- Email: [email protected]
Effects of the antiprogesterone RU 486 in normal women
Effects of the antiprogesterone RU 486 in normal wornen 1. Single-dose administration in the midluteal phase Donna Shoupe, M.D., Daniel R. Mishell, j-; M.D., Pekka Lahteenmaki, M.D. , Oskari Heikinheimo, M.D., Lars Birgerson, M.D., Hosam Madkour, M.D., and Irving M. Spitz, M.D. Los Angeles, California, Neui York, New York, Uppsala, Suieden, and H elsinki, Finland The response to a single oral dose 01the antiprogesterone RU 486 was studied in the midluteal phase in 26 normal women . Each subject received a dose between 50 and 800 mg RU 486 on days 6 to 8 after the luteinizing hormone surge and blood sampies were taken ovsr the lollowing 48 hours. Another group of live patients received a single oral dose 01200 mg RU 486 and blood sampling was extended lor 14 days . Menses were induced in all women but one within 3 days after RU 486 administration. Two distinct patient populations emerged. In nine 01the subjects, there was a single bleeding episode and the treatment cycle was signilicantly shorter (p < 0.05) than the f6110wing cycle. In 1601 these 25 patients a secondbleeding eplsode occurred 19.0 ± 0.8 days after the luteinizing hormone surge. The total treatment cycle was significantly prolonged (p < 0.05) when compared with the following cycle. In the groupwith a single bleeding eolsode, there was a significant decline in follicle-stimulating hormone, estradiol, and progesterone over the 48-hour sampling period, but there was no chanqe in these values in the group with two bleeding episodes. These two groups could not be separated on the basis 01 RU 486 dose or serum levels. After the lour higher doses, there was a dose-dependent rise in serum prolactin. There were no alterations in mean cortisol values with the three lower doses , but there was a signil icant lncrease at 24 and 48 hours after the higher doses . Serum levels 01 RU 486 were maximal between 1 and 4 hoursand the half-Iile of serurn RU 486 was determined to be 24 hours. (AM J OSSTET .GVNECOL 1987;157:1415-20.)
Key words: RU 486 ; contrace ption, go nadotro pins, steroids
The synthesis and development of RU 486 , a 19norprogestin derivative, is conside red a major breakthrough in steroid endocrinolog y' and has opened a new era in fertility control. Beyond its potential use in cont race ptive technalog y, the antiprogestational' and an tiglucocor ticoid'" activities of RU 486 serve as a unique tool for investigation of hormone action. In early re ports, RU 486 has been noted to interrupt th e luteal phase in women':" and in monkeys" and to terminate early pregnancy.t" RU 486 has also bee~ shown to have an affinity for th e glucocor ticoid receptor that is about four time s higher th an that of dexamethasone and has been shown to ha ve antiglucocorFrom the Section of R eproductiue E ndocrinology, Department of Obstetrics and Gynecology, Uniuersity of Southem California , Los Ange les, the Centerfor Biomedical R esearch, Population Couneil, N ew York, the Department of Obstetrics and Gynecology, Uniuersity H ospital, Uppsala, and the Steroid Research Laboratory, Department of Medical Chemistry, Uniuersity of Helsinki. Supp orted in part by grants [rom the Ford and Mellon Foundati ons. Computational assistance was prouided by the CLINFO project fundedby the Diuision of R esearch Resoutces of the National Institutes of H ealth, Grant No . RR-0004 3 . Presented at the Thi rty-second Annual M eeting of the Soeiety [or GynecologicIn uestigation, Phoenix, Arizona, Ma rch 20-23, 1985. R eprint requests: Donna Shoupe, M .D., Women 's H ospital R oom L-946, 1240 No rth Mission R oad, Los Angeles, CA 90033 .
ticoid activity. After a single oral dose of RU 486, there is a transient increase in adrenocorticotropic horrnone.v" The aim of this stud y was to investigate the effect of a single oral dose of RU 486 o n bleeding, gonadotropin, and steroid patterns given to he althy women in the midluteal phase. In addition, the pharmacodynamics of this compound were studied with a recently developed radioimmunoassay.
Material and methods Thirt y-one healthy women, who se ages ranged from 22 to 35 years, were selected for participation in this stud y. They had regular menstrual cyd es (28 ± 3 da ys) and were within 15% of their ide al body weight. They eithe r had been su rgically sterilized or had used a barrier method for contraception, Thc volunteers had not taken any ste ro id medication within th e last 6 months. O n the tenth day of th e men strual cyde, each patient began to undergo dail y blood sarnpling for determinat ion of the day of the luteinizing hormone (LH) surge. Six to eight days after th e LH surge, the volunteers were asked to report at 7:30 A M , after an overnight fast, and to refrain from smoking after midnight for ad ministration of medication. 1415
1416 Shoupe et al.
11.0
Decernber 1987 Am J O bstet Gynecol
E2 210 pg/ml
+
+
LH mlu/ml
5.0 130
3.0
• E2 1
0
2
4
6
• E2 2
110
~.~!-'2~-----' 48
HOURS
0
2
P
4
6
HOURS
~I
10
24
I
48
ng/ml 18.0 + I
FSH mlu/ml
o
2
48
Fig. 1. LH (upper panel) and FSH (lower panel) values afte r a single ora l d ose of RU 486 ra nging from 50 to 800 mg. LH I and FSH1 refer to the patients with one bleeding episode after RU 486 administration and LHz 'and FSHz refer to patients having two bleeding episodes. No significant changes in LH werenoted in either group. Anal ysis of var iance detected a significant de crease in FS H , (p <0.00 1) over the 48-ho u r sam. pling period.
Subjects re ceived either 50 , 100 , 200, 400 , 600. or 800 mg of R U 486 (50 mg tablets supplied by RousselUelaf, Ro mainville, France) a t 8 :00 AM. All groups cornprised four subj ects except for the two highest-dose sched u les where five subjects were stud ied in each group. No smo king or food was permiued for 4 hours after ingestio n of the medication and vital signs were recorded every 30 minutes for 6 hours. Blood sampie s were obtained through an indwelling catheter at - '/ 2 , o + V2, 1,2,4,6, 10,24, a nd 48 hours. A further group of five subjects received 200 mg as a single oral dose and blood sampling was co n tin ued daily for I week and then was performed at 10 and 14 days (ex te nd ed sa m pling group). These blo od sampies were assayed for prolactin, estradiol, progesterone, cortisol, LH, follicle-stimulating hormone ( FS H) , and RU 486 by previou sly d escribed rad ioim m u noassay rnethods.v" Norma l seru m AM co rtisol values are 10 to 30 ILg/dl. Two different radioimmunoassays were used to measure RU 486. The first was the method of Salmon and Mouren. " The seco nd utilizes chromoso rb co lurnn ch ro rna to graphy. " This lauer, more 'specific as say was used only to measure RU 486 level s in the sampies from the extended sam-
6.0 '-r----,--
o
, -- - r - --,..-f'f----r-{ I
2
4
6
HOURS
10
I
i
24
48
Fig. 2. Upper panel: Estradiol (E z) values after a single oral dosof RU 486 ranging from 50 to 800 mg. Ezl refers to patient with one bleedirig episode after RU 486 administration anr E z 2 re fers to those with two bleed ing episodes. Analysis 0 variance detected a significant decrease in EzI (p < 0.05) ove the 48-hour sampling period . Lower panel: Progesteronej'P, values after a single o ra l d ose of RU 486 ranging from 50 to 800 mg . PI refers to pat ients with one bleeding episode after RU 486 ad ministration and P, refers to those with two bleeding epi sodes, Anal ysis of variance detected a significant decrease in P, (p < 0.0 05) over the 48-hour sampling period.
pling group. The antiserum against RU 486 used in both assays was donated by Ro ussel-Uclaf. In addition, SMA-I8 and co m ple te blood co u n t wereperforrned at 0,4, 10, 24 , and 48 hours. . Results were analyzed by analysis of va r ia nce with the use of'BMDP Statistical Software. " One- and two-way analysis of variance and Student's t test were u sed 10 determine statistical differences bet~een gro ups. Results
Clinical features, Menses cornmenced in all patients but one with in 1 to 3 days afte r RU 486 administration (T able I). In nine of these 25 su bj ects (two who received 50 mg, two who received 100 mg, one who received 200 m g, one who received 40 0 rng , and three in the 800 m g group), thi s reprcsented the only bleeding episod e . T h e me an length o f the treatment cyele in these nine subjects was sign ifica n tly less (p <: 0.05) than that of the following cyde (Table 1):
Effecls of RU 486 in normal women. I
Volume 157 Number 6
In th e remaining 16 subjects, a second bleeding episode was reported . In these subjeets the initial bleeding episode occurred at a similar time after the LH surge as in the previous group, but the duration of bleeding was significantly shorter (p < 0.05) . The full duration of the treatrnent cycle in th ose subj eets who had two bleeding episod es (with the onset of the seco nd bleeding episode used as a reference) was 32.9 ± 1.1 days. This was longer (p < 0.05) than the following cyde (Table I). In no ne of these subjeets were an y adverse effects encountered. Furthermore, electrolytes, hepatic fun ction, an d bloo d count d id not change in an y subject. In on e subject who received the 600 mg d ose, bleeding did not occur until 56 days after treatment. Hormonal paramete rs. The hormonal profiles of four patients (two fr om each group) were exduded from analysis becaus~ of incomplete data. In those 14 subj ects who had two bleeding episodes, there was no change in seru m LH , FSH , est radiol, or pro geste rone over th e 48- ho u r sa mpling period. Howeve r, in those seven subjects who had a single bleeding episode, anal ysis of variance showed a significant dedine in estradiol (p < 0.05), FSH (p < 0.001 ), and progesterone (p< 0.005) (Figs. land 2). . There were no significant changes in LH, FSH , estradiol, or progesterone when hor monal res ults were expressed according to th e dosage schedule of RU 486 and independent of the bleeding pattern. Analysis of variance showed that therewas a significant increase in prolactin (p < 0.05) withthe 50 , 200 , 400 , and 800 mg dosages. When compared with the pretreatment levels, the peak values were greatest between 4 and 6 hours (p< 0.05). Values returned to nor mal by 24 hours ( Fig. 3). Ana lysis of variance showed a significant increase (p < 0.05) in mean cortisol values after the 100, 400, 600 , and 800 mg doses. When compared with baseline values , the cortisol levei rose significantly (p < 0.05) at 24 hours .after the 600. and 800 mg doses and at 48 hours with all three higher doses ( Fig. 4). RU 486 levels. When measured directly after diethylether extraction, the serum levels of RU 486 reached a maximum between 1 and 4 hours after ingestion of all doses ( Fig. 5). All RU 486 leve ls were significantly lower with the 50 mg dose than with th e five higher-dose regimens (p < 0.05). The RV486 levels at 6, 24, and 48 hou r s were also significan tly lower after the 100 mg dose than with the four higher dos es (p < 0.05). RU 486 leve ls were the sam e with doses of 200 to 800 mg. With doses of ;;,0200 mg, mean seru m RU 486 levels fell minimally, and even after 48 ho urs values were still above 1.4 fLg/ml. On the other hand , after the 50 and 100 mg doses, RU 486 values did decrease and were under 0.4 fLg/ml at 48 hou rs. Thcre
60 50
1417
- 600 mg ° 600 mg - 40 0 mg
40 30 PRL ng/ml
20 10 30 20 10 0
- 200mg ° 100mg _ 50 mg
0
2
4
6
HOURS
10 12~ 4
18
Fig. 3. Prola ctin (PRL) levels after a single oral dose of RU 486 ranging from 50 to 800 mg. Analysis of variance detected a sign ificant increase in PRL after the 50, 200 , 400 , and 800 mg doses. * p < 0.05 increase over baseline values as determined by the paired t test.
were no differences in the RU 486 levels between the groups with one or two bleeding episodes.. . The serum levels of RU 486 in the extended sampling grou p are shown in Figs. 5 to 6. Chromosorb column chromatography was used in these sam pies and the values were lower tha n th ose with corresponding 200 mg dosage schedule. Never theless, after 4 hours the disappearance cu rves with both assays were parallel. Presumably, the values after chromatography represent · true RU 486 con centrations. The halfdisap pearance tim e was 24 hou rs ( Fig. 6). Comment
. Administration of RU 486 as a single oral dose from 50 to 800 mg in the early lut eal phase was weil tolerated . The induction of menses wirhin 3 days is in accordance with results of p revious studies.v " 15 Thls vaginal bleeding, which occurred des pite high levels of estradiol and progesterone, implies that RU 486 acts directly on the endometrium to produce shedding re gardless of an y effect it may have on hormonal levels. A similar bleeding pattern has also been docurnented in nonpregnant women during concornitant tr eatment with RU 486 and human ch orionic gonadotropirr in the luteal p hase." !' A total of 33% of our subjects dis played only a sin gle
1418 Shoupe et al.
December 1987 Am J Obstet Gynecol
.aOOmg o 600 mg e400 mg
.200mg 0 100 mg e50 mg
Fig. 4. Cortisol values after a single doseof RU 486 rauging from 50 to 800 mg. Analysis of variance detected a significant increase in cortisol (p < 0.05) after the 100, 400 , 600 , and 800 mg dose . * p < 0.05 increase over baseline value s as determined by the paired t test.
5 4
3 2
• 800 mg o 600 mg e 40 0 mg
4 RU 486
V9/ml
3
2 1 .8 .6 .4
.200mg • 200 mg EXTENO o 100mg e 50mg
•
,2
2 Fig. 5~ RU 486 levels after a single oral dose ofRU 486 ranging from 50 to 800 mg. Analysis of variance detected that all points after 50 mg dose and at 6,24, and 48 hours after the 100 mg dose were significantly lower (p < 0.05) than values after the higher doses. The 200 mg ex/end refers to the extended sampling group that is also depicted in Fig. 6.
bleeding episode, whereas in the remaining subjeets a second bleeding episode oecurred. This phenomenon appeared to be independent of the dose or serum concentration of RU 486. The hormonal profile in these two groups was distinetly different. In those subjeets
wiih two bleeding episodes there were no ehanges in LH , estradiol, or progesteronelevels. In addition, ifthe eommeneement of the seeond bleeding episode is regarded as the termination o f the eyde, the luteal phase appeared to be prolonged by RU 486. In contrast, in those subjeets who had asingle bleeding episode there was a progressive signifieant dedine in serurn FSH, estradiol, and progesterone levels over 48 hours of sampling time with a eoneomitant shortening of the luteal phase and eyde length. This suggests that under eertain circumstanees RU 486 ean be luteolytic. Similar observations ha ve been ma de by other workers." 15 Although it is possible that the dedine in steroid levels may represent the fall seen in the lauer part of the normal menstrual cycle, this is unlikely since no corresponding fall occurred in the group with two bleeding episodes. The decrease in the serum progesterone level could be related to a direct action of RU 486 on ovarian steroidogenesis." However, it could also represent an effect on the hypothalamic-pituitary axis. We did observe a decrease in FSH in one group of patients but were unable to detect a decrease in LH secretion. Recently, with more frequent sampling we also ob served a reduction in amplitude and frequency of LH pulses during RU 486 administration in the luteal phase." Other workers have also deseribed a reduction in LH pulse amplitude consequent to RU 486. 15 'Nieman et al." observed menstrual bleeding in norma l women within 72 hours of a single oral dose of RU 486 (10 mg /kg body weight) given in the midluteal phase. In the patients treated with RU 486 on luteal day 7, they re port a decrease in the serurn progesterone level by luteal phase day 10 and the onset of vaginal
Effects of RU 486 in normal women. I
Volume 157 Number 6
1419
3.0 2.0 1.0 0.5 <0_
coE
~::::>0 0:::1..
0.2 0.1 0.05 0.02 0 .01 0.005 0.002 0.001 0
2
4
6
8
10
12
14
DAYS Fig. 6. RU 486 levels in the extended sampling group after a single oral dose of 200 mg RU 486. The half-life of RU 486 is 24 hours.
Table I.' Effects of RU 486 administration Bleeding
episodes I 2
9 16
Onset (days after LH surge)
bleeding
Dass of
Treatment cycle length (days)
Posttreatment cycle length (days)
9.8 :!: 0.6 9.6 :!: 0.5 19.0 :!: 0.8
3.4 ± 0.3 6.0 :!: 0.8t 4.2 ± 0.3
25.2 ± 1.4* 32.9 :!: l.l
34.0:!: 2.1 26.6 :!: 1.7
Values are mean :!: SD. *p < 0.05 when eompared with posureatrnent eyd e length. t p < 0.05 when eompared with group with one b1eeding episode.
bleeding within 72 hours of treatrnent in all patients. They also described two patient groups as regards to the onset of menses, which ma y correlate with our two patient groups. Circulating RU 486 levels measured by immunoassa y showed that this synthetic steroid was absorbed rapidly. Doses of 50 and 100 mg gave lower blood levels than higher doses. However, no c1ear dose response was evident sinee 200 to 800 mg RU 486 gave similar blood levels . The irnmunoassay used to determine these eireulating levels is nonspeeifie and measures metabolites, including the monodemethylated a nd didemethylated eompou nd s as weil as the alcoho lie derivative ." : 12 T hese metabolites seem to have a smaller biologie activity." For thi s reason we developed a more speeifie radioimmunoassay using prelirninary ehromosorb eh romatography. With thi s modification, values were lower but gave a parallel disappearanee eu rve. Values of RU 486 measured by immunoassay after eh romosorb ehro-
matography give values similar to th ose determined by high-pressure liquid ehromatography. 12 The half-li fe of RU 486 was 24 hours. The long half-disappearance of RU 486 suggests that there is binding to plasma proteins, Reeent stud ies have shown that RU 486 binds to an
1420 Shoupe et a!.
tramuscular progesterone after oophorectomy. This would imply a positive effect of progesterone on prolactin secretion, which is in contrast to the transient increase in prolactin we noted after treatment with an antiprogesterone. Healy et apodescribed a decrease in prolactin after treatment with RU 486 in monkeys (10 mg/kg) ." However, these were castrated monkeys who had steroid replacement via estradiol and progesterone capsules, which produce basal hyperprolactinemia. Within 1 hour after parenteral RU 486 administration, these elevated prolactin levels were decreased. Thus the effect of RU 486 on prolactin secretion varies and may depend on the mode of its administration, the dose, the species, and the prevailing steroid concentrations. The reason why two subgroups emerged in this study is not evident. They could be differentiated by both bleeding patterns and hormonal levels. Both groups received RU 486 on days 6 to 8 after the LH surge and there were no differences in RU 486 serum levels between the two groups. Thus these two patterns of response could not be differentiated on the basis of time of treatrnent, dosage of RU 486, or circulating RU 486 levels. RU 486 has been proposed as a once-a-rnonth pill." Our data would support the value of RU 486 for this use. However, the emergence of two patient groups suggests that further characterization of these distinctive patterns is necessary. REFERENCES 1. Teutsch G. Analogues of RU 486 for the mapping of the progestin receptor: synthetic and structural aspects, In : Baulieu EE, Segal SJ, eds. The antiprogesterone steroid RU 486 and human fertility control, New York: Plenum Press, 1985:27-47 . 2. Healy DL, Chrousos G, Schulte HM, Gold PW, Hodgen GD. Increased adrenocorticotropin, cortisol, and arginine vasopressin secretion in primates after the antiglucocorticoid steroid RU 486: dose response relationship. J CIin Endocrinol Metab 1985;60: 1. 3. Gaillard RC, Riondel A, Muller AF, Herrmann W, Baulieu EE. RU 486: asteroid with antiglucocorticoid activity that only disinhibits the human pituitary-adrenal system at a speeific time of day. Proc Natl Acad Sei USA 1984; 81 :3879. 4. Hermann W, Wyss R, Riondel A, et aI. The effects of an antiprogesterone steroid on women: interruption of the menstrual cyde and of early pregnancy. C R Acad Sei [III] 1982;294:933. 5. Shoupe D, Mishell DR, Lacarra M, Gutierrez E, Labteenmaki P, Spitz 1. Endocrinologie effects of the antiprogesterone RU 486 in the luteal phase of normal women . In : Baulieu EE, Segal 5J, eds . The antiprogesterone stero id
December 1987 Am J Obstet Gynecol
RU 486 and human fertility controI. New York: Plenum Press, 1985:285-93. 6. Nieman LK, Choate TM, Chrousos GP, et aI. The progesterone antagonist RU 486 : a potential new contraceptive agent. N EnglJ Med 1987;316:187 . 7. Haspels AA. Interruption of early pregnancy by an antiprogesrational compound, RU 486. EurJ Obstet Gynecol Reprod Biol 1985;20:169. 8. Mishell DR, Shoupe D, Brenner PF, Horenstein J, Lacarra M, Spitz IM. Termination of early gestation with the anti-progesterone, RU 486 . Contraception 1987; 35 :307. 9. Mishell DR, Nakamura RA, Crosignani PG, et aI. Serum gonadotropin and steroid pattern during the normal menstrual cyde. AM J OBSTET GYNECOL 1971;111:60. 10. Goebelsmann U, Bernstein GS, GaleJA, et aI. Serum gonadotropin, testosterone, estradiol and estrone levels prior to and following bilateral vasectomy, In: Teprow IH, Crozier R, eds . Vasectomy: immunologie and pathophysiologic effects in animals and man . New York: Academie Press, 1979: 165. 11. Salmon J, Mouren M. Radioimmunoassay of RU 486. In : Baulieu EE, Segal SJ, eds. The antiprogesterone steroid RU 486 and human fertility controI. New York: Plenum Press, 1985:99-102. 12. Heikinheimo D, Tevilin M, Shoupe D, Croxatto HB, Lahteen maki P. Quanutation of RU 486 after oral adrninistration in human plasma by HPLC and RIA after column chromatography. Contraception 1986;34:613. 13. Dixon WJ. Brown MB, Engleman L. et aI. BMDP Statistical Software. Berkeley: University of California Press, 1983. 14. Croxatto HB, Spitz IM, Salvatierra AM, Bardin CW. The demonstration of the antiprogestin effects of RU 486 when administered to humans during HCG-induced pseudo pregnancy. In : Baulieu EE, Segal SJ, eds . The antiprogesterone steroid RU 486 and human fertility control. New York: Plenum Press, 1985:263-9 . 15. Schaison G, George M, Lestrat N, Reinberg A, Baulieu EE. Effects of the antiprogesterone steroid RU 486 during midluteal phase in normal women. J Clin Endocrinol Metab 1985;61:484. 16. RakoffJJ, Yen SSC. The simultaneous release of prolactin and gonadotropins in response to progesterone adrninistration in estrogen-primed women. J Clin Endocrinol Metab 1979;47 :918 . 17. Dimattina M, Albertsen B, Sayler DE, Loriaux DL, Falk RJ. Effect of the antiprogestin RU 486 on production by cultured human granulosa cellar inhibition of the ovarian 38-hydroxysteroid dehydrogenase. Contraception 1986; 34:199. 18. Shoupe D, Spitz IM, Osborn C, Page M·,Mishell DR, Lobo RA. Effects of progesterone antagonism on gonadotropins at midcyde and during the luteal phase [Abstract]. In : Proceedings of the thirty-third annual meeting of the Soeiety for Gynecologic Investigation, Toronto, Ontario, Canada, March 19-22, 1986. 19. MoguiJewsky M, Philibert D. Biochemical profile of RU 486. In : Baulieu EE, Segal SJ, eds. The antiprogesterone steroid RU 486 and human fertility control, New York: Plenum Press , 1985:87-98. 20. Healy DL, Baulieu EE, Hodgen GD. Induction of menstruation by an antiprogesterone steroid (RU 486) in pri mates: site of action, dose response relationship. and hormonal effects. Fertil Steril 1983;40 :253.
Key words: RU 486 ; contrace ption, go nadotro pins, steroids
The synthesis and development of RU 486 , a 19norprogestin derivative, is conside red a major breakthrough in steroid endocrinolog y' and has opened a new era in fertility control. Beyond its potential use in cont race ptive technalog y, the antiprogestational' and an tiglucocor ticoid'" activities of RU 486 serve as a unique tool for investigation of hormone action. In early re ports, RU 486 has been noted to interrupt th e luteal phase in women':" and in monkeys" and to terminate early pregnancy.t" RU 486 has also bee~ shown to have an affinity for th e glucocor ticoid receptor that is about four time s higher th an that of dexamethasone and has been shown to ha ve antiglucocorFrom the Section of R eproductiue E ndocrinology, Department of Obstetrics and Gynecology, Uniuersity of Southem California , Los Ange les, the Centerfor Biomedical R esearch, Population Couneil, N ew York, the Department of Obstetrics and Gynecology, Uniuersity H ospital, Uppsala, and the Steroid Research Laboratory, Department of Medical Chemistry, Uniuersity of Helsinki. Supp orted in part by grants [rom the Ford and Mellon Foundati ons. Computational assistance was prouided by the CLINFO project fundedby the Diuision of R esearch Resoutces of the National Institutes of H ealth, Grant No . RR-0004 3 . Presented at the Thi rty-second Annual M eeting of the Soeiety [or GynecologicIn uestigation, Phoenix, Arizona, Ma rch 20-23, 1985. R eprint requests: Donna Shoupe, M .D., Women 's H ospital R oom L-946, 1240 No rth Mission R oad, Los Angeles, CA 90033 .
ticoid activity. After a single oral dose of RU 486, there is a transient increase in adrenocorticotropic horrnone.v" The aim of this stud y was to investigate the effect of a single oral dose of RU 486 o n bleeding, gonadotropin, and steroid patterns given to he althy women in the midluteal phase. In addition, the pharmacodynamics of this compound were studied with a recently developed radioimmunoassay.
Material and methods Thirt y-one healthy women, who se ages ranged from 22 to 35 years, were selected for participation in this stud y. They had regular menstrual cyd es (28 ± 3 da ys) and were within 15% of their ide al body weight. They eithe r had been su rgically sterilized or had used a barrier method for contraception, Thc volunteers had not taken any ste ro id medication within th e last 6 months. O n the tenth day of th e men strual cyde, each patient began to undergo dail y blood sarnpling for determinat ion of the day of the luteinizing hormone (LH) surge. Six to eight days after th e LH surge, the volunteers were asked to report at 7:30 A M , after an overnight fast, and to refrain from smoking after midnight for ad ministration of medication. 1415
1416 Shoupe et al.
11.0
Decernber 1987 Am J O bstet Gynecol
E2 210 pg/ml
+
+
LH mlu/ml
5.0 130
3.0
• E2 1
0
2
4
6
• E2 2
110
~.~!-'2~-----' 48
HOURS
0
2
P
4
6
HOURS
~I
10
24
I
48
ng/ml 18.0 + I
FSH mlu/ml
o
2
48
Fig. 1. LH (upper panel) and FSH (lower panel) values afte r a single ora l d ose of RU 486 ra nging from 50 to 800 mg. LH I and FSH1 refer to the patients with one bleeding episode after RU 486 administration and LHz 'and FSHz refer to patients having two bleeding episodes. No significant changes in LH werenoted in either group. Anal ysis of var iance detected a significant de crease in FS H , (p <0.00 1) over the 48-ho u r sam. pling period.
Subjects re ceived either 50 , 100 , 200, 400 , 600. or 800 mg of R U 486 (50 mg tablets supplied by RousselUelaf, Ro mainville, France) a t 8 :00 AM. All groups cornprised four subj ects except for the two highest-dose sched u les where five subjects were stud ied in each group. No smo king or food was permiued for 4 hours after ingestio n of the medication and vital signs were recorded every 30 minutes for 6 hours. Blood sampie s were obtained through an indwelling catheter at - '/ 2 , o + V2, 1,2,4,6, 10,24, a nd 48 hours. A further group of five subjects received 200 mg as a single oral dose and blood sampling was co n tin ued daily for I week and then was performed at 10 and 14 days (ex te nd ed sa m pling group). These blo od sampies were assayed for prolactin, estradiol, progesterone, cortisol, LH, follicle-stimulating hormone ( FS H) , and RU 486 by previou sly d escribed rad ioim m u noassay rnethods.v" Norma l seru m AM co rtisol values are 10 to 30 ILg/dl. Two different radioimmunoassays were used to measure RU 486. The first was the method of Salmon and Mouren. " The seco nd utilizes chromoso rb co lurnn ch ro rna to graphy. " This lauer, more 'specific as say was used only to measure RU 486 level s in the sampies from the extended sam-
6.0 '-r----,--
o
, -- - r - --,..-f'f----r-{ I
2
4
6
HOURS
10
I
i
24
48
Fig. 2. Upper panel: Estradiol (E z) values after a single oral dosof RU 486 ranging from 50 to 800 mg. Ezl refers to patient with one bleedirig episode after RU 486 administration anr E z 2 re fers to those with two bleed ing episodes. Analysis 0 variance detected a significant decrease in EzI (p < 0.05) ove the 48-hour sampling period . Lower panel: Progesteronej'P, values after a single o ra l d ose of RU 486 ranging from 50 to 800 mg . PI refers to pat ients with one bleeding episode after RU 486 ad ministration and P, refers to those with two bleeding epi sodes, Anal ysis of variance detected a significant decrease in P, (p < 0.0 05) over the 48-hour sampling period.
pling group. The antiserum against RU 486 used in both assays was donated by Ro ussel-Uclaf. In addition, SMA-I8 and co m ple te blood co u n t wereperforrned at 0,4, 10, 24 , and 48 hours. . Results were analyzed by analysis of va r ia nce with the use of'BMDP Statistical Software. " One- and two-way analysis of variance and Student's t test were u sed 10 determine statistical differences bet~een gro ups. Results
Clinical features, Menses cornmenced in all patients but one with in 1 to 3 days afte r RU 486 administration (T able I). In nine of these 25 su bj ects (two who received 50 mg, two who received 100 mg, one who received 200 m g, one who received 40 0 rng , and three in the 800 m g group), thi s reprcsented the only bleeding episod e . T h e me an length o f the treatment cyele in these nine subjects was sign ifica n tly less (p <: 0.05) than that of the following cyde (Table 1):
Effecls of RU 486 in normal women. I
Volume 157 Number 6
In th e remaining 16 subjects, a second bleeding episode was reported . In these subjeets the initial bleeding episode occurred at a similar time after the LH surge as in the previous group, but the duration of bleeding was significantly shorter (p < 0.05) . The full duration of the treatrnent cycle in th ose subj eets who had two bleeding episod es (with the onset of the seco nd bleeding episode used as a reference) was 32.9 ± 1.1 days. This was longer (p < 0.05) than the following cyde (Table I). In no ne of these subjeets were an y adverse effects encountered. Furthermore, electrolytes, hepatic fun ction, an d bloo d count d id not change in an y subject. In on e subject who received the 600 mg d ose, bleeding did not occur until 56 days after treatment. Hormonal paramete rs. The hormonal profiles of four patients (two fr om each group) were exduded from analysis becaus~ of incomplete data. In those 14 subj ects who had two bleeding episodes, there was no change in seru m LH , FSH , est radiol, or pro geste rone over th e 48- ho u r sa mpling period. Howeve r, in those seven subjects who had a single bleeding episode, anal ysis of variance showed a significant dedine in estradiol (p < 0.05), FSH (p < 0.001 ), and progesterone (p< 0.005) (Figs. land 2). . There were no significant changes in LH, FSH , estradiol, or progesterone when hor monal res ults were expressed according to th e dosage schedule of RU 486 and independent of the bleeding pattern. Analysis of variance showed that therewas a significant increase in prolactin (p < 0.05) withthe 50 , 200 , 400 , and 800 mg dosages. When compared with the pretreatment levels, the peak values were greatest between 4 and 6 hours (p< 0.05). Values returned to nor mal by 24 hours ( Fig. 3). Ana lysis of variance showed a significant increase (p < 0.05) in mean cortisol values after the 100, 400, 600 , and 800 mg doses. When compared with baseline values , the cortisol levei rose significantly (p < 0.05) at 24 hours .after the 600. and 800 mg doses and at 48 hours with all three higher doses ( Fig. 4). RU 486 levels. When measured directly after diethylether extraction, the serum levels of RU 486 reached a maximum between 1 and 4 hours after ingestion of all doses ( Fig. 5). All RU 486 leve ls were significantly lower with the 50 mg dose than with th e five higher-dose regimens (p < 0.05). The RV486 levels at 6, 24, and 48 hou r s were also significan tly lower after the 100 mg dose than with the four higher dos es (p < 0.05). RU 486 leve ls were the sam e with doses of 200 to 800 mg. With doses of ;;,0200 mg, mean seru m RU 486 levels fell minimally, and even after 48 ho urs values were still above 1.4 fLg/ml. On the other hand , after the 50 and 100 mg doses, RU 486 values did decrease and were under 0.4 fLg/ml at 48 hou rs. Thcre
60 50
1417
- 600 mg ° 600 mg - 40 0 mg
40 30 PRL ng/ml
20 10 30 20 10 0
- 200mg ° 100mg _ 50 mg
0
2
4
6
HOURS
10 12~ 4
18
Fig. 3. Prola ctin (PRL) levels after a single oral dose of RU 486 ranging from 50 to 800 mg. Analysis of variance detected a sign ificant increase in PRL after the 50, 200 , 400 , and 800 mg doses. * p < 0.05 increase over baseline values as determined by the paired t test.
were no differences in the RU 486 levels between the groups with one or two bleeding episodes.. . The serum levels of RU 486 in the extended sampling grou p are shown in Figs. 5 to 6. Chromosorb column chromatography was used in these sam pies and the values were lower tha n th ose with corresponding 200 mg dosage schedule. Never theless, after 4 hours the disappearance cu rves with both assays were parallel. Presumably, the values after chromatography represent · true RU 486 con centrations. The halfdisap pearance tim e was 24 hou rs ( Fig. 6). Comment
. Administration of RU 486 as a single oral dose from 50 to 800 mg in the early lut eal phase was weil tolerated . The induction of menses wirhin 3 days is in accordance with results of p revious studies.v " 15 Thls vaginal bleeding, which occurred des pite high levels of estradiol and progesterone, implies that RU 486 acts directly on the endometrium to produce shedding re gardless of an y effect it may have on hormonal levels. A similar bleeding pattern has also been docurnented in nonpregnant women during concornitant tr eatment with RU 486 and human ch orionic gonadotropirr in the luteal p hase." !' A total of 33% of our subjects dis played only a sin gle
1418 Shoupe et al.
December 1987 Am J Obstet Gynecol
.aOOmg o 600 mg e400 mg
.200mg 0 100 mg e50 mg
Fig. 4. Cortisol values after a single doseof RU 486 rauging from 50 to 800 mg. Analysis of variance detected a significant increase in cortisol (p < 0.05) after the 100, 400 , 600 , and 800 mg dose . * p < 0.05 increase over baseline value s as determined by the paired t test.
5 4
3 2
• 800 mg o 600 mg e 40 0 mg
4 RU 486
V9/ml
3
2 1 .8 .6 .4
.200mg • 200 mg EXTENO o 100mg e 50mg
•
,2
2 Fig. 5~ RU 486 levels after a single oral dose ofRU 486 ranging from 50 to 800 mg. Analysis of variance detected that all points after 50 mg dose and at 6,24, and 48 hours after the 100 mg dose were significantly lower (p < 0.05) than values after the higher doses. The 200 mg ex/end refers to the extended sampling group that is also depicted in Fig. 6.
bleeding episode, whereas in the remaining subjeets a second bleeding episode oecurred. This phenomenon appeared to be independent of the dose or serum concentration of RU 486. The hormonal profile in these two groups was distinetly different. In those subjeets
wiih two bleeding episodes there were no ehanges in LH , estradiol, or progesteronelevels. In addition, ifthe eommeneement of the seeond bleeding episode is regarded as the termination o f the eyde, the luteal phase appeared to be prolonged by RU 486. In contrast, in those subjeets who had asingle bleeding episode there was a progressive signifieant dedine in serurn FSH, estradiol, and progesterone levels over 48 hours of sampling time with a eoneomitant shortening of the luteal phase and eyde length. This suggests that under eertain circumstanees RU 486 ean be luteolytic. Similar observations ha ve been ma de by other workers." 15 Although it is possible that the dedine in steroid levels may represent the fall seen in the lauer part of the normal menstrual cycle, this is unlikely since no corresponding fall occurred in the group with two bleeding episodes. The decrease in the serum progesterone level could be related to a direct action of RU 486 on ovarian steroidogenesis." However, it could also represent an effect on the hypothalamic-pituitary axis. We did observe a decrease in FSH in one group of patients but were unable to detect a decrease in LH secretion. Recently, with more frequent sampling we also ob served a reduction in amplitude and frequency of LH pulses during RU 486 administration in the luteal phase." Other workers have also deseribed a reduction in LH pulse amplitude consequent to RU 486. 15 'Nieman et al." observed menstrual bleeding in norma l women within 72 hours of a single oral dose of RU 486 (10 mg /kg body weight) given in the midluteal phase. In the patients treated with RU 486 on luteal day 7, they re port a decrease in the serurn progesterone level by luteal phase day 10 and the onset of vaginal
Effects of RU 486 in normal women. I
Volume 157 Number 6
1419
3.0 2.0 1.0 0.5 <0_
coE
~::::>0 0:::1..
0.2 0.1 0.05 0.02 0 .01 0.005 0.002 0.001 0
2
4
6
8
10
12
14
DAYS Fig. 6. RU 486 levels in the extended sampling group after a single oral dose of 200 mg RU 486. The half-life of RU 486 is 24 hours.
Table I.' Effects of RU 486 administration Bleeding
episodes I 2
9 16
Onset (days after LH surge)
bleeding
Dass of
Treatment cycle length (days)
Posttreatment cycle length (days)
9.8 :!: 0.6 9.6 :!: 0.5 19.0 :!: 0.8
3.4 ± 0.3 6.0 :!: 0.8t 4.2 ± 0.3
25.2 ± 1.4* 32.9 :!: l.l
34.0:!: 2.1 26.6 :!: 1.7
Values are mean :!: SD. *p < 0.05 when eompared with posureatrnent eyd e length. t p < 0.05 when eompared with group with one b1eeding episode.
bleeding within 72 hours of treatrnent in all patients. They also described two patient groups as regards to the onset of menses, which ma y correlate with our two patient groups. Circulating RU 486 levels measured by immunoassa y showed that this synthetic steroid was absorbed rapidly. Doses of 50 and 100 mg gave lower blood levels than higher doses. However, no c1ear dose response was evident sinee 200 to 800 mg RU 486 gave similar blood levels . The irnmunoassay used to determine these eireulating levels is nonspeeifie and measures metabolites, including the monodemethylated a nd didemethylated eompou nd s as weil as the alcoho lie derivative ." : 12 T hese metabolites seem to have a smaller biologie activity." For thi s reason we developed a more speeifie radioimmunoassay using prelirninary ehromosorb eh romatography. With thi s modification, values were lower but gave a parallel disappearanee eu rve. Values of RU 486 measured by immunoassay after eh romosorb ehro-
matography give values similar to th ose determined by high-pressure liquid ehromatography. 12 The half-li fe of RU 486 was 24 hours. The long half-disappearance of RU 486 suggests that there is binding to plasma proteins, Reeent stud ies have shown that RU 486 binds to an
1420 Shoupe et a!.
tramuscular progesterone after oophorectomy. This would imply a positive effect of progesterone on prolactin secretion, which is in contrast to the transient increase in prolactin we noted after treatment with an antiprogesterone. Healy et apodescribed a decrease in prolactin after treatment with RU 486 in monkeys (10 mg/kg) ." However, these were castrated monkeys who had steroid replacement via estradiol and progesterone capsules, which produce basal hyperprolactinemia. Within 1 hour after parenteral RU 486 administration, these elevated prolactin levels were decreased. Thus the effect of RU 486 on prolactin secretion varies and may depend on the mode of its administration, the dose, the species, and the prevailing steroid concentrations. The reason why two subgroups emerged in this study is not evident. They could be differentiated by both bleeding patterns and hormonal levels. Both groups received RU 486 on days 6 to 8 after the LH surge and there were no differences in RU 486 serum levels between the two groups. Thus these two patterns of response could not be differentiated on the basis of time of treatrnent, dosage of RU 486, or circulating RU 486 levels. RU 486 has been proposed as a once-a-rnonth pill." Our data would support the value of RU 486 for this use. However, the emergence of two patient groups suggests that further characterization of these distinctive patterns is necessary. REFERENCES 1. Teutsch G. Analogues of RU 486 for the mapping of the progestin receptor: synthetic and structural aspects, In : Baulieu EE, Segal SJ, eds. The antiprogesterone steroid RU 486 and human fertility control, New York: Plenum Press, 1985:27-47 . 2. Healy DL, Chrousos G, Schulte HM, Gold PW, Hodgen GD. Increased adrenocorticotropin, cortisol, and arginine vasopressin secretion in primates after the antiglucocorticoid steroid RU 486: dose response relationship. J CIin Endocrinol Metab 1985;60: 1. 3. Gaillard RC, Riondel A, Muller AF, Herrmann W, Baulieu EE. RU 486: asteroid with antiglucocorticoid activity that only disinhibits the human pituitary-adrenal system at a speeific time of day. Proc Natl Acad Sei USA 1984; 81 :3879. 4. Hermann W, Wyss R, Riondel A, et aI. The effects of an antiprogesterone steroid on women: interruption of the menstrual cyde and of early pregnancy. C R Acad Sei [III] 1982;294:933. 5. Shoupe D, Mishell DR, Lacarra M, Gutierrez E, Labteenmaki P, Spitz 1. Endocrinologie effects of the antiprogesterone RU 486 in the luteal phase of normal women . In : Baulieu EE, Segal 5J, eds . The antiprogesterone stero id
December 1987 Am J Obstet Gynecol
RU 486 and human fertility controI. New York: Plenum Press, 1985:285-93. 6. Nieman LK, Choate TM, Chrousos GP, et aI. The progesterone antagonist RU 486 : a potential new contraceptive agent. N EnglJ Med 1987;316:187 . 7. Haspels AA. Interruption of early pregnancy by an antiprogesrational compound, RU 486. EurJ Obstet Gynecol Reprod Biol 1985;20:169. 8. Mishell DR, Shoupe D, Brenner PF, Horenstein J, Lacarra M, Spitz IM. Termination of early gestation with the anti-progesterone, RU 486 . Contraception 1987; 35 :307. 9. Mishell DR, Nakamura RA, Crosignani PG, et aI. Serum gonadotropin and steroid pattern during the normal menstrual cyde. AM J OBSTET GYNECOL 1971;111:60. 10. Goebelsmann U, Bernstein GS, GaleJA, et aI. Serum gonadotropin, testosterone, estradiol and estrone levels prior to and following bilateral vasectomy, In: Teprow IH, Crozier R, eds . Vasectomy: immunologie and pathophysiologic effects in animals and man . New York: Academie Press, 1979: 165. 11. Salmon J, Mouren M. Radioimmunoassay of RU 486. In : Baulieu EE, Segal SJ, eds. The antiprogesterone steroid RU 486 and human fertility controI. New York: Plenum Press, 1985:99-102. 12. Heikinheimo D, Tevilin M, Shoupe D, Croxatto HB, Lahteen maki P. Quanutation of RU 486 after oral adrninistration in human plasma by HPLC and RIA after column chromatography. Contraception 1986;34:613. 13. Dixon WJ. Brown MB, Engleman L. et aI. BMDP Statistical Software. Berkeley: University of California Press, 1983. 14. Croxatto HB, Spitz IM, Salvatierra AM, Bardin CW. The demonstration of the antiprogestin effects of RU 486 when administered to humans during HCG-induced pseudo pregnancy. In : Baulieu EE, Segal SJ, eds . The antiprogesterone steroid RU 486 and human fertility control. New York: Plenum Press, 1985:263-9 . 15. Schaison G, George M, Lestrat N, Reinberg A, Baulieu EE. Effects of the antiprogesterone steroid RU 486 during midluteal phase in normal women. J Clin Endocrinol Metab 1985;61:484. 16. RakoffJJ, Yen SSC. The simultaneous release of prolactin and gonadotropins in response to progesterone adrninistration in estrogen-primed women. J Clin Endocrinol Metab 1979;47 :918 . 17. Dimattina M, Albertsen B, Sayler DE, Loriaux DL, Falk RJ. Effect of the antiprogestin RU 486 on production by cultured human granulosa cellar inhibition of the ovarian 38-hydroxysteroid dehydrogenase. Contraception 1986; 34:199. 18. Shoupe D, Spitz IM, Osborn C, Page M·,Mishell DR, Lobo RA. Effects of progesterone antagonism on gonadotropins at midcyde and during the luteal phase [Abstract]. In : Proceedings of the thirty-third annual meeting of the Soeiety for Gynecologic Investigation, Toronto, Ontario, Canada, March 19-22, 1986. 19. MoguiJewsky M, Philibert D. Biochemical profile of RU 486. In : Baulieu EE, Segal SJ, eds. The antiprogesterone steroid RU 486 and human fertility control, New York: Plenum Press , 1985:87-98. 20. Healy DL, Baulieu EE, Hodgen GD. Induction of menstruation by an antiprogesterone steroid (RU 486) in pri mates: site of action, dose response relationship. and hormonal effects. Fertil Steril 1983;40 :253.