EA046158B1 - COMPOUNDS AND HOW TO USE THEIR TO RELIEF SYMPTOMS ASSOCIATED WITH MENOPAUSE - Google Patents

Description

Technical field of the invention

The present invention relates to hormone replacement therapy, related compounds and related packaging units for the relief of symptoms associated with menopause, wherein such therapy is based on the administration to a female mammal of an esthetrol component in specified daily doses, optionally in combination with a progestogen component.

As detailed hereinafter, such therapy demonstrates statistically significant efficacy along with a favorable side effect profile compared with currently available treatments for alleviating symptoms associated with menopause.

Prerequisites for creating the invention

The term hormone replacement therapy (HRT) is used to describe either the use of free estrogen (for women who have had a hysterectomy) or combined estrogen-progestin therapy (for women who have retained the uterus). The goal of HRT is to relieve menopausal symptoms, primarily vasomotor symptoms (VMS) such as hot flashes. Other diseases and symptoms associated with perimenopause and menopause that respond to estrogen therapy include osteoporosis, vaginal atrophy, and sleep disorders (when associated with hot flashes).

VMS often occurs during the late menopausal transition and early postmenopause. VMS are the most significant complaints during menopause. It is estimated that approximately 75% of women over 50 years of age will suffer from hot flashes (Utian et al., 2005, Comparative controlled trial of a novel oral estrogen therapy, estradiol acetate, for relief of menopause symptoms, Menopause, 12(6): 708-715). Most experience hot flushes for about two years, although about 10% suffer for more than 10 years (Rodstrom et al., 2002, A longitudinal study of the treatment of hot flushes: the population study of women in Gothenburg during a quarter of a century, Menopause, 9(3):156-161). VMS can contribute to physical and psychosocial impairment with subsequent reduction in quality of life and represent one of the main reasons why women may seek medical attention during menopause (Santoro, 2008, Symptoms of menopause: hot flushes, Clin. Obstet. Gynecol. 51 (3):539-548).

The epithelial lining of the vagina and urethra is very sensitive to estrogen, and a lack of estrogen leads to thinning of both epithelia. This leads to vulvovaginal atrophy (VVA) and urinary complaints, which causes symptoms of vaginal dryness, itching, dyspareunia, dysuria, urinary frequency and an increased risk of recurrent urinary tract infections. In early 2014, the International Society for the Study of Women's Sexual Health (ISSWSH) and the North American Menopause Society (NAMS) approved a new terminology, genitourinary syndrome of menopause (GSM), to replace the VVA terminology. The rationale for using this new terminology is that the term VVA was too restrictive, whereas the term GSM was broader and included symptomatic WA as well as lower urinary tract symptoms associated with low estrogen levels (Portman et al., 2014, Genitourinary syndrome of menopause: new terminology for vulvovaginal atrophy from the International Society for the Study of Women's Sexual Health and the North American Menopause Society, Menopause, 21(10):1063-1068). It should be noted that GSM terminology has not yet been adopted in industry guidance issued by the FDA and EMA, and therefore VVA terminology will be used throughout this document.

Estrogen therapy remains the gold standard for reducing menopausal symptoms, particularly VMS. All routes of administration appear to be equally effective in reducing symptoms, but their metabolic effects differ. Estrogens must be administered continuously; past regimens that included estrogen administration from day 1 to day 25 of a calendar month are considered obsolete. It is common for women to experience VMS on days off, with no known benefit to stopping for a few days each month.

With current HRT, all women with a healthy uterus require a progestin, in addition to estrogen, to prevent endometrial hyperplasia and subsequent carcinoma, which occurs after only 6 months of estrogen therapy. The progestin may be administered continuously or sequentially (eg, 10 to 14 days every month or for 14 days every 3 months).

Estrogen therapy is the most effective treatment for VMS during menopause used in the United States and Europe. Following safety concerns described in the Women's Health Initiative's primary publications (Anderson et al., 2004, Effects of conjugated equine estrogen in postmenopausal women with hysterectomy: the Women's Health Initiative randomized controlled trial, JAMA, 291(14):1701-1712 ) and with continued requests from subjects for treatment, clinicians faced a challenge in determining the lowest effective dose of estrogen to relieve menopausal symptoms (Simon et al., 2007, Menopausal hormone therapy for vasomotor symptoms: balancing the risks and benefits with ultra -low doses of estrogen, Expert Opin. Investig. Drugs, 16(12):2005-2020). In addition, it is difficult to develop safer estrogen than those currently used.

In a 2016 publication, Coelingh Bennink et al. (Clinical effects of the fetal estrogen estetrol in a multiple-rising-dose study in postmenopausal women, Maturitas, 2016 Sep; 91:93-100) reported partial

- 1 046158 but a randomized multiple dose escalation study without placebo control in 49 postmenopausal women who received either 2 mg esthetrol or 2 mg estradiol valerate per day for 28 days (randomized portion of the study) followed by a dose increase to 10, 20 and 40 mg esthetrol per day (non-randomized part of the study). We compared the effectiveness of estradiol valerate (2 mg) and two doses of esthetrol alone (2 and 10 mg) in reducing hot flashes in subjects with >35 hot flashes per week at screening. The present inventors reported a reduction in the mean number of hot flashes and sweating as early as 2 mg esthetrol per day, while endometrial thickness was found to be stable in the 2 mg esthetrol group but increased in the 10 mg esthetrol group (briefly).

From this study, since the present inventors did not measure VMS in the 20 and 40 mg esthetrol groups, it will be apparent to one skilled in the art that the present inventors only considered the lower doses (2 and 10 mg) to be suitable for the treatment of VMS. In addition, the practitioner will recognize that a daily dose of 2 mg of esthetrol is as effective as a daily dose of 10 mg of esthetrol in reducing the number of hot flashes per day (see, for example, Fig. 3 in Coelingh Bennink et al., Maturitas, 2016). In addition, the publication reports to the expert that a daily dose of 2 mg does not change endometrial thickness, while a daily dose of 10 mg has a significant effect on endometrial thickness (see, for example, Fig. 2 in Coelingh Bennink et al., 2016 ). Thus, a specialist would be able to conclude that the minimum effective dose of esthetrol for the treatment of VMS is 2 mg per day.

Brief description of the invention

In the context of the foregoing, the present inventors have surprisingly discovered that the minimum effective dose of esthetrol for the relief of VMS in perimenopausal and postmenopausal women is approximately 15 mg daily.

The determination of the minimum effective dose does depend on a number of parameters that take into account not only the average number of VMS per day (as given in Coelingh Bennink et al. in Maturitas, 2016), but also the severity of VMS, as well as a weighted estimate per week, as further defined in this document.

Thus, from the point of view of effectiveness, it was surprisingly found that a daily dose of 15 mg was required to obtain a certain effect on VMS.

In addition, the present inventors have discovered that certain side effects of this treatment, expressed, for example, in the number of treatment-emergent adverse events (TEAEs), or the number of patients withdrawing from the study in each group, or the number of biopsies performed in Each group tends to reach a plateau at a dose of about 10 mg per day and does not worsen with further increases in dose. Surprisingly, it was found that some of these parameters actually returned to the previous values of 10 to 15 mg daily, thus indicating that adverse events may be more tolerable when the dose exceeds 10 mg daily.

Based on this unexpected finding and as a result of measurements taken at daily doses of 20 and 30 mg, the present inventors came to the surprising conclusion that the optimal daily dose of esthetrol for the relief of VMS in postmenopausal women is in the range of 15 to 25 mg. This dose range does provide the opportunity to observe statistically significant effects on VMS, as shown in the examples, while keeping treatment side effects within an acceptable range.

The following numbered paragraphs 1-14 describe embodiments of the present invention.

1. A composition for use in relieving symptoms associated with menopause, wherein said composition contains an esthetrol component and wherein said composition is administered in a daily amount equivalent to about 15 to about 25 mg of esthetrol.

2. A composition for use in the relief of vasomotor symptoms selected from hot flashes, bouts of sweating, night sweats, chills, increased sweating, palpitations and combinations thereof, wherein said composition contains an esthetrol component and wherein said composition is administered in a daily amount equivalent to from about 15 to about 25 mg esthetrol.

3. A composition for use in alleviating the emotional aspects of the menopausal transition selected from depression, irritability, mood swings, insomnia, sleep disturbances, anxiety, nervous tension and combinations thereof, wherein said composition contains an esthetrol component, and wherein said composition is administered in a daily amount, equivalent to about 15 to about 25 mg esthetrol.

4. A composition for use in alleviating physiological aspects of the menopausal transition selected from joint pain, loss of bone density, urinary tract infections, urinary incontinence, vaginal dryness, uterine prolapse, changes in skin texture, weight gain, dyspareunia, cardiovascular disease, diabetes and combinations thereof, where said composition contains esthete

- 2 046158 role component, and wherein said composition is administered in a daily amount equivalent to about 15 to about 25 mg of esthetrol.

5. A composition for use in reducing the frequency of VMS, severity of VMS, weighted assessment of weekly hot flashes, vaginal dryness, dyspareunia and combinations thereof, or for use in improving quality of life in accordance with the MRS and/or MENQOL questionnaires, where the said composition contains an esthetrol component and wherein said composition is administered in a daily amount equivalent to about 15 to about 25 mg of esthetrol.

6. A composition for use according to any of paragraphs 1 to 5 above, wherein the composition is administered daily for at least 1, at least 2, at least 4, at least 6, at least 12 weeks.

7. Composition for use according to any of paragraphs 1-6, wherein a second composition containing a progestogen component is additionally used.

8. Composition for use according to paragraph 7, where the specified progestogenic component is selected from drospirenone, progesterone and dydrogesterone.

9. Composition for use according to paragraph 7, where progesterone is administered in a daily dose of 50 to 200 mg.

10. A composition for use according to any of paragraphs 1 to 6, wherein a second composition containing bazedoxifene is additionally used.

11. A composition for use according to any one of claims 7 to 10, wherein said second composition containing a progestogen component or bazedoxifene is similar to the composition containing an esthetrol component.

12. A composition for use according to any of claims 1 to 11, wherein the esthetrol component is esthetrol, preferably esthetrol monohydrate.

13. A composition for use according to any of the preceding claims, wherein the composition is formulated as a dosage unit for oral administration.

14. A composition for use according to claim 13, wherein the oral dosage unit is formulated to correspond to the daily dosage unit.

The following numbered paragraphs 15-28 describe additional embodiments of the present invention.

15. A method of relieving symptoms associated with menopause, which comprises administering a composition containing an esthetrol component in a daily amount equivalent to about 15 to about 25 mg of esthetrol.

16. A method of relieving vasomotor symptoms selected from hot flashes, bouts of sweating, night sweats, chills, increased sweating, palpitations, and combinations thereof, which comprises administering a composition containing an esthetrol component in a daily amount equivalent to about 15 to about 25 mg esthetrol.

17. A method of alleviating the emotional aspects of the menopausal transition selected from depression, irritability, mood swings, insomnia, sleep disturbances, anxiety, nervous tension, and combinations thereof, which comprises administering a composition containing an esthetrol component in a daily amount equivalent to about 15 to about 25 mg esthetrol.

18. A method of alleviating the physiological aspects of the menopausal transition, selected from joint pain, loss of bone density, urinary tract infections, urinary incontinence, vaginal dryness, uterine prolapse, changes in skin texture, weight gain, dyspareunia, cardiovascular disease, diabetes and their combinations, which involves administering a composition containing an esthetrol component in a daily amount equivalent to about 15 to about 25 mg of esthetrol.

19. A method for reducing the frequency of VMS, the severity of VMS, a weighted assessment of weekly hot flashes, vaginal dryness, dyspareunia and combinations thereof, or improving the quality of life in accordance with the MRS and/or MENQOL questionnaires, which involves the administration of a composition containing an esthetrol component in a daily amount , equivalent to about 15 to about 25 mg esthetrol.

20. The method according to any of paragraphs 15-19 above, wherein the composition is administered daily for at least 1, at least 2, at least 4, at least 6, at least 12 weeks.

21. The method according to any of paragraphs 15-20, wherein a second composition containing a progestogenic component is additionally introduced.

22. The method according to paragraph 21, where the specified progestogenic component is selected from drospirenone, progesterone and dydrogesterone.

23. The method according to paragraph 22, where progesterone is administered in a daily dose of 50 to 200 mg.

24. The method according to any of paragraphs 15-20, where a second composition is additionally introduced, with

- 3 046158 holding bazedoxifene.

25. The method according to any one of paragraphs 21-24, wherein said second composition containing a progestogen component or bazedoxifene is similar to a composition containing an esthetrol component.

26. The method according to any of claims 15-25, wherein the esthetrol component is esthetrol, preferably esthetrol monohydrate.

27. A method according to any of the preceding claims, wherein the composition is formulated as a dosage unit for oral administration.

28. The method according to claim 27, wherein the oral dosage unit is formulated to correspond to the daily dosage unit.

The following numbered paragraphs 29-42 describe additional embodiments of the present invention.

29. The use of an effective amount of an esthetrol component in the manufacture of a composition for the relief of symptoms associated with menopause, wherein the esthetrol component is used in a daily amount equivalent to about 15 to about 25 mg of esthetrol.

30. The use of an effective amount of an esthetrol component in the manufacture of a composition for the relief of vasomotor symptoms selected from hot flashes, bouts of sweating, night sweats, chills, sweating, palpitations, and combinations thereof, wherein the esthetrol component is used in a daily amount equivalent to about 15 to approximately 25 mg esthetrol.

31. The use of an effective amount of an esthetrol component in the manufacture of a composition to alleviate the emotional aspects of the menopausal transition selected from depression, irritability, mood swings, insomnia, sleep disturbances, anxiety, nervous tension, and combinations thereof, wherein the esthetrol component is used in a daily amount equivalent to approximately 15 to approximately 25 mg esthetrol.

32. The use of an effective amount of an esthetrol component in the manufacture of a composition to alleviate physiological aspects of the menopausal transition selected from joint pain, loss of bone density, urinary tract infections, urinary incontinence, vaginal dryness, uterine prolapse, changes in skin texture, weight gain, dyspareunia, cardiovascular diseases, diabetes, and combinations thereof, wherein the esthetrol component is administered in a daily amount equivalent to about 15 to about 25 mg of esthetrol.

33. The use of an effective amount of an esthetrol component in the manufacture of a composition to reduce the frequency of VMS, the severity of VMS, a weighted assessment of weekly hot flashes, vaginal dryness, dyspareunia and combinations thereof, or improve quality of life in accordance with the MRS and/or MENQOL questionnaires where the esthetrol component is used in a daily amount equivalent to about 15 to about 25 mg of esthetrol.

34. Use according to any of paragraphs 29-33 above, wherein the composition is administered daily for at least 1, at least 2, at least 4, at least 6, at least 12 weeks.

35. Use according to any of paragraphs 29-34, wherein a second composition containing a progestogen component is additionally introduced.

36. Use according to paragraph 35, where the specified progestogenic component is selected from drospirenone, progesterone and dydrogesterone.

37. Application according to paragraph 35, where progesterone is administered in a daily dose of 50 to 200 mg.

38. Use according to any one of paragraphs 29-34, wherein a second composition containing bazedoxifene is additionally administered.

39. Use according to any one of paragraphs 35-38, wherein said second composition containing a progestogen component or bazedoxifene is similar to a composition containing an esthetrol component.

40. Use according to any one of claims 29 to 39, wherein the esthetrol component is esthetrol, preferably esthetrol monohydrate.

41. Use according to any of the preceding claims, wherein the composition is formulated as a dosage unit for oral administration.

42. Use according to paragraph 41, where the oral dosage unit is formulated to correspond to the daily dosage unit.

The following numbered paragraphs 43-50 describe additional embodiments of the present invention.

43. A packaging unit containing at least 14, preferably at least 21, even more preferably at least 28 containers for storing separately packaged and separately removable solid oral dosage forms with daily doses, where each container contains at least one a solid dosage form for oral administration with a daily dose containing from about 15 to about 25 mg of esthetrol.

- 4 046158

44. The packaging unit according to paragraph 43, where the packaging unit further contains at least 10, preferably 12, more preferably 14 additional containers for storing separately packaged and separately removable, preferably solid oral dosage forms with daily doses, where each additional container contains at least one, preferably solid, daily dosage oral dosage form containing a progestogen component.

45. Packaging unit according to paragraph 44, where each additional container for storing dosage forms containing a progestogen component is separately visually located next to the container containing a solid dosage form that contains esthetrol, in case these two solid dosage forms need to be administered on the same day .

46. Packaging unit according to paragraphs 44 or 45, where the specified progestogenic component is selected from drospirenone, progesterone and dydrogesterone.

47. The packaging unit of claim 46, wherein said progestogen component is progesterone, and wherein each of said additional containers contains at least one daily dose oral dosage form containing approximately 200 mg of progesterone.

48. The packaging unit according to paragraph 43, where the packaging unit further contains the same number of additional containers for storing separately packaged and separately removable, preferably solid dosage forms for oral administration with daily doses, where each additional container contains at least one, preferably solid dosage form a daily dose oral dosage form containing a progestogen component, where preferably said progestogen component is selected from drospirenone, progesterone and dydrogesterone, where even more preferably said progestogen component is progesterone and wherein said additional container contains at least one oral dosage form with a daily dose containing approximately 100 mg of progesterone.

49. The packaging unit according to paragraph 43, wherein at least 10, preferably 12, more preferably 14 of said solid dosage forms for oral administration with daily doses additionally contain a progestogen component.

50. The packaging unit according to paragraph 43, where each specified solid dosage form for oral administration with a daily dose additionally contains a progestogenic component.

One skilled in the art will appreciate that the embodiments described in numbered claims 44-50 can equivalently be presented as a kit of parts comprising a first packaging unit, for example a blister pack, containing oral daily dosage units containing an esthetrole component, and a second separate a packaging unit, for example a second separate blister pack, which contains daily oral dosage units containing a progestogen component.

One skilled in the art will also appreciate that, within the scope of the present invention, each packaging unit, such as a blister pack, may be numbered or otherwise marked.

Within the scope of the present invention, each packaging unit may be a sealed blister pack with a cardboard, paper, plastic foil backing and enclosed in a suitable box.

In a specific embodiment of the present invention, the packaging unit contains 28 containers or a multiple of 28 containers, such as 2 to 12.

Detailed Description of the Invention

Definitions

The term esthetrol component as used herein covers substances selected from the group consisting of esthetrol, esters of esthetrol, wherein the hydrogen atom of at least one of the hydroxyl groups has been replaced by an acyl radical of a hydrocarbon carboxylic acid, sulfonic acid or sulfamic acid from 1-25 carbon atoms; and their combinations. Even more preferably, the esthetrol component is esthetrol (including esthetrol hydrate). Most preferably, the esthetrol component contained in the dosage unit is esthetrol monohydrate.

The term estrol as used herein refers to 1,3,5(10)-estratriene-3,15alpha,16-alpha,17-beta-tetrol or 15-alpha-hydroxyestriol, as well as hydrates of estratriol, such as esthetrol monohydrate.

The term progestogenic component is defined as a substance that is capable of producing a progestogenic response in vivo, or a precursor that is capable of releasing such a substance in vivo. As a rule, progestogenic components are able to bind to the progestogen receptor.

In the context of the present invention, other compounds can be used, together with the esthetrol component, for administration to women who have a uterus. Expression selective modulators

- 5 046158 estrogen receptor (SERM) refers to a category of such compounds that are considered to be useful additions to the estrol component in the methods of the present invention. A preferred SERM for use in the context of the present invention is bazedoxifene.

It should be understood that in the methods and compositions further described herein, when a progestogen component is mentioned, such reference includes SERMs and, in particular, bazedoxifene.

The expression approximately, as used herein, refers to a measurable value such as a parameter, quantity, time duration, etc., and is intended to cover variations of ±10% or less, more preferably ±5% or less , even more preferably ±1% or less of the specified value as long as such changes are suitable for implementation in the disclosed invention. However, it should be understood that the meaning to which the modifier approximately refers is itself also specifically disclosed.

The term effective amount refers to the amount necessary to obtain a physiological effect. The physiological effect can be achieved with a single dose or repeated doses.

The term perimenopause as used herein (also in the context of a perimenopausal woman and a perimenopausal subject, etc.) refers to the period of life that begins approximately three to four years before menopause and ends one year after the last menstrual period. Perimenopause is characterized by persistently irregular menstrual cycles, extreme fluctuations in hormone levels, frequency of anovulation, and the occurrence of VMS (see, for example: Harlow et al., Menopause, Vol. 19, No. 4, 2012, Executive summary of the Stages of Reproductive Aging Workshop + 10: addressing the unfinished agenda of staging reproductive aging - see, in particular, Fig. 2 in the specified document). During this transition period, the focus of clinical care changes. Although women still need effective contraception during perimenopause, problems including loss of bone mineral density, menstrual cycle changes, and vasomotor instability also need to be addressed. A significant number of women also experience symptoms of depression, such as mood swings, irritability and poor concentration (Bosworth et al., Psychosom. Med., 2001, Jul-Aug; 63(4):603-8, Depressive symptoms, menopausal status, and climacteric symptoms in women at midlife).

The term postmenopause as used herein (also in the context of a postmenopausal woman and a postmenopausal subject, etc.) covers primarily women who are spontaneously postmenopausal, i.e. women who have experienced natural menopause, defined as the permanent cessation of the menstrual cycle, determined retrospectively after the woman has experienced 12 months of amenorrhea without any other apparent pathological or physiological cause. It occurs at an average age of 51.4 years in healthy women and reflects complete or near-complete depletion of ovarian follicles with resulting hypoestrogenemia (with estradiol levels often below 20 pg/ml) and high concentrations of follicle-stimulating hormone (FSH levels >40 IU /l). The term postmenopause also includes menopause as a consequence of premature ovarian failure, surgery (eg, oophorectomy), chemotherapy or radiation therapy for cancer, and certain diseases (eg, infections or hypothyroidism).

The term VMS as used herein (alternatively, in its non-abbreviated form, vasomotor symptom) corresponds to the thermoregulatory disturbances characteristic of menopause. VMS includes hot flashes (sometimes phrased as hot flushes), episodes of sweating such as night sweats, episodes of chills as well as excessive sweating, and episodes of heart palpitations. VMS are episodes of intense heat accompanied by sweating and redness, primarily in the head, neck, chest and upper back. As defined below, VMS is divided into mild, moderate and severe categories.

The term menopause-related symptoms as used herein is used to describe VMS as defined above, but also includes the emotional aspects of the menopausal transition (including, but not limited to, depression, irritability, mood swings, insomnia, sleep disturbances, anxiety and tension) and physiological aspects of the menopausal transition (including, but not limited to, joint pain, loss of bone density, urinary tract infections, urinary incontinence, vaginal dryness, uterine prolapse, changes in skin texture, weight gain and dyspareunia, as well as cardiovascular disease and diabetes).

As used herein, the term quality of life (and the abbreviation QoL) refers to a parameter that can be assessed, for example, by questionnaires such as, by way of example and without limitation, the Menopause Rating Scale (Heinemann et al., 2003, International versions of the Menopause Rating Scale (MRS), Health Qual Life Outcomes, 1:28; Heinemann et al., 2004, The Menopause Rating Scale (MRS) scale: A methodological review. Health Qual Life Outcomes, 2: 5; Heinemann et al., 2004, The Menopause Rating Scale (MRS) as outcome measure for hormone treatment?

- 6 046158 study. Health Qual Life Outcomes, 2:67; as described in more detail in Example 1, Section C below) or the MENQOL questionnaire (MENQOL), Hilditch et al.; Maturitas, 1996; A menopause-specific quality of life questionnaire: development and psychometric properties , 24(3); pp. 161-175).

As used herein, the term BMI (or body mass index in its unabridged form) refers to an index associated with a subject's weight and height, which is calculated by dividing the subject's weight in kilograms by the subject's height in meters squared. A BMI of 27.3 or greater classifies a female subject as overweight, while a BMI of 30 or greater classifies the subject as obese.

Determination of the minimum effective dose.

To determine the minimum effective dose (MED), the first selection criteria required that patients experience at least 7 VMS/day, moderate to severe, or at least 50 VMS/week, moderate to severe, during the week preceding randomization. Second, the placebo group was enrolled in a double-blind manner. Third, the total number of patients was such that statistical significance could be achieved between the individual study arms. Fourth, the absolute change in frequency of moderate to severe VMS per week compared with baseline was considered. Analysis of covariance (ANCOVA) was performed on the ^transformed change in moderate to severe VMS frequency per week from baseline. Finally, change in severity from baseline was assessed. To do this, subjects documented the VMS severity rating system as follows:

rating mild (1) regarding the sensation of heat without sweating;

rating moderate (2) for feeling hot with sweating/able to continue activities; and a rating of severe (3) for feeling hot with sweating/causing cessation of activity.

In addition, a severity score of zero was assigned to patients who experienced 100% attenuation of VMS during the specified week.

From these scores, baseline severity was calculated considering only moderate and severe VMS, such that the total number of moderate VMS within 7 days of the baseline week was multiplied by 2 and added to the total number of severe VMS within 7 days of the baseline week multiplied by 3. Then this was divided by the total number of moderate and severe VMS during the baseline week.

Severity in weeks 4 and 12 was calculated using the following formula for each of these weeks:

the number of mild VMS in 7 days, multiplied by 1;

number of moderate VMS in 7 days multiplied by 2;

number of severe VMS in 7 days multiplied by 3; and adding the three resulting numbers before dividing that sum by the total number of mild, moderate, and severe VMS over the 7 days of the week.

This method was used to obtain data further included in Example 1 below in section A, b) VMS severity.

In an alternative embodiment, VMS severity was assessed as follows: Baseline severity was also calculated considering only moderate and severe VMS, such that the total number of moderate VMS within 7 days of the baseline week was multiplied by 2 and added to the total number of severe VMS during 7 days from the baseline week, multiplied by 3. This was then divided by the total number of moderate and severe VMS during the baseline week.

However, severity in weeks 4 and 12 was calculated using the following formula for each of these weeks:

number of moderate VMS in 7 days multiplied by 2;

number of severe VMS in 7 days multiplied by 3; and adding the two resulting numbers before dividing that sum by the total number of moderate and severe VMS over the 7 days of the week.

In another embodiment, the severity score can be calculated according to the method described in Archer et al., 2014 (A randomized, double-blind, placebo-controlled study of the lowest effective dose of drospirenone with 17A-estradiol for moderate to severe vasomotor symptoms in postmenopausal women; Menopause, 2014; 21(3); p. 227-235). According to this method, the daily severity score is calculated using the following formula for each day for 7 days:

the number of moderate VMS in 1 day, multiplied by 2;

number of severe VMS in 1 day, multiplied by 3;

the two resulting numbers are added together;

the result of the addition is in turn divided by the total number of VMS during the same day (moderate and severe).

The same formula is applied for 7 subsequent days, the 7 resulting numbers are added together and divided by 7 to calculate the average daily severity for the week for VMS, from moderate

- 7 046158 heavy to heavy.

Additionally, the term weighted weekly hot flash score used in this document corresponds to a score that takes into account frequency and severity, which is calculated by using the severity score (as calculated above): [(1 x number of mild VMS) + (2 x number of moderate VMS ) + (3 x number of severe VMS)] over a period of 7 days.

Such weighted estimation was, for example, used in Notelovitz et al. (Initial 17e-Estradiol Dose for Treating Vasomotor Symptoms] Obstetrics and Gynaecology, Vol. 95(5), May 2000, p. 726-731).

This estimate was used to generate the data further included in Example 1 below in Section A, c) Weighted Weekly Tide Estimate.

In alternative embodiments, any combination of the methods described above can be used to assess the severity and/or frequency of VMS over a week.

Hormone replacement therapy.

The present therapy typically involves continuous administration of the esthetrol component over a period of at least 10 days, preferably at least 20 days.

The estetrol component is administered at a daily dose of about 15 to about 25 mg.

In a specific embodiment, the esthetrol component is administered at a daily dose of from about 15 to less than 20 mg.

In another specific embodiment, the esthetrol component is administered at a daily dose of greater than 20 mg to about 25 mg.

In one embodiment, the present therapy is administered to patients who have not undergone a hysterectomy. In a specific embodiment, the present therapy comprises daily administration of approximately 20 mg of the esthetrol component, preferably to patients who have not undergone a hysterectomy.

In cases where the present therapy is administered to a patient who has undergone a hysterectomy, the esthetrol component is preferably administered as the sole active ingredient.

When the present therapy is administered to non-hysterectomized patients, the esthetrol component may be administered as the sole active ingredient or may be administered together with an optional progestogen component. Said optional progestogen component may be administered continuously (i.e., every day in addition to the esthetrol component) or sequentially (wherein sequentially means administration of the progestogen component for, for example, 10-14 days every month or 14 days every 3 months) .

The terms continuous/continuous as used herein mean that the components are administered at relatively regular intervals without (therapeutically) significant interruptions. Naturally, minor interruptions may occur which do not affect the overall effectiveness of the present method, and in fact such deviations are covered by the present invention. In a preferred embodiment, and more arithmetically, an administration regimen is considered continuous if the longest interval between two subsequent administrations is no more than 3.5 times the average interval. Even more preferably, said longest interval is no more than 2.5 times, most preferably no more than 1.5 times the average interval.

In one embodiment, the optional progestogen component is administered through a route other than orally, for example, using an intrauterine device (IUD). In one embodiment, said IUD delivers the progestogen component levonorgestrel. In one such embodiment, the IUD is a Mirena® IUD or a Levosert® IUD.

In one embodiment, the present therapy includes oral, sublingual, buccal or sublabial administration of the esthetrol component. These last 3 modes of administration have the advantages that the esthetrol component does not have to pass through the digestive system and avoids first-pass exposure to the liver. In addition, these routes of administration provide a rapid onset of action.

As used herein, the term sublingual refers to the pharmacological route of administration by which the esthetrol component enters the bloodstream through the tissues under the tongue.

As used herein, the term buccal refers to the pharmacological route of administration by which the esthetrol component enters the bloodstream through the tissues of the buccal vestibule - the area inside the mouth between the buccal mucosa and the teeth/gums.

The term sublabial as used herein refers to the pharmacological route of administration by which the esthetrol component is placed between the lip and the gum.

In the method of the present invention, esthetrol and the progestogen component can be administered in separate dosage units. However, it is also possible and indeed very convenient to combine these two components into one dosage unit.

In the method of the present invention, the combination of progestogen and esthetrol components

- 8 046158 is advantageously administered continuously over a period of at least 10 days.

The present invention can be suitably practiced in the form of a variety of routes of administration that are known to one skilled in the art. Among these methods are methods that use single-phase preparations that contain dosage units with a constant amount of an esthetrol component and an optional progestogen component.

In an embodiment of the present invention where sequential administration of the progestogen component is chosen, it is also possible and convenient to combine the components into one dosage unit on days when two components are administered.

In one embodiment of the present invention, hormone replacement therapy is administered to a perimenopausal subject. In this embodiment, the subject will benefit from relief of VMS through administration of the composition of the present invention, while simultaneously benefiting from the contraceptive effect. In a particular perimenopausal population, contraception is indeed often required, and since VMS occurs during this period of life, the treatment of the present invention uniquely addresses these two needs in one simple treatment.

In another embodiment of the present invention, hormone replacement therapy is administered to a postmenopausal subject.

In a particular embodiment of the present invention, hormone replacement therapy is advantageously administered to obtain the benefit of no effect of the subject's smoking status on the therapy of the present invention. Indeed, it has long been known that smoking significantly reduces serum estrogen concentrations, as reported, for example, in an analysis of clinical studies by Bjarnason et al. (Bjarnason et al.; Climacteric 2012; Acute and long-term estradiol kinetics in smoking postmenopausal women; 15:5; p. 449-454), who found that in the estrogen group, smoking resulted in significantly lower serum estrone levels , and serum estradiol at all time points after randomization, while no differences were observed between smokers and nonsmokers in the placebo group. Bjarnason et al. concluded that smoking reduces serum estrogen levels both at baseline and after 2 hours in postmenopausal women treated with estrogen, and that the effect of smoking on estrogen concentration is fully expressed in women who smoke ten or fewer cigarettes daily and that the effect of smoking on the metabolism of estrogen therapy is constant and not dose dependent for standard smoking intensities.

In this particular embodiment, based on the unexpected discovery that the therapy of the present invention is not affected by the smoking status of the subject, hormone replacement therapy is preferably administered to a population of patients who smoke 5 or more cigarettes daily, a population of patients who smoke 10 or more cigarettes daily, or a population of patients who smoke 15 or more cigarettes daily.

In yet another embodiment of the present invention, hormone replacement therapy is advantageously administered to obtain the benefit of no BMI status effect with respect to the therapy of the present invention. Indeed, it has surprisingly been found that, unlike prior art hormone replacement therapy, the effectiveness of the therapy of the present invention is not affected by the BMI value of the subject. In this particular embodiment, the hormonal therapy of the present invention is preferably administered to a subject whose BMI is 25 or greater, 28 or greater, 30 or greater, 33 or greater, 35 or greater, 37 or greater, or 40 or greater. In this particular embodiment, the hormonal therapy of the present invention is preferably administered to an overweight or obese subject.

The hormone replacement therapy of the present invention has been found to be particularly useful in relieving symptoms associated with menopause while having an extremely limited effect on a large number of hepatic, hemostatic, endocrine and metabolic parameters. Indeed, the case of prior art HRT treatments had a negative impact on these parameters. Surprisingly and very usefully, it has been found that the treatment of the present invention, in contrast, does not change or only minimally changes most parameters.

Compositions.

The estetrole component of the present invention covers substances selected from the group consisting of esthetrol, esters of esthetrol, wherein the hydrogen atom of at least one of the hydroxyl groups has been replaced by an acyl radical of a hydrocarbon carboxylic acid, sulfonic acid or sulfamic acid, consisting of 1-25 carbon atoms ; and their combinations. More preferably, the esthetrol component is esthetrol (including esthetrol hydrate). Most preferably, the esthetrol component contained in the dosage unit is esthetrol monohydrate.

- 9 046158

The estetrol component of the present invention is used in a daily equivalent dose of about 15 to about 25 mg of estetrol monohydrate. In other words, if the esthetrol component is not itself esthetrol monohydrate, then the daily dose of the esthetrol component is adjusted to produce a therapeutic effect equivalent to a daily dose of about 15 to about 25 mg of esthetrol monohydrate.

In a particularly preferred embodiment of the present invention, the pharmaceutical composition of the present invention is intended for daily administration, i.e. it represents a dosage unit with a daily dose.

In the case of oral administration, the oral dosage unit according to the present invention is a solid or semi-solid dosage form such as tablets, capsules, wafers, pellets, pills, powders and granules. The term solid or semi-solid dosage form also includes capsules that contain a liquid, for example an oil, in which the esthetrol component and/or the optional progestogen component is dissolved or dispersed. Tablets and equivalent solid and semi-solid dosage forms may suitably contain materials such as binders (eg, hydroxypropyl methylcellulose, polyvinylpyrrolidone, other cellulosic materials and starch), diluents (eg, lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), agents to improve tablet disintegration (eg starch polymers and cellulosic materials) and lubricants (eg stearates and talc). These tablets and equivalent solid dosage forms can be prepared by wet granulation, for example using an aqueous solution or an organic solution, or by direct compression.

In the case of sublingual, buccal or sublabial administration, the pharmaceutical composition in accordance with the present invention is an oral dispersible dosage unit.

The term orally dispersible dosage unit as used herein refers to a dosage unit that is designed to disintegrate rapidly in the oral cavity upon contact with saliva and to disperse the esthetrole component in saliva so that it is absorbed through the oral mucosa.

In case the dosage unit is an oral dispersible dosage unit, the release rate of the esthetrol component from the dosage unit can be suitably determined using a disintegration test according to Ph. Eur. 2.9.1 (Disintegration of tablets and capsules) and USP <701> (Disintegration), for example using water as the disintegration medium. The oral dispersible solid dosage unit of the present invention, when subjected to the above disintegration test, typically disintegrates in less than 5 minutes, more preferably less than 2 minutes, even more preferably less than 1.5 minutes, even more preferably less than 1 min, even more preferably less than 45 s and most preferably less than 30 s.

If the patient still has a uterus, an additional progestogen component can be administered in addition to the esthetrol component.

Examples of progestogenic components that can be suitably used in accordance with the present invention include levonorgestrel, norgestimate, norethisterone, dydrogesterone, drospirenone, 3-beta-hydroxydesogestrel, 3-ketodesogestrel, 17-desacetylnorgestimate, 19-norprogesterone, acetoxypregnenolone, allylestrenol, amgestone , chlormadinone, cyproterone, demegestone, desogestrel, dienogest, dihydrogesterone, dimethisterone, ethisterone, ethynodiol diacetate, fluorogestone acetate, gastrinone, gestodene, gestrinone, hydroxymethylprogesterone, hydroxyprogesterone, linestrenol, mecyrogestone, medroxyprogesterone, megestrol, melegestrol , nomegestrol, norethindrone, norethinodrel, norgestrel ( including d-norgestrel and dl-norgestrel), norgestrienon, norethisterone, progesterone, quingestanol, (17-alpha)-17-hydroxy-11-methylene-19-norpregna-4, 15-dien-20-yl-3-one, tibolone, trimegestone, algestone acetophenide, nesidero, promegestone, 17-hydroxyprogesterone esters, 19-nor-17-hydroxyprogesterone, 17-alpha-ethynyltestosterone, 17-alpha-ethynyl-19-nortestosterone, d-17-beta-acetoxy- 13-beta-ethyl-17-alpha-ethynylgon-4-en-3-one oxime, 6-beta, 7-beta; 15-beta, 16-betadimethylene-3-oxo-17-pregna-4,9(11)-diene-21, 17-beta-carbolactone or tanproget and precursors of these compounds that are capable of releasing these progestogens in vivo when used in this way.

In addition, the progestogen component used in this method is selected from the group consisting of progesterone, drospirenone, dydrogesterone, precursors of these progestogens, and mixtures thereof.

In one embodiment, the present invention provides a combination composition containing an esthetrol component together with progesterone.

In one embodiment, the present invention provides a combination composition containing an esthetrol component together with drospirenone.

In one embodiment, the present invention provides a combination composition

- 10 046158 item containing the esthetrol component together with dydrogesterone.

When the progestogen component of the present invention is drospirenone, it is preferably used in a daily dose of 0.5 to 10 mg, even more preferably 1 to 4 mg.

When the progestogen component of the present invention is dydrogesterone, it is preferably administered at a daily dose of about 5 to about 10 mg, more preferably at a daily dose of about 5 mg.

In case the progestogenic component of the present invention is progesterone, it is preferably used in a daily dose of 50 to 200 mg. In one embodiment, progesterone is used at a daily dose of 50 to 100 mg if used continuously. In another embodiment, progesterone is administered at a daily dose of 100 to 200 mg when administered sequentially, for example, when administered over approximately 14 days each month.

When using another progestogenic component, the daily dose is adjusted so as to obtain the same pharmacological effect as with a dose of 50 to 200 mg of progesterone.

In a preferred embodiment of the present invention, the composition combines an esthetrol component and an optional progestogen component into one dosage unit, preferably a daily dosage unit. In a more preferred embodiment of the present invention, said daily dose combination dosage unit is an oral dosage combination daily dose unit.

In one embodiment, the present invention provides a combination oral dosage unit with a daily dose containing an esthetrol component and progesterone.

In one embodiment, the present invention provides a combination oral dosage unit with a daily dose containing an esthetrol component and drospirenone.

In one embodiment, the present invention provides a combination oral dosage unit with a daily dose containing an esthetrol component and dydrogesterone.

In a preferred embodiment of the present invention, a daily dose combination oral dosage unit is provided combining esthetrol at a daily dose of approximately 20 mg with progesterone at a daily dose of approximately 100 mg.

In another embodiment of the present invention, the estrol component is administered to a patient who still has a uterus in combination with a selective estrogen receptor modulator (SERM), particularly in combination with bazedoxifene. Preferably, bazedoxifene is administered at a daily dose of about 10 to 50 mg. More preferably, bazedoxifene is administered at a daily dose of approximately 20 mg.

In one embodiment, the present invention provides a combination therapy comprising an esthetrol component together with bazedoxifene.

In one embodiment, the present invention provides a combination oral dosage unit with a daily dose containing an esthetrole component and bazedoxifene.

The present invention has been described above with reference to a number of illustrative embodiments. Modifications and alternative embodiments of certain parts or elements are possible and are included within the scope of protection defined in the appended claims.

Examples

Example 1: Dose finding study to select the daily oral dose of esthetrol (E4) for the treatment of vasomotor symptoms in postmenopausal women.

Study inclusion and duration.

The inclusion lasted approximately 18 months. Individual subjects participated for no more than 27 weeks: no more than 6 weeks before screening and washout, no more than 4 weeks of screening and run-in period, no more than 91 days (13 weeks) of E4/placebo treatment followed by 2 weeks (14 days) of progestin therapy and a follow-up visit 1 week after completion of progestin therapy only in subjects who had not undergone hysterectomy.

The main goal of efficiency.

Determination of the minimum effective dose (MED) of oral E4 dosage by assessing changes in the frequency and severity of moderate to severe vasomotor symptoms (VMS).

Methodology.

This study was a prospective, multicenter, randomized, placebo-controlled, double-blind, dose-finding study.

Subject population.

Eligible subjects were women who had undergone hysterectomy and women who had not.

- 11 046158 hysterectomies, postmenopausal women aged 40 to 65 years inclusive, who experienced at least 7 hot flashes per day, moderate to severe, or at least 50 hot flashes per week, moderate to severe.

Diagnosis and inclusion criteria.

Subjects met all of the following inclusion criteria at the randomization visit. These criteria were assessed during the screening period:

1. Women aged 40 to 65 years, inclusive, who have at least 7 moderate to severe hot flashes per day or at least 50 moderate to severe hot flashes per week in the week prior to randomization.

2. Body mass index (BMI) from 18.0 to 35.0 kg/ ^m2 inclusive.

3. Postmenopausal status is defined as follicle-stimulating hormone (FSH) levels >40 IU/L, as well as:

amenorrhea for at least 12 subsequent months or amenorrhea for at least 6 months with estradiol (E2) < 20 pg/ml, or at least 6 weeks of postoperative bilateral oophorectomy with or without hysterectomy with copy of pathology report or discharge required on letterhead from the attending physician confirming the removal of both ovaries.

4. For women without hysterectomy: preserved uterus with bilayer endometrial thickness <5 mm on TVUS.

5. Negative pregnancy test.

6. Good physical and mental health, as judged by the Principal Investigator (PI) based on medical, surgical, and gynecologic history, physical examination, pelvic examination, clinical laboratory data, and vital signs.

7. Before admission to the study, the subject provided signed and dated written informed consent.

8. The subject is able to understand and comply with the protocol requirements, instructions and restrictions established by the protocol.

Exclusion criteria.

Potential study subjects were excluded if one of the following exclusion criteria was present at the randomization visit. These criteria were assessed during the screening period:

1. For women without a hysterectomy: uterine disease or condition, including:

a) thickness of the two-layer endometrium >5 mm according to TVUS;

b) the presence of fibroid(s) that complicates the assessment of the endometrium using TVUS;

c) history of uterine cancer or its presence;

d) presence of endometrial hyperplasia;

f) the presence of an endometrial polyp with hyperplastic or malignant epithelium.

2. Unspecified vaginal bleeding in the past 12 months.

3. Any history of malignancy, with the exception of basal cell (excluded if observed within the previous 2 years) or squamous cell carcinoma (excluded if observed within the previous year) skin carcinoma. Any clinically significant findings on breast examination and/or mammography suggestive of breast malignancy that would require additional clinical testing to rule out breast cancer (however, simple cysts confirmed by ultrasound were accepted). Note: A screening mammogram was required if the subject did not have written documentation of a mammogram performed within the past 9 months.

4. Pap smear from the cervix with evidence of pathology in subjects who have not undergone hysterectomy (written documentation of a previous test within 18 months or a test at a screening study), with evidence of cervical dysplasia higher than low-grade squamous intraepithelial lesion (LSIL) ). The study included women diagnosed with atypical squamous cells of undetermined significance (ASCUS).

5. Systolic blood pressure (BP) outside the range of 90 to 140 mmHg. Art., diastolic blood pressure outside the range from 60 to 90 mm Hg. Art. and/or heart rate outside the range of 40 to 100 bpm. Subjects with mild to moderate hypertension who were controlled by a stable antihypertensive regimen were included in the study if they met the inclusion/exclusion criteria.

6. Any clinically significant abnormalities detected on the 12-lead ECG.

7. History of venous or arterial thromboembolic disease (eg, deep vein thrombosis, pulmonary embolism, stroke, myocardial infarction, angina, etc.), known coagulopathy, or abnormal coagulation factors.

8. Diabetes mellitus with poor glycemic control in the past 6 months, defined by laboratory glucose levels outside the normal range and glycated hemoglobin above 7%.

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9. Dyslipoproteinemia predisposes to atherosclerotic cardiovascular disease (ASCVD). If a subject had an ASCVD score of <5% over 10 years, calculated using the ASCVD risk score (ACC/AHA Cardiovascular Risk Assessment Guide, 2013), they were not included in the study. In all cases, LDL cholesterol >190 mg/dL or plasma triglyceride level >400 mg/dL were exclusion criteria.

If the subject was receiving lipid-lowering therapy, it must have been treated at a stable dose for at least 1 month prior to screening and the same eligibility criteria must have been used.

10. Smoking >0 cigarettes/day or consuming >1 ml/day of nicotine-containing e-liquid.

11. History of gallbladder disease or the presence of this disease, if cholecystectomy was not performed.

12. Systemic lupus erythematosus.

13. Multiple sclerosis.

14. Acute or chronic liver disease.

15. Acute or chronic renal failure, including severe renal failure.

16. Uncontrolled thyroid disorders.

17. Subject has a history of major depressive episode or post-traumatic stress disorder (PTSD) for 2 years or a history of other major mental disorder at any time (eg, schizophrenia, bipolar disorder, etc.).

18. Use of drug(s) containing estrogen or progestin. An introductory period is required before the withdrawal period if used:

a) vaginal hormonal products (rings, creams, gels) - elimination for at least 4 weeks;

b) transdermal estrogen or estrogen/progestin - elimination of at least 4 weeks;

c) oral estrogen and/or progestin - elimination of at least 4 weeks;

d) intrauterine progestin therapy - withdrawal for at least 4 weeks.

Current users of progestin implants or those receiving estrogen-only injectable drug therapy were ineligible for participation unless treatment had been discontinued more than 3 months previously. Current users receiving estrogen pellet therapy or progestin injectable drug therapy were ineligible unless treatment had been discontinued more than 6 months previously.

19. Use of non-hormonal treatments to reduce hot flashes. For non-hormonal prescription and over-the-counter treatments for hot flashes (eg, the antidepressants paroxetine, escitalopram, venlafaxine, desvenlafaxine, and clonidine; or phytoestrogens, black sheep, etc.), a washout period of 1 week was required before the run-in period. If one of these treatments was taken concomitantly with an estrogen or progestin-containing drug, the washout periods could be combined and not necessarily performed sequentially.

20. Use of medications that may interfere with VMS endpoint performance within 28 days prior to the run-in period. This included (but was not limited to): SSRIs [selective serotonin reuptake inhibitors], SNRIs [serotonin norepinephrine reuptake inhibitors], dopaminergic or antidopaminergic drugs, or gabapentin.

21. History of allergy or allergy to the investigational product or drugs of this class, or history of allergy to a drug or other allergy that in the opinion of the investigator constituted a contraindication to the subject's participation.

22. History of allergies or intolerance to any component of the study product.

23. History of alcohol or substance abuse or dependence within 12 months as determined by the investigator, i.e. the subject drank excessive amounts of alcohol, abused drugs, or had a condition that could compromise the subject's ability to meet the requirements of the study in the opinion of the investigator.

24. Sponsor or Contract Research Organization (CRO) staff or investigator's department staff and relatives associated with the study.

25. Subjects with porphyria and subjects with a known or suspected clinically significant systemic disease, unstable medical condition, life-threatening illness, or current history of malignancy that, in the opinion of the investigator, may pose a risk to the subject.

26. Participation in another clinical trial of an investigational drug within 1 month (30 days) or receipt of an investigational drug within the past 3 months (90 days).

27. Declared by the researcher to be unsuitable for any reason.

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Number of subjects.

Principle regarding patients to be treated.

This principle states that the effect of a treatment policy can best be assessed on the basis of the subject's intention to treat (i.e., the planned treatment regimen) rather than the actual treatment prescribed. As a result, subjects assigned to a treatment group should be observed, assessed and analyzed as members of that group, regardless of their compliance with the planned course of treatment.

In addition, the intent-to-treat principle implies that the primary analysis should include all randomized subjects.

Maintaining the original randomization in the analysis is important to prevent bias and provide a reliable basis for statistical tests. In many clinical trials, use of the full battery of assays provides a conservative strategy. In many cases, it can also provide estimates of treatment effects that are more likely to reflect the effects observed in subsequent practice.

In the present study, the treatment population included a total of 257 patients.

Subjects were randomly assigned to one of 5 treatment groups in a 1:1:1:1:1 ratio. Randomization was stratified by center.

Research visits

A week Visit Notation used in all tables, enumerations and figures Part of the study Week from -7 to -4 Visit 1 (before screening) VI Screening Week from -4 to -3 Visit 1a (screening) Via Screening Week -1 Visit 2 (baseline) V2 Randomization Week 5 Visit 3 V3 Treatment period Week 13 Visit 4 (EOT) V4 End of treatment Week 16 Visit 5 (EOS) V5 End of the study

Test product and reference product, dose and route of administration

All treatments (esthetrol, hereinafter E4, capsule [2.5, 5, 10, 15 mg]) were administered once daily (QD) per os for at least 12 subsequent weeks until the last biological assessment was performed ( maximum 91 days).

Placebo, 1 capsule administered QD per os for at least 12 subsequent weeks until final biological assessments were performed (maximum 90 days).

If TVUS revealed an endometrial double layer >15 mm thick and/or a non-hysterectomized woman reported abnormal uterine bleeding (as determined by the gynecologist in terms of estrogen therapy), she underwent an endometrial biopsy and received progestin (10 mg). dydrogesterone) once daily until the end of week 11 in a sequential manner (i.e., a 14-day progestin treatment period followed by a 14-day progestin rest period), in addition to E4/placebo treatment. If the endometrial biopsy demonstrated endometrial hyperplasia, the subject's participation was immediately discontinued and treatment of the hyperplasia was carried out according to local guidelines. If abnormal uterine bleeding occurred again after the first normal endometrial biopsy, a thorough gynecological examination and TVUS were performed. If necessary, according to the decision of the gynecologist, a second endometrial biopsy was performed.

After the E4/placebo treatment period, all subjects without hysterectomy (including those previously treated with a progestin) received progestin therapy for 14 days with 10 mg dydrogesterone QD.

Results.

A. Vasomotor parameters for each of the 5 treatment groups.

a) VMS frequency.

i) Absolute change (average change from baseline) in VMS frequency over a week, ranging from moderate to severe.

a) Weekly for each group

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A week 2.5 mg 5 mg 10 mg 15 mg Placebo Average meaning SD (standard deviation) Its average value is not SD Its average value is not SD Its average value is not SD Its average value is not SD 0 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1 -17.28 19.71 -15.04 17.36 -13.37 19.59 -14.86 16.45 -16.65 16.24 2 -25.10 23.91 -21.68 19.26 -23.47 24.74 -29.19 20.25 -27.38 23.16 3 -32.46 26.15 -24.38 21.36 -31.34 25.21 -36.30 23.23 -30.16 23.09 4 -35.89 31.57 -27.57 22.47 -36.38 22.62 -41.43 21.60 -32.94 23.14 5 -37.16 33.27 -30.70 23.05 -39.07 21.55 -44.34 20.95 -34.70 23.01 6 -39.98 35.44 -38.02 22.13 -42.56 21.18 -46.58 19.83 -37.18 21.25 7 -42.44 36.96 -38.23 21.73 -43.30 22.18 -48.51 19.34 -38.55 21.85 8 -43.62 38.43 -38.55 23.54 -44.46 23.30 -48.35 19.75 -38.13 21.69 9 -45.19 36.91 -39.34 23.51 -45.19 24.22 -49.62 18.97 -39.25 22.74 10 -45.54 37.93 -39.83 23.79 -45.93 23.58 -48.88 20.04 -41.12 21.82 eleven -44.74 39.30 -41.46 23.59 -45.90 23.57 -49.92 19.42 -42.50 22.68 12 -45.04 38.91 -40.60 24.37 -47.21 22.87 -50.94 18.38 -42.97 22.31

To analyze the data recorded in this study, treatment groups were compared using ANCOVA (analysis of covariance) for change in frequency of moderate to severe VMS over the week from baseline to weeks 4 and 12. The ANCOVA model included treatment (trt1), and study center (SITEPOOL) as a fixed effect, and baseline (reference point) as a covariate.

b) Covariate value.

The table below provides a comparison for all treatment groups.___________

Time point of analysis (N) Covariate p value 4 trtl 0.0164 4 Starting point <0.0001 4 SITEPOOL 0.2214 12 trtl 0.0384 12 Starting point <0.0001 12 SITEPOOL 0.2706

When examined, the effect due to study center was considered to be not very important, and a second ANCOVA was performed excluding study sites.

No side effects

Time point of analysis (N) Covariate p value 4 trtl 0.0130 4 Starting point <0.0001 12 trtl 0.0254 12 Starting point <0.0001

c) The table below presents the mean change from baseline by week and treatment.

- 15 046158

Time point of analysis (N) Planned treatment (mg) Adjusted LS average Lower 95% confidence limit Upper 95% confidence limit 4 0 -33.7959277 -40.006133 -27.585722 4 2.5 -32.2601479 -38.742792 -25.777504 4 5 -27.4372730 -34.135693 -20.738853 4 10 -35.3672857 -41.716169 -29.018402 4 15 -43.5996056 -50.256441 -36.942770 12 0 -43.7344395 -50.150527 -37.318352 12 2.5 -40.1310220 -46.828579 -33.433465 12 5 -40.6802008 -47.600688 -33.759714 12 10 -45.8376026 -52.396965 -39.278240 12 15 -53.8273074 -60.704831 -46.949784

No side effects

Time point of analysis (N) Planned treatment (mg) Adjusted LS average Lower 95% confidence limit Upper 95% confidence limit 4 0 -33.6519764 -39.821576 -27.482377 4 2.5 -32.6798943 -39.056255 -26.303533 4 5 -27.8705668 -34.540176 -21.200958 4 10 -35.8741722 -42.156516 -29.591828 4 15 -44.3569892 -50.965323 -37.748656 12 0 -43.9304437 -50.292513 -37.568374 12 2.5 -40.6840423 -47.259323 -34.108761 12 5 -41.0122585 -47.889936 -34.134581 12 10 -46.5333871 -53.011718 -40.055056 12 15 -54.9073270 -61.721817 -48.092837

All statistical tests were supported by reporting the adjusted least squares LS mean (LS-adjusted mean: group means after controlling for a covariate; also called marginal means or estimated marginal means) and 95% confidence intervals for the respective treatment effects . These adjusted LS means and confidence intervals are based on the statistical models used for the analyses.

Confidence intervals imply that if the same population is sampled multiple times and interval estimates are performed on each occasion, the resulting intervals will bracket the true population parameter about 95% of the time.

d) The table below shows differences from placebo by week and treatment.___________

Time point of analysis (N) Higher dose (mg) Lower dose (mg) Adjusted average value LS Lower 95% confidence limit Upper 95% confidence limit p value 4 2.5 0 1.535780 -9.602669 12.674228 0.99183 4 5.0 0 6.358655 -4.958071 17.675380 0.44910 4 10.0 0 -1.571358 -12.573355 9.430639 0.99067 4 15.0 0 -9.803678 -21.033229 1.425873 0.10653 12 2.5 0 3.603418 -7.904295 15.111130 0.86103 12 5.0 0 3.054239 -8.637660 14.746138 0.92091 12 10.0 0 -2.103163 -13.469900 9.263574 0.97581 12 15.0 0 -10.092868 -21.694703 1.508967 0.10838

- 16 046158

No side effects

Analysis time t (N) Higher dose (mg) Lower dose (mg) Adjusted average LS value Lower 95% confidence limit Upper 95% confidence limit p value 4 2.5 0 0.972082 -10.164048 12.108212 0.99860 4 5.0 0 5.781410 -5.579628 17.142447 0.53888 Analysis time t (N) Higher dose (mg) Lower dose (mg) Adjusted average LS value Lower 95% confidence limit Upper 95% confidence limit p value 4 10.0 0 -2.222196 -13.242056 8.797664 0.96728 4 15.0 0 -10.705013 -21.979289 0.569264 0.06834 12 2.5 0 3.246401 -8.237137 14.729940 0.89864 12 5.0 0 2.918185 -8.797276 14.633647 0.93259 12 10.0 0 -2.602943 -13.966584 8.760697 0.94911 12 15.0 0 -10.976883 -22.602878 0.649111 0.07057

These tables show that a daily dose of 15 mg provided a nearly statistically significant difference from placebo at 4 weeks (p-value 0.10653 in the first statistical analysis and 0.06834 in the analysis without study site) and at 12 weeks (p-value 0 .10838 in the first statistical analysis and .07057 in the analysis without study center).

ii) Relative change (% from baseline) in VMS frequency per week, ranging from moderate to severe.

a) Weekly for each group.

A week 2.5 mg 5 mg 10 mg 15 mg Placebo Average value SD Its average value is not SD Average value SD Average value SD Average value SD 0 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 1 -23% 23% -23% 28% -21% 29% -22% 33% -26% 25% 2 -34% 27% -33% 29% -37% 37% -46% 34% -41% 35% 3 -44% 29% -38% 33% -48% 39% -57% 34% -45% 33% 4 -49% 33% -43% 34% -55% 35% -67% thirty% -49% 32% 5 -50% 33% -47% 34% -60% 33% -72% 28% -52% 32% 6 -54% 36% -57% 31% -65% 33% -76% 26% -56% thirty% 7 -57% 36% -57% thirty% -66% 34% -79% 25% -58% thirty% 8 -58% 38% -57% 32% -68% 35% -79% 26% -58% 31% 9 -61% 36% -59% 32% -69% 36% -81% 24% -60% 31% 10 -61% 37% -60% 32% -70% 35% -80% 27% -63% thirty% AND -60% 39% -63% 31% -70% 34% -82% 25% -65% 31% 12 -61% 38% -62% 32% -72% 33% -84% 23% -65% thirty%

This table shows that a daily dose of 15 mg resulted in a greater than 80% reduction in the frequency of moderate to severe VMS compared with baseline.

b) Covariate value

The table below shows comparisons across all treatment groups.

- 17 046158

c) The table below presents the mean relative change (%) from baseline by week and treatment.__________________________________________________________

Time point of analysis (N) Planned treatment (mg) Adjusted LS average Lower 95% confidence limit Upper 95% confidence limit 4 0 -0.49080693 -0.578953 -0.402661 4 2.5 -0.48482309 -0.576836 -0.392811 4 5 -0.42247695 -0.517552 -0.327402 4 10 -0.54456428 -0.634678 -0.454450 4 15 -0.64721795 -0.741703 -0.552733 12 0 -0.64237949 -0.726371 -0.558388 12 2.5 -0.60565210 -0.693328 -0.517976 12 5 -0.60259514 -0.693189 -0.512001 12 10 -0.70041629 -0.786283 -0.614549 12 15 -0.80089486 -0.890927 -0.710863

No side effects

Time point of analysis (N) Planned treatment (mg) Adjusted LS average Lower 95% confidence limit Upper 95% confidence limit 4 0 -0.48986685 -0.577115 -0.402619 4 2.5 -0.49532213 -0.585494 -0.405151 4 5 -0.43132298 -0.525642 -0.337004 4 10 -0.55503454 -0.643877 -0.466192 4 15 -0.66014529 -0.753597 -0.566693 12 0 -0.65107969 -0.735113 -0.567046 12 2.5 -0.62210640 -0.708956 -0.535256 12 5 -0.61436957 -0.705214 -0.523525 12 10 -0.71732171 -0.802891 -0.631752 12 15 -0.82338275 -0.913392 -0.733373

d) Table below with placebo by week and treatment.

Moment of time Higher dose Lower dose Adjusted LS average Lower 95% confidence limit Upper 95% confidence limit value

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a(N) analysis (mg) (mg) 4 2.5 0 0.005984 -0.152112 0.164079 0.99995 4 5.0 0 0.068330 -0.092296 0.228956 0.68478 4 10.0 0 -0.053757 -0.209916 0.102401 0.81738 4 15.0 0 -0.156411 -0.315800 0.002977 0.05622 12 2.5 0 0.036727 -0.113917 0.187372 0.93696 12 5.0 0 0.039784 -0.113271 0.192840 0.92218 12 10.0 0 -0.058037 -0.206836 0.090762 0.74506 12 15.0 0 -0.158515 -0.310392 -0.006639 0.03771

From this table it can be seen that a daily dose of 15 mg produces a statistically significant difference from placebo at 12 weeks (p-value 0.03771) and a nearly statistically significant difference from placebo at 4 weeks (p-value 0.05622).

It is particularly surprising to observe the low p-values obtained for the 15 mg dose compared with the elevated p-values obtained for the 10 mg dose.

No side effects

Time point of analysis (N) Higher dose (mg) Lower dose (mg) Adjusted average LS value Lower 95% confidence limit Upper 95% confidence limit p value 4 2.5 0 -0.005455 -0.162937 0.152027 0.99996 4 5.0 0 0.058544 -0.102119 0.219206 0.78717 4 10.0 0 -0.065168 -0.221006 0.090670 0.69797 4 15.0 0 -0.170278 -0.329714 -0.010843 0.03206 12 2.5 0 0.028973 -0.122708 0.180654 0.97301 12 5.0 0 0.036710 -0.118034 0.191454 0.94271 12 10.0 0 -0.066242 -0.216339 0.083855 0.65761 12 15.0 0 -0.172303 -0.325866 -0.018741 0.02210

This table shows that in a statistical analysis excluding local effects, a daily dose of 15 mg provided a statistically significant difference from placebo at 4 weeks (p=0.03206) and 12 weeks (p=0.02210).

It is particularly surprising to observe the low p-values obtained for the 15 mg dose compared with the elevated p-values obtained for the 10 mg dose.

Frequency changes across respondent groups in VMS frequency per week, ranging from moderate to severe

The incidence of VMS was also studied by grouping patients according to their degree of response.

The first group of patients who demonstrated a response of 50% or greater (relative change from baseline) was recruited. According to this analysis, at week 12, the 15 mg daily dose group contained 91.8% of respondents and the placebo group contained 65.5% of respondents. The difference between the two groups had a p-value less than 0.01, whereas the difference between the 10 mg daily dose group and the placebo group was not statistically significant (p-value >0.1).

A second group of patients who demonstrated a response of 75% or greater was prepared. According to this analysis, at week 12, the 15 mg daily dose group contained 77.6% of respondents and the placebo group contained 43.6% of respondents. The difference between the two groups had a p-value less than 0.001, whereas the difference between the 10 mg daily dose group and the placebo group was not statistically significant (p-value > 0.05).

b) VMS severity.

i) Absolute change (average change from baseline) in severity of VMS over a week, ranging from moderate to severe.

a) Weekly for each group.

2.5 mg 5 mg 10 mg 15 mg Placebo A week Average value Average value Average value Average value Average value 0 0 0 0 0 0

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1 -0.1542 -0.098 -0.2028 -0.072 -0.1638 2 -0.2112 -0.1199 -0.3007 -0.2853 -0.2746 3 -0.3246 -0.1574 -0.3983 -0.4842 -0.2676 4 -0.3373 -0.2341 -0.4842 -0.5897 -0.3327 5 -0.3984 -0.1683 -0.5107 -0.7432 -0.4146 6 -0.5014 -0.3212 -0.6086 -0.7035 -0.45 7 -0.4562 -0.2787 -0.643 -0.8112 -0.4645 8 -0.5359 -0.3239 -0.6644 -0.8854 -0.52 9 -0.5465 -0.3249 -0.6864 -0.9092 -0.5522 10 -0.6052 -0.3667 -0.709 -0.9628 -0.5514 eleven -0.5564 -0.4161 -0.666 -1.0123 -0.6551 12 -0.6279 -0.4007 -0.6941 -1.0425 -0.6604

To better analyze the data recorded in this study, treatment groups were compared using ANCOVA on the change in VMS severity, from moderate to severe, from baseline to mild, moderate to severe VMS at week 4 and week 12 for each active treatment compared to placebo. For women who experienced 100% attenuation of VMS at week 4 and/or week 12, a value of zero was assigned. The ANCOVA model included treatment (trt1) as a fixed effect and baseline (baseline) as a covariate.

b) Covariate value.

The table below shows comparisons across all treatment groups.

Time point of analysis (N) Covariate p value 4 trtl 0.0119 4 Starting point 0.3781 12 trtl 0.0032 12 Starting point 0.7990

c) The table below presents the mean change from baseline by week and treatment.

Time point of analysis (N) Planned treatment (mg) Adjusted LS average Lower 95% confidence limit Upper 95% confidence limit 4 0 -0.327801 -0.470747 -0.184854 4 2.5 -0.341092 -0.486518 -0.195666 4 5 -0.238552 -0.393041 -0.084063 4 10 -0.482536 -0.627769 -0.337303 4 15 -0.588755 -0.739764 -0.437746 12 0 -0.658280 -0.868694 -0.447865 12 2.5 -0.629592 -0.843656 -0.415528 12 5 -0.402567 -0.629971 -0.175163 12 10 -0.693404 -0.907183 -0.479624 12 15 -1.042087 -1.264369 -0.819805

d) The table below shows differences from placebo by week and treatment.

Time point of analysis (N) Higher dose (mg) Lower dose (mg) Adjusted average LS value Lower 95% confidence limit Upper 95% confidence limit p value 4 2.5 0 -0.013291 -0.268767 0.242185 0.9998 4 5.0 0 0.089249 -0.174507 0.353004 0.8253 4 10.0 0 -0.154735 -0.409222 0.099751 0.3767 4 15.0 0 -0.260954 -0.520749 -0.001159 0.0486 12 2.5 0 0.028687 -0.347368 0.404743 0.9992 12 5.0 0 0.255713 -0.132530 0.643955 0.3062 12 10.0 0 -0.035124 -0.409723 0.339475 0.9981 12 15.0 0 -0.383807 -0.766221 -0.001394 0.0489

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From this table it can be seen that a daily dose of 15 mg produces a statistically significant difference from placebo at 4 weeks (p-value 0.0486) and at 12 weeks (p-value 0.0489). In summary, the 15 mg dose significantly improved the severity of VMS at weeks 4 and 12 compared with placebo.

Regarding also the severity parameter, the difference between the 10 mg and 15 mg doses is impressive: this is expressed primarily in the mean changes from baseline presented in the table from section c) above, where, for example, after 12 weeks the adjusted mean LS value for The 10 mg was -0.69 (versus -0.66 found for the placebo group), while it was -1.04 for the 15 mg daily dose group. This clear difference was supported by an almost 8-fold improvement in p-value at 4 weeks when switching from 10 to 15 mg per day and a more than 20-fold improvement in p-value after 12 weeks when switching from 10 to 15 mg per day.

ii) Relative change (% from baseline) in severity of VMS over a week, ranging from moderate to severe.

a) Weekly for each group.

A week 2.5 mg 5 mg 10 mg 15 mg Placebo Average value Average value Average value Average value Average value 0 0% 0% 0% 0% 0% 1 -7% -4% -8% -3% -7% 2 -9% -5% -13% -12% -12% 3 -14% -7% -17% -20% -eleven% 4 -15% -10% -20% -24% -14% 5 -17% -7% -21% -31% -18% 6 -22% -14% -26% -thirty% -19% 7 -20% -12% -27% -34% -20% 8 -24% -14% -28% -37% -22% 9 -24% -14% -28% -38% -23% 10 -26% -15% -29% -40% -23% eleven -25% -18% -25% -42% -28% 12 -28% -17% -28% -44% -27%

This table shows that a daily dose of 15 mg resulted in a more than 40% reduction in severity

VMS, moderate to severe, compared with baseline.

b) Covariate value.

The table below provides a comparison for all groups. treatment.

Time point of analysis (N) Covariate p value 4 trtl 0.0126 4 Starting point 0.6594 12 trtl 0.0031 12 Starting point 0.1651

c) The table below presents the mean relative change from baseline by week and treatment.

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Time point of analysis (N) Planned treatment (mg) Adjusted LS average Lower 95% confidence limit Upper 95% confidence limit 4 0 -0.140987 -0.200215 -0.081759 4 2.5 -0.145345 -0.205600 -0.085090 4 5 -0.095556 -0.159566 -0.031546 4 10 -0.201564 -0.261739 -0.141388 4 15 -0.242707 -0.305276 -0.180139 12 0 -0.279744 -0.368327 -0.191161 12 2.5 -0.275038 -0.365158 -0.184919 12 5 -0.164881 -0.260616 -0.069145 12 10 -0.286159 -0.376158 -0.196159 12 15 -0.437476 -0.531055 -0.343897

d) The table below shows differences from placebo by week and treatment.

Moment of time and analysis of a(N) Higher dose (mg) Lower dose (mg) Adjusted LS average Lower 95% confidence limit Upper 95% confidence limit p value 4 2.5 0 -0.004358 -0.110211 0.101495 0.9999 4 5.0 0 0.045431 -0.063853 0.154714 0.7006 4 10.0 0 -0.060577 -0.166020 0.044866 0.4281 4 15.0 0 -0.101720 -0.209363 0.005922 0.0702 12 2.5 0 0.004705 -0.153612 0.163022 1.0000 12 5.0 0 0.114863 -0.048585 0.278310 0.2522 12 10.0 0 -0.006415 -0.164119 0.151289 0.9999 12 15.0 0 -0.157732 -0.318726 0.003261 0.0568

From this table it can be seen that a daily dose of 15 mg produces an almost statistically significant difference from placebo after 12 weeks (p-value 0.0568). A daily dose of 15 mg improved VMS severity at weeks 4 and 12 compared with placebo, whereas a daily dose of 10 mg here again was again no different from placebo, particularly at 12 weeks.

c) Weighted estimate of weekly tides.

i) Absolute change (average change from baseline) in the weighted score over a week.

a) Weekly for each group.___________________________________________

2.5 mg 5 mg 10 mg 15 mg Placebo A week Average value Average value Average value Average value Average value 0 2.4151 1.8297 2.3773 2,219 2.0545 1 -40.805 -33.7092 -31.4591 -33.1745 -38.203 2 -58.6163 -49.1319 -54.0849 -67.6983 -64.1272 3 -74.8798 -56.9697 -73.5189 -85.1915 -70.45 4 -82.75 -64.8344 -87.2673 -98.8329 -77.936 5 -84.6062 -71.6181 -93.4648 -105223 -83073 6 -89.9842 -88.2337 -101.646 -110.268 -89.6742 7 -94.2468 -88.2989 -103.024 -115.263 -92.9248 8 -97.1084 -89.2039 -104.835 -115.656 -91.6603 9 -101.429 -90.923 -107.578 -118.578 -94.9918 10 -100.18 -91.4798 -109.514 -116.853 -99.4484 eleven -97.6016 -95.8365 -109.532 -119.365 -102.771 12 -98.5424 -93.5117 -111.764 -121.777 -104.292

b) Covariate value.

The table below shows comparisons across all treatment groups.

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Time point of analysis (N) Covariate p value 4 trtl 0.0128 4 Starting point <0.0001 12 trtl 0.0107 12 Starting point <0.0001

c) The table below presents the mean change from baseline by week and treatment.

Time point of analysis (N) Planned treatment (mg) Adjusted LS average Lower 95% confidence limit Upper 95% confidence limit 4 0 -78.267781 -93.531339 -63.004223 4 2.5 -75.370737 -91.064629 -59.676845 4 5 -66.878234 -83.400001 -50.356467 4 10 -85.563378 -101.119727 -70.007030 4 15 -106.330686 -122.645517 -90.015855 12 0 -104.682568 -120.535991 -88.829145 12 2.5 -89.857250 -106.157637 -73.556864 12 5 -95.915504 -113.075759 -78.755249 12 10 -109.760136 -125.917663 -93.602609 12 15 -130.594733 -147.540056 -113.649411

d) Table below with placebo by week and treatment.

Time point of analysis (N) Higher dose (mg) Lower dose (mg) Adjusted average value LS Lower 95% confidence limit Upper 95% confidence limit p value 4 2.5 0 2.897044 -24.495634 30.289722 0.9970 4 5.0 0 11.389547 -16.739876 39.518971 0.7205 4 10.0 0 -7.295597 -34.556270 19.965075 0.9147 4 15.0 0 -28.062905 -55.994318 -0.131492 0.0485 12 2.5 0 14.825318 -13.625957 43.276593 0.5176 12 5.0 0 8.767064 -20.449428 37.983557 0.8780 12 10.0 0 -5.077568 -33.391737 23.236601 0.9785 12 15.0 0 -25.912165 -54.922995 3.098664 0.0951

ii) Relative change (% from baseline) in weighted score per week.

a) Weekly for each group.

2.5 mg 5 mg 10 mg 15 mg Placebo A week Average Average Average Average Average meaning meaning meaning meaning meaning 0 0% 0% 0% 0% 0% 1 -22% -22% -20% -19% -24% 2 -32% -32% -35% -43% -40% 3 -43% -37% -47% -56% -43% 4 -47% -43% -54% -66% -47% 5 -48% -46% -58% -70% -51% 6 -51% -56% -64% -74% -55% 7 -53% -56% -64% -78% -57% 8 -55% -56% -66% -78% -57% 9 -57% -58% -67% -80% -58% 10 -57% -59% -68% -79% -62% eleven -56% -61% -69% -80% -64% 12 -57% -60% -70% -82% -65%

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b) Covariate value.

The table below shows comparisons across all treatment groups.

Time point of analysis (N) Covariate p value 4 trtl 0.0108 4 Starting point 0.4181 12 trtl 0.0024 12 Starting point 0.0593

c) The table below presents the mean relative change from baseline by week and treatment.

Time point of analysis (N) Planned treatment (mg) Adjusted LS average Lower 95% confidence limit Upper 95% confidence limit 4 0 -0.474107 -0.562044 -0.386170 4 2.5 -0.473966 -0.564382 -0.383549 4 5 -0.426177 -0.521362 -0.330991 4 10 -0.541886 -0.631510 -0.452262 4 15 -0.650317 -0.744310 -0.556323 12 0 -0.644566 -0.728624 -0.560509 12 2.5 -0.579457 -0.665884 -0.493029 12 5 -0.599251 -0.690237 -0.508264 12 10 -0.699351 -0.785021 -0.613681 12 15 -0.812214 -0.902061 -0.722368

d) The table below shows differences from placebo by week and treatment.

Time point of analysis (N) Higher dose (mg) Lower dose (mg) Adjusted average LS value Lower 95% confidence limit Upper 95% confidence limit p value 4 Ί,5 0 -0.473966 -0.564382 -0.383549 1.0000 4 5.0 0 -0.426177 -0.521362 -0.330991 0.8833 4 10.0 0 -0.541886 -0.631510 -0.452262 0.6745 4 15.0 0 -0.650317 -0.744310 -0.556323 0.0267 12 2.5 0 -0.579457 -0.665884 -0.493029 0.6744 12 5.0 0 -0.599251 -0.690237 -0.508264 0.8871 12 10.0 0 -0.699351 -0.785021 -0.613681 0.7861 12 15.0 0 -0.812214 -0.902061 -0.722368 0.0276

From this table it can be seen that a daily dose of 15 mg produces a statistically significant difference from placebo at 4 weeks (p-value 0.0267) and at 12 weeks (p-value 0.0276).

It is particularly surprising to observe the low p-values obtained for the 15 mg dose compared with the elevated p-values obtained for the 10 mg dose.

B. Vasomotor parameters for the 10 and 15 mg groups compared with placebo and ineffective doses (2.5 and 5 mg), grouped together.

Based on the results obtained in section A above, it was apparent that the two lowest doses tested (2.5 mg per day and 5 mg per day) did not demonstrate effectiveness. Therefore, a further analysis of the results was prepared in which the data for these two doses were grouped with the placebo dose and compared with the 10 mg and 15 mg doses.

1. Relative change (% from baseline) in VMS frequency per week, moderate to severe

a) Weekly for each group.

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A week 10 mg 15 mg Placebo with 2.5 and 5 mg Average value SD Average value SD Average value SD 0 0% 0% 0% 0% 0% 0% 1 -21% 29% -22% 33% -24% 25% 2 -37% 37% -46% 34% -35% 31% 3 -48% 39% -57% 34% -42% 32% 4 -55% 35% -67% thirty% -47% 33% 5 -60% 33% -72% 28% -50% 33% 6 -65% 33% -76% 26% -56% 32% 7 -66% 34% -79% 25% -58% 32% 8 -68% 35% -79% 26% -58% 34% 9 -69% 36% -81% 24% -60% 33% 10 -70% 35% -80% 27% -61% 33% AND -70% 34% -82% 25% -63% 34% 12 -72% 33% -84% 23% -63% 33%

b) Covariant value.

Time point of analysis (N) Covariate p value 4 trtl 0.0039 4 Starting point 0.3935 4 SITEPOOL 0.3308 12 trtl 0.0017 12 Starting point 0.1244 12 SITEPOOL 0.0957

c) Pairwise comparisons with placebo (including ineffective doses of 2.5 and 5 mg).

Time point of analysis (N) 4 4 Planned treatment (mg) 0 10 Adjusted LS average -0.46779276 .0.54424142 Lower 95% confidence limit -0.521579 -0.634204 Upper 95% confidence limit -0.414007 -0.454279 4 15 -0.64752092 -0.741851 -0.553191 12 0 -0.61799510 -0.669162 -0.566828 12 10 -0.70075417 -0.786337 -0.615172 12 15 -0.80058459 -0.890322 -0.710847

d) Differences from placebo (including ineffective doses of 2.5 and 5 mg)

Time point of analysis (N) Higher dose (mg) Lower dose (mg) Adjusted average LS value Lower 95% confidence limit Upper 95% confidence limit p value 4 10.0 0 -0.076449 -0.193421 0.040523 0.26148 4 15.0 0 -0.179728 -0.301777 -0.057679 0.00218 12 10.0 0 -0.082759 -0.194036 0.028518 0.17979 12 15.0 0 -0.182589 -0.298696 -0.066483 0.00099

As mentioned above in section A), it is particularly striking to observe the low p-values obtained for the 15 mg daily dose compared to the elevated p-values obtained for the 10 mg daily dose, demonstrating the unique attenuation obtained with the 15 mg daily dose. mg.

2. Relative change (% from baseline) in the weighted hot flash score per week.

a) Weekly for each group.

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A week 10 mg 15 mg Placebo with 2.5 and 5 mg Average value SD Average value SD Average value SD 0 0% 0% 0% 0% 0% 0% 1 -20% 28% -22% 32% -24% 25% 2 -34% 36% -45% 32% -36% thirty% 3 -46% 39% -56% 32% -42% 31% 4 -53% 34% -66% 29% -46% 32% 5 -57% 32% -70% 27% -49% 33% 6 -62% 32% -72% 26% -54% 32% 7 -64% 32% -75% 25% -55% 31% 8 -66% 34% -76% 26% -56% 32% 9 -66% 34% -78% 25% -57% 31% 10 -68% 34% -77% 27% -59% 32% eleven -68% 33% -79% 26% -60% 33% 12 -69% 32% -81% 24% -60% 32%

b) Covariant value.

Time point of analysis (N) Covariate p value 4 trtl 0.0037 4 Starting point 0.5294 4 SITEPOOL 0.3783 12 trtl 0.0015 12 Starting point 0.0770 12 SITEPOOL 0.0487

c) Pairwise comparisons with placebo (including ineffective doses of 2.5 and 5 mg)

Time point of analysis (N) Planned treatment (mg) Adjusted LS average Lower 95% confidence limit Upper 95% confidence limit 4 0 -0.45362100 -0.506219 -0.401023 4 10 -0.51368261 -0.601866 -0.425499 4 15 -0.63242707 -0.724866 -0.539989 12 0 -0.59132448 -0.640861 -0.541788 12 10 -0.67757463 -0.760625 -0.594524 12 15 -0.76832743 -0.855386 -0.681269

d) Differences from placebo (including ineffective doses of 2.5 and 5 mg)

Time point of analysis (N) Higher dose (mg) Lower dose (mg) Adjusted average LS value Lower 95% confidence limit Upper 95% confidence limit p value 4 10.0 0 -0.060062 -0.174760 0.054636 0.41666 4 15.0 0 -0.178806 -0.298114 -0.059498 0.00178 12 10.0 0 -0.086250 -0.194272 0.021772 0.14044 12 15.0 0 -0.177003 -0.289367 -0.064639 0.00097

As mentioned above in Section A, this table shows that a daily dose of 15 mg produces a statistically significant difference from placebo at 4 weeks (p-value 0.00178) and at 12 weeks (p-value 0.00097).

In addition, it was particularly surprising to observe the low p-values obtained for the 15 mg dose compared with the increased p-values obtained for the 10 mg dose.

C. Menopause Rating Scale

The Menopause Rating Scale (MRS) is a health-related quality of life scale that measures the severity of age/menopause-related complaints by assessing the symptom profile (Heinemann et al., 2003, International versions of the Menopause Rating Scale (MRS) , Health Qual Life Outcomes, 1:28; Heinemann et al., 2004, The Menopause Rating Scale (MRS) scale: A methodological review. Health Qual Life Outcomes, 2:5; Heinemann et al., 2004, The Menopause Rating

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Scale (MRS) as outcome measure for hormone treatment? A validation study. Health Qual Life Outcomes, 2:67).

The score increased item by item with increasing severity of subjectively perceived complaints for each of the 11 items (severity was expressed from 0 to 4 points for each item). By checking these five possible severity boxes for each item on the questionnaire, the respondent was expressing his or her personal perception. The total MRS score ranged from 0 (asymptomatic) to 44 (highest complaint). Minimum/maximum scores varied across three dimensions, depending on the number of complaints assigned to the corresponding symptom dimension (Heinemann et al., 2003, Health Qual Life Outcomes, 1:28):

1) physiological symptoms: from 0 to 16 points (4 symptoms - depression, irritability, anxiety, exhaustion);

2) somato-vegetative symptoms: from 0 to 16 points (4 symptoms - sweating/flushing, complaints about the heart, sleep disturbances, complaints about pain in the joints and muscles);

3) urogenital symptoms: from 0 to 12 points (3 symptoms - sexual problems, complaints about urination, vaginal dryness).

Total MRS Score

A week 2.5 mg 5 mg 10 mg 15 mg Placebo Average value SD Average value SD Average value SD Average value SD Average value SD Baseline 16.5 7.2 16.5 7.3 17.6 7.6 16.4 8.1 18.2 8.9 Week 4 10.3 5.6 10.5 6.7 I,7 6.8 8.7 6.2 12.8 8.0 Week 12 9.5 6.7 and about 7.7 9.7 6.9 8.1 5.8 I,4 7.8

The Menopause Rating Scale (MRS) indicates an overall improvement in quality of life, with the strongest effect for the 15 mg dose. At this dose, a statistically significant effect was observed compared to placebo at week 4 with a p-value of 0.0113 and a near-statistically significant effect was observed at week 12 with a p-value of 0.0694.

D. Genitourinary symptoms (GSM)

Changes in the following GSM symptoms from baseline to week 12 (self-reported VVA) were recorded.

a) Vaginal dryness (feeling of dryness or burning in the vagina; absent = 0, mild = 1, moderate = 2 or severe = 3):

E4 dose (mg) Baseline (mean ± SD) Week 12 (mean ± SD) p-value compared to placebo 2.5 1 ± 0.90 0.5 ± 0.77 0.3345 5 1.3 ±0.94 0.7 ±0.86 0.1202 10 1 ± 0.93 0.5 ± 0.75 0.0798 15 1.1 ± 1.04 0.5 ± 0.68 0.0291* Placebo 1.3 ± 1.10 0.9 ± 1.02

*p<0.05 compared to placebo at week 12.

b) Irritation/itching of the vagina and/or vulva (feeling of abnormal irritation or tenderness in the vagina; none = 0, mild = 1, moderate = 2 or severe = 3): ________________________________________________________________________________

E4 dose (mg) Baseline (mean ± SD) Week 12 (mean ± SD) /^-value compared to placebo 2.5 0.7 ± 0.96 0.3 ± 0.60 0.1717 5 0.6 ± 0.90 0.4 ± 0.65 0.9618 10 0.7 ±0.87 0.3 ± 0.64 0.2487 15 0.5 ± 0.85 0.4 ± 0.70 0.931 Placebo 0.8 ± 0.92 0.5 ± 0.77

c) Dysuria (pain or difficulty urinating; absent = 0, mild = 1, moderate = 2 or severe = 3):

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E4 dose (mg) Baseline (mean ± SD) Week 12 (mean ± SD) /^-value compared to placebo 2.5 0.2 ±0.56 0 ± 0.19 0.2942 5 0.2 ±0.56 0±0.20 0.3488 10 0.2 ±0.58 0.1 ±0.23 0.3386 15 0.3 ±0.58 0.3 ± 0.61 0.643 Placebo 0.2 ± 0.60 0.3 ±0.55

d) Vaginal pain associated with sexual activity (no pain during intercourse = 0, mild = 1, moderate = 2 or severe = 3):

E4 dose (mg) Baseline (mean ± SD) Week 12 (mean ± SD) /7-value compared to placebo 2.5 0.6 ±0.77 0.3 ± 0.73 0.0763 5 1 ± 1.07 0.5 ± 0.78 0.0246* 10 0.6 ±0.77 0.2 ±0.36 0.0004** 15 0.7 ±0.90 0.3 ± 0.54 0.0006** Placebo 1 ± 1.14 0.7 ± 1.01

*p<0.05 versus placebo at week 12;

**p<0.001 compared to placebo at week 12.

f) Vaginal bleeding associated with sexual activity (blood loss during sexual intercourse; present = 1 vs. absent = 0):_________________________________

E4 dose (mg) Baseline Week 12 Presence (%) Absence (%) Presence (%) Absence (%) p-value compared to placebo 2.5 0 100 0 100 0.9958 5 6.4 91.5 ^# 0 100 0.903 10 0 100 0 100 0.9955 15 0 97.9 ^# 4.2 95.8 0.9308 Placebo 3.6 92.7 ^# 3.6 96.4

Some patients had no sexual activity.

The development of VVA symptoms indicates a general improvement, with the strongest effect occurring at a daily dose of 15 mg. For vaginal pain associated with sexual activity, significant differences from placebo were observed at doses of 5, 10 and 15 mg per day with p-values of 0.0246, 0.0004 and 0.0006, respectively. However, vaginal dryness, which is generally considered the most bothersome symptom, was significantly reduced only with the 15 mg daily dose, with a p-value of 0.0291.

E. Measurements Related to Treatment Side Effects.

1. Number of patients who underwent biopsy.

Treatment group Placebo 2.5 mg 5 mg 10 mg 15 mg Number of patients undergoing biopsy 4 4 5 AND 9

2. Adverse events (AE).

Treatment group Placebo 2.5 mg 5 mg 10 mg 15 mg Number of AEs that appeared during treatment (TEAE) 71 61 63 95 82 Percentage of patients with severe TEAE 9.1 7.7 10.6 7.4 6.1 Percentage of patients with TEAE that resulted in study discontinuation 3.6 1.9 6.4 5.6 4.1

From the above table, it can be seen that patients in the 15 mg group had less TEAE than patients in the 10 mg group. In the 10 mg group, patients who had AEs had an average of 3.2 AEs per patient. By comparison, in the 15 mg group, patients who had AEs had an average of 2.6 AEs. Generally,

- 28 046158 these data show that a daily dose of 15 mg provided significant attenuation of VMS without causing additional side effects in patients. In addition, fewer biopsies were required in the 15 mg/day group than in the 10 mg/day group.

This was confirmed by the following statistical analysis. Using a Poisson regression model with a random effect for patient and treatment group as a covariate to model TEAE counts across treatment groups, it can be shown that there is no statistical difference between treatment groups (p-value 0.099). Second, a chi-square test was used to assess whether the prevalence of patients reporting TEAE was similar in each treatment group. No statistical difference was found between treatment groups (p-value 0.575).

3. Patients leaving the study._________________________________

Treatment group Placebo 2.5 mg 5 mg 10 mg 15 mg Number of patients leaving the study 14 9 eleven 15 8

F. Measurements were carried out at daily doses of 15 and 20 mg.

To better evaluate the possibility of increasing the daily dose beyond the minimum effective dose of 15 mg per day, a number of parameters were monitored in a study in which esthetrol was administered at an increased dose of 20 mg per day.

1. Triglyceride levels, (mmol/l).

Treatment Parameter Baseline End of treatment* Exact value Exact change from baseline E4, 15 mg Average value 1.32 1.61 0.20 SD 0.64 0.94 0.82 Sample mean 1.21 1.40 0.15 E4, 20 mg Average value 1.43 1.53 0.10 SD 0.47 0.63 0.44 Sample mean 1.40 1.40 0.10

^The end of treatment occurred after 28 days for the 20 mg dose and after 12 weeks for the 15 mg dose.

2. Glucose level (mmol/l).

Treatment Parameter Baseline End of treatment* Exact value Exact change from baseline E4, 15 mg Average value 4.77 4.79 0.02 SD 0.37 0.58 0.56 Sample mean 4.72 4.77 0.05 E4, 20 mg Average value 5.53 5.44 -0.10 SD 0.54 0.50 0.22 Sample mean 5.60 5.60 -0.15

^The end of treatment occurred after 28 days for the 20 mg dose and after 12 weeks for the 15 mg dose.

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3. Cholesterol level (mmol/l).

Treatment Parameter Baseline End of treatment* Exact value Exact change from baseline E4, 15 mg Average value 5.51 5.63 0.12 SD 0.83 0.97 0.73 Sample mean 5.62 5.71 0.08 E4, 20 mg Average value 6.27 6.09 -0.18 SD 0.90 0.96 0.65 Average 6.25 6.10 -0.20 sample value

*End of treatment occurred after 28 days for the 20 mg dose and after 12 weeks for the 15 mg dose.

4. HDL-cholesterol level (mol/l). _______________________________________

Treatment Parameter Baseline End of treatment* Exact value Exact change from baseline E4, 15 mg Average value 1.73 1.89 0.16 SD 0.46 0.41 0.24 Sample mean 1.66 1.81 0.17 E4, 20 mg Average value 1.68 1.97 0.29 SD 0.37 0.36 0.14 Sample mean 1.60 1.90 0.35

*End of treatment occurred after 28 days for the 20 mg dose and after 12 weeks for the 15 mg dose.

5. LDL-cholesterol level (mmol/l).

Treatment E4, 15 mg Parameter Average value SD Average value Baseline 3.05 0.85 3.22 End of treatment* Exact value 3.14 0.95 3.25 Exact change from baseline 0.09 0.45 0.10 samples E4, 20 mg Average value 3.94 3.43 -0.51 SD 0.98 1.05 0.56 Sample mean 4.00 3.45 -0.65

*End of treatment occurred after 28 days for the 20 mg dose and after 12 weeks for the 15 mg dose.

From the above 5 tables, it can be noted that these lipid parameters and glucose levels were not significantly different when using a daily dose of 20 mg instead of a daily dose of 15 mg.

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6. C-terminal telopeptide (CTX-1) (ng/l).

Treatment Parameter Baseline End of treatment* Exact value Exact change from baseline E4, 15 mg Average value 416.0 320.9 -95.0 SD 286.16 359.65 442.18 Sample mean 339.0 197.5 -142.5 E4, 20 mg Average value 422.50 274.9 -147.60 SD 134.8 95.3 70.6 Sample mean 416.5 267.50 -138.0

*End of treatment occurred after 28 days for the 20 mg dose and after 12 weeks for the 15 mg dose.

CTX-1 is a specific marker of bone resorption.

The table above shows that a daily dose of 15 mg led to a slight decrease in bone resorption, and this effect was more pronounced with a daily dose of 20 mg after 28 days of treatment.

G. Measurements were carried out with daily doses of 15 and 30 mg.

The following treatments were assigned to healthy women (aged 15 to 50 years inclusive) according to the randomization code.

Placebo (n=16).

15 mg group: single oral dose of 15 mg esthetrol/3 mg drospirenone (n=10) followed, after 14 days of withdrawal, by multiple oral doses of 15 mg esthetrol/3 mg drospirenone (n=10) once daily for 14 days.

30 mg group: single oral dose of 30 mg esthetrol/3 mg drospirenone (n=10) followed, after 14 days of withdrawal, by multiple oral doses of 30 mg esthetrol/3 mg drospirenone (n=10) once daily for 14 days.

Adverse events were recorded from the time of first hospitalization until the end of the follow-up visit (from 37 to 42 days after the first day of treatment).___________________

Treatment-emergent AEs (TEAEs) depending on the study drug Placebo 15 mg 30 mg Total 75% 80% 70% Depends 31% 50% 50% Does not depend 75% 50% 50%

% = number of subjects reporting one or more AEs as a percentage of the total number of subjects in the corresponding treatment group.

Overall, single-dose and once-daily oral esthetrol/drospirenone for 14 days ranging from 15 mg esthetrol/3 mg drospirenone to 30 mg esthetrol/6 mg drospirenone were safe and well tolerated by healthy women in this study. With increasing single and multiple doses of esthetrol/drospirenone (the dose was doubled), there was no increase in the percentage of subjects reporting TEAE or the number of TEAE.

Thus, it is reasonable to consider hormone replacement therapy to relieve symptoms associated with menopause, with a daily dose of esthetrol ranging from 15 mg, the minimum effective dose, to 20 or even 25 mg, which will allow a better assessment of the benefit-risk ratio. Increasing the dose of esthetrol beyond the minimum effective dose of 15 mg per day will indeed provide even better effectiveness in attenuating VMS and in parameters such as bone resorption, while maintaining an excellent safety profile (including, without limitation, adverse events, as presented above in sections E), 2) and G), and in particular the lipid and glucose parameters presented in section F) 1) to 5) above. Increasing the dose of esthetrol beyond the minimum effective dose of 15 mg per day will also allow for faster attenuation when patients begin hormone replacement therapy.

Claims (12)

1. Use of the composition in the relief of symptoms associated with menopause selected from the group consisting of psychological symptoms associated with menopause selected from the group consisting of insomnia, sleep disturbance, anxiety, nervous tension and combinations thereof in the subject, or muscle pain and/or joints in subjects who do not have breast cancer, wherein said composition contains an esthetrol component and wherein said composition is administered in a daily amount equivalent to 15 to 25 mg of esthetrol. 2. Use according to claim 1, wherein the symptoms associated with menopause are muscle and/or joint pain in subjects who do not have breast cancer, and wherein the composition is administered in a daily amount equivalent to 20 to 25 mg of esthetrol. 3. Use according to any one of claims 1, 2, where the composition is administered in a daily amount equivalent to 20 mg of esthetrol. 4. Use according to any one of claims 1 to 3, wherein the composition is administered daily for at least 4 weeks, preferably at least 12 weeks. 5. Use according to any one of claims 1 to 4, wherein a second composition containing a progestogenic component is additionally used. 6. Use according to claim 5, wherein said progestogenic component is selected from drospirenone, progesterone and dydrogesterone. 7. Application according to claim 6, where progesterone is administered in a daily dose of 50 to 200 mg. 8. Use according to any one of claims 1 to 7, wherein a second composition containing bazedoxifene is additionally used. 9. Use according to claim 8, wherein said second composition contains a progestogen component or bazedoxifene. 10. Use according to any one of claims 1 to 9, wherein the esthetrol component is esthetrol, preferably esthetrol monohydrate. 11. Use according to any one of claims 1 to 10, wherein the composition is formulated as a dosage unit for oral administration. 12. Use according to claim 11, wherein the oral dosage unit is formulated to correspond to the daily dosage unit.