US 2005.0070488A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0070488A1 Coelingh Bennik et al. (43) Pub. Date: Mar. 31, 2005

(54) ESTROGENIC COMPOUNDS IN prises the oral administration of an estrogenic component COMBINATION WITH PROGESTOGENIC and a progestogenic component to a mammal in an effective COMPOUNDS IN amount to prevent or treat Symptoms of hypoestrogenism, HORMONE-REPLACEMENT THERAPY wherein the estrogenic component is Selected from the group (76) Inventors: Herman Jan Tijmen Coelingh Bennik, consisting of Substances represented by the above formula in Driebergen (NL); Evert Johannes which formula R, R2, R, R independently are a hydrogen Bunschoten, Heesch (NL); Christian atom, a hydroxyl group or an alkoxy group with 1-5 carbon Franz Holinka, New York, NY (US) atoms; each of Rs, R, R-7 is a hydroxyl group; and no more than 3 of R, R2, R, R are hydrogen atoms; precursors Correspondence Address: capable of liberating a Substance according to the aforemen William Logsdon tioned formula when used in the present method; and Webb Ziesenheim Logsdon Orkin & Hanson mixtures of one or more of the aforementioned Substances 700 Koppers Building and/or precursors. Another aspect of the invention concerns 436 Seventh Avenue a pharmaceutical kit comprising oral dosage units that Pittsburgh, PA 15219-1818 (US) contain the aforementioned estrogenic component and a (21) Appl. No.: 10/495,707 progestogenic component as well as an androgenic compo nent. (22) PCT Filed: May 23, 2002 (86) PCT No.: PCT/NL02/00332 (30) Foreign Application Priority Data Nov. 15, 2001 (EP)...... O1204377.4 Feb. 21, 2002 (EP)...... O2O75695.3 Publication Classification 51)1) Int. Cl.'C.7 ...... A61K 31/5656; A61K 31/704 (52) U.S. Cl...... 514/26; 514/182 (57) ABSTRACT One aspect of the present invention relates to a method of hormone replacement in mammals, which method com US 2005/0070488 A1 Mar. 31, 2005

ESTROGENIC COMPOUNDS IN COMBINATION organism: normal levels make a decisive contribu WITH PROGESTOGENC COMPOUNDS IN tion to a woman's well-being. Notwithstanding the wide HORMONE-REPLACEMENT THERAPY Spread use of in HRT methods, there are still Some unsolved problems. Known estrogens, in particular the bio TECHNICAL FIELD OF THE INVENTION genic estrogens (i.e. estrogens naturally occurring in the human body), show serious pharmacokinetic deficits. Bio 0001. The present invention relates to a method of hor genic estrogens Such as , , estrone Sulphate, mone replacement in mammals. More particularly the inven esters of estradiol and become bioavailable only to a tion is concerned with a method of hormone replacement very low degree when taken orally. This degree may vary So that comprises the oral administration to a mammal of a much from perSon to person that general dosage recommen combination of an estrogenic component and a progestoge dations cannot be given. Fast elimination of these estrogens nic component in an effective amount to prevent or treat from the blood is another related problem. For instance, for Symptoms of hypoestrogenism. the main human biogenic estrogen 17B-estradiol the half-life is around 1 hour. As a result, between Separate (daily) BACKGROUND OF THE INVENTION administration events, blood Serum levels of Such biogenic estrogens tend to fluctuate considerably. Thus, Shortly after 0002 In hormone replacement therapy (HRT), some administration the Serum concentration is usually Several times also referred to as estrogen replacement therapy, times higher than the optimum concentration. In addition, if estrogens are administered to prevent or treat Symptoms the next administration event is delayed, Serum concentra resulting from estrogen deficiency or hypoestrogenism. tions will quickly decrease to a level where the estrogen is Hypoestrogenism can occur in both females and males, and no longer physiologically active. can lead to disorders and ailments Such as Osteoporosis (loss of bone mass), arteriosclerosis, climacteric Symptoms Such 0007. The most important synthetically altered estrogenic as hot flushes (flashes), Sweats, urogenital atrophy, mood is 17O-ethinyl estradiol (EE). This estrogen is hardly disturbances, insomnia, palpitations. Estrogen deficiency used in HRT methods because prolonged administration of has also been associated with cognitive disturbances and EE has been associated with an increased risk of throm Alzheimer's disease. boembolism, which is deemed to be particularly detrimental in menopausal and post-menopausal females. Apart from 0.003 Hypoestrogenism, and in particular chronic EE, has been used in a few cases, mestranol is a hypoestrogenism, is frequently observed in (peri-)meno “prodrug” that is metabolised to EE in the organism. When pausal and post-menopausal women. However, it can also applied orally to humans, EE has a much better bioavail result from hypogonadism or castration, as well as from ability than the biogenic estrogens mentioned above, but its primary ovarian failure, treatment of e.g. breast cancer with and -releasing hormone oral bioavailability varies to a large extent from individual analogue treatment of benign gynaecological diseaseS Such to individual. Several authors have pointed to this as well as as endometriosis, adenomyosis, uterine fibroids (leiomyo to the fact that concentrations in the blood proved to be mas), dysmenorrhoea, menorrhagia and metrorrhagia. highly fluctuating after oral application of this Substance. 0008. In addition to pharmacokinetic problems, the 0004 HRT employs continuous administration of effec known estrogens also show pharmacodynamic deficits. After tive amounts of an estrogen for prolonged periods of time. resorption from the intestinal lumen, orally applied active The administration of estrogens has been associated, how ingredients enter the organism via the liver. This fact is of ever, with endometrial proliferation in Women and it is now Specific importance for estrogenic agents as the liver is a widely accepted that “unopposed” estrogen therapy Substan target organ for estrogens, oral intake of estrogens results in tially increases the risk of endometrial cancer (Cushing et Strong estrogenic effects in the liver. The Secretion activity al., 1998. Obstet. Gynecol.91, 35-39; Tavani et al., 1999. that is controlled by estrogens in the human liver includes Drugs Aging, 14, 347-357). There is also evidence of a increased synthesis of transport proteins CBG, SHBG, TBG, Significant increase in breast cancer with long-term (10-15 Several factors that are important for the physiology of blood years) use of estrogen therapy (Tavani et al., 1999. Drugs clotting, and lipoproteins. If biogenic estrogens are intro Aging, 14, 347-357; Pike et al., 2000. , 65, 659 duced to the female organism while avoiding passage 664). through the liver (e.g. by transdermal application), the liver 0005. In order to counteract the negative effects of unop functions mentioned remain largely unchanged. Therapeu posed estrogen therapy, adjunctive treatment is tically equivalent doses of biogenic estrogens, when applied nowadays commonly applied. Regular progestogen admin orally, result in clear responses of hepatic parameters, Such istration is believed to inhibit the continual estrogen Stimu as increase of SHBG, CBG, angiotensinogen and HDL (high lation of the endometrium through an anti-proliferative density lipoprotein). These hepatic effects of estrogens are effect and appears to reduce the incidence of endometrial also observed when formulations (so-called carcinoma in post-menopausal women receiving estrogen ) are used. Ethinyl estradiol and dieth replacement therapy (Beral et al., 1999. J. Epidemiol. Bio ylstilbestrol (DES) have an even greater hepatic estrogenic Stat., 4, 191-210). Such an adjunctive treatment, generally ity. Elger et al., J. Steroid Biochem. Molec. Biol. (1995), using Synthetic , is given either in continuous 55(3/4), 395-403, have reported that EE or DES have much combined regimens with estrogen, or added Sequentially, higher hepato-cellular than Systemic estrogenicity: in rela typically for about 14 days each month, to continuous tion to FSH-secretion inhibitory activity these estrogens are estrogen treatment. 4-18 times more active in the liver than . 0006 Endogenous and exogenous estrogens fulfil impor 0009. The aforementioned deficits are of considerable tant central nervous and metabolic functions in the female clinical significance when commonly known biogenic and US 2005/0070488 A1 Mar. 31, 2005

Synthetic estrogens are applied. Consequently, there is an as Endocrinologica, 98, 73-80: “ agonistic yet unmet need for estrogens that do not display these potency is 2% of the magnitude observed for 17B deficits and which can suitably be administered orally in estradiol in in vitro cell proliferation”. HRT methods to effectively replace endogenous ovarian Secretion of estradiol, i.e. to treat or prevent Symptoms of 0017 Holinka et al., 1980. Comparison of effects of hypoestrogenism. estetrol and with those of estriol and estra diol on the immature rat uterus. Biol. Reprod. 22, 913-926: “Subcutaneously administered estetrol has SUMMARY OF THE INVENTION Very weak uterotrophic activity and is considerable leSS 0.010 The inventors have surprisingly found that these potent than 17B-estradiol and estriol'. objectives are met by that are repre 0018 Holinka et al., 1979. In vivo effects of estetrol on sented by the following formula the immature rat uterus. Biol. Reprod. 20, 242-246: “Subcutaneously administered estetrol has very weak uterotrophic activity and is considerable less potent than 17 B-estradiol and estriol'. 0019 Tseng et al., 1978. Heterogeneity of Saturable estradiol binding Sites in nuclei of human endometrium. Estetrol studies. J. Steroid Biochem. 9, 1145-1148: “Relative binding of estetrol to estrogen receptors in the human endometrium is 1.5% of 17 B estradiol'. 0020 Martucci et al., 1977. Direction of estradiol metabolism as a control of its hormonal action uterotrophic activity of estradiol metabolites. Endocrin. 101, 1709-1715: “Continuous administration of estetrol from a Subcutaneous depot shows very weak 0.011 in which formula R, R2, R, R independently are uterotrophic activity and is considerably less potent a hydrogen atom, a hydroxyl group or an alkoxy group with than 173-estradiol and estriol'. 1-5 carbon atoms, each of Rs, R, R-7 is a hydroxyl group; 0021 Tseng et al., 1976. Competition of estetrol and and no more than 3 of R, R2, R, R are hydrogen atoms. ethynylestradiol with estradiol for nuclear binding in 0012. A known representative of this group of estrogenic human endometrium. J. Steroid Biochem. 7, 817-822: substances is 1.3.5 (10)-estratrien-3, 15C, 16C, 17B-tetrol, “The relative binding constant of estetrol binding to the also known by the names of estetrol, Oestetrol and 15C.- in the human endometrium is 6.25% hydroxyeStriol. Estetrol is an estrogen that is produced by compared to 17B-estradiol (100%)”. the fetal liver during human pregnancy. Unconjugated 0022 Martucci et al., 1976. Uterine estrogen receptor estetrol levels in maternal plasma peak at about 1.2 ng/ml at binding of catecholestrogens and of estetrol (1,3,5(10)- term pregnancy and are about 12 times higher in fetal than estratriene-3,15alpha, 16alpha, 17beta-tetrol). Steroids, in maternal plasma (Tulchinsky et al., 1975. J. Clin. Endo 27, 325-333: “Relative binding affinity of estetrol to rat crinol. Metab., 40, 560-567). uterine cytosol estrogen receptor is 0.5% of 17 B-estra 0013. In 1970, Fishman et al., “Fate of 15o-hydrox diol (100%). Furthermore, the relative binding affinity yestriol-3H in Adult Man”, J. Clin Endocrinol Metab (1970) of estetrol to rat uterine nuclear estrogen receptor is 31, 436-438, reported the results of a study wherein tritium 0.3% of 17B-estradiol (100%)”. labeled 15C-hydroxyestriol (estetrol) was administered 0023 All of the above publications have in common that intravenously to two adult women. It was found that the the authors have investigated the estrogenic potency of estetrol was rapidly and completely excreted in urine as the estetrol. Without exception they all conclude that estetrol is glucosiduronate and that Virtually no metabolism except for a weak estrogen. In Some of the cited articles the estrogenic conjugation took place. potency of estetrol has been found to be lower than that of 0014) Between 1975 and 1985 several researchers have another biogenic estrogen, namely, 17B-estradiol, which is investigated the properties of estetrol and reported on its considered to be a relatively weak estrogen (e.g. compared estrogenic potency and uterotrophic activity. The most rel to ethinyl estradiol). With these findings in mind, it is not evant publications that were issued during this period are Surprising that the interest in estetrol has dwindled Since the mentioned below: early eighties and that no publications on the properties of estetrol have been issued since. 0.015 Levine et al., 1984. Uterine vascular effects of estetrol in nonpregnant ewes. Am. J. Obstet. Gynecol., 0024 U.S. Pat. No. 5,468,736 (Hodgen) describes a 148:73, 735-738: “When intravenously administered in method of hormone replacement therapy involving the nonpregnant ewes, estetrol is 15 to 30 times less potent administration of estrogen together with an amount of than estriol and 17 B-estradiol in uterine vasodilation”. antiprogestin (antiprogestogen), which inhibits estrogen induced endometrial proliferation in Women. In Example 3 0016) Jozan et al., 1981. Different effects of oestradiol, the combined use of estetrol and lilopristone is mentioned. Oestriol, Oestetrol and of oestrone on human breast No clues are given in the examples as to the mode and cancer cells (MCF-7) in long term tissue culture. Acta frequency of administration or regarding the dosage level US 2005/0070488 A1 Mar. 31, 2005

employed. A disadvantage associated with the use of anti tion in an amount effective to Suppress ovarian progestogens, Such as lilopristone, is the risk of inducing estrogen and progesterone production and adminis abnormal endometrial morphology, i.e. cystic hyperplasia, tering an estrogenic composition in an amount effec as has been observed in Women who received an anti tive to prevent Symptoms of estrogen deficiency. progestogen treatment against endometriosis (Murphy et al., 0032) WO 00/73416 (Yifang et al.): A method for 1995. Fertil. Steril, 95, 761-766). regulating the fertility of a host, comprising contact 0025 U.S. Pat. No. 5,340,586 (Pike et al.) is concerned ing host Ovarian cells with a safe and effective with compositions and methods which are effective to treat amount of a pharmaceutical composition comprising Oophorectomised Women, wherein an effective amount of an an antisense oligonucleotide that is complementary estrogenic composition and an androgenic composition are to the nucleotide Sequence of the follicle Stimulating provided over a period of time. In the US-patent it is stated hormone (FSH) receptor. The possibility of com that natural and Synthetic estrogenic compositions that can bined administration of Such an antisense oligo be used include natural estrogenic hormones and congeners, nucleotide with an estrogenic Steroid is mentioned in including but not limited to estradiol, , the application. , , estrone, diethylstil bestrol, piperazine estrone Sulfate, ethinyl estradiol, meStra 0033. The benefits of the present invention may be rea nol, , estriol, estriol hemisuccinate, lised without the co-administration of anti-progestogens, , , pinestrol and estrone potassium Sul LHRH compositions, GnRH compositions and/or antisense fate, and furthermore that equine estrogens, Such as equile oligonucleotides that are complementary to the nucleotide linin, equilelinin Sulfate and estetrol, may also be employed. sequence of the follicle stimulating hormone (FSH) receptor Except for the exhaustive inventory of known estrogens, no as proposed in the aforementioned patents. Also, the present other reference to estetrol (which is erroneously referred to invention may suitably be applied in individuals who have as an equine estrogen) is made in this US-patent. not been oophorectomised, or in whom the risk of endome trial Stimulation by estrogenic compositions is not mini 0026. The same exhaustive list of estrogens is found in mised or absent, other than through the co-administration of the following patent documents: a progestogen. Furthermore the present method does not 0027 U.S. Pat. No. 4,762,717 (Crowley): A contra require the use of a slow release formulation as is dictated ceptive method comprising the Sequential adminis by most of the aforementioned publications. tration of (1) a combination of luteinizing hormone 0034. It is noted that none of the aforementioned publi releasing hormone (LHRH) and estrogen and (2) a cations describe the oral administration of estetrol. The only combination of LHRH and estrogen and progesto modes of administration described therein are intravenous gen. and Subcutaneous (depot) administration. For each of these 0028 U.S. Pat. No. 5,130,137 (Crowley): A method modes of administration it can be concluded that the per of treating benign ovarian Secretory disorder com formance of estetrol is very much inferior to that of e.g. prising the Sequential administration of (1) a com 17B-estradiol. Given that there was no reason to assume that bination of luteinizing hormone releasing hormone a different outcome might be obtained in case of oral (LHRH) and estrogen and (2) a combination of administration, it is not Surprising that oral administration of LHRH and estrogen and progestogen. estetrol has not been pursued and that no reports to this effect 0029 U.S. Pat. No. 5,211,952 (Spicer et al.): A can be found in the prior art. contraceptive method comprising administering a 0035) In view of the low estrogenic potency of the gonadotropin hormone releasing hormone (GnRH) estetrol-like Substances that are employed in accordance composition in an amount effective to inhibit OVu with the invention, it is Surprising that these Substances can lation and administering estrogen and progestogen to effectively be used in HRT methods, particularly in HRT maintain Serum levels above a defined minimum methods that employ oral administration of Such Substances. level. Although the inventors do not wish to be bound by theory, 0030 U.S. Pat. No. 5,340,584 (Spicer et al.): A it is believed that the unexpected efficacy of orally admin method for preventing conception or for treating istered estetrol-like Substances results from the combination benign gynaecological disorders comprising admin of unforeseen favourable pharmacokinetic (ADME) and istering a GnRH composition for a first period of pharmacodynamic properties of these Substances. time in an amount effective to SuppreSS ovarian 0036 AS regards the pharmacokinetic properties of the estrogen and progesterone production, Simulta present estrogenic Substances the inventors have discovered neously administering an estrogenic composition in that their oral bioavailability is Surprisingly high and that an amount effective to prevent Symptoms of estrogen their in vivo half-life is considerably longer than that of deficiency and Simultaneously administering a other biogenic estrogens. Thus, even though estetrol and progestogen in an amount effective to maintain estetrol-like Substances have relatively low estrogenic Serum level of Said progestogen at a level effective to potency, they may effectively be employed in an oral HRT decrease endometrial cell proliferation. method because their low potency is compensated for by a 0031 U.S. Pat. No. 5,340,585 (Pike et al.): A relatively high oral bioavailability in combination with a method of treating benign gynaecological disorders high metabolic Stability, as demonstrated by a long half-life. in a patient in whom the risk of endometrial Stimu 0037. An important advantage of oral administration of lation by estrogenic compositions is minimised or estetrol and estetrol-like Substances resides in the fact that absent, comprising administering a GnRH composi the hepatic effects of estetrol-like Substances are deemed to US 2005/0070488 A1 Mar. 31, 2005 be minimal Since they are hardly metabolised during the So of hypoestrogenism, wherein the estrogenic component is called “first pass'. The first-pass effect of drugs given orally, Selected from the group consisting of: refers to the process of drug degradation by the liver during 0044 substances represented by the following formula a drug's transition from initial ingestion to circulation in the blood stream. 0.038 Another advantageous property of the present R7 estrogenic Substances resides in the fact that R6 binding globulin (SHBG) hardly binds these estrogenic Substances, meaning that, in contrast to most known estro gens, Serum levels are representative for bio-activity and independent of SHBG levels. R1 Rs 0039. Yet another important benefit of the present estro genic Substances is derived from their relative insensitivity R2 to interactions with other drugs (drug-drug interactions). It is well known that certain drugs may decrease the effec R R4 tiveness of estrogens, Such as ethinyl estradiol, and other drugs may enhance their activity, resulting in possible increased side-effects. Similarly estrogens may interfere 0045 in which formula R, R2, R, R indepen with the metabolism of other drugs. In general, the effect of dently are a hydrogen atom, a hydroxyl group or an other drugs on estrogens is due to interference with the alkoxy group with 1-5 carbon atoms; each of Rs, R, absorption, metabolism or excretion of these estrogens, R, is a hydroxyl group; and no more than 3 of R, R, whereas the effect of estrogens on other drugs is due to R, R are hydrogen atoms, competition for metabolic pathways. 0046 precursors capable of liberating a substance according to the aforementioned formula when used in 0040. The clinically most significant group of estrogen the present method; drug interactions occurs with drugs that may induce hepatic 0047 and mixtures of one or more of the aforemen microSomal enzymes which may decrease estrogen plasma tioned Substances and/or precursors. The term "oral levels below therapeutic level (for example, anticonvulsant administration” as used in here also encompasses oral agents, phenyloin, primidone, barbiturates, carbamazepine, gavage administration. ethoSuximide, and methoSuximide; antituberculous drugs Such as rifampin; antifungal drugs such as griseofulvin). The 0048. The HRT method according to the invention may present estrogenic Substances are leSS dependent on up- and advantageously be used to treat all known forms of downregulation of microsomal liver enzymes (e.g. P450's) hypoestrogenism, e.g. hypoestrogenism associated with and also are less sensitive to competition with other P450 (peri-)menopausal and post-menopausal women, hypoestro Substrates. Similarly, they do not interfere Significantly in genism resulting from hypogonadism or castration, as well the metabolism of other drugs. as hypoestrogenism resulting from primary ovarian failure, treatment of e.g. breast cancer with aromatase inhibitor and 0041. The conjugates of most estrogens, as formed in the gonadotropin-releasing hormone analogue treatment of e.g. liver, are excreted in the bile and may be broken down by gut benign gynaecological diseases. Examples of manifestations bacteria in the colon to liberate the active hormone which of hypoestrogenism that can effectively be treated or pre can then be reabsorbed (enterohepatic recirculation). There vented with the present method in both females and males are clinical reports that Support the View that enterohepatic include Osteoporosis, arteriosclerosis, cognitive distur recirculation of estrogens decreases in women taking anti bances and Alzheimer's disease. The method may also bioticS Such as amplicillin, tetracycline, etc. Conjugated advantageously be used in the (prophylactic) treatment of forms of the present estrogenic Substances are hardly climacteric Symptoms Such as hot flushes (flashes), Sweats, excreted in the bile, meaning that they are Substantially urogenital atrophy, mood disturbances, insomnia and palpi insensitive to drugs that do influence the enterohepatic tations. The present method is particularly Suited for treating recirculation of other estrogens. or preventing osteoporosis and climacteric Symptoms. 0042. The above observations serve to explain why the 0049. The term “estrogenic component” as used through estrogenic Substances of the invention hardly Suffer from out this document encompasses Substances that are capable drug-drug interactions and thus produce a very consistent, of triggering an estrogenic response in Vivo, as well as i.e. predictable, impact. Thus, the efficacy of the estrogenic precursors that are capable of liberating Such an estrogenic Substances of the invention is highly reliable. component in Vivo when used in accordance with the present invention. In order for estrogenic components to trigger Such DETAILED DESCRIPTION OF THE a response they normally have to bind to an estrogen INVENTION receptor, which receptors are found in various tissues within the mammalian body. The term “progestogenic component' 0043. Accordingly one aspect of the present invention is defined as a Substance that is capable of triggering an relates to a method of hormone replacement in mammals, progestogenic response in Vivo or a precursor which is which method comprises the oral administration of an capable of liberating Such a Substance in Vivo. Usually estrogenic component and a progestogenic component to a progestogenic components are capable of binding to a mammal in an effective amount to prevent or treat Symptoms progeStogen receptor. US 2005/0070488 A1 Mar. 31, 2005

0050. It is noted that the present invention not only 0062) The estrogenic substances according to the formula encompasses the use of estrogenic and progestogenic com encompass various enantiomerS Since the carbon atoms that ponents Specifically mentioned in this application, but also carry hydroxyl-Substituents Rs, Re and R7 are chirally active. metabolites of these hormones that display comparable in In one preferred embodiment, the present estrogenic Sub vivo functionality. In this context it is observed that, for stance is 15C.-hydroxy substituted. In another preferred instance, levonorgestrel is a metabolite of norgestimate and embodiment the substance is 16C.-hydroxy substituted. In that estriol is a metabolite of 17beta-estradiol. Both these yet another preferred embodiment, the Substances is 17 B progestogens and estrogens have found application in con hydroxy substituted. Most preferably the estrogenic Sub traceptive formulations and/or hormone replacement stances are 15C, 16C, 17 B-trihydroxy substituted. therapy. The term "estrogenic Substances as used in this document does not encompass tritium (H) labeled estro 0063. In another preferred embodiment of the present genic Substances Such as tritium labeled estetrol. invention R represents a hydroxyl group or an alkoxy group. In another preferred embodiment the groups R, R 0051. The present estrogenic substances are distinct from and R represent hydrogen atoms, in which case, if R, Rs. both the biogenic and Synthetic estrogens that are commonly Re and R7 are hydroxyl groups, the Substance is 1,3,5 applied in pharmaceutical formulations in that they contain (10)-estratrien-3, 15,16,17-tetrol. A preferred isomer of the at least 4 hydroxyl groups. The present Substances are latter substance is 1,3,5 (10)-estratrien-3, 15C, 16C, 17 B Special in that the 5 membered ring in the Steroid skeleton tetrol (estetrol). comprises 3 hydroxyl substituents rather than 0-2. 0064. The invention also encompasses the use of precur 0.052 Known estrogens that contain at least 4-hydroxyl Sors of the estrogenic Substances that constitute the active groupS and derivatives thereof are: component in the present method. These precursors are 0053) 1, 3, 5(10)-estratrien-2, 3, 15c., 16C, 17 B-pentol capable of liberating the aforementioned estrogenic Sub 2-methyl ether stances when used in the present method, e.g. as a result of metabolic conversion. These precursors are preferably 0054) 1, 3, 5(10)-estratrien-2, 3, 15C, 16C, 17B-pentol Selected from the group of androgenic precursors as well as 2-methyl ether derivatives of the present estrogenic Substances. Suitable examples of androgenic precursors include that 0.055 1, 3, 5(10)-estratrien-2, 3, 16C, 17 B-tetrol can be converted into the present estrogenic Substances 0056 1, 3, 5(10)-estratrien-3, 4, 16C, 17 B-tetrol 4-me through in Vivo aromatisation. Examples of derivatives of thyl ether the present estrogenic Substances that can Suitably be used as precursors include Such Substances wherein the hydrogen 0057) 1, 3, 5(10)-estratrien-3, 15C, 16C, 17 B-tetrol atom of at least one of the hydroxyl groups has been Substituted by an acyl radical of a hydrocarbon carboxylic, 0.058 1, 3, 5(10)-estratrien-3, 15c., 16C, 17 B-tetrol Sulfonic acid or Sulfamic acid of 1-25 carbon atoms, tet tetra acetate rahydrofuranyl, tetrahydropyranal; or a Straight or branched 0059) 1, 3, 5(10)-estratrien-3, 15 B, 16.f3, 17 B-tetrol chain glycosidic residue containing 1-20-glycosidic units tetra acetate per residue. 0060 Preferably, the estrogenic Substance applied as the 0065 Typical examples of precursors which can Suitably active component in the present composition is a natural be used in accordance with the invention are esters that can estrogen, i.e. an estrogen that is found in nature and espe be obtained by reacting the hydroxyl groups of the estro cially in mammals. Even more preferably, the estrogenic genic Substances with Substances that contain one or more Substance is a So called biogenic estrogen, i.e. an estrogen carboxy (MOOC-) groups, wherein M" represents a that occurs naturally in the human body, a precursor of a hydrogen or (akali)metal cation. Hence, in a particularly biogenic estrogen or mixtures thereof. Because biogenic preferred embodiment, the precursors are derivatives of the estrogens are naturally present in the fetal and female body, estrogenic Substances, wherein the hydrogen atom of at least Side-effects are not expected to occur, particularly not if the one of the hydroxyl groups in Said formula has been Sub Serum levels resulting from the exogenous administration of stituted by -CO-R, wherein R is a hydrocarbon radical Such estrogens do not Substantially exceed naturally occur comprising from 1-25 carbon atoms. Preferably R is hydro ring concentrations. Since estetrol Serum levels in the fetus gen, or an alkyl, alkenyl or aryl radical comprising from are Several times higher than those found in pregnant 1-20 carbon atoms. females and knowing that the fetus is particularly Vulner 0066. The present method usually employs uninterrupted able, estetrol is deemed to be a particularly Safe biogenic oral administration of the estrogenic component during a estrogen. Side-effects are not expected to occur, particularly period of at least 10 days, preferably of at least 20 days. The not if the Serum levels resulting from the exogenous admin term “uninterrupted as used in here, means that the estro istration of Such estrogens do not Substantially exceed genic component is administered at relatively regular inter naturally occurring (fetal) concentrations. With Synthetic vals, with no (therapeutically) Significant interruptions. estrogens Such as ethinyl estradiol there is a (dose depen Naturally, minor interruptions may occur that do not affect dent) risk of undesirable side-effects, Such as thromboem the overall effectiveness of the present method, and indeed bolism, fluid retention, nausea, bloating, cholelithiasis, Such aberrations are encompassed by the present invention. headache and breast pain. In a preferred embodiment, and more arithmetically, the 0061. In a preferred embodiment of the present invention administration regimen is deemed to be continuous if the the estrogenic Substance contains 4 hydroxyl groups. Also, longest interval between 2 Subsequent administrations is not in the aforementioned formula, R preferably represents a more than 3.5 times as long as the average interval. Even hydrogen atom. In Said formula preferably at least 2, more more preferably Said longest interval is not more than 2.5 preferably at least 3 of the groups R, R, R and R. times, most preferably not more than 1.5 times as long as the represent a hydrogen atom. average interval. US 2005/0070488 A1 Mar. 31, 2005

0067. The benefits of the present invention are most 0074 Yet another embodiment of the invention, which pronounced when the estrogen component is used in longer concerns a Sequential method without a significant pause, is term hormone replacement therapy So as to minimise the characterised in that it comprises the uninterrupted oral negative effects of chronic hypoestrogenism. Therefore, the administration of the estrogenic component during a period method of hormone replacement therapy, preferably, com of at least 28 days, preferably at least 60 days, and in that, prises administering the estrogenic component for a period following the combined administration of the estrogenic of at least 1 month, more preferably of at least 3 months. component and the progestogenic component, the estrogenic 0068. In the present method, the estrogenic and progesto component and no progestogenic component are adminis genic component may be administered in Separate oral tered during 3-18 consecutive days, preferably during 5-16 dosage units. However, it is also possible and indeed very consecutive days and the resulting decrease in Serum con convenient to combine these two components into a single centration of the progestogenic component should normally oral dosage unit. be Sufficient to induce menses. 0075. In the present methods uninterrupted administra 0069. The invention may suitably be reduced to practice tion of the estrogenic component may usually occur at in the form of a variety of HRT methods that are known to intervals of between 6 hours and 7 days, preferably of the person skilled in the art. Amongst these methods are the between 12 hours and 3 days. The relatively high in vivo So called “combined' methods. The combined methods half-life of the present estrogenic components in comparison make use of preparations that contain a combination of an to most known estrogens makes it feasible to employ oral estrogen and a progestogen. The combined methods have in administration intervals that are significantly longer than 1 common that they are based on a regimen which involves day. For practical reasons, and particularly with a view to administration of the aforementioned combined preparation, user compliance, it is preferred to orally administer the followed by an administration-free interval of about 7 days estrogenic component as well as the progestogenic compo whereby withdrawal bleeding, Simulating the natural nent at least once daily, most preferably once daily. menses, occurs. Thus 21 day intervals of hormone admin 0076. In all of the aforementioned methods it is preferred istration alternate with 7 days during which no hormones are to orally administer the estrogenic component and the administered. progestogenic component at least once daily during a period 0070. As an alternative to the aforementioned combined of at least 10, preferably of at least 20 days. In case of a methods, the So called “Sequential method has been pro Sequential method without pause or a continuous combined posed. Typical of the Sequential method is that it comprises method it is preferred to orally administer the estrogenic two consecutive phases, i.e. one phase during which estro component and/or the progestogenic component at least gen and no progestogen is administered and another phase once daily during a period of at least 30 days, more during which a combination of estrogen and progestogen is preferably of at least 60 days, most preferably of at least 150 administered. The first Sequential methods, like the afore days. Uninterrupted Sequential methods, which employ con mentioned combined methods, made use of an administra tinuous estrogen administration, are characterised by excel tion free interval of about 7 days. More recently, sequential lent cycle control. methods have been proposed which do not include an 0077. The general concerns about the so called unop administration-free (or placebo) period, meaning that estro posed administration of estrogen, i.e. administration of gen is administered throughout the full cycle and that estrogen without co-administered progestogen might cause progestogen is co-administered during only part of that hyperplasia of the endometrium, are leSS applicable to the cycle. WO95/17895 (Ehrlich et al.) describes such an estrogenic components of the present invention. Therefore, uninterrupted Sequential method. in a particularly preferred embodiment, the present HRT 0071. Yet another example of an HRT method which is method is executed in accordance with a Sequential method encompassed by the present invention is the So called without pause. “continuous combined' method, which is a particular ver 0078 Good results can be obtained with the present Sion of the combined method that uses uninterrupted com method if the estrogenic component is orally administered in bined administration of a progestogenic and an estrogenic an amount of less than 1 mg per kg of bodyweight per day, component during a prolonged period of time, e.g. more than preferably of less than 400 ug per kg of bodyweight per day, 50 days. In contrast to ordinary combined and Sequential more preferably of less than 200 ug per kg of bodyweight methods, no regular menses occur in the continuous com per day. In order to achieve a Significant impact from the bined method as the continuous administration of progesto administration of the present estrogenic component, it is gen in the indicated amounts induces amenorrhoea. advisable to orally administer in an amount of at least 1 lug 0.072 In one embodiment of the invention, which relates per kg of bodyweight per day. Preferably, the orally admin to the continuous combined method, the present method istered amount is at least 2 ug per kg of bodyweight per day. comprises the uninterrupted oral administration of the com More preferably, the orally administered amount is at least bination of the estrogenic component and the progestogenic 5 ug per kg of bodyweight per day. component during a period of at least 28, preferably at least 0079. In the present method, particularly when used in 60 days. humans, the estrogenic component is usually administered 0073. In another embodiment of the invention, which in an average dosage of at least 0.05 mg per day, preferably relates to Sequential and combined methods that employ a of at least 0.1 mg per day. The maximum dosage is normally Significant administration-free interval, the method of the kept below 40 mg per day, preferably below 20 mg per day. invention comprises an interval of at least 2 days, preferably The normally employed dose of the progestogenic compo from 3-9 days, most preferably from 5-8 days, during which nent is equivalent to an average oral dosage of 30-750 ug no progestogenic component and no estrogenic component levonorgestrel per day, preferably to an average oral dosage is administered and wherein the resulting decrease in Serum of 50-400 ug levonorgestrel per day. concentration of the progestogenic component and the estro 0080. In the present method, the estrogenic component is genic component induces menses. preferably administered in an amount effective to achieve a US 2005/0070488 A1 Mar. 31, 2005

blood Serum concentration of at least 1 nanogram per litre, 6, more preferably from 8-20 and preferably 9-13 carbon more preferably of at least 10 nanogram per litre, most atoms. The androgens that can be used most advantageously preferably at least 100 nanogram per litre. Generally the in the present method are DHEA and/or unde resulting blood Serum concentration of the estrogenic com Canoate. ponent will not exceed 100 ug per litre, preferably it will not 0086. It is noted that, for instance, DHEA and testoster exceed 50 ug per litre, more preferably it will not exceed 25 one undecanoate are precursors of testosterone and that Said pig per litre. precursors per Se exhibit virtually no affinity for 0081. In accordance with the present invention the receptors in the female body. The effectiveness of the progestogenic component is advantageously administered in androgens within the method of the invention is determined an amount which is equivalent to a daily oral dosage of 0.3 by their functionally active form, which may well be dif to 20 uglevonorgestrel per kg of bodyweight, preferably of ferent from the form in which they are administered. 0.5-5 ug levonorgestrel per kg of bodyweight. 0087. In a preferred embodiment the androgen is pro 0082) Examples of progestogens which may suitably be Vided in an amount equivalent to a daily oral dosage of 5 to used in accordance with the present invention include: 250 mg DHEA, which is equivalent to a daily oral dosage of progesterone, levonorgestrel, norgestimate, , 1 to 50 mg . More preferably the dydrogesterone, droSpirenone, 3-beta-hydroxydeSogeStrel, androgen is provided in an amount which is equivalent to a 3-keto desogestrel (=etonogestrel), 17-deacetyl norgesti daily oral dosage of 10-120 mg DHEA. Most preferably the mate, 19-norprogesterone, acetoxypregnenolone, allyleStre androgen is administered in an amount which is equivalent nol, anageStone, chlormadinone, cyproterone, demegestone, to a daily oral dosage of 20-60 mg DHEA. deSogeStrel, dienogest, dihydrogesterone, dimethisterone, ethisterone, ethynodiol diacetate, flurogestone acetate, gas 0088. In order to obtain the desired impact from the trinon, gestodene, , hydroxymethylprogesterone, present method it is advisable to administer the dosage units hydroxyprogesterone, (=lynoestrenol), in an amount which leads to an increase in blood Serum medrogestone, medroxyprogesterone, megestrol, androgen level of no more than 5 nmole testosterone equiva melengestrol, nomegestrol, norethindrone (=norethisterone), lent per litre, preferably less than 3 nmole testosterone norethynodrel, norgestrel (includes d-norgestrel and equivalent per litre and most preferably less than 1.5 mole dl-norgestrel), norgestrienone, normethisterone, progester testosterone equivalent per litre. one, quingestanol, (17alpha)-17-hydroxy-11-methylene-19 0089. The present method preferably does not employ a norpregna-4,15-diene-20-yn-3-one, , trimegestone, gonadotropin hormone releasing hormone composition as algestone acetophenide, nestorone, promegestone, 17-hy described in the aforementioned patents U.S. Pat. No. 5,211, droxyprogesterone esters, 19-nor-17 hydroxyprogesterone, 952, U.S. Pat. No. 5,340,584 and U.S. Pat. No. 5,340,585. 17alpha-ethinyl-testosterone, 17alpha-ethinyl-19-nor-test Similarly, the present method preferably does not employ a osterone, d-17beta-acetoxy-13beta-ethyl-17alpha-ethinyl luteinizing hormone releasing hormone composition as gon-4-en-3-one oXime and precursors of these compounds described in U.S. Pat. No. 4,762,717 and U.S. Pat. No. that are capable of liberating these progestogens in vivo 5,130,137. Furthermore, the present method preferably does when used in the present method. Preferably the progesto not comprise the co-administration of an anti-progestogen as gen used in the present method is Selected from the group described in U.S. Pat. No. 5,468,736. The method may also consisting of progesterone, desogestrel, etonogeStrel, Suitably be applied without the co-administration of an gestodene, dienogest, levonorgestrel, norgestimate, nore antisense oligonucleotide that is complementary to the thisterone, droSpire none, trimegeStone, dydrogesterone, pre nucleotide Sequence of the follicle Stimulating hormone cursors of these progestogens and mixtures thereof. (FSH) receptor (WO 00/73416). 0.083. The present method also encompasses the co 0090 The present method is preferably not used in administration of active principles in addition to the Oophorectomised females or in females in whom endome progestogenic and estrogenic component. For instance, trial Stimulation by estrogenic compositions is minimised or androgens may advantageously be co-administered in order absent, other than by combined administration of a progesto to prevent Symptoms of hypoandrogenicity. Thus, a pre gen and an estrogen, e.g. as a result of hysterectomy. ferred embodiment of the invention comprises the co-ad ministration of an androgenic component. The androgenic 0091 Another aspect of the invention relates to a phar component is Suitably co-administered in an effective maceutical kit comprising at least 20 oral dosage units that amount to SuppreSS Symptoms of hypoandrogenicity. contains the estrogenic component as defined herein before Hypoandrogenicity has been associated with mood distur and/or the progestogenic component and/or the androgenic bances, unfavourable changes in haemostatic parameters component as described herein before, wherein at least 10 units contain between 0.01 and 20 mg, preferably between and lack of bone mass. 0.05 and 10 mg of the estrogenic component, at least 10 0084. The term “androgenic component” is defined as a units contain the progestogenic component in an amount Substance that is capable of triggering an androgenic equivalent to 30-750 tug levonorgestrel and at least 10 response in Vivo or a precursor which is capable of liberating dosage units contain the androgenic component in an Such a Substance in Vivo. Usually androgenic components amount equivalent to 5-250 mg . are capable of binding to an androgen receptor. 0092. In the present kit, the estrogenic component may 0085 Androgenic components that may suitably be conveniently be combined with the progestogenic compo employed in the present method may be Selected from the nent and the androgenic in a single dosage unit. Accordingly, group consisting of dehydroepiandrosterone (DHEA), dana the kit preferably comprises at least 10 dosage units which Zol, gestrinone, testosterone esters, precursors capable of contain between 0.01 and 20 mg of the estrogenic compo liberating these androgens when used in the present method nent, the progestogenic component in an amount equivalent and mixtures thereof. Preferably the testosterone esters to 30-750 ug levonorgestrel and the androgenic component employed comprise an acyl group which comprises at least in an amount equivalent to 5-250 mg dehydroepiandroster US 2005/0070488 A1 Mar. 31, 2005 one. A pharmaceutical kit that is particularly Suitable for use ceutical procedures. The active ingredient(s) are combined in combined and Sequential methods will usually comprise with a pharmaceutically acceptable excipient and converted 20-35 oral dosage units, wherein 10-35 units contain a into a pharmaceutically acceptable form for oral adminis combination of the estrogenic component and the progesto tration, e.g. a tablet, capsule, cachet, pellet, pill, powder or genic component in the indicated amounts, 6-25 units con granules. The excipient may include appropriate pharma tain no progestogenic component and the estrogenic com ceutical carrierS Such as diluents, binders and lubricants. For ponent in the indicated amounts, and 0-8 units contain no example gums, Starches and Sugars are commonly used as estrogenic component and no progestogenic component. pharmaceutical carriers. Tablets and other oral dosage units 0093. A pharmaceutical kit that is particularly suitable for can Suitably contain materials Such binders (e.g. hydrox use in a continuous combined regimen or a combined ypropylmethyl cellulose, polyvinyl pyrrolidine, other cellu regimen comprises at least 20 oral dosage units which either losic materials and Starch), diluents (e.g. lactose and other contain the combination of the progestogenic and the estro Sugars, Starch, dicalcium phosphate and cellulosic materi genic component or neither of these two components (pla als), disintegrating agents (e.g. starch polymers and cellu cebo's) and of which dosage units at least 15, preferably at losic materials) and lubricating agents (e.g., Stearates and least 20 contain the combination of the estrogenic compo talc). nent and the progestogenic component and 0-8 contain no 0099] The active ingredient(s) may comprise from about estrogenic component and no progestogenic component. If 0.01% by weight to about 50% by weight of the formulation Such a kit is to be used in a continuous combined method, in the dosage unit, the remainder consisting of excipient. The the kit may advantageously comprise at least 28, preferably active ingredient(s) are compounded with the chosen carrier at least 60 dosage units, all of which dosage units contain the and in for example the case of a tablet form, placed in a combination of the estrogenic component and the progesto tablet moulding apparatus to form the tablets. Alternatively genic component in the amounts indicated above. the compounded material may be incorporated as a powder 0094. In case the present kit is meant to be used in an or granules in a capsule. Various other options that may HRT method that employs an administration free intervalso Suitably be used in accordance with the present invention are as to induce menses (e.g. a combined method or a sequential well known to the perSon Skilled in the pharmaceutical art. method with pause) the kit will usually comprise at least 3 0100. The present invention is further illustrated by the units, preferably at least 5 units that contain no estrogenic following examples, which, however, are not to be construed component and no progestogenic component. as limiting. The features disclosed in the foregoing descrip 0.095. In a particularly preferred embodiment of the tion, in the following examples and in the claims may, both invention the present kit is designed for use in a sequential separately and in any combination thereof, be material for method. Such a kit will usually comprise 20-35 oral dosage realising the invention in diverse forms thereof. units wherein 10-32 units contain the combination of the estrogenic component and the progestogenic component, EXAMPLES and 3-18 units contain the estrogenic component and no progestogenic component. Particularly preferred is a kit that Example 1 is designed for use in a Sequential method without a Sig nificant pause. In Such a kit, which will usually comprise 0101 Vaginal cornification was chosen as a tissue-spe 20-35 oral dosage units, 10-20 units contain a combination cific and estrogen-Sensitive endpoint to determine the estro of the estrogenic component and the progestogenic compo genicity of estetrol (E4), after oral administration, in nent, 10-18 units contain the estrogenic component and no hypoestrogenic rats. 17O- (EE) and vehicle progestogenic component and at most 1 unit contains no (10% ethanol/sesame oil) served as controls in these bioas estrogenic component and no progestogenic component. SayS. 0096. The pharmaceutical kits according to the present 0102 Uterine weight increase in the rat is more com invention will normally contain only one or more of the monly used as a measure of estrogenicity. However, uterine following types of oral dosage units: units that contain the weight also responds to progesterone, testosterone, and other combination of the estrogenic and the progestogenic com agents not characteristically regarded as estrogens. In the ponent; units that contain the estrogenic component and no early 1920s it was discovered that follicular fluid from the progestogenic component; and units that effectively function pig ovary contained a factor(s) that caused cornification/ as placebo's. Preferably the kit comprises at least 20 units keratinization of the vaginal epithelium in the rat (Allen and that contain the combination of the estrogenic and the Doisy, 1923, JAMA, 81,819-821; Allen and Doisy, 1924, progestogenic component or the estrogenic component and Am. J. Physiol., 69,577-588). The so-called vaginal corni no progestogenic component. fication response in rats Subsequently provided a bioassay 0097. If the present kit is to be used in a combined or for testing estrogenicity. Vaginal epithelial cornification/ Sequential protocol, the oral dosage units are preferably keratinization in ovariectomized rats can be produced only arranged within the kit in a fixed Sequence corresponding to by compounds considered to be true estrogens (Jones et al., the intended order of administration. Data indications may 1973, Fert. Steril. 24, 284-291). Vaginal epithelial cornifi be provided on the packaging. The packaging may be a tube cation/keratinization represents, therefore, a highly Selective or box or a Strip. The box may be circular, Square, or endpoint to determine the potency of estrogens (Reel et al., otherwise shaped with the tablets being accommodated 1996, Fund. Appli. Toxicol. 34, 288-305). Separately therein for ease of administration. Date indica 0103) Adult intact female CD rats were ovariectomized to tions may appear adjacent to each tablet corresponding with induce estrogen deficiency. Vaginal lavages were performed the days on which each tablet is to be taken. Some indication daily for Seven days to ensure that the rats demonstrated of the Sequence in which the tablets are to be taken prefer castrate vaginal Smears (predominance of leukocytes in the ably appears on the packaging regardless of its form. vaginal Smear, and Similar in appearance to a diestrous 0.098 Generally speaking, the oral dosage units in the vaginal Smear). Castrate vaginal Smears are indicative that present kit are prepared according to well known pharma complete ovariectomy was achieved. Treatment commenced US 2005/0070488 A1 Mar. 31, 2005 following completion of the 7 days of Smearing (day 0-first 0106 Three months old female Sprague-Dawley rats day of dosing). Animals were dosed, once daily for 7 were either Sham-operated (Sham) or ovariectomized consecutive dayS. Daily vaginal lavages continued to be (OVX) one day prior to commencement of the dosing study. obtained for 7 days after dosing was initiated in order to Animals were anesthetized using a ketamine/Xylazine anes detect vaginal cornification, as an indication of an estrogenic thetic mixture and underwent a bilateral Ovariectomy or response. A drop of vaginal washings was placed on a glass were Sham-treated. A Section of hair on the dorsal Surface Slide and examined by light microScopy to detect the pres was shaved and an incision made overlying the lumbar ence or absence of cornified epithelial cells. Vaginal lavages region of the Spine. The skin was separated from the were obtained prior to dosing on days 0-6 and prior to underlying fascia So that a Second incision could be made necropsy on day 7. through the abdominal musculature approximately caudal to 0104. The vaginal cornification bioassay was performed the kidneys. The ovaries were then exteriorized and removed in order to determine the estrogenic profile of E4 when given and the musculature was closed with a single Suture. The orally (po) to ovariectomized adult rats. EE was used as a skin incision was closed using Surgical Staples. positive control. The vehicle (10% ethanol/sesame oil) Served as the negative control. Steroids were dissolved in 0107 Ten animals per treatment group were orally dosed absolute ethanol and then brought to the final concentration once per day for four consecutive weeks. The dosing com with Sesame oil (10% ethanol in Sesame oil). A vaginal menced 1 day after Surgical removal of the ovaries and was estrogenic response occurred in all rats (8/8) given 50 administered by oral gavage using a Syringe and Stainless Aug/kg/day EE po by day 7 (Table 1). Similarly, vaginal Steel gavage needle at doses of 0.1 mg/kg/day EE, or either epithelial cornification was observed in all rats (8/8) treated 2.5, 0.5 or 0.1 mg/kg/day E4. Vehicle control was daily po with either 0.1, 0.3, 1.0, or 3.0 mg/kg/day E4 by day 7 administered to one group of OVX-animals and Sham (Table 1), whereas animals treated with the vehicle did not operated rats. After treatment, anesthetized rats were Sub exhibit vaginal epithelial cornification (0/8). Even in rats jected to cardiac puncture and asphyxiated by CO inhala given relatively low doses of E4 (e.g. 0.1 and 0.3 mg/kg/ tion. Tibiae and femura were removed, cleaned of Soft-tissue day), the onset of vaginal cornification (defined as the and fixed and stored in 70% ethanol/saline at 4°C. (tibia) or amount of animals responding at days 1-3 of the study) was saline at 4 C. (femura) until further analysis. as fast as in EE-treated animals (Table 1), demonstrating estetrol’s Superb bioavailability characteristics after oral 0108) Ex vivo peripheral Quantitative Computed Tomog administration. raphy (pOCT) was performed on the excised left tibiae using

TABLE 1. Vaginal estrogenic response in Ovariectomized rats treated orally (po) with 17C-ethinyl estradiol (EE) or estetrol (E4). Data are expressed as the number of rats showing vaginal cornification over the number of rats (ratio) treated. Number of Rats Exhibiting Estrogenic Response/ Number of Rats Treated Treatment Dosing Day of Study Group route Day 0 Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 0.05 mg/kg/day po Of8 1f8 3/8 8/8 8/8 8/8 8/8 8/8 EE Vehicle po Of8 Of8 Of8 Of8 Of8 Of8 Of8 Of8 Control (2 ml/kg/day) 0.1 mg/kg/day po Of8 Of8 1f8 7/8 8/8 8/8 8/8 8/8 E4 0.3 mg/kg/day po Of8 Of8 1f8 7/8 8/8 8/8 8/8 8/8 E4 1.0 mg/kg/day po Of8 Of8 4/8 8/8 8/8 8/8 8/8 8/8 E4 3.0 mg/kg/day po Of8 Of8 678 8/8 8/8 8/8 8/8 8/8 E4

Example 2 a Stratec XCT-RM and associated software (Stratec Mediz intechnik GmbH, Pforzheim, Germany, Software version 0105 The ovariectomized aged rat was used as a model 5.40). The scans were performed at 12% of the total length for the human disease osteoporosis. This is an established from the proximal end of the tibiae. The positions were animal model, recommended by the United States Food and Verified using Scout views and one 0.5-mm Slice perpen Drug Administration (FDA), to evaluate and assess potential dicular to the long axis of the tibial Shaft was acquired from agents for Osteoporosis prevention and therapy. The anti each site. The Scans were analyzed using a threshold for resorptive efficacy of estetrol (E4) was tested by ex vivo delineation of the external boundary. The total and trabecu measuring total and trabecular bone mineral density and lar bone mineral content, area and density at each Site were bone Strength after 4 weeks of treatment at necropsy. 17C.- determined. Mean values are shown in Table 2. Furthermore, ethinyl-estradiol (EE) and vehicle (1% ethanol/arachidis oil) pOCT data for mean total bone mineral density are depicted Served as controls in this bioassay. in FIG. 1. US 2005/0070488 A1 Mar. 31, 2005 10

TABLE 2 pOCT densitometry data from the proximal tibiae of Sham- and OVX-rats orally (po) treated with 17c- ethinyl estradiol (EE), estetrol (E4) or vehicle. Data are expressed as the mean values obtained for each group (n = 10). Mean Total Bone Mean Trabecular Bone Treatment Mineral Mineral Group Dosing Content Area Density Content Area Density (n = 10) route (mg/mm) (mm) (mg/cm) (mg/mm) (mm) (mg/cm) SHAM+ Vehicle po 9.36 14.10 664.O7 1.49 6.34 235.48 OVX + Vehicle po 8.76 14.47 606.61 1.10 6.51 169.63 OVX + 0.1 mg/kg/day po 9.66 13.87 697.48 181 6.24 290.16 EE OVX + 0.1 mg/kg/day po 8.46 14.41 588.62 O.96 6.48 145.46 E4 OVX + 0.5 mg/kg/day po 9.74 14.80 660.57 1.60 6.65 243.31 E4 OVX + 2.5 mg/kg/day po 9.61 13.59 707.11 1.89 6.12 309.58 E4

0109 Comparison of the pCCT densitometry data from orally treated with E4 (Table 2, FIG. 1). As compared to the proximal tibiae of Sham-operated and OVX-rats dem hypoestrogenic OVX-rats receiving vehicle treatment alone, 0.5 and 2.5 mg/kg/day E4 prevented bone resorption as onstrated a consistent loSS of total and trabecular bone in the exemplified by total bone mineral density levels equivalent OVX-group, as expected (Table 2, FIG. 1). Furthermore, to sham-operated rats (FIG. 1). Furthermore, the anti-resorp there was a consistent dose-dependent increase for each of tive activity achieved with the highest dose of E4 (2.5 the parameters associated with total and trabecular bone mg/kg/day) was equivalent to the effect seen with the mineral content and bone mineral density in the animals positive control EE. US 2005/0070488 A1 Mar. 31, 2005 11

7O O s

6 5 O S S 6O O O c

9A o n Sham- OVX- OVX-E0.1 OVX-E40,1 OVX-E40.5 OVX-E4 2.5 vehicle Vehicle Tg/kg/day mg/kg/day mg/kg/day mg/kg/day DOSe

Figure 1: Total bone mineral density from the proximal tibiae of Sham- and OVX-rats orally (po) treated with 17a-ethinyl estradiol (EE), estetrol (E4) or vehicle for 4 consecutive weeks. Data are expressed as the mean values obtained for each group (n=10). US 2005/0070488 A1 Mar. 31, 2005 12

0110) Ex vivo evaluation of bone biomechanical strength was perfomed with an indentation test at the distal femura. TABLE 3-continued Prior to mechanical testing femura were rinsed in cold Saline Indentation testing of the distal femur of Sham- and OVX-rats and carefully cleaned of any remaining adherent Soft tissue. orally (po) treated with 17c- ethinyl estradiol (EE), estetrol (E4) or vehicle. Data are expressed as the mean values A 3-mm Segment of the distal femoral metaphysis was cut obtained for each group (n = 10). directly proximal to the femoral condyle with a low-speed Treatment Maximum Ultimate diamond Saw under constant Saline irrigation. The load was Group Dosing load Stiffness Energy strength applied with a cylindrical indenter (with a flat testing face of (n = 10) route (N) (N/mm) (m) (N/mm2) 1.6 mm diameter) to the center of marrow cavity on the OVX - 2.5 po 13.07 173.12 O.68 6.94 distal face of the Segment. The indenter was allowed to mg/kg/day E4 penetrate the cavity at a constant displacement of 6 mm/min to a depth of 2 mm before load reversal. 0111. The maximum load, stiffness and energy absorbed were determined from load displacement curves. Ultimate TABLE 3 Strength was calculated by dividing the maximum load by Indentation testing of the distal femur of Sham- and OVX-rats the indenter area. Mean values of maximum load, Stiffness, orally (po) treated with 17c- ethinyl estradiol (EE), estetrol energy and ultimate Strength are shown in Table 3. Further (E4) or vehicle. Data are expressed as the mean values more, mean ultimate Strength values are depicted in FIG. 2. obtained for each group (n = 10). AS compared to Sham-operated rats, the mechanical Strength Treatment Maximum Ultimate of cancellous bone appeared to be markedly reduced in Group Dosing load Stiffness Energy strength OVX-rats treated with vehicle alone (Table 3, FIG. 2). (n = 10) route (N) (N/mm) (m.J) (N/mm2) Reductions in maximum load, Stiffness, energy and ultimate SHAM+ Vehicle po 8.61 131.96 O.48 4.57 strength were -68%, -68%, -27% and -68%, respectively, OVX + Vehicle po 2.77 42.08 O.21 1.47 clearly accompanying the bone mineral density loSS in OVX -- 0:1 po 9.05 169.12 O.53 4.80 estrogen deficient rats. Oral treatment of hypoestrogenic mg/kg/day EE OVX -- 0:1 po 1.50 28.00 O.09 O.80 OVX-rats with E4 prevented the declines in maximum load, mg/kg/day E4 Stiffness, energy and ultimate Strength, in a dose-dependent OVX + O.5 po 7.25 132.57 O.31 3.85 manner (Table 3, FIG. 2). In addition, the efficacy achieved mg/kg/day E4 with the highest dosis of E4 (2.5 mg/kg/day) even appears Superior to that of the positive control EE (Table 3, FIG. 2). US 2005/0070488 A1 Mar. 31, 2005 13

8 a 7 E S 6 al 5 4 s 3 2 s 1 0 Sham- OVX-Vehicle OVX-E 0.1 OVX-E4 0.1 OVX-E4 O.5 OVX-E42.5 Vehicle mg/kg/day mg/kg/day mg/kg/day mg/kg/day Dose

Figure 2: Mean ultimate strength of the distal femur of Sham- and OVX-rats orally (po) treated with 17a ethinyl estradiol (EE), estetrol (E4) or vehicle for 4 consecutive weeks. Data are expressed as the mean values 5 obtained for each group (n=10). US 2005/0070488 A1 Mar. 31, 2005

Example 3 Separated from the underlying fascia So that a Second 0112 The morphine-dependent ovariectomized (OVX) incision could be made through the abdominal musculature rat was used as a model for postmenopausal hot flush. The approximately caudal to the kidneys. The Ovaries were then potency of estetrol (E4) to prevent tail skin temperature exteriorized and removed and the musculature was closed rises, normally accompanied by a drop in core body tem with a single Suture. The skin incision was closed using parature, after naloxone-induced opiate withdrawel was Surgical Staples. Six rats per treatment group were dosed tested. 17O-ethinyl-estradiol (EE) and vehicle (hydroxy pro once per day for eight consecutive days prior to and includ pyl-beta-cyclodextrin 20% wit/vol) served as controls in this ing the day of naloxone-induced opiate withdrawal (the hot bioassay. flush Session). The dosing commenced three days after 0113. The most common and characteristic symptom of Surgical removal of the ovaries and was administered by oral human menopause is the hot flush, which is experienced by gavage using a Syringe and StainleSS Steel gavage needle. over 70% of menopausal females. While the exact mecha Morphine dependency was induced by implantation of Sub nism underlying this vasomotor instability is unknown, the cutaneous pellets containing 75-mg morphine. The first characteristic features of the hot flush appear to reflect a pellet was implanted five days before the hot flush session centrally mediated adaptation to a progressive decline in the under a light inhalation anesthesia. Three days before the hot levels of estrogens. In Women experiencing the hot flush the flush Session, two additional morphine pellets were Symptoms are manifested by 1) rapid, regional elevations in implanted under the Same conditions. skin temperature; 2) a decrease in core body temperature; 3) 0116 For the hot flush manipulations the animals were an increased heart rate with no change in blood preSSure, and placed in a test cage. Following a 5-10 minute adaptation 4) closely timed Surges in release of luteinizing hormone period, the rats were anesthetized with ketamine HCl (LH) and B-endorphin. approximately 10 minutes prior to the hot flush Session. A 0114. The morphine-dependent ovariectomized (OVX) temperature Sensitive electrode was fixed to the Ventral rat model has been proposed by Several investigators Surface of the tail with tape and the electrode was connected (Katovich et al., 1986, Maturitas, 67-76; Merchenthaler et al. to a multi-channel temperature recorder. The tail-skin tem 1998, Maturitas, 307-316) as an animal model for the hot perature was recorded until it was stable and the animals flush. During opiate withdrawal with the morphine antago were then injected with naloxone HCl (1 mg/kg). The nist naloxone, tail skin temperature (TST) rises and this rise temperature recordings then continued for a period of 60 is accompanied by a drop in core body temperature. In minutes and the temperature was reported at 5-minute addition, the temperature changes are accompanied by intervals. At the completion of the hot flush Session, all Surges in LH and a transient tachycardia. These events are animals were killed using CO asphyxiation followed by Similar in magnitude and temporal nature to those observed cervical dislocation. in the menopausal hot flush. 0117 AS expected, vehicle control was ineffective in 0115 8-week-old OVX rats were treated orally (po) with preventing the naloxone-induced TST increases in the mor estetrol (E4), 17o-ethinyl estradiol (EE) or vehicle control phine addicted OVX rats (FIG. 3). 17O-ethinyl estradiol (hydroxy propyl-beta-cyclodextrin 20% w/vol) for seven (EE), at the single dose tested of 0.3 mg/kg/day, prevented consecutive days prior to, and on the morning of naloxone the naloxone-induced TST increases in the morphine induced opiate withdrawal in morphine-dependent animals. addicted OVX rats (FIG. 3). Oral treatment with estetrol Three days prior to the commencement of dosing, animals (E4) showed a clear dose-dependendent effect (FIG. 3). The were anesthetized using a ketamine/Xylazine anesthetic mix three highest doses of E4 (0.3, 1.0 and 3.0 mg/kg/day) all ture and underwent a bilateral ovariectomy. A Section of hair attenuated the TST, with the highest dose (3.0 mg/kg/day) on the dorsal Surface was shaved and an incision made having a Suppressive response Similar to the potent oral overlying the lumbar region of the Spine. The skin was estrogen, 17O-ethinyl estradiol (EE). US 2005/0070488 A1 Mar. 31, 2005 15

30 Vehicle 29 l o' Estetrol (0.1 mg/kg/day) 28 r. Estetrol (0.3 mg/kg/day) - N . Estetrol (1 mg/kg/day) 27 : i ? N \ .. Estetrol (3 mg/kg/day)

26 5 / Y. 17-ethinyl estradiol (0.3 mg/kg/day)

Y

-5 0 5 10 1s 20 25 30 35 40 45 50 55 60 time (min) post-naloxone

10 Figure 3: The effects of estetrol (E4) and 17o-ethinyl estradiol (EE) on the naloxone induced hot flush response in female ovariectomized rats. US 2005/0070488 A1 Mar. 31, 2005

Example 4 0.122 Human SHBG was purified from transgenic mouse 0118 To evaluate the oral bioavailability of estetrol (E4) serum, as described previously (Avvakumov GV et al., 2000. and to determine the elimination half-life, Single oral (po) J Biol Chem 275: 25920-25925). The human SHBG pre and Subcutaneous (Sc) dose studies were performed in pared in this way was assessed to be >99% pure by poly female Sprague Dawley rats followed by frequent blood acrylamide gel electrophoresis under denaturing conditions. Sampling over a 24 hours interval. Its Steroid-binding characteristics are indistinguishable from 0119 Female Sprague Dawley rats were equipped with a SHBG in human serum (Avvakumov GV et al., 2000. J Biol permanent Silatic heart catheter, as described by KuiperS et Chem 275: 25920-25925). The in vitro assay involved the al. (1985, Gastroenterology, 88, 403-411). Rats were use of the purified human SHBG and HDHT or H allowed to recover from Surgery for 5 days and were than estradiol as labeled ligands. Human SHBG was treated for administered 0.05, 0.5, or 5 mg/kg E4 in 0.5 ml arachidis oil. 30 min at room temperature with a dextran-coated charcoal For Sc administration, E4 was injected in the neck area using (DCC) suspension in phosphate buffered saline (PBS) to a 1 ml Syringe and 20 g needle. Forpo administration of E4, remove any Steroid ligand. After centrifugation (2,000xg for rats were lightly anaesthesized with halothene/NO/O and 10 min) to sediment the DCC, the Supernatant containing the E4 was directly applied intragastrically using a plastic human SHBG was diluted in PBS to a concentration of 1 nM Stomach intubator. Blood Samples were Subsequently col based on its Steroid binding capacity. lected via the heart catheter in heparinized tubes at 0.5, 1, 2, 0123 Duplicate aliquots (100 ul) of this human SHBG 4, 8 and 24 hours. Erythrocytes were removed by centrifu Solution were then incubated with an equal volume of either gation at 5000xg for 10 minutes at 4 C. and blood plasma HDHT or Hestradiol at 10 nM, together with 100 ul of was stored at -20° C. After thawing the plasma samples, PBS alone or the same amount of PBS containing increasing liquid-liquid extraction (hexane and diethyl ether) was concentrations of unlabeled Steroid ligands as competitors in employed to prepare the E4-containing plasma Samples for polystyrene test tubes. After incubation for 1 h at room HPLC analysis (Perkin Elmer 200) and tandem mass spec temperature the reaction mixtures were placed in an ice bath trometry using a PE Sciex 3000 tandem mass spectrometer for a further 15 min. Aliquots (600 ul) of an ice cold and APCI interface. With each sample batch, a calibration suspension of DCC were then added to each tube, and after curve with 6 calibrators was recorded. The calibration curve a brief 2 Seconds mixing, each tube was incubated in an ice was calculated using linear regression (correlation coeffi bath for either 10 min or 5 min depending on whether cient >0.98), which permitted quantitation of plasma con HDHT or Hestradiol were being used as labeled centrations. For each rat plasma, Sampled at different time ligands, respectively. The unbound ligands adsorbed to DCC intervals, data were collected. were then removed by centrifugation (2,000xg for 15 min at 0120 Plasma E4 concentration data were analysed with 4 C), and the amounts of Hlabeled ligands bound to “WinNonLin, edition 3.1” and involved pharmacokinetic SHBG were counted in 2 ml ACS Scintillation cocktail using parameters for C, half-life and AUC. Especially, in liquid Scintilation spectrophotometer. The average using the lower and intermediate dose levels of 0.05, 0.5 amounts of Hlabeled ligands bound to SHBG at each mg/kg, E4 demonstrated an oral bioavailability equal to the concentration of competitor (B) were expressed as a per bioavailability obtained with sc administration (80-100%). centage of the average amounts of Hlabeled ligands At the highest dose level tested, 5.0 mg/kg E4, absorption bound to SHBG in the absence of competitor (Bo), and were kinetics gave rise to an oral bioavailability approximating plotted against the concentration of competitor in each assay 30-60% of Sc administered E4. Interestingly, E4 demon tube. strated a relatively long half-life of 2-3 hours, enabling the 0.124. The results of the competitive binding assays are detection of bioactive levels of unconjugated E4 at all time depicted in FIG. 4. AS is clearly apparent from these points over a 24 hour interval. competitive binding assays, estetrol does not bind at all to Example 5 human SHBG when tested with either HDHT or H estradiol as labeled ligands. This is in marked contrast with 0121 An established competitive steroid-binding assay reference Steroids ethinylestradiol, 173-estradiol, testoster (Hammond and Lahteenmaki. 1983. Clin Chem Acta one and 5C-, which, in this order, Show 132:101-110) was used to determine the relative binding an increased relative binding affinity for human SHBG. affinity of estetrol (E4), 17O-ethinylestradiol (EE2), 17B Importantly, estetrol binding to SHBG was negligible when estradiol (E2), testosterone (T) and 5C.-dihydrotestosterone compared with the other estrogens tested, ethinylestradiol (DHT) for human sex Hormone Binding Globulin (SHBG). and 17(3-estradiol. US 2005/0070488 A1 Mar. 31, 2005 17

100

80

6O

40

10 100 1000 OOOC Steroid competitor (nM)

100 1000 OOOO Steroid competitor (nM) Figure 4: Competitive displacement of HODHT (panel A) and (H)estradiol (panel B) from the human sex hormone-binding globulin steroid binding site. The unlabeled steroid ligands used as competitors were as follows: estetrol (E4), 17a-ethinylestradiol (EE2), 17B-estradiol (E2), testosterone (T) and 5a dihydrotestosterone (DHT) US 2005/0070488 A1 Mar. 31, 2005

Example 6 mixtures of one or more of the aforementioned Substances 0.125 The present estrogenic components may suitably and/or precursors. be processed, together with additives, excipients and/or 19. The method according to claim 18, wherein the flavouring agents customary in galenic pharmacy, in accor Symptoms of hypoestrogenism are Selected from the group dance with the conventional methods into the usual forms of consisting of Osteoporosis, arteriosclerosis, climacteric administration. For oral administration, Suitable are, in par Symptoms, cognitive disturbances and Alzheimer's disease. ticular, tablets, dragees, capsules, pills, Suspensions, or Solu 20. The method according to claim 18, wherein R tions. represents a hydroxyl group or an alkoxy group. 21. The method according to claim 18, wherein at least 3 0.126 Estetrol tablets: 1,000 tablets of 185 mg, contain of the groups R, R, R and R represent hydrogen atoms. ing 1.5 mg estetrol and 0.15 mg levonorgestrel, are produced 22. The method according to claim 18, wherein the from the following formulation: method comprises the uninterrupted oral administration of the estrogenic component during a period of at least 10 dayS. 23. The method according to claim 22, wherein the Estetrol 1.500 g method comprises the uninterrupted oral administration, Levonorgestrel 0.150 g during a period of at least 10 days, of a combination of the Polyvinylpyrrolidone (Kollidon 25 (R) ex BASF) 13.500 g estrogenic component and a progestogenic component. Lactose 135.645 g Microcrystalline cellulose (Avicel PH 101 (E)) 26.250 g 24. The method according to claim 23, wherein the Glyceryl palmitostearate (Precirol (R) 2.775 g method comprises the uninterrupted oral administration of Anhydrous colloidal silica (Aerosil 200 (R) 1.000 g Crospovidone (Polyplasdone XL (R) 4.000 g the combination of the estrogenic component and the Coloring agent 0.180 g progestogenic component during a period of at least 28 dayS. 25. The method according to claim 23, wherein the method comprises an interval of at least 2 days during which 0127 Tablets that additionally contain 50 mg dehydroe no progestogenic component and no estrogenic component piandrosterone may be prepared from a Similar formulation. is administered and wherein the resulting decrease in Serum concentration of the progestogenic component and the estro 1-17. (Cancelled) genic component induces menses. 18. A method of hormone replacement in mammals, 26. The method according to claim 23, wherein the which method comprises oral administration of an estro method comprises the uninterrupted oral administration of genic component and a progestogenic component to a mam the estrogenic component during a period of at least 28 days mal in an effective amount to prevent or treat Symptoms of and wherein, following the combined administration of the hypoestrogenism, the estrogenic component being Selected estrogenic component and the progestogenic component, the from the group consisting of: Substances represented by the estrogenic component and no progestogenic component are following formula: administered during 3-18 consecutive days and the resulting decrease in Serum concentration of the progestogenic com ponent induces menses. 27. The method according to claim 18, wherein the method comprises the at least once daily oral administration of the estrogenic component and/or the progestogenic com ponent during a period of at least 10 dayS. 28. The method according to claim 18, wherein the estrogenic component is orally administered in an amount of less than 1 mg per kg of bodyweight per day. 29. The method according to claim 18, wherein the estrogenic component is orally administered in an amount of at least 1 lug per kg of bodyweight per day. R R4 30. The method according to claim 18, wherein the progestogenic component is administered in an amount which is equivalent to a daily oral dosage of 0.3 to 20 lig in which formula R, R2, R, R independently are a hydro levonorgestrel per kg of bodyweight. gen atom, a hydroxyl group or an alkoxy group with 1-5 31. A pharmaceutical kit comprising at least 20 oral carbon atoms; each of Rs, Re, R, is a hydroxyl group; and dosage units that contain the estrogenic component as no more than 3 of R, R2, R, R are hydrogen atoms, defined in claim 18 and/or a progestogenic component precursors capable of liberating a Substance according to and/or an androgenic component, wherein at least 10 units the aforementioned formula when used in the present contain between 0.01 and 20 mg of the estrogenic compo method, which precursors are derivatives of the Sub nent, at least 10 units contain the progestogenic component stances represented by the formula, wherein the hydro in an amount equivalent to 30-750 ug levonorgestrel and at gen atom of at least one of the hydroxyl groups in Said least 10 dosage units contain the androgenic component in formula has been Substituted by an acyl radical of a an amount equivalent to 5-250 mg dehydroepiandrosterone. hydrocarbon carboxylic, Sulfonic or Sulfamic acid of 32. The pharmaceutical kit according to claim 31, com 1-25 carbon atoms, tetrahydrofuranyl, tetrahydropyra prising at least 10 oral dosage units that contain between nal; or a Straight or branched chain glycosidic residue 0.01 and 20 mg of the estrogenic component, the progesto containing 1-20 glycosidic units per residue, and genic component in an amount equivalent to 30-750 ug US 2005/0070488 A1 Mar. 31, 2005 19 levonorgestrel and the androgenic component in an amount geStrinone, testosterone esters, precursors capable of liber equivalent to 5-250 mg dehydroepiandrosterone. ating these androgens when used in the present method and 33. The pharmaceutical kit according to claim 31, wherein mixtures thereof. the androgenic component is Selected from the group con sisting of dehydroepiandrosterone (DHEA), , k . . . .