Comparative Studies of the Ethynyl Estrogens Used in Oral Contraceptives: Effects with and without Progestational Agents on Plasma Cortisol and Cortisol Binding in Humans, Baboons, and Beagles*

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Baboons. The statistical analysis showed no difference between ethynylestradiol and mestranol at the same dose, nor between the two dose levels (1 and 4 J-Lg/kg) of the same estrogen (P = 0.05) (Table 1). The data were therefore combined and the means and standard deviations, by cycle, are shown in Table 2. Figure 2 (top) shows the plasma cortisol levels for the animals ultimately assigned to progestins A (norethindrone acetate), B (norgestrel), and C (megestrol acetate). For the first four treatment cycles, these three groups are essentially replicates, and document good reproducibility. The high values obtained in baboons probably reflect the stress effect of transient restraint and Sernylan sedation . However, the method of obtaining blood was standard throughout the study, and therefore the stress was also presumably standardized. After one cycle of estrogen administration, the mean plasma cortisol level rose from 43.0 to 55.4 J-Lg/dl. This was statistically signifi-

r GOLDZlEHER ET AL.

1186

TABLE 2. Total Plasma Cortisol Levels, by Cycle, for All Estrogen Regimens Combined, in Baboons and Beagles" Baboons Treatment cycle

Beagles

TC level

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TC level

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0 1 2 4 5 6 8 11

59 60 60 60 60 60 59 59

43.0 55.4 52.8 56.6 63.0 52.2 50.2 47.5

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58 54 52 60 59 57 57 57

4.3 3.4 3.0 5.3 5.4 5.5 5.0 4.4

± ± ± ± ± ± ± ±

2.7 2.6 2.7 3.5 3.9 5.0 3.4 3.0

"Results for human subjects, being dose-related, are presented separately in Figure 1.

cant. The TC level remained at the same elevation throughout the phase of estrogen therapy. Upon the addition of progestin, there was a further statistically significant rise, followed by a consistent decline, and a return to a level not significantly different from the initial value by cycle 8. Three cycles without treatment produced no further significant fall in the total plasma cortisol level. Thus, a prompt and stable rise was produced by estrogen; superimposition of progestins caused a biphasic response consisting of an elevation, followed by a rapid decline to pretherapy levels. There was no significant difference between the three progestins. Beagles. Once again, analysis showed no statistically significant difference between estrogens, by type or by dose, on the TC level, which is very low in this species (Table 2). As with the baboon data, the TC levels are displayed for the groups of animals destined to receive a particular progestational agent, without regard to the type or dose of estrogen administered (Fig. 2, bottom). 70

November 1977 The initial TC levels of the three sets were very similar (3.3, 4.7, and 4.9 j.tg/dl); the expanded ordinate scale must be kept in mind. Initiation of estrogen therapy produced a decrease in TC in the first cycle which was statistically significant. This was followed by a gradual rise to pretreatment levels or above, by the last estrogen cycle. The addition of norethindrone acetate (A) caused a further, statistically significant, rise in TC (4.0 to 6.0 j.tg/dl) which persisted throughout treatment. The absolute rise is, of course, relatively small. Pretreatment values were restored after 3 months without treatment. Norgestrel (B) appeared to have no effect on the elevated TC level; interestingly, this elevation did not revert entirely to normal by the end of the study. Megestrol acetate (C) caused a prompt and sustained decrease in TC (5.2 to 3.4 j.tg/dl) which also persisted into the post-treatment period.

Cortisol Binding Human Subjects. The percentage cortisol binding (PCB) in the IUD subjects averaged 87.6% ± 1.9%, and the initial value for all test subjects averaged 88.4% ± 2.8% (n = 160). By the end of the first cycle, PCB had risen to 92.7% ± 1.5% and remained at this level throughout the entire treatment. There was no difference between ethynylestradiol and mestranol, or between estrogen doses, and no progestational effect was observed. Animal Studies. The level of PCB in the two species is shown in Figure 3. It is slightly higher in the baboon than in the dog (83.3% ± 2.2% versus 80.9% ± 3.8%). The reaction to estrogen is similar in the two species. The dose or type of estrogen made no significant difference in the degree of binding, suggesting that a maximal effect was obtained with all four regimens. Binding

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COMPARATIVE STUDIES OF ETHYNYL ESTROGENS

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rose significantly in the first cycle of estrogen treatment, peaked by the second cycle, and leveled off thereafter at 86.5% ± 2.6% for the baboon and. 82.9% ± 2.5% for the dog. Thereafter, a species difference was observed. In the baboon, treatment with progestational agents was associated with a continuing rise in PCB until cycle 6, foIlowed by a decline to significantly subnormal values even in the post-treatment sample. The beagle, on the other hand, showed a downward trend immediately, although the fall was not statistically significant in the first progestin cycle. It continued and was still apparent in the posttreatment sample. Analysis of the responses to the various progestins showed only a somewhat stronger depressant effect in the first two norgestrel cycles.

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Human Subjects. The level of free plasma cortisol (FC) in the IUD population averaged 1.7 ± 1.0 JLg/dl (n = 147); the pretreatment value for the test group was 1.9 ± 0.9 JLg/dl (n = 160). As is shown in Table 3, there was no difference between the effect of ethynylestradiol and mestranol at equal doses, and there was no dose-related effect over the range studied. Even when all of the data for cycles 1, 3, and 6 for both estrogens were combined, the FC level at 50 JLg/day (2.4 ± 1.2 JLg/dl, n = 130) did not differ from that at 80 JLg/day (2.4 ± 0.9 JLg/dl, n = 186) nor from that at 100 JLg/day(2.6 ± 0.9 JLg/dl,n = 79). Theeffectofestrogen (Table 4; Fig. 4) was already maximal by the end of the first cycle, having risen from 1.9 ± 0.9 JLg/dl to 2.5 ± 0.9 JLg/dl (n = 150, P < 0.01). The values at cycle 3 (2.5 ± 1.1 JLg/dl, n = 137), cycle 6 (2.3 ± 1.0 JLg/dl, n = 108), and cycle 12 (2.3 ± 1.0 JLg/dl, n = 103) were not significantly different. The effect of the various progestational compounds on the estrogen-stimulated level ofFC was examined statistically for each estrogen at each TABLE 4. Free Plasma Cortisol Levels, by Cycle, for All Estrogen Regimens Combined, in Human Subjects, Baboons, and Beagles Treatment cycle

Human subjects

Treatment cycle

pgldl

0 1 3 6 12

H3 2.5 2.5 2.3

± ± ± ±

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2.3 ± 1.0

Baboons pgldl

0 1 2 4 5 6 8 11

7.2 8.2 7.0 7.6 7.7 5.7 6.7 8.7

± 0.4 ± 0.3 ± 0.4 ± 0.2 ± 0.2 ± 0.3 ± 0.5 ± 0.7

Beagles pgldl

-0.82 0,63 0.56 0.90 0.96 1.0 0.96 0.94

± 0.18 ±-O.07 ± 0.01 ± 0.04 ± 0.24 ± 0.10 ± 0.16 ± 0.14

GOLDZIEHER ET AL.

1188 10

BABOON B

November 1977 nificant decline appeared during the first two cycles of estrogen treatment; this reverted to normal levels by the end of the fourth cycle. The addition of progestational compounds had no significant effect.

DISCUSSION

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dose level, and the FC level at cycle 6 was compared with that at cycle 12. No significant differences between the estrogen regimens were observed. When the data from all of the regimens were combined, the FC levels for those destined to receive norethindrone acetate (2.4 ± 1.0 /Lg/dl, cycle 6) did not change significantly with treatment (2.1 ± 0.7 /Lg/dl, cycle 12). Similarly, there was no effect of treatment with norgestrel (2.2 ± 0.9 /Lg/dl versus 2.2 ± 1.0 /Lg/dl) , nor with megestrol acetate (2.2 ± 0.9 /Lg/dl versus 2.6 ± 1.1 /Lg/dl. It is clear that the effects of the progestational compounds on total cortisol and percentage binding compensate one another so that the net effect of each progestational compound on free cortisol is negligible. Animal Studies. As is shown in Table 3, there was no difference between ethynylestradiol and mestranol at either dose level, and there was no difference between the effects of 1 /Lg/kg versus 4 /Lg/kg on FC: The data were therefore combined, as shown in Table 4 and Figure 4, which also shows the human data. The six cycles of estrogen treatment in women are graphed to compare with four cycles in the experimental animals, and cycle 12 in humans is compared with cycle 8 in the animals. Baboon FC levels rose significantly in the first cycle of estrogen treatment, then fell significantly at once, to return to base line levels. There was no further change during estrogen treatment, and none in the first cycle of progestin treatment. In the second progestin cycle, FC fell significantly below control levels, rose again by the end of treatment, and rebounded significantly above control levels 3 months after discontinuation of treatment. There was no difference in the effect of the three progestational agents, and all showed a similar pattern. In the beagle, the levels of FC were extremely low under control conditions. A statistically sig-

Both human subjects and baboons showed an increase in total plasma cortisol upon the administration of estrogens. The effect is known to be prompt, and stabilizes within the 1st month. The increase in TC levels in humans from 13 to 30 /Lg/dl, on the average, is very similar to that reported by others 6, 12. 17,18 and is proportionally somewhat greater than that in baboons. The latter species, however, already had a stress-induced elevation of total cortisol (43 /Lg/dl) at the start of the study, and comparisons are therefore difficult. The dog, on the other hand, showed an entirely different response, with a decrease during the first two cycles of estrogen treatment and a significant elevation above base line only at the end of the fourth estrogen cycle. In none of the three species could a difference between the two kinds of estrogen, at the same dosage, be demonstrated. This is consistent with our previous studies on human target tissues (endometrium, hypothalamic-pituitary system I3 ,19) but differs from the results of animal assays (for a review, see reference 20) and from the report by Schwartz and Hammerstein lO on the transcortin-binding capacity of hypoestrogenic human subjects given various estrogens or contraceptive preparations. These careful studies, on a very small number of patients (four per dose), showed that mestranol had 64% as much activity as ethynylestradiol, with 95% confidence limits of 39% to 86%. Care was taken to ensure similar bioavailability of the compounds; furthermore, normal women given commercial contraceptive formulations differing in the type, but not the dose, of estrogen used gave similar results. The reasons for the difference between our results and those of Schwartz and Hammerstein 10 are difficult to identify, since the type of subject and the techniques used, as well as the event measured, were different. Possibly the use of hypoestrogenic women permitted the dose-response curves to fall in a region where differences could be discriminated. There was no difference in the effect produced by different dose levels of these estrogens in baboons or beagles, and only a small, statistically demonstrable, difference between 50 and

Vol. 28, No. 11

COMPARATIVE STUDIES OF ETHYNYL ESTROGENS

100 jLg/day in human subjects. This finding is consistent with observations on albumin, ceruloplasmin, haptoglobin, and orosomucoid, which show a dose-response relationship which begins to plateau at about 50 jLg/day (roughly equivalent to 1 jLg/kg). Much larger doses have been shown to have greater effects, and higher levels, of course, are seen in pregnancy. The type and dose of estrogen made no difference in the percentage of cortisol bound, which increased from 88% to 93% within the first cycle in human subjects, and stabilized at this level. In the baboon, a rise from 83% to 87%, on the average, required two cycles of treatment. The effects in the beagle were similar, which is remarkable in view of the concomitant decrease in total plasma cortisol. It must be recalled, however, that the circulating cortisol levels in this species are very low to begin with,21 and the relationship of numerical changes to physiologically significant events is uncertain. The unbound cortisol undoubtedly accounts for the major share of the biologic activity of this steroid. In keeping with studies on combination type oral contraceptives, we found a small, but statistically significant, increase in human free plasma cortisol (1.8 ± 0.9 jLg/dl to 2.5 ± 0.9 jLg/dl) in the first cycle; the level remained stable thereafter, and was independent of the dose or type of estrogen administered, confirming our other observations on the relative potency of the two estrogen types and on the plateau of the dose-response curve. The degree of change is less than that seen by Doe et aLB in late pregnancy, using 200 jLg/day, and by Lindholm and Schultz-Moller,18 using doses similar to ours. Many of the treatment values overlap the normal range. The baboon showed only a transient rise in FC in the first cycle, after which the values returned to the normal range for the duration of estrogen treatment. In the beagle, the levels were extremely small to begin with (0.8 ± 0.2 jLg/dl), and changes of a few tenths of a microgram per decaliter are extremely difficult to interpret. The effect of progestational compounds on plasma cortisol dynamics is complex. Some of the 19-norsteroids are contaminated with minute but biologically important quantities of ethynyl estrogens, and others, such as norethynodrel, have intrinsic estrogenic properties. The metabolic conversion of these compounds to estrogens has also been suggested and apparently discounted. Some plasma proteins, such as albumin, may be decreased, while others, such as ceruloplasmin,

1189

may be increased. Moreover, the results of various investigations differ: Ylostalo et al,22 and Dale and Spivey23 observed effects of medroxyprogesterone acetate on albumin, total protein, and (X- and f3globulins at high doses, while Settlage et al,24 found no change with 10 mg/day. Pekkarinen and Pulkkinen 12 found a higher TC with megestrol acetate plus ethynylestradiol than with lynestrenol or norethindrone acetate plus ethynylestradiol. Schwartz and Hammerstein,IO on the other hand, found no effects of megestrol, norgestrel, norethindrone, or norethindrone acetate on the transcortin-binding capacity of hypo estrogenic subjects. In our studies, both norethindrone acetate and norgestrel decreased TC, whereas megestrol acetate had no effect in human subjects. In the baboon, all three progestins first increased, then decreased, TC; in the beagle, norethindrone acetate produced an increase, norgestrel had no effect, and megestrol acetate caused a decrease. In this instance, species differences are quite clear. None ofthe three progestins altered the slightly elevated level of FC in human subjects, and no effect could be detected on the very low level of FC in beagles. In the baboon, all three compounds produced a biphasic change in FC, consisting of a substantial drop in the second estrogen-pIus-progestin cycle, with a return almost to the normal FC level after two more cycles. Since these results are the product of replicate experiments carried out sequentially in a few animals at a time, it is extremely unlikely that they are a random event or the result of a methodologic flaw. The fall in FC occurred just after a cycle in which TC rose to a value higher than that produced by estrogen alone; i.e., the free cortisol, which did not rise along with the TC and percentage binding, was now dropping to subnormal levels as the TC was returning to the estrogen-induced plateau. Within the next two cycles, the TC dropped toward the control level while the FC rose almost to the pretreatment level. The significance and relationship of these changes is obscure, but it is important to note that neither change is observed in human subjects. Thus, while the baboon liver responds to estrogens with synthesis of CBG, as does that of man, the response to synthetic progestins differs from that of man; this must be taken into account in selecting appropriate animal models for pharmacologic studies. On the other hand, the beagle, which has a low plasma cortisol level to begin with, does not respond to estrogen with an increased

1190

GOLDZIEHER ET AL.

synthesis of CBG and an elevation of TC; in fact, TC falls rather than rises, while the FC is transiently depressed as well. Clearly, the hepatic response to estrogen, with respect to the synthesis of CBG, is very different from that of man. The response to added progestins also differs, for megestrol acetate produced a drop in TC in contrast to the response in humans, where no change was observed, and norethindrone acetate produced a rise, as contrasted to the fall in humans. Some of this may be a dose-related phenomenon, since it has been shown that compounds closely related to megestrol, such as chlormadinone acetate, are far more slowly metabolized by the dog than by man, in effect vastly increasing their potency. It may be concluded that the beagle is an inappropriate surrogate for man in the study of estrogenic effects on liver protein synthesis; it has already been shown to be inappropriate for studies of the metabolism and peripheral effects of 17acetoxy progestins. 25 The baboon appears to be a suitable model for estrogenic effects, but some effects of progestational compounds differ from those observed in man, and this matter will have to be elucidated before the validity of the experimental model in this context can be determined. Acknowledgments. We wish to express our appreciation for the expert technical assistance of Mr. C. Celaya, Mr. I. Gomez, Mr. D. Garg, anq Ms. J. Mandel, and for the quality-controlled tablets of ' the various steroids prepared by Wyeth Laboratories.

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