiol in women from different populations. Contraception. 23:487-496. 5. Goldzieher JW, Dozier TS, de la Pena A. 1980 Plasma levels and pharmacokinetics of ethinyl estrogens in various populations. I. Ethinyl estradiol. Contraception. 21:1-16. 6. Mishell DR. 1989 Correcting misconceptions about oral contraceptives. Am J Obstet Gynecol. 161:1385-1389. 7. Zacur HA, Linkins S, Chang V, Smith B, Kimball AW, Burkman RT. 1991 Ethinyl estradiol and norethindrone radioimmunoassay following sephadex LH-20 column chromatography. Clin Chim Acta. 204:209-215. 8. Stanczyk FZ, Gale JA, Goebelsmann U, Nerenberg C, Matin S. 1980 Radioimmunoassay of plasma ethinyl estradiol in the presence
ET AL.
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of circulatory norethindrone. Contraception. 22:457-470. 9. Stanczyk F, Brenner P, Mishell DR, Ortiz A, Gentzschein EKE, Goebelsmann V. 1978 A radioimmunoassay for norethindrone (NET): measurement of serum NET concentrations following ingestion of NET containing oral contraceptive steroids. Contraception. 18:615-632. RT, Zacur HA, Kimball AW, Kwiterovich I’, Bell WR. 10. Burkman 1989 Oral contraceptives, lipids, and lipoproteins, I. Variations in mean levels by oral contraceptive type. Contraception. 40:553-561. 11. Burkman RT, Zacur HA, Kimball AW, Kwiterovich P, Bell WR. 1989 Oral contraceptives, lipids, and lipoproteins. II. Relationship to plasma steroid levels and other status, Contraception. 40:675689. 12. Hvidberg EF. 1990 Why do we need pharmacokinetic studies. Am J Obstet Gynecol. 163:316-318. 13. Goldzieher JW. 1990 Selected aspects of the pharmacokinetics and metabolism of ethinyl estradiol and their clinical implications. Am J Obstet Gynecol. 163:318-322. 14. Back DJ, Breckenridge AM, Crawford FE, MacIver M, Orme MLE, Rowe PH. 1981 Interindividual variation, drug interactions with normal steroid contraceptives. Drugs. 21:46-61. 15. Kuhl H. 1990 Pharmacokinetics of oestrogens, progestogens. Maturitas. 12:171-197. 16. Orme MLE, Back DJ. 1990 Factors affecting the enterohepatic circulation of oral contraceptive steroids, Am J Obstet Gynecol. 163:2146-2152. 17. Orme MLE, Back DJ, Breckenridge AM. 1983 Clinical pharmacokinetics of oral contraceptive steroids. Clin Pharmacokinet. 8:95136. 18. Kaufman JM, Thiery M, Vermeulen A. 1981 Plasma levels of ethinyl estradiol (EE) during cyclic treatment with combined oral contraceptives, Contraception. 24:589-602. 19. Back DJ, Bates M, Breckenridge AM, et al. 1981 The pharmacokinetics of levonorgestrel and ethinyl estradiol in women-studies with ovran and ovranette. Contraception. 23:229-239. M, Nieuweboer B, Wendt H, Speck U. 1979 Investigations 20. Humpel of pharmacokinetics of ethinyl estradiol to specific consideration of a possible first pass effect in women. Contraception, 19:421-432. 21. Pasqualini JR, Castellet R, Portois MC, Hill JL, Kinel FA. 1977 Plasma concentrations of ethinyl oestradiol and norethindrone after oral administration to women. J Reprod Fertil. 49:189-193. AM, Crawford FE, et al. 1978 Kinetics of 22. Back DJ, Breckenridge norethindrone in women. II. Single dose kinetics, Clin Pharmacol Ther. 24:448-453. 23. Kiriwat 0, Fotherby K. 1983 Pharmacokinetics of oral contraceptive steroids after morning or evening administration, Contraception. 27:53-160. 24. Newburger J, Goldzieher JW. 1985 Pharmacokinetics of ethinyl estradiol: a current view. Contraception. 32:33-44. 25. Stanczyk FZ, Mroszczak EJ, Ling T, et al. 1983 Plasma levels and pharmacokinetics of norethindrone and ethinyl estradiol administered in solution and as tablets to women. Contraception. 28:241251. 26. Stanczyk F, Lobo RA, Chiang ST, Woutersz TB. 1990 Pharmacokinetic comparison of two triphasic oral contraceptive formulations containing levonorgestrel and ethinyl estradiol. Contraception. 41:39-53. 27. Balikian HM, Brodie AH, Dale SL, Melby JC, Tait JF. 1968 Effect of posture on the metabolic clearance rate, plasma concentration, and blood production rate of aldosterone in man. J Clin Endocrinol Metab. 28:1630-1640. 28. Baird DT, Horton R, Longcope C, Tait JF. 1969 Steroid dynamics under steady-state conditions. Recent Prog Horm Res. 25:611-664. 29. Flood C, Hunter SA, Lloyd CA, Longcope C. 1973 The effects of posture on the metabolism of androstenedione and estrone in males, J Clin Endocrinol Metab. 36:1180-1188. ST, Gibson M. 1989 The effect of a meal on circulating 30. Nakajima steady-state progesterone levels. J Clin Endocrinol Metab. 69:917919.
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