METABOLITES IN HUMANS
`
`OSKARI HEIKINHEIMO, KIMMo KONTULA*, HORACIO CROXATTOT, IRVING Spirzi,
`TAPANI LUUKKAINEN and PEKKA LAHTEENMAKI§
`Steroid Research Laboratory, Department of Medical Chemistry, University of Helsinki, Helsinki,
`Finland, *Department of Obstetrics and Gynecology, University of Helsinki, Helsinki, Finland, TInstituto
`Chileno De Medicina Reproductiva, Santiago, Chile and ICenter
`for Biomedical Research, The
`Population Council, New York, NY 10021, U.S.A.
`
`(Received 18 August 1986)
`
`Summary—Using Chromosorb® chromatography and HPLC, we measured the plasma concentrations of
`RU 486, and its monodemethylated (RU 42633), didemethylated (RU 42848) and alcoholic non-
`demethylated (RU 42698) metabolites up to 72 h following oral ingestion of 100 mg of RU 486 by five
`female volunteers. The peak plasma level of RU 486 (4.5 umol/l) occurred within 1 h after ingestion of
`the compound; at this point significant amounts of the metabolites were also present in the plasma. After
`the initial redistribution within 6h the plasma concentrations of RU 486 and three of its metabolites
`measured remained stable for 24 h. Concentrations of the monodemethylated metabolite exceeded those
`of the parent steroid during the time period measured, whereas the concentrations of the didemethylated
`and alcoholic metabolites were lower than those of RU 486, but still notable. At 72 h the concentrations
`of all the four steroids were still in the micromolar range. The relative binding affinities of these metabolites
`to human endometrial and myometrial progesterone receptors as well as to human placental glu—
`cocorticoid receptors were determined in vitra. The affinity of RU 486 for the human uterine progesterone
`receptor (Kd = 1.3 x 10“9M for RU 486) was higher than that of progesterone but lower than that of
`ORG-2058, a potent synthetic progestin. The relative binding affinities of the monodemethylated,
`alcoholic and didemethylated metabolites to the progesterone receptor were 21, 15 and 9%, respectively,
`compared with the parent compound RU 486; each was lower than that of progesterone (43%). RU 486
`had an approx. 4-fold higher relative binding affinity to the glucocorticoid receptor than dexamethasone.
`Interestingly, the relative binding affinities of the metabolites studied to the human glucocorticoid receptor
`exceeded those of dexamethasone or cortisol. Compared with the parent compound RU 486, they were
`61, 48 and 45% for the monodemethylated, alcoholic and didemethylated metabolites, respectively; each
`was higher than that of dexamethasone (23%). The affinity of dexamethasone to the human glucocorticoid
`receptor was 1.6 x 10‘9 M. These data indicate that the pool of certain metabolites of RU 486 may
`contribute to a significant extent
`to the antiprogestagenic (23—33%) and even greater extent to the
`antiglucocortieoid (47—61%) effects of RU 486.
`
`INTRODUCTION
`
`RU 486 is a recently described l9-nor-steroid deriva—
`tive with considerable antiprogestagenic and anti-
`glucocorticoidal properties [1, 2]. When given during
`the luteal phase of the menstrual cycle, RU 486 is able
`to induce uterine bleeding [I]. In preliminary clinical
`studies RU 486 induced abortion in approx. 80% of
`the subjects when given between weeks 5~8 of preg-
`nancy, at a daily dose of 200mg for 4 days [3,4].
`Recently, using RU 486, Nieman et a].
`reported
`successful
`symptomatic
`treatment of Cushing’s
`syndrome [5].
`The dimethylaminophenyl side-chain at carbon 11
`
`
`Steroid Research Laboratory, De-
`§Correspondence1
`partment of Medical Chemistry, University of Helsinki,
`Siltavuorenpenger 10 A, SF-OOI70 Helsinki, Finland.
`Presented in part at the X11 Meeting of the International
`Study Group for Steroid Hormones, Rome, Italy, 1985.
`
`for antiprogestagenic
`important
`of RU 486 is
`action [6]. For all mammalian progesterone receptors
`investigated, RU 486 has a higher atfinity than
`progesterone [4, 7, 8]. The relative binding affinity of
`RU 486 for the glucocorticoid receptor is either equal
`to [7] or greater
`than [4]
`that of dexamethasone.
`Synthetic steroids may have biologically active me-
`tabolites. Recently, Deraedt et al.[9] identified micro-
`molar plasma concentrations of a monodemethylated
`metabolite after oral ingestion of RU 486. Our earlier
`studies indicate the presence of additional
`noreactive metabolites [10].
`Deraedt et a1. studied the metabolism of RU 486
`in rats and found that the monodemethylated, di-
`demethylated and alcoholic metabolites all retain
`antiglucocorticoidal and antiprogestagenic activity
`that correlated with the binding affinity to both
`progesterone and glucocorticoid receptors [9].
`Since RU 486 has a high potential for clinical use,
`the biological activity of its major metabolites is of
`
`279
`
`

`

`relative binding
`their
`of RU 486. Furthermore,
`affinities for human placental glucocorticoid and
`uterine (myometrial and endometrial) progesterone
`receptors in vitra were compared with those of refer-
`ence steroids.
`
`EXPERIMENTAL
`
`Chemicals
`
`(l7fi-hydroxy-llfi-(4—dimethylamino-
`RU 486
`pheny1)-17a-(l—propynyl)-estra—4,9’dien-3—one),
`the
`monodemethylated metabolite RU 42633
`(178-
`hydroxy- 1 1,8-(4-monomethylaminophenyl) - l7ot-(l —
`propynyl)—estra-4,9—dien~3—one),
`the didemethylated
`metabolite RU 42848 (17fl-hydroxy—llfl-(4—amino-
`phenyl)—17or -(1-propynyl)—estra-4,9-dien-3-one),
`the
`alcoholic metabolite RU 42698 (17,8-hydroxy-llfi-
`(4-dimethylaminophenyl)-l 705 -(l -propynol)—estra-4,9~
`dien-3-one) and [6,7-3H]RU 486 (sp. act. 37 Ci/mmol)
`were kindly donated by the Roussel—Uclaf Research
`Center, Romainville, France. The molecular struc~
`tures of the compounds are presented in Fig.
`l.
`Progesterone (4—pregnene-3,20-dione), dexametha—
`sone (9-fluoro-16oc-methyl-l1fi,l7ot,21-trihydroxy-1,4—
`pregnadiene-3,20-dione)
`and cortisol
`(1113,1121-
`trihydroxy-4—pregnene-3,20-dione) were purchased
`from Steraloids Inc, Wilton, NH, USA. ORG-
`2058 (16oz-ethyl-2l~hydroxy-l9-nor—4-pregnene-3,20-
`
`dione) was obtained from Organon Int, 035, The
`
`
`Netherlands. [6,7-3H] dexamethasone [ )XM] (sp. act.
`
`ethanolamine, titriplex III (EDTA), and HPLC col-
`umn Hibar LiChrosorb RP-18 (250 x 4mm int. dia)
`were purchased from Merck, Darmstadt, West Ger-
`many. Tris~HCl, dithiothreitol and Chromosorb® W-
`NAW 60/80 Mesh were from Sigma, St Louis, MI,
`U.S.A. Norit A was purchased from Amend, Irving-
`ton, NJ, U.S.A., and dextran T70 from Pharmacia,
`Uppsala, Sweden. Ammonium sulfate was purchased
`from Schwartz/Mann and scintillation fluid YA—
`riatuike (70% pseudochumene) was obtained from
`Yliopiston Apteekki, Helsinki, Finland.
`
`Human samples
`
`Plasma samples were collected from five healthy
`female volunteers after oral ingestion of 100 mg RU
`486 in mid-luteal phase of their cycle. Uteri were
`obtained from patients undergoing hysterectomy for
`uterine fibroids. The last menstrual period of the
`patients had occurred approx. 2 weeks prior to
`operation. Only non-myomatous uterine tissue was
`used for the experiments described below. Placentas
`were obtained from women undergoing elective
`Caesarean section.
`
`HPLC studies
`
`The Chromosorbg columniHPLC-method de-
`
`scribed before [10] was modified. Disposable Pasteur
`pipettes were packed with 3ml of Chromosorbi":
`W-NAW 60/80 Mesh/20% ethylene glycol. A plasma
`sample was applied to the column,
`
`OH
`
`"*CEC—C HZOH
`
`
`
`
`RU 42698
`
`H3C\N
`H3C/
`
`H C
`\N
`3
`H3C/
`
`
`
`
`RU42848
`
`MOLECULAR STRUCTURES OF RU486,RU 42633, RU42848
`AND RU 42698
`
`Fig.
`
`1. Molecular structures of RU 486 and its monodemethylated (RU 42633), didemethylated (RU
`42848) and alcoholic non-demethylated (RU 42698) metabolites.
`
`

`

`of RU 486 was methanol—water—triethanolamine,
`90: 10005, pumped at a rate of 1.5 ml/min; and for
`the assay of
`the
`three metabolites, methanol—
`water—acetic
`acid‘diethyl
`ether—triethanolamine,
`75:45:30:7.5:0.05, pumped at a rate of 2.2 ml/min.
`
`Preparation of tissue samples
`
`The uterine samples were processed as described by
`Haukkamaa [l l] and placental tissues as described by
`Kontula et a], for adrenal cortical tissue [12]. Cytosol
`samples were prepared by high-speed centrifugation
`of tissue homogenates. To remove endogenous ste—
`
`roids from the cytosol samples a Dextran-coated
`
`
`charcoal ( CC) suspension containing 0.5% Norit
`A, 0.005% Dextran T70 and 0.1% gelatin in 50 mM
`TrisvHCl-bufier, pH 7.4, was prepared. An aliquot of
`DCC suspension (the volume corresponding to the
`cytosolic preparation to be stripped) was centrifuged
`at 3000g for 10 min. The supernatant was discarded
`and the cytosol preparation was added to the char-
`coal pellet. The tubes were vortex-mixed and incu-
`bated for 10min at +4°C. After centrifugation at
`3000 g for 10 min, the stripped cytosol samples were
`used for the competitive protein binding assays.
`
`Competitive receptor binding assays
`
`All assays were performed in duplicate or triplicate
`in disposable glass test tubes and were repeated at
`least
`3
`times. For progesterone receptor studies,
`varying amounts
`(final concentrations,
`10’10 to
`lO'SM) of the steroids investigated (RU 486, RU
`42633, RU 42848, RU 42698, ORG—2058 and pro-
`gesterone) together with 10“7 M cortisol (to block
`binding to corticosteroid-binding globulin and to the
`glucocorticoid receptor), were pipetted into the tubes
`and evaporated to dryness. One—hundred microliters
`of cytosol (diluted to such an extent that approx 50%
`of the tritiated ligand was bound in the absence of
`any competitor) and 0.03 uCi of [3H]ORG—2058
`(pipetted in 100111 of 50 mM Tris containing 1%
`ethanol; final concentration 2.8 nM) were added, the
`tubes were vortexed-mixed and then incubated over-
`
`night at +4OC. After incubation, 200 pl of DCC
`suspension was added to each tube and the contents
`vortex-mixed. After 10 min at +4OC, The tubes were
`centrifuged for 5min at 3000 g. The supernatants
`(containing the bound fraction of the tritiated ligand)
`were transferred to polyethylene counting vials to-
`gether with 3 ml of scintillation fluid and were coun-
`ted for 5min in a liquid scintillation 1212 Minibeta
`counter (Wallac, Turku, Finland). The relative bind-
`ing affinities of the difierent compounds to the pro-
`gesterone receptor were calculated at the 50% com—
`petition level according to Korenman [13].
`
`[3H]ORG-2058.
`
`Scatchard—plot analysis
`
`To verify the glucocorticoid receptor—nature of the
`steroid-binding component in placental cytosol, the
`dissociation constant
`(Kd) of its interaction with
`[3H]DXM was measured. Aliquots
`charcoal-stripped placental cytosol were incubated,
`in a total volume of 0.2 ml, with varying concen-
`trations (03—300 nM) of [3H]DXM dissolved in
`50 mM Tris-buffer. The extent of non-specific binding
`of [3H]DXM was estimated from a parallel set of
`tubes also containing 10’5 M non-radioactive DXM.
`The tubes were incubated overnight at +4OC. 0.25 ml
`of DCC was added to separate bound and unbound
`steroids. Further steps were carried out as described
`above for the competitive receptor binding assays.
`The binding data (corrected for non-specific binding)
`were analyzed according to Scatchard [14].
`To measure the Kd of RU 486 for the human
`uterine progesterone receptor, a partially purified
`progesterone receptor preparation from human myo-
`metrial cytosol was first prepared as described by
`Kontula et al.[15]. Before use,
`[3H]RU 486 was
`purified using the Chromosorb® technique [10]. The
`rest of the analysis was essentially as described above,
`except that partially purified progesterone receptor
`preparation and [3H]RU 486 were used instead of
`placental cytosol and [3H]DXM, respectively, and
`non-radioactive RU 486 was used instead of DXM
`
`for the correction for non-specific binding. No excess
`of cortisol was used.
`
`RESULTS
`
`The u.v.-absorption spectra of the synthetic metab-
`olites and their behavior in our HPLC system were
`analyzed. All
`the synthetic metabolites shared a
`common u.v.-absorption maximum at 304 nm. Each
`also had a characteristic u.v.-absorption maximum:
`RU 42633 at 250 nm, RU 42848 at 240 nm and RU
`42698 at 258 nm. Their retention times in our HPLC
`system were 4min 36s, 3min 56s and 2min 49 s,
`respectively.
`Plasma concentrations (mean ~1- SEM) of RU 486
`and of
`its monodemethylated (RU 42633), di-
`demethylated (RU 42848) and non-demethylated al-
`coholic (RU 42698) metabolites, after oral ingestion
`of 100 mg of RU 486 by five female volunteers, are
`depicted in Fig. 2. Peak plasma concentrations of RU
`486 (4.5 umol/l) were reached Within 1 h after inges—
`tion of the drug. The concentrations of the mono—
`demethylated metabolite (RU 42633) and hydroxy-
`lated alcoholic metabolite (RU 42698) also reached
`
`

`

`‘6moL/1.mu4:-orm«1
`
`o
`.—
`
`1
`
`\T
`A
`
`\K
`«vi
`\T—._._i\.___:\
`1.1....3.
`~
`.
`~2r-......
`.
`...;....-«r~-5>-\\-3‘6
`
`arise-"A" ’ T"‘:'"t"":re
`2
`4
`6
`"
`24
`48
`‘
`72
`(Hours)
`
`Fig. 2. Plasma concentrations (mean+ SEM) of RU 486,
`RU 42633, RU 42848 and RU 42698, after oral ingestion of
`100 mg of RU 486 by five female volunteers.
`
`peak concentrations within 1+2h suggesting rapid
`first pass metabolism of RU 486. Plasma concen-
`trations of
`the didemethylated metabolite (RU
`42848) increased slowly between 6 and 24 h, maxi-
`mum concentrations were measured 24 h after inges-
`tion of RU 486. After initial redistribution of 6 h the
`
`plasma concentrations of RU 486 and three of the
`metabolites assayed plateaued for 24h or more.
`Concentrations of the monodemethylated metabolite
`exceeded those of the parent RU 486. Plasma concen-
`trations of the didemethylated and the alcoholic
`metabolite were lower than those of RU 486 but still
`
`notable. Importantiy, both RU 486 and the three
`metabolites were still present in micromolar concen-
`trations at 72 h.
`
`The binding of RU 486 and its metabolites to
`human progesterone receptor in vitro was studied
`
`
`
`MEAN
`
`Kd 1.3-169M
`
`
`
`o
`
`nmm/
`20
`15
`1O
`05
`SPEClFlC BiNDlNG OF (3H)'RU486
`
`[3H]RU 486 Scatchard plot analysis of human
`Fig. 3.
`myometrial progesterone receptor. Mean Kd 1.3 x 10‘9 M.
`
`Bound
`Free
`
`MEAN Kd1,6~ 169M
`
`02
`
`
`01
`
`\
`
`01
`spacmc
`
`OTA'nmol/l
`03
`02
`BINDING OF(3H1~DXM
`
`373
`100
`
`ORG-2058
`RU 486
`Progesterone
`RU 42633
`RU 42698
`
`RU 42848 9
`
`systematic names
`*For
`mental.
`TRelative to RU 486 (= 100%).
`
`see Experi-
`
`using both human endometrial and myometrical cy-
`tosol. The relative binding affinities were identical
`and therefore combined. The Kd (mean of three
`separate experiments) of the binding of RU 486 to the
`human myometrial
`progesterone
`1.3 X 10'9M (Fig. 3). The relative binding affinity
`of RU 486 to the human progesterone receptor was
`higher than that of progesterone but lower than that
`of the potent synthetic progestin ORG—2058. All the
`metabolites of RU 486 studied had a lower afiinity to
`the progesterone receptor than progesterone itself.
`The relative binding affinities of ORG-2058, pro—
`gesterone and the three metabolites of RU 486 to the
`progesterone receptor are given in Table 1.
`The binding of RU 486 and its metabolites to the
`human glucocorticoid receptor in vitro was studied
`using human placental cytosol. Figure 4 shows a
`representative Scatchard—plot of the interaction be-
`tween the placental glucocorticoid receptor and triti—
`ated DXM. The mean Kd in four experiments was
`1.6 x 10'9M. Competition studies revealed that all
`three major metabolites of RU 486, along with the
`parent compound, had higher affinities for the glu-
`cocorticoid receptor than the potent glucocorticoids
`dexamethasone and cortisol. Table 2 gives the relative
`affinities of the steroids tested for the human placen-
`
`tal glucocorticoid receptor (mean values of 5 separate
`experiments).
`
`DISCUSSION
`
`Synthetic steroid derivatives may have biologically
`active metabolites. Radioimmunoassays often lack
`
`Table 2. The relative binding affinities of
`the
`steroids
`investigated for human
`glucocorticoid receptor
`Relative
`affinity
`
`100
`
`Compound*
`RU 486
`RU 42633
`RU 42698
`RU 42848
`Dexamethasone
`Cortisol
`
`4.
`Fig.
`human
`
`[3H]dexamethasone Scatchard plot analysis of
`placental
`glucocorticoid
`receptor. Mean
`Kd
`1.6 x 10’9M.
`
`systematic names,
`*For
`mental.
`JrRelative to RU 486 (= 100%).
`
`see Experi«
`
`

`

`to
`have developed methods
`assay [16, 17]. We
`specifically measure plasma concentrations of RU
`486 and its three most proximal metabolic products
`using Chromosorbi‘j-column chromatography and
`HPLC. The HPLC method described previously [9]
`had to be improved since it did not separate the
`monodemethylated metabolite from the alcoholic
`metabolite. Our results show that after ingestion of
`100 mg of RU 486 by human female volunteers,
`at
`least
`three metabolites
`of RU 486,
`the
`monodemethylated (RU 42633),
`didemethylated
`(RU 42848) and alcoholic non—demethylated (RU
`42698) forms, are circulating in micromolar concen-
`trations, i.e. close to that of the parent compound for
`72 h. When measured by a specific Chromosorbl‘i-
`HPLC-method the plasma concentrations of RU 486
`did not differ significantly when the single oral dose
`of RU 486 was increased from 100 to 800 mg[10].
`This suggests rapid distribution of RU 486 into the
`tissues, and rapid first-pass metabolism of RU 486.
`Oral administration of [3H]RU 486 resulted in re-
`markable extravascular diffusion in rats as reported
`by Deraedt et al.[9]. Studies employing specific HPLC
`method will reveal whether there is a change in the
`ratios between RU 486 and its metabolites after the
`
`administration of different oral and parenteral doses
`of RU 486. In general the receptor binding ability of
`a steroid gives an indication, although not proof, of
`its biological activity. Deraedt et a]. determined the
`relative binding affinities of RU 486, RU 42633, RU
`42848 and RU 42698 to cytosolic progesterone and
`glucocorticoid receptors. Oral administration of RU
`486, RU 42633, RU 42848 or RU 42698 in rats
`resulted in abortion or inhibited the thymolytic effect
`of dexamethasone thus demonstrating their anti-
`progestational
`and
`antiglucocorticoidal
`nature,
`respectively [9]. Their results indicate that the alco-
`holic metabolite might have a higher biological activ-
`ity in relation to receptor binding as compared with
`the monodemethylated metabolite. The relative bind-
`ing affinities of RU 486 and its three metabolites to
`the human glucocorticoid and progesterone receptors
`were determined, using dexamethasone and ORG-
`2058, respectively, as reference steroids. Before ac-
`cepting the previously characterized progesterone [l 1]
`and glucocorticoid receptor[12] systems as models,
`the saturability and high affinity of the binding was
`confirmed in each case (Figs 3 and 4). In previous
`studies, RU 486 has been shown to display a
`binding affinity greater than that of progesterone
`in
`all
`the mammalian
`progesterone
`receptors
`investigated [4, 7]. Variations
`in
`the
`reported
`affinities [4, 7, 8] may
`be
`explained
`by
`species
`differences
`in
`the
`characteristics
`of
`steroid
`
`reveals features suggesting high affinity binding to
`progesterone receptor [18]. The antiprogestagenic
`properties of RU 486 are thought to be due to the
`dimethylaminophenyl side chain at carbon 11[6].
`Demethylation of this side chain decreases its hydro—
`phobicity, and also decreases the binding affinity of
`mono- and didemethylated metabolites to 21 and
`9%, respectively (Table 1). Hydroxylation of the side
`chain at carbon 17 decreases the binding affinity of
`the compound from 100% (RU 486) to 15% [RU
`42698) (Table 1).
`Based on the relative receptor binding affinities of
`the metabolites (Table 1) and their plasma concen-
`trations (Fig. 2), it is possible to estimate the con-
`tribution of
`the metabolite pool
`progestational action of RU 486. The theoretical
`contribution of the prevailing metabolite pool to the
`antiprogestational activity of RU 486 after ingestion
`of 100 mg of RU 486 amounts to about 23% at 1 h
`but as high as 33% at 24 h.
`Comparatively little is known about the relative
`affinity of RU 486 for human glucocorticoid recep—
`tors. However, in comparison with published clinical
`and experimental studies [2, 4, 7], the high affinities of
`RU 486 and of its metabolites to the human glu-
`cocorticoid receptor (Table 2) are not surprising.
`However, it must be kept in mind that competition
`studies performed at Out—4°C in cell-free conditions
`do not necessarily correctly reflect
`the situation at
`+37OC and in the whole organism[19]. The the-
`oretical contribution of the metabolites of RU 486 to
`
`the antiglucocorticoidal action of RU 486 was calcu-
`lated. This was based on the relative receptor binding
`affinities (Table 2) and plasma concentrations (Fig. 2)
`of the metabolites. These results suggests that 1 and
`24 h after the intake of 100 mg of RU 486, the three
`metabolites would represent 47 and 61%,
`spectively, of the total antiglucocorticoid activity of
`RU 486.
`
`Despite the high affinity binding of RU 486 and its
`metabolites to the human glucocorticoid receptor in
`vitro, previous clinical experience suggests that large
`single doses of RU 486 (2400 mg) are needed to
`promote antiglucocorticoid effects
`Chronic treatment with 25—200 mg/day of RU 486,
`doses sufficient to produce uterine bleeding in 80%
`or more cases, did not result in any apparent anti—
`glucocorticoidal effects [1, 3]. This may be partly ex-
`plained by the fact that the concentrations of plasma
`cortisol are at least one order of magnitude higher
`than that of plasma progesterone, even during the
`luteal phase of the menstrual cycle. The commonly
`used clinical parameters of antiglucocorticoid activ-
`ity,
`i.e. plasma ACTH and cortisol concentrations,
`
`

`

`in viva [2], might be explained by the
`and cortisol
`higher bioavailability of DXM (32% non-protein
`bound
`in
`plasma,
`ref.
`21)
`or
`higher
`hypothalamic/pituitary uptake of DMX compared to
`RU 486. In view of the fact that plasma concen-
`trations of RU 486 are not elevated by increasing the
`oral dose of RU 486 from 100 to 800 mg, all associ-
`ated with micromolar
`concentrations of
`anti-
`
`glucocorticoid steroids (Fig. 2, ref.10), it still remains
`an enigma why systemic antiglucocorticoidal effects
`are Virtually never seen at RU 486 doses below
`400 mg.
`In conclusion, the remarkable binding affinities of
`the metabolites of RU 486 to human progesterone
`and glucocorticoid receptors suggest an important
`role of these metabolites, along with the parent
`compound, as regards the antisteroidal action of RU
`486. This also justifies further metabolic studies after
`administration of varying oral or parenteral doses of
`RU 486.
`
`Acknowledgements—This work was undertaken as part of
`the contraceptive development program sponsored by the
`International Committee for Contraception Research of the
`Population Council, Inc., New York. The financial support
`provided by the Ford Foundation, the Mellon Foundation
`and the Pehr Oscar Klingendahl Foundation is gratefully
`acknowledged. The content does not necessarily reflect the
`policy of any of the funding sources. We thank Ms Marjatta
`Tevilin for her expert
`technical help and Ms Tellervo
`Hiljanen for her kind assistance.
`
`REFERENCES
`
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`3. Haspels A. A; Interruption of early pregnancy by an
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`hebd. Acad. Seanc. Sci. Paris 294 (1982) 933—938.
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`Metab. 61 (1985) 53&540.
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`Contraception 28 (1983) 77—85.
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