Investigations on interactions at the uterine level between progestogen and
oestrogen have been published by Bhakoo (1977). The test system was the rat uterus
and compounds used included progesterone, NG and chlormadinone acetate (CMA).
Exposure to progestogen antagonised the synthesis of a specific uterine protein
induced by oestradiol treatment. Androgens and corticosteroids were without efTect
Briggs (1980) has reported that progesterone administration induces specific
cytosol receptors for progesterone in the mammary glands and uterus of adult beagle
bitches, but not in the mammary tissues or uterus of adult female rats .
Following uptake of asteroid hormone by its cytosol receptor, the complex
migrates into the cell nucleus, where binding occurs to specific acceptor sites (or
'second receptors') on chromatin (Baulieu, 1978; Baxter & Funder, 1979). The nature
of these nuclear acceptor sites is poorly understood, though they ofTer a further
parameter where major species difTerences may exist.
Urinary recovery of labelled-EE is significantly less than for an equivalent dose of
labelied oestradiol in most laboratory animals as weil as in humans (Karnyab,
Fotherby & Steele , 1969; Abdel-Aziz & Williams, 1970; Kulkarni & Goldzieher,
1970; Reed, Fotherby & Steele, 1972). In bile-duct cannulated rats, about 50% ofthe
radioactivity was EE-3-glucuronide, while 35% was EE-3-sulphate (Steinetz, Meli ,
Giannina & Beach, 1967). These conjugates are also the principal urinary
metabolites. The importance of biliary excretion has also been demonstrated in
humans (Cargill, Steinetz, Gosnell, Beach, Meli, Fujimoto & Raynolds, 1969; Reed et
al., 1972; Fotherby, 1973), and significant amounts of EE metabolites appear in
faeces. Approximately 40% of the dose is excreted in faeces, compared to 60% in
urine. The overall recovery from both sources has been reported to be 91 ± 9%
(Speck , Wendt, Schulze, Jentsch, 1976). Biliary excretion is also important in the
Mention has already been made of the demethylation of MEE in most species to
form EE, though small amounts of MEE-17-g1ucuronide and 2-hydroxy-MEE are
also formed (Williams, Helton & Goldzieher, 1975a; Williams, Longcope &
Chenault & Goldzieher, 1975; Nilsson & Nygren, 1978; Ranney, 1977; Bolt , 1979).
Oral administration of MEE or EE to normal women leads to aseries of minor
metabolites. The principal hydroxylated derivative is 2-hydroxy-EE, some of which
is converted to 2-methoxy-EE and both excreted as sulphate and glucuronide
conjugates (Abdel-Aziz & Williams, 1969; Williams et al., 1975a ,b). Up to 64% of
oral administered EE has been reported to be hydroxylated in the 2-position by some
individuals (Bolt , Kappus & Bolt, 1974), though the mean value of the group was
29% (Bolt, Bolt & Kappus, 1977). Irreversible tissue binding ofsmall amounts of EE
may involve this catechol metabolite (Kappus, Bolt & Remmer, 1973). Metabolites
identified in small amounts in the urinary polar fraction include 4-hydroxy-EE,
6a-hydroxy-EE and 16ß-hydroxy-EE (Cargill et al., 1969; Williams et al., 1975a ;
MEE and EE als o undergo D-homoa nnulatio n to yie ld D-homoestrone and
D-homoestradiol-17 aß. The mechanism of this reaction may involve oxidative
attack on the ethynyl group, followed by ring enlargement and oxidative elimination
of the co-situated C atom (Abdel-Aziz & Williams, 1969). This reaction occurs
The extent of de-ethynylation of EE and MEE is controversial. According to
Kulkarni & Goldzieher (1970) and Williams et al. (l975b), between 15 and 20 % of
human urinary glucuronide metabolites of EE were de -ethynylated (oestrone ,
oestradiol, oestriol and 2-methoxy-oestradiol) , however, Williams et al. (l975a)
found only I to 2% de -ethynylation in women given labelIed EE or MEE.
While several major centres have published papers on the ph armacokinetics ofEE
and MEE in humans, comparison oftheir results is difficult due to use ofboth male
and female subjects, and the adm inistration of either pure steroids or commercial
preparations. It is also apparent that the number ofsubjects in each study has been
smalI, wh ile other confounding factors include differences in methods for caIculating
volumes ofdistribution, cle arance rates, and the various kinetic models used .
Fotherby & Warren (1976) found no difference in bioavailability ofEE in tablet or
capsule presentation with a variety of vehicles. When administered in tablet or
solution form EE is ab sorbed in humans with great rapidity , with a half-life of
absorption of 12-20 min. There is however significant metabolism during absorption
through the intestina l wall and during the first liver pass (Speck et al., 1976;
Goldzieher, Dozier, de la Pena, Ojo, Lean, Chinnatarnby, Basnayake & Ko etsawang,
Due to th e rapid absorption, peak plasma concentration is attained within 2 h a fter
ingestion of the compound, though there are ver y large individual variations in the
concentration found . Pasquileani, Ca steIlet, Portois, Hili & Kincl (1977) reported an
av erage value of 422 ± 128pg litre'" (1.42 ± 0.43pmol litreJ], though seven of their
twelve subjects exc eeded 250pg mI-I (0.85pmol litre"). In contrast, in male subjects
Warren & Fotherby (1973) found peak values ranged from 800-1000pg mi-I
The recent study by Goldzieh er et al. (l980a) has compared plasma concentrations
and pharmacokinet ics of EE in women resident in a variety of different countries. In
these investigations single oral doses of EE ranging from 35-1001Jg were given to a
total of98 women resident in Nigeria, Singapore, Sri Lanka, Thailand, and th e USA.
Usin g a highly specific radioimmunoassay, plasma levels offree EE were determined
and appropriate ph armokinetic pa rameters caIculated. W ith doses of 50-801Jg EE,
two-thirds ofNorth American women showed detectable plasma EE concentrat ion s
at 24 h. All parameters of the tri-exponential kinetic curve were ca Iculated for
women in the Sri Lankian study and for one ofthe U SA investigations, wh ile partial
kinetic da ta were obtained for other countries. Strikingly lower plasma EE levels
were consistently observed in Niger ian women, wh ile Thai women showed the
highest concentrations, even when co rrected for differences in body surface area.
In these investigations, the half-life ofthe absorption phase ranged fro m 4-22 m in ,
litre rrr' , while the total body clearance was between 38 and 69 litre rn? h-I.
A high degree of correlation (r = 0.95) was reported between the peak plasma EE
concentration and area under the curve of plasma EE values. The origin of these
substantial differences in th e kinetics of EE between women located in various
countries remains to be identified, but could be of considerable c1inical significance,
and greatly complicates the comparison of pharmacokinetics in the human with
Table 1 Human pharmacokineticsoforal MEEand EE*
Urinary metabolites conjugated (%)
A comparison of the values obtained in this last mentioned investigation with
reported by Speck et al. (1976), and of 7 h estimated by Warren & Fotherby (1973).
Similarly the estimate ofthe elimination phase half-life of6-14 h by Goldzieher et al.
(l980a) is shorter than the 27 h reported by Speck et al. (1976) and 48 h reported by
Warren & Fotherby (1973) (Table 1).
It is recognized that there is prompt partial conversion by intestine and Iiver of EE
to its sulphate (Bird & Clarke, 1975), which is known to have a relatively long
biological half-life and which accounts for the slow elimination phase of EE .
Unfortunately the metabolism and pharmacokinetics ofEE-3-sulphate have not been
/.-OH-050____________ MESTRANOL OUINESTROL
Figure 3 Metabolism of contraceptive oestrogens*
Investigations of EE pharmacokinetics in animals are unfortunately very limited.
Kulkarni (1976) and Kulkarni, Avila & O'Leary (1977) have published information
in the baboon. In this species the elimination half-life was 31-46 h during the Irrst
cycle of exposure in three animals and 51-60 h in the third cycle of treatment. No
such difference in metabolic c1earance between users and non-users ofO.C. has been
found in investigations in women by Longcope & Williams (1977) or by Mills, Lin,
Braselton, Ellegood & Mahesh (1976).
Most investigators agree that MEE is rapidly absorped from the digestive tract and
the peak plasma concentrations are attained in the majority ofsubjects at 1-2 h. At
24 h post-ingestion, 58% of subjects were reported to have detectable MEE plasma
levels following a 50~ dose, and 79% after 100~ dose (Goldzieher et al., 1980b).
These findings are similar to those ofLongcope & Williams (1977).
Plasma MEE concentration one hour after oral administration to women has been
reported to be between 0.005 and 0.23% ofthe dose per litre (Bolt & Bolt, 1974) and
between 0.1 and 0.4% per litre (Goldzieher et al., 1980a). In the latter international
multicentre study, the ratio of plasma EE (following oral EE) to plasma MEE
(following oral MEE) ranged from about one in Srilankan women to four in Thai
women. As the groups were smalI, it is not yet known whether these represent true
biological differences between the various populations. The total body c1earance of
MEE ranged from 60-282 litres h', while that ofEE derived from MEE ranged from
. A comparison of areas under the plasma response curves showed
no significant difference between EE derived from MEE and EE given as such. This is
further confirrnation that EE and MEE are bioequivalent over the usual dose range
The 3-methoxy group of MEE renders the compound more lipophilic than EE, so
that the tissue pool of MEE is larger than for the same dose of EE, due to storage in
fats (Appelgren & Karlsson, 1971; Bolt & Remmer, 1972; Bolt & Bolt, 1974). An even
more lipophilic compound is quinestrol (QST), which is the 3-cyclopentylether of
EE . Dealkylation ofQST to release EE occurs to varying degrees in different species.
It is a minor pathway in the rabbit (Layne & Williams, 1967), but the principal
pathway in humans (Williams, Layne, Hobkirk, Nilsen & Blahey, 1967) and the rat
(Meli, Steinetz, Giannina, Cargill & Manning, 1968). Amongst the human biliary
metabolites of QST are glucuronides of EE, QST, and 6a-hydroxy-QST, together
with unidentified dealkylated derivatives (Cargill et al., 1969). Urinary excretion of
labelIed metabolites following the administration of (lH]-QST to women has been
studied by Zuleski et al. (1978). Unchanged QST was not detected, but small amounts
ofunconjugated EE and EE-glucuronide were found.
(a) Noreth isterone (NET) and related compounds
There are several animal studies of radioactive NET. Kamjab, Littleton & Fotherby
(1967) found 45 to 57% of the label was excreted in urine within 48 h following
i.v. injection of [14C]-NET into rabbits. The plasma half-life was about 2 h. The
urinary metabolites were conjugates of NET, together with other more polar
compounds. There was \ittle or no conversion of NET containing a ['4C]-ethynyl
Unlike the rabbit, the principal route of excretion of NET in the rat is via bile.
Hanasono & Fischer (1974) reported recovery of80% ofthe label within 8 h from bile
duct-cannulated rats given (lH]-NET. MetaboIites in bile were conjugates that
participated in an enterohepatic circulation. Kappus & Remmer (1975) have shown
that rat liver microsomes convert NET in vitro to a compound (believed to be
NET-4 ,5-epoxide) that binds covalently to tissue proteins. The gut wall ofthe rat also
metabolises NET (Back, Breckenridge, Crawford, McIver, Orme, Rowe & Smith,
Structurally related progestogens include noreth ynodrel (NEO), lynestrenol
(LYN), and eth ynodiol diacetate (EOA), together with the esters norethisterone
acetate (NEA) and norethisterone enanthate (NEE) (Figure 4).
The earliest study ofNEO is that of Arai , Golab, Layne & Pincus (1962), who used
[lH]-NEO in rabbits, some ofwhich had bile duct cannulas. In this latter group, mean
reco very ofradioactivity was 21% in urine, 17% in faeces , and 33% in bile, compared
with 50% in ur ine and 16% in faeces ofintact rabbits. The principal metabolite in bile
was 3P-dihydro-NEO, together with NET and 10p-hydroxylated derivatives. Urine
also contained these same compounds as conjugates, together with what were thought
to be de-ethynylated metabolites, though less than 10% oforal NET is de-ethynylated
(Littleton et al., 1968). In bile duct-cannulated rats, Hanasono & Fischer (1974) found
Fotherby & Warren (\976) have compared the bioavailability ofnorethisterone in
various forms by measuring blood levels following administration of either the usual
tablets, or gelatine capsules containing various vehicles. No significant ditTerences
were seen and the authors conclude that rapid intestinal absorption occurs from
either type of preparation and the nature of the vehicle is unimportant. Metabolism
in the gut wall and liver reduces bioavailability to about 65% ofthe oral dose.
Similar conclusions were reached by Okerholm, Paterson, Keeley, Smith &
ETHYNODIOL- 3- ACETATE ETHYNODIOL-17-A CETATE
NEE 1 ------- NET • DE-ETHYNYLATED METABOUTES 'CA? ",OROmAOED METABOU ITS
3ß ,5ß -TETRAHYDRO-NET 30 ,5ß -TETRAHYDRO- NET 3ß, 50-TETRAHYDRO-NET 30.50- TETRAHYDRO-NET
Figure 4 Metabolism of norethisterone and related progestogens"
levels in men receiving a single dose of 5mg NET were 20.3 ± l.71Jg litre-I (68 ± 5.7
nmol litre-1) compared to 42 .0 ± 6.81Jg litre-1 (\41 ± 22.8nmol litre-1
perhaps due to the high er concentration ofsex hormone-binding globulin, to wh ich
NET shows significant affmity, in adult female plasma. In this study plasma half-life
ofNET (IOmg dose) in men was 4.6 ± 0.5 to 5.5 ± 0.6 h.
Pasqualini et al. (\977) have measured plasma concentrations of NET in warnen
was 7 h. At most, 2 to 3% ofthe administered dose was present in plasma at the peak
concentration, and had declined to about 0.5% by 24 h.
Using 10 women with normal men strual histories, aged 20-37 yea rs, who had not
taken any O.c. for at least I year prior to the study, invest igations were made and a
single i.v . injeetion teehnique reported the metabolie c1earanee rate of NET to be
The authors eomment that NET disappeared rapidl y from the circulation, but
metabolites appeared to persist. The mean radioacti ve half-life of [3H]-NET was
on blood radioactivit y, with no indi eation of a plateau , thou gh on diseontinuation
the mctabolites slowl y c1 cared with a half-life of circa 70 h. The rnajor metabolites
were a ll ring A-reduced compounds, including th e 3 a . 5 n : 3 a . 5ß and 3 ß , 5 a
derivatives. In the blood an additional metabolite was identified as 5ß-din ydroNET.
Early studies on the metabolism of NET in humans followed the exeretion of the
radioaetive label. Layne, Golab, Arai & Pineus (1963) gave oral doses of [3H]-NET
and measured urinary exeretion. In 7 days after oral administration ofthe compound
50-70% of JH appeared in the urine, and the rnajor fraction of the label was
eonjugated as glueuronides. Fotherby, Karnjab, Littleton & Klopper (1966) did a
similar study using [J4C]-NET. More than 90% ofthe urinary metabolites were found
to still possess the 17-ethynyl group. Fotherby & Klopper (1968a) also reported
studies on the metabolism of [14C]-NET in women. In seven subjects given 14C-NET
intravenously, 40-80% of the 14C appeared in the urine in 5 da ys. Only 3% of the
radioaeti vit y was free stcro id, 15% was present as sulphat e co njuga tes, and about 50%
was present as glucuronides. Th ese workers confirmed th at thcre was little or no
de- eth ynylation ofthe eo mpo und and thc half-l ife ofplasma 14C was 19 h.
Detailed identification of norethisterone metabolites was provided by Murata
(1968), who gave [3H]-NET orally and analyzed ur ine for metabolites. About 9% of
urinary JH was free , 25% was eonjugated as glucuronides, and 40 % was present as
sulphates. Separation of the metabolites by chromatography and their identification
resulted in the deseription ofseveral metabolites more polar th an norethisterone. In
general, the metabolie transformations were saturation of th e double bond and
reduetion ofthe 3-keto to a hydroxyl group.
A thorough study of the metabolism of norethisterone has been earried out b y
Gerhards (1971). They gave [3H] or [J4C]-NET orally to three women and iso lated
labelIed drug. The disappearance of pla sm a radioactivity showed a half-life of 9 h,
while the half-life of NET was 2 h. Pla sma radioactivity was rapidly converted to
polar metabolites. Sulphates composed 80-90% of the conjugates, and of these 3 ß ,
5 ß-tetrahydro-NET was the major metabolite. Urinary radioactivity amounted to
40-50% of the dose, and 50-65% of urinary JH or 14C was present as sulphate
A detailed account on the use ofcombined gas ehromatography-mass spectrometry
for the ident ification in human urine and blood of NET and its metabolites is given
by Braselton, Lin , Mills, Ellegood & Mahesh (1977). A healthy female volunteer
received 25mg NET daily for 4 da ys. A composite anal ysis was made of a mixture
containing 2.5 % of each of the 4 da ys urine. Of the identified compounds, 3 a ,
5 a-tetrahydro-NET, 3 a , 5ß-tetrahydro-NET were present as free steroids, and as
sulphate and glucuronide eonjugates. Other tetrahydroconjugates, as weil as NET
itself, were present only as mixed conjugates. A number of other urinary metabolites
were det eeted, but not quantified , These include both the sulphate and glucuronide of
A comparison ofthe metabolites in blood and urine reveals that the major plasma
component is unchanged NET, together with its sulphate and glucuronide. The
major plasma metabolite is the 5ß, 3a-derivative (as sulphate), with smaller
amounts of the 5 a, 3 a -sulphate, and 5 ß, 3 a -glucuronide, In contrast, the major
urinary derivative is the 5ß, 3 a-derivative (mainly as sulphate and glucuronide). No
free NET is present, though there are small amounts of NET sulphate and
In a separate experiment, plasma levels of NET metabolites were measured in a
woman receiving a commercial oral contraceptive (2mg NET + 1001Jg MEE). The
following results were obtained on a blood specimen taken 3 h after ingestion ofthe
tenth pill ofthe cycle (day 14):
Total 274.5 (932 nmol litre'")
Only traces of glucuronides were detected. It should be noted that the values
obtained by GCMS are significantly higher than those obtained by radioimmunoassays.
The same group (Braselton, Lin, Ellegood, Mills & Mahesh, 1979) have studied
blood levels offree, sulphate, and glucuronide conjugates ofNET, and its metabolites
in a female volunteer who received six consecutive daily doses of2.5mg NET, and in
four female volunteers undergoing chronic treatment with 2mg NET and 100~
MEE). Blood levels were quantified by GCMS. During treatment for 6 days with
2.5mg daily, the 3 h blood levels ofNET and ring A reduced metabolites increased in
a stepwise fashion. During long-terrn treatment the concentrations of NE , NE
sulphate, and the conjugates ofthe ring A reduced metabolites were seen to build up
to a peak at approximately the midpoint of the treatment phase of each cycle , and
Bedolla-Tovar, Rahman, Cekan & Diczfalusy (1978) have assessed the specificity
for two ring-A reduced metabolites. Results of serum NET concentration as
determined using this new radioimmunoassay are presented in four groups, each of
three women, receiving either Img NET + 50~ MEE, O.5mg NET + 35~ EE , or
O.35mg NET administered alone without oestrogen. Measurements were made on
the fifth day of treatment. Peak serum concentrations of NET were observed within
0.5 to 4 h following oral intake, and thereafter fell precipitously throughout the
remainder ofthe day. Mean values ofseru m NET observed 3 h after ingestion ofthe
first and second as weil as the fourth and fifth daily dose of each oral contraceptive
are presented. Two different commercially available forms of 1mg NET + 501Jg MEE
were used . The first ofthese gave a mean serum NET concentration for doses 1 and 2
of 6.86j.Jg litre"! (23 .2 nmol litre") and for doses 4 and 5 9.331Jg litre-' (3l.3nmol
litre:"). This difference is statistically significant at P < 0.05 . None of the other
preparations showed statistically significant differences between the peak NET
concentrations for doses 1 and 2 as compared with peaks for doses 4 and 5. The
corresponding results for the other three O.c. were as follows: the Irrst value is for the
peak for doses 1 and 2, while the second is the mean value for doses 4 and 5. Results
for the second commercial formulation containing Img NET + 501Jg MEE were
5.721Jg litre'" (l9 .02nmol litre:'), 8040IJg litre'" (28 .2nmol litre"), while 0.5mg
NET + 351Jg EE gave 5.361Jg litre-' (18.lnmol litre"), 6.211Jg litre-! (2Q.9nmol litre-I
The progestogen-only mini-pill containing 0.35mg NET gave 3.151Jg litre-I
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