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Estetrol Review - Profile and Potential Clinical Applications
Estetrol Review - Profile and Potential Clinical Applications
Pantarhei Bioscience, Zeist, The Netherlands; *PharmConsult1, New York, NY, USA;
{
Karolinska Institute, Stockholm, Sweden
ABSTRACT
In this review paper, the existing information on the human fetal steroid estetrol (E4) has
been summarized. In the past, E4 was considered as a weak estrogen and interest
disappeared. However, recent new research has demonstrated that E4 is a potent, orally
bioavailable, natural human fetal selective estrogen receptor modulator, since it acts in
the rat as an estrogen on all tissues investigated except breast tumor tissue, where it has
estrogen antagonistic properties in the presence of estradiol. Based on its safety data, its
pharmacokinetic properties, its pharmacological profile and the results of first human
studies, E4 may be suitable as a potential drug for human use in applications such as
hormone replacement therapy (vaginal atrophy, hot flushes), contraception and
For personal use only.
osteoporosis. Additional areas worth exploring are the treatment of breast and prostate
cancer, hypoactive sexual desire disorder and topical use (wrinkles) in women, auto-
immune diseases, migraine, cardiovascular applications and the treatment of selected
obstetric disorders.
Correspondence: Professor H. J. T. Coelingh Bennink, Pantarhei Bioscience, PO Box 464, 3700 AL Zeist, The Netherlands
REVIEW ARTICLE
ª 2008 International Menopause Society
DOI: 10.1080/13697130802073425
Estetrol review Coelingh Bennink, Holinka and Diczfalusy
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Figure 1 Structural formulae of estrone (E1), estradiol, (E2), estriol (E3) and estetrol (E4)
E4 does not appear to enter the enterohepatic Estetrol produced a number of biological
circulation. changes in the rodent uterus, such as weight in-
For personal use only.
Estetrol was detected in maternal urine as early crease, progesterone receptor stimulation, enzyme
as 9 weeks of pregnancy12,13. It was found at high induction, and histological and ultrastructural
levels in maternal plasma during the second changes31. It also bound to the human endometrial
trimester of pregnancy, with steadily rising con- estrogen receptor. The biochemical, histological
centrations of unconjugated E4 to about 1 ng/ml and ultrastructural responses of the immature
(43 nmol/l) toward the end of pregnancy14,15. rat uterus to E4 revealed a tendency toward
Conjugated E4 levels were seven times higher than cell differentiation, in contrast to the typical
unconjugated levels16. The levels of unconjugated mitotic responses that were observed after E2
E4 in fetal plasma at parturition were about 12–19 administration32.
times those in maternal plasma17,18. Amniotic After 20 years of experimental work, E4
fluid levels were about one-third of fetal plasma research was virtually abandoned and ended in
levels and five to six times higher than maternal the mid-1980s. Consensus at that time was that,
plasma levels17,19. Maternal urinary excretion in first, E4 is a weak estrogen and, second, E4 cannot
late pregnancy varied between 0.5 and 2.3 mg/ be used as a marker of fetal well-being during
day13,20–22. pregnancy due to the high inter- and intra-
For follow-up and survey of pregnancy pathol- individual variations in plasma levels14,33.
ogy, E4 levels were not appropriate due to the However, it seems unlikely that an estrogenic
large intra- and inter-individual variations in steroid produced in such significant quantities by
plasma levels23–27. the male and female human fetal liver during
Competitive receptor binding studies for nucle- pregnancy would have no physiological signifi-
ar binding in human proliferative endometrium28 cance. Therefore, in 2001, a project was started at
and in rat uterine cytosol29 revealed low estrogen Pantarhei Bioscience to investigate the properties
receptor binding affinity for E4, relative to that of E4 with state-of-the-art technologies.
of E2.
The effects of E4 when compared to those of
estrone (E1), E2, and E3 on progesterone receptor SYNTHESIS AND
levels and growth in the human breast cancer cell PHARMACEUTICAL PROPERTIES
line MCF-7 revealed that both E3 and E4 behave A new route of synthesis has been developed for
as E2 agonists but require substantially higher E4 by Pantarhei, starting with the commercially
concentrations to achieve the effects of E230. available E134. This new route results in accep-
48 Climacteric
Estetrol review Coelingh Bennink, Holinka and Diczfalusy
table yields of E4 of very high purity (498%) different36. Metabolites produced by rat hepato-
without contamination with E2. It permits the cytes were not found with human hepatocytes and
synthesis of E4 on a (semi)-industrial scale suitable vice versa. In rat hepatocytes, phase I metabolism
for GMP (Good Manufacturing Production) for is most important. This may result in active
human use35. This new method overcomes the metabolites in the rat, whereas inactivation by
disadvantages of previous methods. glucuronidation and sulfation, i.e. phase II meta-
Pharmaceutical studies revealed that E4 is bolism, are the pathways observed in human
chemically very stable even under non-optimal hepatocytes36. This confirms that, in the human,
storage conditions34. It has high water solubility E4 is an end-stage product of metabolism and has
and might be slightly hygroscopic. Estetrol has no active metabolites.
an octanol–water parturition coefficient (Pow) of Estetrol at a high concentration of 10 mmol/l did
about 1.5, making it about a 100-fold less not inhibit the major cytochrome P450 enzymes
lipophylic than E2 or ethinylestradiol. As a CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP
Pow of 2 is considered optimal for passage 3A436. Estradiol and ethinylestradiol significantly
through the blood–brain barrier, E4 might be inhibited CYP2C19. Ethinylestradiol had a strong
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expected to have effects on the central nervous inhibitory effect on CYP3A4, whereas E2 stimu-
system. lated this enzyme significantly and E4 had some
It is concluded that, with this new method, E4 stimulatory effect36. These results suggest that E4
can be synthesized consistently with high purity may exhibit less interference with concomitantly
and without important contaminations. Estetrol administered drugs (drug–drug interaction) com-
appears to have very favorable properties for the pared to ethinylestradiol and E2.
development of a pharmaceutical product. The ERa-dependent effect of the steroids E2, E3,
E4 and ethinylestradiol on sex hormone binding
globulin (SHBG) production was investigated
RECEPTOR BINDING AND TARGET using the HepG2 and Hep89 cell lines39. Estetrol
INTERACTION
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Figure 3 Mean (+standard deviation) plasma concentrations of estetrol after oral (p.o.) or subcutaneous (s.c.)
administration of a single dose of 0.5 mg/kg estetrol to female rats (n ¼ 3). *Significantly different from vehicle:
p50.01
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Figure 4 Mean estetrol plasma levels (+standard deviation) after a single oral dose of 1, 10 and 100 mg estetrol in
postmenopausal women
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Estetrol review Coelingh Bennink, Holinka and Diczfalusy
Figure 5 Mean (+standard deviation) ultimate strength (N/mm2) at the distal femora after 4 weeks of once-daily
treatment with estetrol (E4) (0.1, 0.5 or 2.5 mg/kg/day) or ethinylestradiol (EE) (0.1 mg/kg/day) compared to vehicle
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well-validated animal model, E4 is a potential drug These results suggest that E4 may be effective
for the prevention of osteoporosis in postmenopau- for the treatment of hot flushes and other
sal women. It seems worthwhile also to investigate vasomotor symptoms in peri- and postmenopausal
the potential efficacy of E4 for the treatment of women.
osteoporosis and osteoporotic fractures.
VAGINA, UTERUS AND
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52 Climacteric
Estetrol review Coelingh Bennink, Holinka and Diczfalusy
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Figure 6 The effects of estetrol (E4) and 17a-ethinylestradiol (EE) on the naloxone-induced hot flush response in
female ovariectomized rats
For personal use only.
Figure 7 Number of animals with vaginal cornification over a 7-day treatment period with orally administered
estetrol (E4) (0.1, 0.3, 1.0 or 3.0 mg/kg/day), ethinylestradiol (EE) (0.05 mg/kg/day), or vehicle
Estetrol appeared to have a dose-dependent rats: vaginal epithelium, myometrium and endo-
proliferative estrogenic effect on the rat endome- metrium. The potency of E4 was approximately
trium after 4 weeks’ treatment37. Estetrol was 20-fold lower compared to ethinylestradiol.
found to be less potent than ethinylestradiol, since It is concluded that E4 may be suitable for the
the order of increasing potency per mg/kg/day was treatment of urogenital atrophy and the accom-
estimated as follows: 0.1 mg E4 50.5 mg E4 panying clinical complaints such as vaginal
50.1 mg ethinylestradiol 52.5 mg E4. dryness and dyspareunia in estrogen-deficient
In summary, estrogenic activity of E4 was women. Since E4 has a proliferative effect on the
demonstrated in three tissues in ovariectomized endometrium, in women with a uterus, measures
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Estetrol review Coelingh Bennink, Holinka and Diczfalusy
to protect against endometrial hyperplasia and Based on these results, one may hypothesize
cancer should be taken. that E4 could be a natural antagonist of the
abundantly available estrogens during human
pregnancy. These findings provide the basis for
BREAST the potential clinical development of E4 as a
Rats treated with DMBA (7,12 dimethylbenz(a)- natural selective estrogen receptor modulator
anthracene) develop estrogen-responsive breast (natural SERM) and antagonist for the treatment
tumors. Two prevention studies and one interven- of breast cancer. The estrogen antagonistic effect
tion study were performed in this animal model on breast tumor tissue would be a major
with E443. In the prevention studies, the effect advantage compared to presently used estrogen-
was investigated of oral doses of E4 over a dose containing drugs.
range of 0.5–3.0 mg/kg. The intervention study
used oral doses of 1, 3 and 10 mg/kg E4.
The antiestrogen tamoxifen was used as reference OVULATION INHIBITION
compound in all three studies; ovariectomy The effectiveness of E4 as an ovulation inhibitor
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and ethinylestradiol, at doses pharmacologically was studied in regularly cycling female rats and
equipotent to E4, acted as control treatments in compared to ethinylestradiol44,45. The animals
one prevention study and in the intervention were treated orally twice daily for 4 consecutive
study. days, starting on the day of estrus, with E4 (0.03,
When DMBA-induced rats were co-treated with 0.1, 0.3, 1.0 or 3.0 mg/kg), or ethinylestradiol
E4 for 8 weeks, this resulted in a dose-dependent (0.0003, 0.001, 0.003, 0.01 or 0.03 mg/kg) or
reduction in the number and size of tumors, an vehicle control. The primary endpoint was the
effect that appeared equally effective as tamoxifen number of ovulated oocytes in the genital tract.
treatment or ovariectomy and was not seen with Estetrol at the twice-daily dose of 0.3 mg/kg and
ethinylestradiol. When E4 was administered to above inhibited ovulation. This effect was statis-
tically significant (p 5 0.05). The comparator,
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Figure 8 Intervention study. Mammary tumor count per animal (þ SEM) at baseline and 4 weeks after ovariectomy
(OVX) or daily oral treatment with vehicle, tamoxifen (TAM) or estetrol (E4)
54 Climacteric
Estetrol review Coelingh Bennink, Holinka and Diczfalusy
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Figure 9 Estimation of ED50 for ovulation inhibition in 4-day cycling rats treated twice daily with the indicated oral
doses of ethinylestradiol (EE) or estetrol (E4)
In the human pharmacokinetic study performed failed due to the high inter- and intra-individual
with single doses of 0.1, 1, 10 and 100 mg E4, the variations in plasma levels23–27.
effect on plasma levels of luteinizing hormone In 2001, Pantarhei Bioscience started its re-
For personal use only.
(LH) and follicle stimulating hormone (FSH) was search activities on E4. The motivation was the
also studied. LH levels were suppressed dose- simple consideration that it seems unlikely that
dependently. A profound and sustained inhibition nature would endow the human fetus with the
of FSH levels, lasting over 7 days, was observed in ability to produce this steroid in such significant
the 100 mg dose group (FSH was not measured in amounts without a sound reason.
the other dose groups). It was concluded that E4 The new research has revealed that, first of all, E4
has a profound central inhibitory and dose- seems a safe compound with selective binding to
dependent effect on gonadotropins, expected to both estrogen receptors36 and some preference for
contribute to the contraceptive effect of E4. the ERa above the ERb. No toxicity was observed at
In summary, based on these and other results high dose levels in pharmacological studies in the rat,
reported in this review, E4 might be a potential with maximum doses of 10 mg/kg/day for 4 weeks43
candidate to replace ethinylestradiol in combined and in single-dose studies in the human, with a
oral contraceptives. maximum dose of 100 mg38. Based on its chemi-
cal34, pharmaceutical34 and metabolic proper-
ties14,33,37,38, it seems possible to develop E4 as a
CONCLUSIONS drug for human use. Especially its high and dose-
Estetrol is a steroid synthesized exclusively by related oral bioavailability in the rat37 and the
the human fetal liver during pregnancy. After its human38, the absence of binding to SHBG39 and the
discovery in 1965 by Egon Diczfalusy and co- long elimination half-life of 28 h in the human38 may
workers at the Karolinska Institute in Stock- enable its use as an oral once-a-day drug.
holm, Sweden, basic research was performed on In well-validated and predictive rat models, E4
E4 until about 1984. At that time there was behaves as an estrogen agonist in all tissues
consensus that E4 is a weak estrogen and investigated, i.e. bone37, vagina42, myometrium42,
interest in this steroid discontinued. The judge- endometrium42 and brain (hot flush41 and ovula-
ment of low potency of E4 was primarily based tion inhibition44,45), except for breast tumor tissue
on low ER binding affinity, ranging from 0.3% where this steroid acts as an estrogen antagonist in
(rat)29 to 6.25% (human)28, and 2–3% potency the presence of E243. Interaction with liver
compared to E2 in a series of in vitro and in function in in vitro models demonstrates, first,
vivo experiments30–32. Efforts to use E4 as a slow metabolism36, explaining the long half-life;
marker of fetal well-being during pregnancy second, absence of cytochrome P450 inhibition36,
Climacteric 55
Estetrol review Coelingh Bennink, Holinka and Diczfalusy
which may implicate less drug–drug interaction; order and topical use (wrinkles) in women,
and, third, no stimulation of SHBG synthesis39, migraine, cardiovascular applications and the
which suggests a potentially lower risk of venous treatment of selected obstetric disorders.
thromboembolism when used as a drug in the Although there are sound reasons to test the use
human, a serious side-effect of all known estro- of E4 in all these conditions, it seems unlikely that
gens and synthetic SERMs. E4 will be efficacious in all these disorders and
Based on its pharmacological profile, E4 can be diseases. However, the present data on E4 allow
classified as a natural human fetal SERM. Contrary the conclusion that the pharmacological profile of
to the conclusion in the past, E4 seems to be a potent E4 is not a weak estrogen but a potent steroidal
steroid. In the pharmacological studies, E4 was 10– SERM with potential applications in the human.
20 times less potent compared to ethinylestradiol, The question remains about the physiological
the most potent estrogen available. Single doses of role of E4 during human pregnancy since this has
E4 strongly suppressed LH and FSH in postmeno- not been studied and is unknown. The facts are
pausal women. The difference between the past and that E4 is a steroid synthesized exclusively by the
the present conclusions can be explained by (lack of) human fetal liver during pregnancy1–6. Data on
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knowledge of the metabolic properties of E4. Past file show that E4 is not synthesized by pregnant
studies were all in vitro or short in vivo experiments rats and mares. Estetrol is already present in urine
and adequate ADME studies demonstrating the of pregnant women at 9 weeks of gestation12,13.
favorable pharmacokinetics of E4 were performed Estetrol plasma levels increase exponentially dur-
only recently37,38. ing pregnancy and the term fetus synthesizes a
Estetrol may be useful for a series of potential high amount of E4 up to 3 mg/day. Unanswered
clinical applications including the prevention and questions are, for example:
treatment of osteoporosis and hormone replace-
ment therapy in women, especially for the treat- (1) Why is E4 present during pregnancy?
ment of vaginal atrophy and hot flushes. Estetrol (2) How is 15a-hydroxylation during pregnancy
For personal use only.
References
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2. Gurpide E, Schwers J, Welch MT, Vande Hagen AA, Diczfalusy E. Isolation of 15a-
Wiele RL, Lieberman S. Fetal and maternal hydroxyl-oestriol from pregnancy urine and
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Estetrol review Coelingh Bennink, Holinka and Diczfalusy
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oral estetrol in early postmenopausal women. 44. Coelingh Bennink HJT, Skouby S, Bouchard P,
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For personal use only.
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