Estetrol for use in the prevention and treatment of alzheimer's disease

Abstract

The present invention relates to a composition comprising an estetrol component for use in the prevention and treatment of menopause-associated Alzheimer's disease symptoms. The composition described herein displays favorable properties when compared to existing estrogen-based compositions that aim to alleviate estrogen deficiency symptoms. Also described are related uses and methods of treatment comprising administration of the composition.

Description

[0001] ESTETROL FOR USE IN THE PREVENTION AND TREATMENT OF ALZHEIMER’S DISEASE

[0002] FIELD OF THE INVENTION

[0003] The present invention broadly relates to the field of medicine, and more particularly to hormone treatment of female subjects to prevent or treat cognitive diseases such as Alzheimer’s disease. Specifically, the invention relates to a composition comprising estetrol for use in treating or preventing Alzheimer’s disease.

[0004] BACKGROUND OF THE INVENTION

[0005] Alzheimer's disease (AD) is the main cause of dementia in the elderly population, and the 5thleading cause of mortality. In 2020, the disease affected about 44 million people worldwide and this prevalence is estimated to double by 2050 due to the rapid increase in average population age (Dumurgier and Sabia, Rev. Prat. 2020).

[0006] The cognitive symptoms of Alzheimer’s disease begin subtle and progress gradually over a period of many years. Typical early symptoms include deficits in episodic memory, such as an impaired recollection of recent experiences or recently received information. The microscopic hallmarks of Alzheimer’s disease are neurofibrillary tangles and amyloid plaques. Neurofibrillary tangles occur within the cell bodies of affected neurons in the cerebral hemisphere and brainstem, and consist largely of paired helical filaments, the latter containing as main constituent cytoskeletal protein Tau that has been excessively phosphorylated. Amyloid plaques are formed through the aggregation and accumulation of amyloid-beta, a peptide derived from the amyloid precursor protein, within extracellular spaces between nerve cell bodies (Henderson, J Steroid Biochem Mol Biol, 2014).

[0007] Recent studies directed to sex-specific mechanisms of Alzheimer’s disease have identified the female sex, and more particularly the menopause transition to be a risk factor for developing the disease. More specifically, translational imaging studies of the brain have demonstrated that perimenopausal and postmenopausal women exhibit decreased metabolic activity and increased brain amyloid-beta deposition as compared to women that are not perimenopausal. A similar observation could be made when comparing perimenopausal and post-menopausal women to age-matched men (Sheyer et al., J Prev Alzheimers Dis, 2018). Moreover, surgically-induced menopause was observed to influence cognitive decline and Alzheimer amyloid formation (Bove et al., Neurology, 2014).

[0008] Related to the above, retrospective studies have demonstrated the beneficial effect of certain menopausal hormone therapies in reducing the risk of neurodegenerative diseases such as Alzheimer’s disease (Kim et al., Alzheimers Dement (NY), 2021). Given the impact that Alzheimer’s disease imposes on society, there is an unmet need for therapeutic strategies to treat the disease, as well as preventive measures that can be taken to reduce the risk of disease development or progression. Further, there is a great interest in identifying specific subject groups that respond well to certain hypothesized Alzheimer therapies and / or preventive measures.

[0009] SUMMARY OF THE INVENTION

[0010] By means of extensive experimentation, the inventors have observed that estetrol components (such as estetrol, estetrol esters, and estetrol monohydrate) display favourable characteristics over other estrogens used in the art as active pharmaceutical ingredient in hormone replacement strategies. More particularly, the inventors have obtained results that indicate that estetrol is more potent in the prevention and therapy of Alzheimer’s disease than other estrogens. Moreover, multiple additional beneficial properties of estetrol over other estrogens are known such as its improved safety profile (low hepatic effects and hemostasis balance, suggesting a lower risk of thrombosis and a likely lower risk of developing breast cancer; Gerard, Celine, and Jean-Michel Foidart. “Estetrol: From Preclinical to Clinical Pharmacology and Advances in the Understanding of the Molecular Mechanism of Action.” Drugs in R&D vol. 23,2 (2023): 77-92. doi: 10.1007 / s40268-023-00419-5).

[0011] As is evident from the Example section, the beneficial effects of estetrol, in some cases surpassing those of other estrogens, in the context of Alzheimer’s disease include but are not limited to neuroprotection, protection against amyloid beta toxicity, reduction / modulation of Tau phosphorylation, reduced production of Reactive Oxygen Species (ROS) and promotion of neuronal bioenergetics.

[0012] The invention therefore provides in the following aspects:

[0013] Aspect 1. A composition for use in the prevention and / or treatment of Alzheimer’s disease symptoms, wherein said composition comprises from about 15 mg to about 25 mg of an estetrol component and wherein preferably said composition is administered once daily.

[0014] Aspect 2. Use of a composition for the manufacturing of a medicament for preventing and / or treating Alzheimer’s disease symptoms, wherein said composition comprises from about 15 mg to about 25 mg of an estetrol component and wherein preferably said composition is administered once daily.

[0015] Aspect 3. Use of a composition comprising an estetrol component for preventing and / or treating Alzheimer’s disease symptoms, wherein said composition comprises from about 15 mg to about 25 mg of an estetrol component and wherein preferably said composition is administered once daily.

[0016] Aspect 4. A method of treating Alzheimer’s disease symptoms in a subject, wherein said method comprises the step of administering once daily a composition comprising from about 15 mg to about 25 mg of an estetrol component. Aspect 5. The composition for use according to aspect 1, the use according to aspects 2 or 3, or the method according to aspect 4, wherein the estetrol component is estetrol, preferably estetrol monohydrate.

[0017] Aspect 6. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the subject has a genetic predisposition to develop Alzheimer’s disease.

[0018] Aspect 7. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the subject is a carrier of the APOEs4 allele, has a mutation in the APP gene, a mutation in the PSEN1 gene, a mutation in the PSEN2 gene, or any combination thereof.

[0019] Aspect 8. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the subject is a carrier of the APOEs4 allele.

[0020] Aspect 9. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the Alzheimer disease is menopause-associated Alzheimer’s disease, preferably wherein the Alzheimer's disease is early-stage Alzheimer's disease, preferably wherein the early Alzheimer’s disease is pre-clinical stage Alzheimer’s disease or earliest clinical stage of Alzheimer's disease.

[0021] Aspect 10. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the subject is a menopausal, perimenopausal, or post-menopausal subject.

[0022] Aspect 11. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the Alzheimer’s disease symptoms are selected from the group consisting of: impaired memory, verbal memory decline, depression, anxiety, anger, irritability, disturbed sleep patterns, insomnia, paranoia, problem solving difficulties, difficulties of executing complex tasks, impaired sound judgement, personality changes, reduced ability to organizing and expressing thoughts, or any combination thereof.

[0023] Verbal memory decline can encompass both subjective memory complaints (SMC) or mild cognitive impairment (MCI) and can be assessed using a list-learning verbal memory task and / or functional magnetic resonance imaging (fMRI) to assess the magnitude of activation of network functional connectivity (prefrontal and hippocampal regions), e.g. using the Wechsler Memory Scale-Revised (WMS-R) and / or fMRI activity level.

[0024] Insomnia or disturbed sleep patterns can be assessed using e.g., the (modified or simplified) Pittsburgh Sleep Quality Index (a self-report questionnaire that assesses sleep quality over a period of time) or by measuring the change from baseline to week 12 in sleep efficiency levels (i.e., ratio between the time a person spends asleep, and the total time dedicated to sleep reported as a percentage) using actigraphy.

[0025] Aspect 12. The composition for use, the use, or the method according to any one of the preceding aspects, wherein said composition reduces the average Alzheimer’s disease progression as measured by one or more Alzheimer’s disease rating scales by at least about 5%, preferably by at least about 10%, preferably by at least about 15%, preferably by at least about 20%, preferably by at least 30%, preferably by at least 40%, preferably by at least 50%, when compared to a population considered to have Alzheimer’s disease that has not received any treatment for Alzheimer’s disease.

[0026] Aspect 13. The composition for use, the use, or the method according to any one of the preceding aspects, wherein said composition has a neuroprotective effect against amyloid beta toxicity.

[0027] Aspect 14. The composition for use, the use, or the method according to any one of the preceding aspects, wherein said composition reduces the toxicity of amyloid beta by at least about 5%, preferably at least about 10%, preferably at least about 15%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably from about 50% to about 75% when compared to amyloid beta toxicity in absence of a neuroprotective agent.

[0028] Aspect 15. The composition for use, the use, or the method according to any one of the preceding aspects, wherein said composition reduces Reactive Oxygen Species (ROS) generation.

[0029] Aspect 16. The composition for use, the use, or the method according to any one of the preceding aspects, wherein said composition reduces the ROS generated by oxidative stress by at least about 5%, preferably at least about 10%, preferably at least about 15%, preferably at least about 20%, preferably at least about 30% when compared to ROS generation in absence of an estetrol component.

[0030] Aspect 17. The composition for use, the use, or the method according to any one of the preceding aspects, wherein said composition reduces the degree of Tau protein phosphorylation, preferably wherein said composition reduces the relative abundance of phosphorylated Tau protein (pTau).

[0031] Aspect 18. The composition for use, the use, or the method according to any one of the preceding aspects, wherein said composition reduces the absolute amount of pTau in a subject tissue or subject sample by at least about 1%, preferably by at least about 2.5%, preferably by at least about 5%, preferably by at least about 10%.

[0032] Aspect 19. The composition for use, the use, or the method according to any one of the preceding aspects, wherein said composition reduces the relative abundance of pTau in a subject tissue or subject sample by at least about 1%, preferably by at least about 2.5%, preferably by at least about 5%, preferably by at least about 10%.

[0033] Aspect 20. The composition for use, the use, or the method according to any one of the preceding aspects, wherein said composition reduces the formation of one or more phosphorylated Tau protein (pTau) selected from the group comprising of: pTaul81, pTau202, pTau214, pTau262, pTau396.

[0034] Aspect 21. The composition for use, the use, or the method according to any one of the preceding aspects, wherein said composition improves cell viability upon amyloid beta lesion damage. Aspect 22. The composition for use, the use, or the method according to any one of the preceding aspects, wherein said composition improves cell viability upon amyloid beta lesion damage when compared to cell viability upon amyloid beta lesion damage in presence of an estrogen that is not an estetrol component, preferably wherein the estrogen is estradiol.

[0035] Aspect 23. The composition for use, the use, or the method according to any one of the preceding aspects, wherein said composition reduces lactate dehydrogenase (LDH) release upon amyloid beta lesion damage.

[0036] Aspect 24. The composition for use, the use, or the method according to any one of the preceding aspects, wherein said composition reduces LDH release when compared to LDH release upon amyloid beta lesion damage in presence of an estrogen that is not an estetrol component, preferably wherein the estrogen is estradiol.

[0037] Aspect 25. The composition for use, the use, or the method according to any one of the preceding aspects, wherein said composition increases the mitochondrial membrane potential by at least about 10%, preferably by at least about 15%, more preferably by at least about 20%.

[0038] Aspect 26. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the composition is formulated as an oral, sublingual, buccal, or sublabial dosage unit, preferably wherein the composition is formulated as an oral dosage unit.

[0039] Aspect 27. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the subject is a female menopausal, perimenopausal, or post-menopausal subject.

[0040] Aspect 28. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the estetrol component is estetrol or an ester thereof.

[0041] Aspect 29. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the composition comprises from about 15 mg to 25 mg of an estetrol component and wherein said composition is administered once daily, preferably wherein the composition comprises from about 15 mg to about 25 mg of an estetrol component and wherein said composition is administered once daily.

[0042] Aspect 30. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the composition comprises from about 15 mg to 20 of an estetrol component and said composition is administered once daily, preferably wherein the composition comprises from about 15 mg to about 20 mg of said estetrol component and wherein said composition is administered once daily.

[0043] Aspect 31. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the composition comprises about 15 mg of an estetrol component and wherein said composition is administered once daily, preferably wherein the composition comprises about 15 mg of an estetrol component, and wherein said composition is administered once daily.

[0044] Aspect 32. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the composition comprises about 20 mg of an estetrol component and wherein said composition is administered once daily, preferably wherein the composition comprises about 20 mg of an estetrol component and wherein said composition is administered once daily.

[0045] Aspect 33. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the composition further comprises a progestogenic component.

[0046] Aspect 34. The composition for use, the use, or the method according to aspect 33, wherein the progestogenic component is selected from the group consisting of: progesterone, drospirenone, norethisterone, norethisterone-acetate (NETA), dydrogesterone, levonorgestrel (LNG), etonogestrel, norgestrel, nomegestrol, nomegestrol-acetate (NOMAC), trimegestone, nestorone, dydrogesterone, gestodene, desogestrel, norgestimate, cyproterone acetate, dienogest, and chlormadinone.

[0047] Aspect 35. The composition for use, the use, or the method according to aspect 33 or 34, wherein said progestogenic component is selected from the group comprising drospirenone, progesterone, or dydrogesterone.

[0048] Aspect 36. The composition for use, the use, or the method according to any one of aspects 33 to 35, wherein the progestogenic component is drospirenone, preferably of from about 0.25 mg to about 10 mg of drospirenone, more preferably from about 1 mg to about 4 mg of drospirenone, more preferably about 3 mg of drospirenone, or wherein said progestogenic component is administered in an amount equivalent to from about 0.25 mg to about 10 mg of drospirenone, preferably in an amount equivalent to from about 1 mg to about 4 mg of drospirenone, more preferably in an amount equivalent to about 3 mg drospirenone.

[0049] Aspect 37. The composition for use, the use, or the method according to any one of aspects 33 to 36, wherein the progestogenic component is progesterone, preferably from about 25 mg to about 300 mg of progesterone, more preferably about 100 mg to about 200 mg of progesterone, or wherein said progestogenic component is administered in an amount equivalent to from about 25 mg to about 300 mg of progesterone, preferably in an amount equivalent to from about 100 mg to about 200 mg.

[0050] Aspect 38. The composition for use, the use, or the method according to any one of aspects 33 to 37, wherein the progestogenic component is dydrogesterone, preferably of from about 1 mg to about 20 mg dydrogesterone, more preferably from about 5 mg to about 10 mg dydrogesterone, or wherein said progestogenic component is administered in an amount equivalent to from about 1 mg to about 20 mg dydrogesterone, preferably in an amount equivalent to from about 5 mg to about 10 mg dydrogesterone. Aspect 39. The composition for use, the use, or the method according to any one of aspects 1 to 32, wherein the composition further comprises bazedoxifene.

[0051] Aspect 40. The composition for use, the use, or the method according to any one of aspects 1 to 32, wherein the estetrol component is the single (i.e. only) pharmaceutically active ingredient in the composition.

[0052] Aspect 41. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the subject has a BMI of 30 or higher, more preferably of 35 or higher, wherein the subject is smoking, or wherein the subject is hepatically impaired.

[0053] Aspect 42. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the composition is formulated to correspond to a daily dosage unit.

[0054] Aspect 43. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the composition is formulated to correspond to an oral dosage unit, preferably an oral dosage unit that is specifically formulated for oral ingestion.

[0055] Aspect 44. The composition for use, the use, or the method according to any one of the preceding aspects, wherein the composition is used in a once-a-day multiple dose regimen.

[0056] Aspect 45. In any one of the aspects defined herein, said dosage unit or composition may be presented as a kit-of-parts containing a packaging unit, e.g. a blister pack, containing the daily oral dosage units comprising the estetrol component. The skilled person will additionally know that, within the scope of the present invention, each packaging unit, e.g. blister pack, may be numbered or otherwise marked.

[0057] Within the scope of the invention, each such packaging unit may be a sealed blister pack with a cardboard, paperboard, foil plastic backing and enclosed in a suitable cover.

[0058] Also envisaged in any one of the aspects of such packaging units as defined herein are bottles, such as glass bottles, more particularly brown glass bottles. The material of the bottle is not particularly limiting. In preferred embodiments, the bottle is a glass bottle characterized by a color capable of reducing or preventing degradation of the contents of the bottle by e.g. UV light while maintaining a degree of transparency that allows for visual inspection of the contents of said bottle. Suitable colors include without limitation amber, cobalt, or vintage green.

[0059] In a particular embodiment of the kit-of-parts according to aspect 45, the packaging unit comprises 28 containers or a multitude of 28 containers, such as 2 to 12 times 28 containers.

[0060] The above and further aspects and preferred embodiments of the invention are described in the following sections and in the appended claims. The subject matter of the appended claims is hereby specifically incorporated in this specification. BRIEF DESCRIPTION OF THE FIGURES

[0061] Figure 1. Effect of Test Item (TI) Estetrol (E4) and Reference Ttem (RI) Estradiol (E2) on primary cortical neurons in growth factor withdrawal assay. Cells were treated with TI Estetrol (E4) at 5 concentrations or RI Estradiol (E2) at 1 concentration (10 nM) and lesioned by removal of B-27 supplement (growth factor) for 28 h. (A) Cell viability was assessed using MTT assay, (B) cell toxicity was determined using LDH assay. Data are shown as % of vehicle control (%VC) and presented as mean + standard error of mean (SEM); n=6. Group differences were evaluated by One-way ANOVA followed by Dunnett's multiple comparisons test versus the lesion control (LC); *p<0.05; **p<0.01; ***p<0.001.

[0062] Figure 2. Effect of Test Item (TI) Estetrol (E4) and Reference Item (RI) Estradiol (E2) on Api-42 toxicity in SH-SY5Y cells. Cells were treated with either (A) TI Estetrol (E4) at 5 concentrations or (B) RI Estradiol (E2) at 3 concentrations and simultaneously lesioned with 5 pM Api-42 for 48 h. Cell viability was assessed using MTT assay. Data are shown as % of vehicle control (%VC) and presented as mean + standard error of mean (SEM); n=6. Group differences were evaluated by One-way ANOVA followed by Dunnett's multiple comparisons test versus the lesion control (LC); *p<0.05; **p<0.01; ***p<0.001.

[0063] Figure 3. Effect of Test Item (TI) Estetrol (E4) and Reference Item (RI) Estradiol (E2) on A 1-42 toxicity in primary hippocampal neurons. Cells were treated with TI Estetrol (E4) at 5 concentrations or RI Estradiol (E2) at 1 concentration (10 nM) and lesioned with 10 pM Api-42 for 6 days. (A) Cell viability was assessed using MTT assay, (B) cell toxicity was determined using LDH assay. Data are shown as % of vehicle control (%VC) and presented as mean + standard error of mean (SEM); n=6. Group differences were evaluated by One-way ANOVA followed by Dunnett's multiple comparisons test versus the lesion control (LC); *p<0.05; **p<0.01; ***p<0.001.

[0064] Figure 4. Effect of Test Item (TI) Estetrol (E4) and Reference Item (RI) Estradiol (E2) on reactive oxygen species (ROS) using DCFDA assay in H2O2 treated SH-SY5Y cells. Cells were treated with TI Estetrol (E4) at 5 concentrations or RI Estradiol (E2) at 1 concentration (10 nM) and treated with H2O2 for 3 h Data are given as (A) DCFDA assay fluorescent signal (RFU) or (B) percent of the lesion control (%LC) and displayed as bar graphs with group means +SEM (n=6 per group). Statistics: Oneway ANOVA with Dunnett's multiple comparisons test (post hoc test) compared to lesion control (LC) *p<0.05; **p<0.01; ***p<0.001.

[0065] Figure 5. Effect of Test Item (TI) Estetrol (E4) and Reference Items (RIs) 1 Estetrol (E2) and 2 CHIR on levels of total Tau and six phosphorylated tau sites (pTaul81, pTau202, pTau214, pTau231, pTau262 and pTau396) in cell extracts of SH-SY5Y-hTau441(P301L) cells. Cells were treated with TI Estetrol (E4) at 5 concentrations, RI.l Estradiol (E2) at 1 concentration (10 nM) or RI.2 CHIR at 1 concentration (2.5 pM) for 24 h. Data are shown as pg / pg total protein for total Tau (A) or arbitrary units (AU) for all phospho sites (B-G) and displayed as bar graphs with group means +SEM (n=4-6 per group). Statistics: One-way ANOVA with Dunnett's multiple comparisons test (post hoc test) compared to vehicle control (VC) *p<0.05; **p<0.01; ***p<0.001.

[0066] Figure 6. Effect of Test Item (TI) Estetrol (E4) and Reference Items (RIs) 1 Estetrol (E2) and 2 CHIR on levels of total Tau and six phosphorylated tau sites (pTaul81, pTau202, pTau214, pTau231, pTau262 and pTau396) in cell extracts of primary cortical neurons from hTau mice. Cells were treated with TI Estetrol (E4) at 5 concentrations, RI. l Estradiol (E2) at 1 concentration (10 nM) or RI.2 CHIR at 1 concentration (2.5 pM) for 24 h. Data are shown as pg / pg total protein for total Tau (A) or in relation to total Tau for all phospho sites (B-G) and displayed as bar graphs with group means +SEM (n=4-6 per group). Statistics: One-way ANOVA with Dunnett's multiple comparisons test (post hoc test) compared to vehicle control (VC) *p<0.05; **p<0.01; ***p<0.001.

[0067] Figure 7. Effect of a single treatment with E4,a repeated treatment with E4 or a repeated treatment with E2 on cell viability and ATP level in control cells. Cells were treated once with E4 at 4 concentrations (A), with E4 at 5 concentrations with treatment renewal every 24h (B), or with E2 at 4 concentrations with treatment renewal every 24h (C), for a total of 24h, 48h or 72h. Cell viability was assessed by MTT assay (left) and ATP level by ATP bioluminescence assay (right). Data are presented as mean ±SEM normalized to 100% of the vehicle condition. In addition, each individual data is presented (gray open circles, N=15-30 replicates per condition). (A) Samples from left to right for each time point: Vehicle; 10 nM E4; 100 nM E4; 1000 nM E4; 10000 nM E4. (B) Samples from left to right for each time point: Vehicle; 10 nM E4; 100 nM E4; 1000 nM E4; 10000 nM E4; 50000 nM E4. (C) Samples from left to right for each time point: Vehicle; 10 nM E2; 100 nM E2; 1000 nM E2; 10000 nM E2.

[0068] Figure 8. Effect of a repeated treatment with E4 and E2 on ATP level and the mitochondrial membrane potential in control cells. Cells were treated with E4 at 2 concentrations or E2 at 1 concentration for 48h with treatment renewal after 24h. (A) Assessment of ATP level. Data are presented as mean ±SEM normalized to 100% of the vehicle condition. In addition, each individual data is presented (gray open circles, N=35-40 replicates per condition). (B) Assessment of mitochondrial membrane potential (MMP). Data are presented as mean ±SEM normalized to 100% of the vehicle (Veh) condition. In addition, each individual data is presented (gray open circles, N=45-50 replicates per condition). Samples from left to right for (A) and (B): Vehicle, 100 nM E4, 1000 nM E4, 100 nM E2.

[0069] Figure 9. Effect of a repeated treatment with E4 and E2 on reactive oxygen species level in control cells. Cells were treated with E4 at 2 concentrations or E2 at 1 concentration for 48h with treatment renewal after 24h. (A) Assessment of total superoxide anion radicals’ level. Data are presented as mean ±SEM normalized to 100% of the vehicle (Veh) condition. In addition, each individual data is presented (gray open circles, N=36-45 replicates per condition). (B) Assessment of the mitochondrial superoxide anion radicals’ level. Data are presented as mean ±SEM normalized to 100% of the vehicle (Veh) condition. In addition, each individual data is presented (gray open circles, N=48-60 replicates per condition). DHE: dihydroethdium, MS: MitoSOX. *p<0.05, One-way ANOVA + Dunnett’s multiple comparison test versus Veh.

[0070] Figure 10. Effect of a repeated treatment with E4 and E2 on cell viability, ATP level and the mitochondrial membrane potential in P301L cells. Cells were treated with E4 at 2 concentrations or E2 at 1 concentration for 48h with treatment renewal after 24h. (A) Assessment of cell viability. Data are presented as mean ±SEM normalized to 100% of the vehicle (Veh) condition. In addition, each individual data is presented (gray open circles, N=36-42 replicates per condition). (B) Assessment of ATP level. Data are presented as mean ±SEM normalized to 100% of the vehicle (Veh) condition. In addition, each individual data is presented (gray open circles, N=36-42 replicates per condition). (C) Assessment of the mitochondrial membrane potential (MMP). Data are presented as mean ±SEM normalized to 100% of the vehicle (Veh) condition. In addition, each individual data is presented (gray open circles, N=36-42 replicates per condition). *p<0.05, ***p<0.01, One-way ANOVA + Dunnett’s multiple comparison test versus Veh.

[0071] DETAILED DESCRIPTION

[0072] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0073] The terms “comprising”, “comprises” and “comprised of’ as used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms also encompass “consisting of’ and “consisting essentially of’, which enjoy well-established meanings in patent terminology.

[0074] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. This applies to numerical ranges irrespective of whether they are introduced by the expression “from ... to ... ” or the expression “between . . . and ... ” or another expression.

[0075] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, preferably + / -5% or less, more preferably + / - 1% or less, and still more preferably + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed. Whereas the terms “one or more” or “at least one”, such as one or more members or at least one member of a group of members, is clear per se, by means of further exemplification, the term encompasses inter alia a reference to any one of said members, or to any two or more of said members, such as, e.g. any >3, >4, >5, >6 or >7 etc. of said members, and up to all said members. In another example, “one or more” or “at least one” may refer to 1, 2, 3, 4, 5, 6, 7 or more.

[0076] The discussion of the background to the invention herein is included to explain the context of the invention. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge in any country as of the priority date of any of the claims.

[0077] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. All documents cited in the present specification are hereby incorporated by reference in their entirety. In particular, the teachings or sections of such documents herein specifically referred to are incorporated by reference.

[0078] Unless otherwise defined, all terms used in disclosing the invention, including technical and scientific terms, have the meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By means of further guidance, term definitions are included to better appreciate the teaching of the invention. When specific terms are defined in connection with a particular aspect of the invention or a particular embodiment of the invention, such connotation or meaning is meant to apply throughout this specification, i.e. also in the context of other aspects or embodiments of the invention, unless otherwise defined. For example, embodiments directed to products are also applicable to corresponding features of methods and uses.

[0079] In the following passages, different aspects or embodiments of the invention are defined in more detail. Each aspect or embodiment so defined may be combined with any other aspect(s) or embodiment(s) unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[0080] Reference throughout this specification to “one embodiment”, “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention, and form different embodiments, as would be understood by those in the art. For example, in the appended claims, alternative combinations of claimed embodiments are encompassed, as would be understood by those in the art.

[0081] Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to standard handbooks as well as to the general background art referred to herein and to the further references cited therein.

[0082] The term “estetrol component”, as used throughout this document, encompasses substances selected from the group consisting of estetrol, esters of estetrol, esters of estetrol wherein the hydrogen atom of at least one of the hydroxyl groups has been substituted by an acyl radical of a hydrocarbon carboxylic, sulfonic acid or sulfamic acid of 1-25 carbon atoms, estetrol hydrates such as estetrol monohydrate; and combinations thereof. It is understood that when estetrol is mentioned throughout any section of this specification, any estetrol-containing component (i.e. compound or (mono)hydrate) and / or estetrol derivative (such as an estetrol ester) is also envisaged. More preferably, in the context of the present disclosure, a particularly preferred estetrol component suitable for the dosage units, medical uses and methods of treatment described herein is estetrol (including estetrol hydrates). Most preferably, said estetrol component is estetrol monohydrate.

[0083] The term “estetrol” as used herein refers to 1,3,5 (10)-estratrien-3,15alpha,16alpha,17beta-tetrol or 15alpha-hydroxyestriol as well as hydrates of estetrol, e.g. estetrol monohydrate. “Estetrol”, or short “E4” is an estrogen steroid produced by the foetal human liver (PubChem CID: 27125). Estetrol may be described as a 3 -hydroxy steroid corresponding to 17beta-estradiol wherein the 15a and 16a positions are substituted for two additional hydroxy groups. It is known that estetrol is an estrogen receptor agonist (Coelingh Bennink et al., Climacteric, 2008). The estetrol may be chemically synthetised, synthesised by the use of (mutant) recombinant enzymes, or synthesised by any combination thereof. It is therefore evident that the terms “estetrol” and “estetrol components” equally encompasses further chemically modified estetrol. Estetrol may be indicated in the art by its molecular formula: C18H24O4, or by structural formula (I).

[0084] Formula (I) In preferred embodiments the estetrol component is estetrol or an ester thereof. In further embodiments, the estetrol component is estetrol monohydrate. A skilled person appreciates that estetrol monohydrate corresponds to estetrol containing one molecule of water, and that the core structural formula of estetrol does not differ from Formula (I). By means of illustration and not limitation, the structural formula of estetrol monohydrate is indicated by Formula (II):

[0085] Formula (II)

[0086] The term “subject”, or “patient” as used herein refers to female human subjects, preferably peri- and / or post-menopausal female subjects. The female subject envisaged herein may be subjects in need of, or deemed in need of a treatment for alleviating estrogen deficiency symptoms (such as menopause- associated symptoms), or predicted to be in need of such a treatment in a foreseeable future point in time due to for example entering the peri -menopausal stage of life.

[0087] “Alzheimer’s disease“ as used herein and commonly abbreviated as “Alzheimer’s”, “Alzheimer”, or “AD” refers to its generally accepted meaning as used in the art. Alzheimer’s disease as referred to herein therefore relates to a neurodegenerative disorder characterized by amyloid-beta (Ap) -containing extracellular plaques and Tau-containing intracellular neurofibrillary tangles. Alzheimer’s disease is typically associated with amnestic cognitive impairment but can also be presented by a non-amnestic cognitive impairment. Short-term memory difficulty is the most common impairment in Alzheimer, but other symptoms generally include impairments in expressive speech, visuospatial processing and executive (mental agility) functions. The severity of these cognitive impairments in patients with Alzheimer’s disease varies greatly.

[0088] At a preclinical stage, Alzheimer’s disease is specifically linked with verbal memory decline or delay, one of the earliest signs. Verbal memory decline or delay can be assessed using e.g. the International Shopping List Task (ISLT) or the Wechsler Memory Scale-Revised (WMS-R) test or similar memory testing tests. The earliest symptomatic stage of cognitive impairment is generally referred to in the art as “Mild Cognitive Impairment” (MCI), which corresponds to the impairment of a single cognitive domain or the impairment of multiple cognitive domains to at least a mild extent whilst functional capacities are relatively preserved. Subjective memory complaints (SMC) represent a type of complaint made by individuals with cognitive symptoms or complaints but who do not yet have a clear impairment on objective psychometric testing. Such individuals may be at increased risk of dementia, with studies showing that patients with SMCs have a higher rate of progression to mild cognitive impairment (MCI) or AD than those without. Upon further progression of Alzheimer’s disease the symptoms are routinely simply referred to as “dementia”, which is generally accepted as the disease stage wherein the cognitive impairment has reached a sufficient magnitude to impair independence and affect daily life. A skilled person appreciates that Alzheimer’s disease seldomly occurs in isolation in elderly subjects. Hence, it is to be understood that multi-aetiology dementia (interchangeably indicated throughout the art as ‘mixed dementia’) is also envisaged in the context of the present specification.

[0089] On a molecular level, the two main components indicative for Alzheimer’s disease subjects are amyloid plaques comprising amyloid beta and neurofibrillary tangles consisting of excessively phosphorylated tau protein.

[0090] “Amyloid beta”, interchangeably indicated in the art by abbreviations such as “Abeta”, “PA”, “PAP”, or “Ap”, refers to peptides of 36-43 amino acids present in extracellular deposits in brain tissue of Alzheimer’s disease subjects. Amyloid beta is derived by proteolysis from amyloid precursor protein (APP). According to the amyloid hypothesis, the soluble oligomeric forms of the amyloid beta peptide appear to be the causative agents in the development of Alzheimer's disease, with amyloid beta oligomers acting as the main toxic amyloid beta form (Zhao et al., Int J Mol Sci, 2012). Truncated and post-translationally modified forms of amyloid beta have been described. For example, Ap40 is a short form of Ap, and the more fibrillogenic isoform Ap42 is a longer form. Further examples of Ap include but are not limited to, N-truncated Api l-42, Api l-42-Pyro, Ap3-42-Pyro, and Api-42-E22Q-Dutch mutation (described in e.g. Levy et al., Science, 1990; and Van Broeckhoven et al., Science, 1990).

[0091] In a more general context, the term “amyloid”, interchangeably used with terms such as “amyloid fibres” and “amyloid deposits” corresponds to a generic term for a tertiary structure that is formed by misfolding or aggregation of any of several different proteins and that comprises an ordered arrangement of P-sheets stacked perpendicular to a fibre axis forming insoluble fibrous protein aggregates (as defined in e.g. Sunde et al., J Mol Biol, 1997). Amyloids have common morphologic properties. For example, amyloids stain with specific dyes such as Congo red and have a characteristic red-green birefringent appearance in polarized light after staining. Additionally, amyloids share common ultrastructural features and common x-ray diffraction and infrared spectra (Morris and Serpell, Methods Mol Biol, 2012). Shearing and / or fragmentation of mature fibrils into fibrillar oligomers has been described, which may in turn again aggregate to form mature fibrils.

[0092] As indicated above, particularly preferred subjects in the context of the present disclosure are menopausal, perimenopausal, or post-menopausal subjects. It is known that menopause involves a diverse range of neurological symptoms such as but not limited to vasomotor symptoms (e.g. hot flushes), disturbed sleep patterns, pain, depression, and cognitive dysfunction (Nelson, Lancet, 2008). Each of these have been described as risk factors for Alzheimer’s disease. Menopause has also been associated with the emergence of estrogen receptor splice variants, changes in protein expression, alterations in receptor degradation, and possible epigenetic reconfigurations, which all contribute to reduced estrogen levels and reduced receptor activity. Preclinical studies have indicated that during these changes, certain mechanisms required for estrogen activation of cerebral glucose metabolism rates (CMRglc) and suppression of the ketogenic pathways may start to function in a suboptimal manner. This subsequently results in an adaptive starvation reaction, increasing the fatty acid metabolism for the generation and utilization of ketone bodies by mitochondria as an alternative fuel. Hypometabolism, reduced mitochondrial function and subsequent oxidative damage each have been described to promote accumulation of amyloid beta, therefore increasing risk of developing Alzheimer’s disease (Mattson and Magnus, Nat Rev Neurosci, 2006).

[0093] Through experimentation and as exemplified in the Examples of the present specification, the inventors have observed that estetrol components (such as estetrol and estetrol monohydrate) display favourable characteristics over other estrogens used in the art as active pharmaceutical ingredient in hormone replacement strategies. More particularly, the inventors have obtained results that indicate that in contrast to reports using other estrogens, estetrol components have a preventing effect on Alzheimer disease which cannot be observed when using other estrogens. Additional beneficial effects of estetrol over other estrogens in the context of Alzheimer’s disease include but are not limited to improved protection to Reactive Oxygen Species (ROS) generation, cell viability, ATP production, mitochondrial membrane potential (MMP), and reduction of several phosphorylated Tau protein (pTau) species.

[0094] Therefore, in a first aspect the invention relates to a composition for use in the prevention and / or treatment of Alzheimer’s disease in a subject, wherein the composition comprises an estetrol component. More particularly, the aspect relates to a composition for use in the prevention and / or treatment of Alzheimer’s disease symptoms, wherein said composition comprises from about 15 mg to about 25 mg of an estetrol component, preferably wherein said composition is administered once daily.

[0095] Additionally, the invention envisages use of a composition for the manufacturing of a medicament for preventing and / or treating Alzheimer’s disease symptoms in a subject, wherein said composition comprises from about 15 mg to about 25 mg of an estetrol component and preferably wherein said composition is administered once daily. Additionally, the use of a composition comprising an estetrol component for preventing and / or treating Alzheimer’s disease symptoms in a subject, wherein said composition comprises from about 15 mg to about 25 mg an estetrol component and wherein said composition is preferably administered once daily. Additionally, a method of treating Alzheimer’s disease symptoms in a subject is envisaged, wherein said method comprises the step of administering a composition comprising from about 15 mg to about 25 mg an estetrol component, preferably once daily. Preferably, the Alzheimer’s disease is menopause-associated Alzheimer’s disease. Hence, in preferred embodiments the composition is administered to a subject to (additionally) alleviate menopause- associated symptoms distinct to menopause-associated Alzheimer’s disease symptoms.

[0096] The term “daily” indicates that the recited amounts are the cumulative amount that is administered to a subject per day. A skilled person understands that if the estetrol component is administered only once per day (i.e. daily), that the amount of estrogen administered in that single administration will be the daily dose. Alternatively, a skilled person appreciates that if the estetrol component is administered more than once per day (e.g. 2 times or 3 times) the daily amount will correspond to the sum of estetrol component administered during each administration event within a total time window of 24 hours. In embodiments where different estetrol components are comprised in the composition (e.g. estetrol monohydrate and an estetrol ester), it is within the capacities of a skilled person to verify the amount of estetrol each estetrol component corresponds to. Preferred embodiments within the context of the invention comprise the administration of a single estetrol component, such as but not limited to estetrol monohydrate.

[0097] The term “subject”, or “patient” as used herein refers to female human subjects, preferably peri- and / or post-menopausal female subjects. The female subject envisaged herein may be subjects in need of, or deemed in need of a treatment for alleviating estrogen deficiency symptoms (such as menopause- associated symptoms which may include menopause-associated Alzheimer’s disease symptoms), or predicted to be in need of such a treatment in a foreseeable future point in time due to for example entering the peri -menopausal stage of life.

[0098] A skilled person is aware that terms such as “quantity”, “amount” and “level” are synonyms and have a well-defined meaning in the art. The terms as used herein may particularly refer to an absolute quantification of a molecule such as a steroid, in (a sample taken from) a subject, or to a relative quantification of a molecule or analyte in a sample, i.e., relative to another value such as relative to a reference value as taught herein, or to a range of values indicating a base-line of a certain parameter. These values or ranges of values may be obtained from one single subject or from a group of subjects (i.e. at least two subjects).

[0099] The terms “treatment” or “treat” are to be interpreted as both the therapeutic treatment of a symptom, disease or condition that has already developed, leading to (clinical) manifestations, as well as prophylactic or preventive measures, wherein the goal of the treatment is to prevent, lessen, or reduce the chances of incidence of an undesired affliction, such as to prevent occurrence, development and progression of symptoms, (clinical) conditions related to menopause. Beneficial or desired clinical results may include, without limitation, alleviation of one or more symptoms, improvement of one or more biological markers, diminishment of the extent (i.e. a reduction in severity) of the Alzheimer’s disease symptoms, stabilized (i.e. not worsening) of Alzheimer’s disease symptoms, delay or slowing of the manifestation of Alzheimer’s disease symptoms, and the like.

[0100] “Prevention” or “prevent” as used in the context of the invention refers to an aversion of manifestation of a condition or disease image in a subject, i.e. the establishment of preventive measures or prophylactic measures. Preventive treatment refers to treatments wherein the object is to avoid a subject’s body or an element thereof to show (worsening of) symptoms of an undesired physiological or psychological change induced by menopause. As used herein, the term “prevent” includes both preventing symptoms from occurring and preventing symptoms of worsening.

[0101] As used herein, the terms "therapeutic treatment" or "therapy" and the like, refer to treatments wherein the aim is to change a subjects body or a part of a subjects body from an undesired physiological state, disease or disorder which is caused by aging, to a desired state, such as a less severe state (e.g., amelioration, or even back to its normal, healthy state (e.g., restoring the health, the physical integrity and the physical well-being of a subject), to keep it (i.e., not worsening) at said undesired physiological status (e.g., stabilization), or slow down progression to a more severe or worse state compared to said undesired physiological change or disorder). Measurable lessening includes any statistically significant decline in a measurable marker or symptom. Statistically significant as used herein refers to p values below 0.05, which is a commonly accepted cut-off score in statistical analysis as a skilled person appreciates.

[0102] “Estrogen deficiency symptoms” have been described in the art and encompass any symptom a subject can experience due to estrogen deficiency (i.e. “hypoestrogenism”, or “estrogen deficiency syndrome”). In a preferred embodiments of the invention, the estrogen deficiency symptoms are occurring in a context of menopause and hence the symptoms may be referred to as menopause-associated symptoms. The expression “alleviating menopause-associated symptoms” as used throughout the present specification has a well-established meaning within the technical field and indicates treatment of any unwanted physical or psychological manifestation that accompanies menopause, or the onset of menopause. Thus, the composition disclosed herein is typically used as a (therapeutic) treatment for menopause-associated symptom, i.e. as a composition that is administered to a subject in a context of therapy. Also envisaged is the use of the composition described herein for preventing the onset of estrogen deficiency symptoms or more specifically menopause-associated symptoms such as menopause-associated Alzheimer’s disease symptoms.

[0103] In embodiments wherein the subject is already experiencing one or more menopause-associated symptoms, the subject may be a hormone deregulated subject. In the context of the present invention, the deregulation is determined or established by comparison of the hormone level of the subject with a representative value of an adult, healthy female subject. The cause of the hormone deregulation is particularly limiting for the present disclosure, and may be of a natural cause (such as menopausal hormonal changes) but may equally be caused by a pathologic condition or deficiency. In certain embodiments, the hormone deregulation is an estrogen deregulation, such as an 17p-estradiol deregulation. In such embodiments, the 17p-estradiol differs at least 15%, preferably at least 25%, more preferably at least 50% from a representative 17p-estradiol value for female adult subjects. In cases wherein the endogenous estrogen production of the female subject is reduced, the subject may be considered to be a subject suffering from estrogen-deficiency syndrome.

[0104] The term "17p-estradiol" refers to (17beta)-estra-l,3,5(10)-triene-3,17-diol and may interchangeably be indicated by the terms “estradiol”, “oestradiol”, or “E2”, and is an estrogen endogenously produced by the human body. More particularly, estradiol is the predominant estrogen hormone produced by the human ovaries during the initial half of the menstrual cycle (i.e. the follicular phase). Estradiol is also involved in the maintenance of bone density, reduction of vasomotor symptoms, and maintenance of the normal structure of the female genital organs in menopausal subjects.

[0105] Embodiments concerning subjects characterised by an estrogen depletion (i.e. hypoestrogenism, or estrogen deficiency syndrome) are envisaged by the invention. While the invention is primarily directed to alleviating menopause-associated symptoms, particularly menopause-associated Alzheimer’s disease symptoms, a skilled person will appreciate that Alzheimer’s disease symptoms can also present themselves in a female subject that has a medical condition which is not menopause but nonetheless results in similar symptoms, or even the same set of symptoms. Hence, the invention is equally directed to alleviating estrogen deficiency symptoms that may present themselves in a person that is not undergoing menopause, and / or is not predicted by an imminent onset of menopause. The cause of the hypoestrogenism is not particularly limited and may therefore be caused by the non-limiting causes of menopause, hypogonadism, castration, primary ovarian failure, and aromatase inhibitor or gonadotropin-releasing hormone analogue breast cancer treatment.

[0106] In certain embodiments, the subject is a female menopausal, perimenopausal, or postmenopausal subject. In certain embodiments, the subject is a menopausal, perimenopausal, or postmenopausal subject having an 17p-estradiol level of less than 100 pg / ml, preferably less than 50 pg / ml, preferably less than 30 pg / ml, more preferably less than 20 pg / ml, more preferably less than 20 pg / ml, most preferably less than 10 pg / ml. In alternative embodiments, the subject is a menopausal, perimenopausal, or postmenopausal subject that is characterised by having follicle-stimulating hormone concentrations of at least 20 milli-intemational units per millilitre (mlU / ml), preferably at least 25 mlU / ml, more preferably at least 30 mlU / ml, more preferably at least 35 mlU / ml, most preferably at least 40 mlU / ml.

[0107] “Menopausal subjects”, used interchangeably in the art with “post-menopausal subjects” or “climacteric subjects” are female subjects that that have not had menstrual bleeding for a year which is accompanied by a decrease or discontinuation in hormone production by the ovaries (such as 17p-estradiol).

[0108] According to the US FDA, the criteria for post-menopausal status are: At least 12 months of spontaneous amenorrhea; or

[0109] • At least 6 months of spontaneous amenorrhea with serum FSH levels >40 mIU:mL; or

[0110] • At least 6 weeks postsurgical bilateral oophorectomy with or without hysterectomy.

[0111] Alternatively worded, “menopause” may be described as a biological condition characterised by impairment or cessation of ovarian primary function. Menopause may be accompanied by a broad range of clinical symptoms which are variable in severity such as but not limited to vasomotor dysfunction, vaginal dryness, mood changes, sleep disturbances, insomnia, urinary incontinence, cognitive changes, somatic complaints, and sexual dysfunction. Methodologies to diagnose menopause have been described in the art and are therefore known to a person skilled in the art (Nelson, Menopause, Lancet, 2008).

[0112] “Perimenopause” refers to a period of life which begins approximately three to four years prior to menopause and ends one year after the final menstrual period, and is characterised by persistent irregular menstrual cycles, extreme fluctuations in hormonal levels, frequent anovulation and the appearance of vasomotor symptoms (Harlow et al., Executive summary of the Stages of Reproductive Aging Workshop + 10: addressing the unfinished agenda of staging reproductive aging, Menopause, 2012). The term “post menopause” or “postmenopausal” is indicative for female subjects that are characterised by a permanent cessation of menstrual periods. This permanent cessation is determined retrospectively after an observation of 12 months of amenorrhea without any other obvious pathological or physiological cause. The term “post menopause” also includes menopause as the consequence of premature ovarian failure, surgery (ovariectomy for example), chemotherapy or radiotherapy for cancer, and certain diseases (for example, infections or hypothyroidism).

[0113] Thus, in the context of the present invention the female subject is a menopausal, perimenopausal, or post-menopausal subject.

[0114] Preferably, the subject is a female subject of adult age. More preferably, the subject is a female subject of middle age or elder age. Yet more preferably, the subject is a female subject of at least 40 years of age, preferably of at least 50 years of age, preferably of at least 55 years of age, more preferably of at least 60 years of age, more preferably of at least 65 years of age. Alternatively, the subject may be a female subject of between 40 and 90 years, preferably of between 45 and 85 years of age, preferably of between 50 and 80 years of age, more preferably of between 55 and 75 years of age, more preferably of between 60 and 70 years of age or between 65 and 75 years of age. In certain embodiments, the female subject is at most 90 years of age, preferably at most 85 years of age, more preferably at most 80 years of age, more preferably at most 75 years of age, more preferably at most 70 years of age, more preferably at most 65 years of age, more preferably at most 60 years of age. “Diagnosed with”, “diagnosing”, and diagnosis are indicative for a process of recognising, deciding on, or concluding on a disease, condition, or (adverse side effect) in a subject on the basis of symptoms and signs and / or from results of various diagnostic procedures (such as, for example, from knowing the presence, absence and / or quantity of one or more biomarkers of or (pre)clinical symptoms characteristic for the diagnosed disease or condition).

[0115] “Diagnosis of’ one or more Alzheimer’s disease symptoms may particularly mean that the subject has at least one Alzheimer’s disease symptom as adjudged by a skilled medical practitioner. Although a subject presents one or more conventional symptoms or signs indicative thereof, an absence of symptoms may ultimately be diagnosed.

[0116] "Prognosticating" in the context of the invention is indicative for anticipation on the progression of one or more Alzheimer’s disease symptoms in a subject and the prospect (e.g. the probability, duration, and / or extent) of recovery, and / or the severity of experiencing or amelioration of said one or more Alzheimer’s disease symptoms. The term may encompass anticipation of not further worsening or aggravating of such, preferably within a given time period. The term "a poor prognosis of the disease or condition typically encompasses an anticipation of a substandard recovery and / or unsatisfactorily slow recovery, or no recovery at all, or further worsening of the one or more Alzheimer’s disease symptoms.

[0117] Related to the foregoing, "predicting" or "prediction" generally refers to a statement, declaration, indication or forecasting of a disease or condition in a subject not (yet) showing any, or a limited, clinical manifestation of one or more (menopause-associated) Alzheimer’s disease symptoms. A prediction of one or more (menopause-associated) Alzheimer’s disease symptoms in a subject may indicate a probability, chance, or risk that said subject will develop said clinical manifestation, condition, or (adverse) side effect, for example within a certain time period after diagnosis of the one or more (menopause-associated) Alzheimer’s disease symptoms. Said probability, chance or risk may be indicated as any suitable qualitative or quantitative expression, wherein non-limiting examples of a quantitative expression include absolute values, ranges or statistics. Alternatively, probabilities, chances, or risks may be indicated relative to a suitable control subject or group of control subject (i.e. a control subject population (such as, e.g., relative to a general, normal or healthy subject or subject population)). Therefore, any probability, chance or risk may be advantageously indicated as increased or decreased, upregulated or downregulated, as fold-increased or fold-decreased relative to a suitable control subject or subject population, or relative to a baseline value which may be derived from either a control subject (population), textbook reference values. It is evident that when a population of subjects is used to define the baseline value, said baseline value will be a centre size of one or more values (parameters) of a population, such as the mean or median of said value. A skilled person further appreciates that monitoring may be applied in the course of a medical treatment of a subject. Such monitoring may be comprised, e.g., in decision making whether a patient may be discharged from a controlled clinical or health practice environment, needs a change in treatment or therapy, or requires hospitalisation.

[0118] In each of the embodiments disclosed herein, the composition aims to alleviate menopause-associated Alzheimer’s disease symptoms by providing a composition comprising from about 15 mg to about 25 mg of an estetrol component, preferably to be administered once daily. This implies that the estetrol component in the composition is present in a pharmaceutically effective amount.

[0119] “A pharmaceutically effective amount” refers to an amount necessary to obtain a physiological effect and may indicate a therapeutically effective amount and / or a prophylactically effective amount. The physiological effect may be achieved by a single dose or by multiple doses.

[0120] A “therapeutically effective amount” or “therapeutically effective dose” indicates an amount of estetrol component that when administered brings about a clinical positive response with respect to treatment of a subject afflicted by (menopause-associated) Alzheimer’s disease symptoms.

[0121] Similarly, a “prophylactically effective amount” or “prophylactically effective dose” refers to an amount of estetrol component that inhibits or delays the onset of clinical manifestation of condition as being sought by a researcher, veterinarian, medical doctor or other clinician.

[0122] A skilled person is aware that terms such as “quantity”, “amount” and “level” are synonyms and have a well-defined meaning in the art and appreciates that these may particularly refer to an absolute quantification of an estetrol component which is considered an effective amount for the applications described herein, or to a relative quantification of the estetrol component. Suitable values or ranges of values may be obtained from one single subject or from a group of subjects (i.e. at least two subjects).

[0123] In certain embodiments, the subject has (i.e. is characterised by) a genetic predisposition to develop Alzheimer’s disease. The term “genetic predisposition” is well known to a skilled person and indicates an increased chance or likelihood of developing a particular disease based on the presence of one or more genetic variants and / or a family history suggestive of an increased risk of the disease. Numerous genetic predispositions for developing Alzheimer have been described throughout the art (as summarised in e.g. Neuner et al., Neurobiol Dis, 2021). The exact genetic predisposition is not particularly limiting for the present invention, and therefore the subject may be a genetically predisposed subject for developing Alzheimer’s disease having a mutation in one or more genes selected from the group consisting of: GLIS1, ADAMTS4, CR1, PSEN2, PPB, BINI, INPP5D, HESX1, TF, GMNC, CLNK, HS3ST1, UNC5C, MEF2C, SERPINB1, HLA, TREM2, NCR2, CD2AP, NME8, COBL, AKAP9, ZCWPW1, NYAP1, PILRA, EPHA1, CNTNAP2, CLU / PTK2B, GLIS3, DAPK1, ECHDC3, FRA10AC1, SPI1, BDNF, MS4A cluster, PICALM, CNTN5, SORL1, GRIN2B, PCDH8, SLC10A2, FERMT2, PSEN1, SLC24A4, RIN3, ADAM10, APH1B, IQCK, KAT8, IL34, WWOX, PLCG2, ZNF232, SCIMP, ABI3, BZRAP1-AS1, ACE, DSG2, ALPK2, CTDP1, ABCA7, NOTCH3, PLD3, APOE, CD33, PRNP, CASS4, APP, and AD AMTS 1. Optionally, the subject may be a genetically predisposed subject for developing Alzheimer’s disease having a mutation in one or more genes selected from the group consisting of: APOE, APP, PSEN1, and PSEN2. Optionally, the subject may be a genetically predisposed subject for developing Alzheimer’s disease having a mutation in APOE.

[0124] Preferred subjects in the context of the present invention are subjects that carry the APOEs4 allele. The polymorphic APOE gene encodes the fat-binding protein apolipoprotein E (APO-E). Different APOE alleles differ from each other by a limited number of amino acids, but have distinct structural and functional implications. It is generally accepted that the APOEs4 constitutes a considerable genetic risk factor for developing Alzheimer’s disease. Without wishing to be bound by theory, the inventors hypothesize that particularly female subjects carrying the APOEs4 allele benefit favorably from treatment with an estetrol-comprising composition as described herein.

[0125] The degree, or progression status of the Alzheimer’s disease of the subject is not particularly limiting for the invention. Hence, both mild cognitive impairment (pre-Alzheimer’s stage), early-stage Alzheimer’s disease (mild), middle-stage Alzheimer’s disease (moderate), and late-stage Alzheimer’s disease (severe) are envisaged in the present context. Alternative subject classifications include preclinical Alzheimer's disease stage, subjective memory complaints (SMCs), verbal memory decline or delay, mild cognitive impairment (MCI) due to Alzheimer's disease, mild dementia due to Alzheimer's disease, moderate dementia due to Alzheimer's disease, severe dementia due to Alzheimer's disease.

[0126] The compositions, uses, and methods described herein are envisaged to have a general favorable effect on Alzheimer’s disease symptoms, and therefore an amelioration of different hallmark symptoms is encompassed by the expression “Alzheimer’s disease symptoms”. By means of illustration and not limitation, said symptoms include impaired or declined memory, verbal memory decline, depression, anxiety, anger, irritability, insomnia, paranoia, problem solving difficulties, difficulties of executing complex tasks, impaired sound judgement, personality changes, reduced ability to organizing and expressing thoughts, or any combination thereof.

[0127] The subjects described herein that experience, or are expected to experience, Alzheimer’s disease symptoms may be diagnosed by means of any suitable (pre)clinical Alzheimer rating scale known to a skilled person. Therefore, illustrative examples of suitable rating scales include Clinical Dementia Rating Scale- Sum of Boxes (CDR-SB), the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog), the Alzheimer's Disease Cooperative Study-Instrumental Activities of Daily Living scale (ADCS-iADL), the Mini-Mental State Examination (MMSE), the Functional Activities Questionnaire (FAQ), and the integrated Alzheimer's Disease Rating Scale (iADRS) (Wessels et al., Alzheimers Dement (NY), 2022). Pre-clinically, different cognitive tests have been developed over the years to detect cognitive decline among older adults, i.e. testing one or more of attention, memory, visuospatial, language, and reasoning skills. Further modified or updated iterations of said scales are envisaged by the present disclosure. In certain embodiments, the compositions described herein reduce the average Alzheimer’s disease progression as measured by one or more Alzheimer’s disease rating scale by at least about 5%, preferably by at least about 10%, preferably by at least about 15%, preferably by at least about 20%, preferably by at least 30%, preferably by at least 40%, preferably by at least 50% when compared to a population considered to have Alzheimer’s disease that is not receiving any treatment for Alzheimer’s disease.

[0128] As demonstrated by the Examples of the present disclosure, the compositions described herein have a neuroprotective effect. For example, the compositions described herein have a neuroprotective effect against toxicity caused by amyloid beta. In certain embodiments, the compositions described herein reduce toxicity of amyloid beta by at least about 5%, preferably at least about 10%, preferably at least about 15%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably from about 50% to about 75% when compared to amyloid beta toxicity in absence of a neuroprotective agent. In certain embodiments, the compositions described herein reduce toxicity of amyloid beta by at least about 5%, preferably at least about 10%, preferably at least about 15%, preferably at least about 20%, preferably at least about 30%, preferably at least about 40%, preferably at least about 50%, preferably from about 50% to about 75% when compared to amyloid beta toxicity tested in presence of an estrogenic component that is not an estetrol component (e.g. estradiol). In certain embodiments, the compositions described herein reduce toxicity of amyloid beta by at least about 5%, preferably at least about 10%, preferably at least about 15%, preferably at least about 20%, preferably at least about 30% preferably at least about 40%, preferably at least about 50%, preferably from about 50% to about 75% when compared to amyloid beta toxicity tested in presence of estradiol.

[0129] As further demonstrated by the Examples of the present disclosure, the compositions described herein reduce Reactive Oxygen Species (ROS) generation. A skilled person is familiar with the term “Reactive Oxygen Species” and appreciates that the term refers to reactive chemicals formed from diatomic oxygen (O2). Optionally, the compositions described herein reduce the ROS generated by oxidative stress by at least about 5%, preferably at least about 10%, preferably at least about 15%, preferably at least about 20%, preferably at least about 30% when compared to ROS generation in absence of an estetrol component (or any other external antioxidant moiety). Optionally, the compositions described herein reduce the ROS generated by oxidative stress by at least about 5%, preferably at least about 10%, preferably at least about 15%, preferably at least about 20%, preferably at least about 30% when compared to ROS generation in presence of an estrogenic component that is not an estetrol component (e.g. estradiol). In any of the embodiments described herein, the ROS generation may be tested by means of H2O2 treatment. Oxidative stress, which refers to an imbalance between oxidants and antioxidant mechanisms, is known to fulfil an essential role in Tan hyperphosphorylation, polymerization, and toxicity. It has been described in the art that cells characterized by an overexpression of Tan protein are more susceptible to oxidative stress. Furthermore, oxidative stress is considered to have a role in the abnormal phosphorylation and polymerization of Tau, resulting in Tau protein aggregation. Therefore, the compositions described herein reduce the degree of tau protein phosphorylation.

[0130] The Tau protein has been well characterized throughout the art and is known to have at least six isoforms produced by alternative splicing of the microtubule-associated protein Tau gene. The main physiological function of Tau protein is to stabilize the internal skeleton of neurons in the brain, assisting in cell division, and is additionally involved in the transportation of material among cells. In Alzheimer disease and other neurodegenerative pathologies, Tau oligomerization and their inclusion in amyloid structures act as pathological marker. Monomers of Tau protein are highly soluble proteins that are intrinsically disordered proteins. Tau oligomers are primarily composed of monomeric or dimeric subunits of tau and act as intermediate structures that can induce conformational changes in the structure of the tau and attach to the oligomeric structure, resulting in the formation of stacked beta-sheet strands and forming insoluble paired helical filaments. Tau accumulation results in brain cell damage, but the exact underlying process remains undetermined.

[0131] In Alzheimer’s disease and other “tauopathies”, tau is abnormally phosphorylated. More particularly, Tau phosphorylation in the brain of a subject diagnosed to have Alzheimer’s disease is characterized by at least a three-fold increase in phosphorylation which is hypothesized to be a major cause of the observed loss of microtubule binding. Indeed, hyperphosphorylation of the Tau protein at different sites may detach Tau from microtubules and the consequential impediment of intracellular traffic, which eventually results decreased activity and viability of neurons.

[0132] As indicated above, the compositions described in the present disclosure reduce the degree of Tau phosphorylation. Optionally, the compositions described herein reduce the absolute amount of phosphorylated Tau (pTau) in a subject tissue or subject sample. Preferably, the compositions described herein reduce the absolute amount of pTau in a subject tissue or subject sample by at least about 1%, preferably by at least about 2.5%, preferably by at least about 5%, preferably by at least about 10%. Preferably the compositions described herein reduce the absolute amount of pTau in a subject tissue or subject sample by from about l%to about 10%. Alternatively, the compositions described herein reduce the rate of phosphorylation of pTau in a subject tissue or subject sample by at least about 1%, preferably by at least about 2.5%, preferably by at least about 5%, preferably by at least about 10%. Preferably the compositions described herein reduce the rate of phosphorylation of pTau in a subject tissue or subject sample by from about 1% to about 10%. Alternatively or additionally, the compositions described herein reduce the relative abundance of phosphorylated Tau protein (pTau). Preferably, the compositions described herein reduce the relative abundance of pTau in a subject tissue or subject sample by at least about 1%, preferably by at least about 2.5%, preferably by at least about 5%, preferably by at least about 10%. Preferably the compositions described herein reduce the relative abundance of pTau in a subject tissue or subject sample by from about 1% to about 10%.

[0133] The particular reduced pTau protein is not particularly limiting for the invention and may therefore include phosphorylation of any Serine, Threonine, Tyrosine, or any combination thereof. By means of illustration and not limitation, the pTau protein may be one or more pTau proteins selected from the group consisting of: pTaul81, pTau202, pTau214, pTau262, pTau396, or any combination thereof. Hence, in certain embodiments, the compositions described herein reduce the cumulative amount of pTaul81, pTau202, pTau214, pTau262, and pTau396 in a subject tissue or subject sample by at least about 2.5%, preferably by at least about 5%, preferably by at least about 10%, preferably by at least about 15%, preferably by at least about 20%, preferably by at least about 25%, preferably by at least about 30%, more preferably by at least about 40%, most preferably by at least about 50%. In alternative embodiments, the compositions described herein reduce the cumulative amount of one or more of pTaul81, pTau202, pTau214, pTau262, and pTau396 in a subject tissue or subject sample by at least about 2.5%, preferably by at least about 5%, preferably by at least about 10%, preferably by at least about 15%, preferably by at least about 20%, preferably by at least about 25%, preferably by at least about 30%, more preferably by at least about 40%, most preferably by at least about 50%. In yet alternative embodiments the compositions described herein reduce the cumulative amount of one or more of pTau 181, pTau202, pTau214, pTau262, and pTau396 in a subj ect tissue or subj ect sample by at least about 2.5%, preferably by at least about 5%, preferably by at least about 10%, preferably by at least about 15%, preferably by at least about 20%, preferably by at least about 25%, preferably by at least about 30%, more preferably by at least about 40%, most preferably by at least about 50%.

[0134] By means of illustration and not limitation, suitable samples to measure pTau levels include cerebrospinal fluid samples and blood samples.

[0135] A further benefit of the compositions described herein is the ability to improve cell viability upon amyloid beta induced cell or tissue damage (amyloid beta lesion damage), i.e. cell toxicity upon treatment with amyloid beta peptides. Preferably, the compositions described herein improve cell viability in case of amyloid beta induced cell or tissue damage when compared to cell viability in case of amyloid beta lesion damage in presence of an estrogen that is not an estetrol component (e.g. estradiol). In preferred embodiments, the composition improves the cell viability upon amyloid beta lesion damage with at least 5%, preferably at least 10%, more preferably at least 15%, most preferably at least 20% when compared to the cell viability upon amyloid beta lesion damage in presence of an estrogen that is not an estetrol component, preferably wherein the estrogen is estradiol.

[0136] Optionally, the compositions described herein reduce lactate dehydrogenase (LDH) release upon amyloid beta lesion damage. Preferably, the compositions described herein reduce LDH release when compared to LDH release upon amyloid beta lesion damage in presence of an estrogen that is not an estetrol component, preferably wherein the estrogen is estradiol. In preferred embodiments, the composition reduces LDH release upon beta amyloid damage by at least about 5%, preferably at least about 10%, more preferably at least 15%, most preferably at least 20% when compared to the cell viability upon amyloid beta lesion damage in presence of an estrogen that is not an estetrol component, preferably wherein the estrogen is estradiol.

[0137] The compositions, uses, and methods described herein have a favourable effect on adenosine 5'- triphosphate (ATP) production. Hence, optionally the compositions described herein increase the mitochondrial membrane potential by at least about 10%, preferably by at least about 15%, more preferably by at least about 20% when compared to the cell viability upon amyloid beta lesion damage in presence of an estrogen that is not an estetrol component, preferably wherein the estrogen is estradiol.

[0138] The term “mitochondrial membrane potential” is known to a skilled person and refers to the potential originating from redox transformations associated with the activity of the Krebs cycle. The mitochondrial membrane potential serves as an intermediate form of energy storage which is harnessed by the enzyme ATP synthase to produce ATP. The redox transformations generate both an electrical potential and a proton gradient, and together they form the transmembrane potential of hydrogen ions.

[0139] The composition of the present invention is particularly suited for formulation as an oral dosage unit, as evidenced by the Examples enclosed herewith. Preferably, the oral dosage unit for use according to the invention is swallowed, i.e. ingested and passes through the ingestive system of the subject. Alternatively worded, the dosage unit described herein is administered orally. More preferably, the oral dosage unit for use according to the invention is swallowed as a whole without any prolonged presence in the oral cavity than required for swallowing. In an alternative embodiment, However, equally envisaged are dosage units formulated towards alternative administration methods such as but not limited to sublingual, buccal, or sublabial dosage units. In contrast to oral administration, these bypass the first-pass effect, i.e. a phenomenon in which a drug gets metabolized at a specific location in the body (gastric, hepatic,...) that results in a reduced concentration of the active drug upon reaching its site of action or the systemic circulation. A sublingual, buccal or sublabial dosage unit thus differs from an oral dosage unit according to the invention.

[0140] “Dosage unit”, interchangeably used with “dosage form” herein and in the art indicates a physical preparate that is suitable for administration to a subject, without the necessity to adapt the drug product prior to administration, i.e. the final beneficial product. A dosage unit therefore indicates a ready-to- administer composition. The term is not limiting for any other particulars of the treatment, such as frequency of administration and / or any characteristic of the dosage unit (taste, appearance, size, etc.). “Oral dosage unit” encompasses any dosage unit that is intended to and / or suitable for administration to a subject by means of the oral cavity. (Immediate or near-immediate) ingestion of the dosage unit is envisaged but not a limitation for the oral dosage unit of the invention, as detailed further below. The composition or dosage unit may be administered once daily, or a discrete number of times during a 24 hour period. In certain embodiments the oral dosage unit is specifically formulated for administration by ingestion. Hence, “oral dosage unit” as used herein refers to a dosage unit which is administered orally.

[0141] In the context of the present invention, each dosage unit preferably comprises from about 15 mg to about 25 mg of an estetrol component as pharmaceutically acceptable ingredient, or comprises from about 15 mg to about 25 mg of estetrol, preferably estetrol monohydrate. The presence of an estetrol component as pharmaceutically acceptable ingredient does not exclude the presence of one or more further pharmaceutical ingredients and / or pharmaceutically acceptable ingredients in the dosage unit. The term “pharmaceutically acceptable” as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof. Non-limiting suitable excipients are described further throughout the disclosure.

[0142] As detailed above, the composition which is subject of the present invention comprises from about 15 mg to about 25 mg of an estetrol component. Optionally, the composition is administered at a daily amount of estetrol component equivalent to from about 15 mg to about 25 mg of estetrol monohydrate. Preferably, the composition comprises from about 15 mg to about 20 mg of estetrol and is administered daily. More preferably, the composition comprises from about 15 mg to about 20 mg of estetrol monohydrate and is administered daily.

[0143] In further embodiments, the composition comprises from about 15 mg to about 20 mg of estetrol component, preferably wherein the estetrol component is estetrol, estetrol monohydrate, or an ester of estetrol. Preferably the composition comprises from about 15 mg to about 25 mg of estetrol monohydrate, more preferably from about 15 mg to about 20 mg estetrol monohydrate. In alternative embodiments, the composition comprises from about 15 mg to about 25 mg of estetrol or an ester thereof, preferably from about 15 mg to about 20 mg of estetrol or an ester thereof.

[0144] Optionally, the composition comprises about 17 mg of estetrol component, or an amount of estetrol component equivalent to a daily dose of about 17 mg of estetrol component. In further embodiments, the composition comprises about 17 mg of estetrol monohydrate, or an amount of estetrol component equivalent to a daily dose of about 17 mg of estetrol monohydrate. In alternative further embodiments, the composition comprises about 17 mg of estetrol or ester thereof, or an amount of estetrol component equivalent to a daily dose of about 17 mg of estetrol or an estetrol ester. In alternative embodiments, the composition comprises from about 12 mg to about 28 mg, from about 13 mg to about 27 mg, from about 14 mg to about 26 mg, from about 15 mg to about 25 mg, from about 16 mg to about 24 mg, from about 17 mg to about 23 mg, from about 18 mg to about 22 mg, or from about 19 mg to about 21 mg, or from 13 mg to 17 mg, or from 14 mg to 16 mg, or from 18 mg to 22 mg, or from 19 mg to 21 mg of an estetrol component, or an amount of estetrol component equivalent to a daily dose of from about 12 mg to about 28 mg of estetrol, from about 13 mg to about 27 mg, from about 14 mg to about 26 mg, from about 15 mg to about 25 mg, from about 16 mg to about 24 mg, from about 17 mg to about 23 mg, from about 18 mg to about 22 mg, or from about 19 mg to about 21 mg, or from 13 mg to 17 mg, or from 14 mg to 16 mg, or from 18 mg to 22 mg, or from 19 mg to 21 mg of an estetrol component. In preferred alternative embodiments, the composition comprises from about 12 mg to about 28 mg, from about 13 mg to about 27 mg, from about 14 mg to about 26 mg, from about

[0145] 15 mg to about 25 mg, from about 16 mg to about 24 mg, from about 17 mg to about 23 mg, from about

[0146] 18 mg to about 22 mg, or from about 19 mg to about 21 mg, or from 13 mg to 17 mg, or from 14 mg to

[0147] 16 mg, or from 18 mg to 22 mg, or from 19 mg to 21 mg of estetrol monohydrate, or an amount of estetrol component equivalent to a daily dose of from about 12 mg to about 28 mg, from about 13 mg to about 27 mg, from about 14 mg to about 26 mg, from about 15 mg to about 25 mg, from about 16 mg to about 24 mg, from about 17 mg to about 23 mg, from about 18 mg to about 22 mg, or from about

[0148] 19 mg to about 21 mg, or from 13 mg to 17 mg, or from 14 mg to 16 mg, or from 18 mg to 22 mg, or from 19 mg to 21 mg of estetrol monohydrate. In yet alternative embodiments, the composition comprises from about 12 mg to about 28 mg, from about 13 mg to about 27 mg, from about 14 mg to about 26 mg, from about 15 mg to about 25 mg, from about 16 mg to about 24 mg, from about 17 mg to about 23 mg, from about 18 mg to about 22 mg, or from about 19 mg to about 21 mg, or from 13 mg to 17 mg, or from 14 mg to 16 mg, or from 18 mg to 22 mg, or from 19 mg to 21 mg of estetrol or an ester thereof, or an amount of estetrol component equivalent to a daily dose of from about 12 mg to about 28 mg, from about 13 mg to about 27 mg, from about 14 mg to about 26 mg, from about 15 mg to about 25 mg, from about 16 mg to about 24 mg, from about 17 mg to about 23 mg, from about 18 mg to about 22 mg, or from about 19 mg to about 21 mg, or from 13 mg to 17 mg, or from 14 mg to 16 mg, or from 18 mg to 22 mg, or from 19 mg to 21 mg of estetrol or an ester thereof.

[0149] Optionally, the composition comprises about 15 mg of estetrol component (e.g. from 13 to 17 mg or from 14 to 16 mg), or an amount equivalent to about 15 mg of estetrol. Optionally, the composition may comprise about 15 mg of estetrol. In further optional embodiments the composition comprises about 15 mg of estetrol monohydrate.

[0150] Alternatively, the composition comprises about 20 mg of estetrol component (e.g. from 18 to 22 mg or from 19 to 21 mg), or an amount equivalent to about 20 mg of estetrol. Optionally, the composition may comprise about 20 mg of estetrol. In further optional embodiments the composition comprises about 20 mg of estetrol monohydrate. The estetrol component may be comprised as a multitude of particles in the composition. The particle size of the estetrol component is not particularly limiting. By means of illustration and not limitation, suitable particle sizes may be expressed by means of particle-size distribution values such as but not limited to D(10), D(50), and D(90). A skilled person is well aware how to interpret these parameters. “Particle-size distribution”, commonly abbreviated as “PSD” is a numerical value expressing a relative amount of particles according to size, wherein the relative amount of particles is preferable expressed by mass. For example, the DIO or Dv(10) value signifies the point in the size distribution, up to and including which, 10% of the total volume of the sample (i.e. a collection of particles) is contained. For example, a DIO of 10 pm means that 10% of the sample has a size of maximum 10 pm. The D10, D50, and D90 values are routinely used in the art to calculate the span of a sample, which indicates the width of the size distribution. The span is calculated according to the following formula: (D90 - D10) / D50. A skilled person appreciates that representative particle-size distribution values can only be derived from a representative sample.

[0151] Optionally, the estetrol particles have a D(10) from about 0.5 pm to about 10 pm, preferably from about 1 pm to about 5 pm, more preferably of from about 1.5 pm to about 2.5 pm. Optionally, the estetrol particles have a D(50) of less than 20 pm, preferably less than 12 pm, more preferably from about 5 pm to about 15 pm, preferably from about 6 pm to about 12 pm, more preferably from about 7 pm to about 11 pm, most preferably from about 8 pm to about 12 pm. Optionally, the estetrol particles have a D(90) from about 15 pm to about 50 pm, preferably from about 20 pm to about 30 pm, more preferably from about 22 pm to about 28 pm.

[0152] A multitude of measurement techniques are available for determining particle-size distribution values and include sieve analysis, air elutriation analysis, photo analysis, optical counting, electro resistance counting, sedimentation, laser diffraction, laser obscuration, time of transition, acoustic spectroscopy, ultrasound attenuation microscopy, by means of a cascade impactor, or any combination thereof. Unless explicitly mentioned otherwise, the particle-size distribution values of the present disclosure are obtained by laser diffraction analysis. Laser diffraction analysis, interchangeably annotated in the art by laser diffraction spectroscopy, is a particle measurement technology based on interpretation of laser diffraction patterns passed through an object. Laser diffraction is capable to measure the geometrical dimensions of a particle. Laser diffraction protocols have been described in detail in the art on numerous occasions (e.g. as reviewed in detail in a context of particle analysis in Eshel et al., Soil Science Society of America Journal, 2004).

[0153] The compositions and dosage units described herein are envisaged for use in treating or preventing Alzheimer’s disease symptoms. Evidently, this includes any closely related indications which have a considerable chance of being diagnosed interchangeably with Alzheimer’s disease, or indications that are generally considered to be a pre-clinical stage of Alzheimer’s disease. Such indications include by means of illustration mild cognitive impairment (MCI), subjective memory complaints (SMCs), verbal memory decline, and the like.

[0154] Hence, the invention equally envisages a composition for use in the prevention and / or treatment of mild cognitive impairment symptoms, wherein said composition comprises an estetrol component and wherein said composition is administered at a daily amount equivalent to from about 15 mg to about 25 mg of estetrol.

[0155] It is evident that any of the compositions and dosage units may suitably contain one or more pharmaceutically acceptable excipients. The term “pharmaceutically acceptable” as used herein is consistent with the art and means compatible with the other ingredients of a pharmaceutical composition and not deleterious to the recipient thereof.

[0156] The compositions and dosage units described herein may comprise in addition to the estetrol component one or more additionally active pharmaceutical ingredients that are considered to beneficial in treating or preventing one or more Alzheimer’s disease symptoms. For example, the compositions and dosage units described herein may be used in combination with, or comprise, one or more cholinesterase inhibitors. Illustrative cholinesterase inhibitors include without limitation galantamine, rivastigmine, and donepezil. Alternatively, the compositions and dosage units described herein may be used in combination with, or comprise, one or more immunotherapeutic agents. Illustrative examples include without limitation lecanemab and aducanumab. Yet alternatively, the compositions and dosage units described herein may be used in combination with, or comprise, one or more an N-methyl-D-aspartate (NMDA) antagonists.

[0157] The oral dosage unit described herein may be a solid or semi solid dosage unit such as a tablet, a capsule, a cachet, a pellet, a pill, powder, or granules, or any combination thereof. For example, the oral dosage unit subject of the invention may be a tablet comprising estetrol component-containing granules or a capsule comprising estetrol component-containing granules. The term "solid or semi-solid dosage unit" also encompasses capsules that contain a liquid, e.g. an oil, in which the present estetrol component and / or the optional progestogenic component is dissolved or dispersed.

[0158] Tablets and equivalent solid and semi-solid dosage units can suitably contain materials such as binders (e.g. hydroxypropylmethyl cellulose, polyvinyl pyrrolidone (povidone, PVP), other cellulosic materials and starch), diluents (e.g. lactose (monohydrate) and other sugars, starch (e.g. maize starch), dicalcium phosphate and cellulosic materials), disintegrating agents (e.g. starch polymers and cellulosic materials (e.g. sodium starch glycolate) and lubricating agents (e.g., (magnesium) stearates and talc). These tablets and equivalent solid dosage units may be prepared by any suitable means, which have been described in detail in the art (e.g. Kaur, Int Res J Pharm, 2012). Non-limiting examples of processing the estetrol component when manufacturing the dosage unit include wet granulation, e.g. using an aqueous solution or an organic solution, direct compression, 3D printing, or by coating carrier particles with the estetrol component using an organic or inorganic solvent.

[0159] As described above, the composition, and hence the (oral) dosage unit may comprise one or more suitable excipients. The term “excipient” as used interchangeably herein and in the art with “carrier” may be indicative for any solvent, diluent, buffer (including but not limited to neutral buffered saline, phosphate buffered saline, or optionally Tris-HCl, acetate or phosphate buffers), solubiliser (including but not limited to Tween 80 or Polysorbate 80), colloid, dispersion medium, vehicle, fdler, chelating agent (including but not limited to EDTA or glutathione), amino acid, protein, disintegrant, binder, lubricant, wetting agent, stabiliser, emulsifier, sweetener, colorant, flavoring, aromatiser, thickener, any agent suitable to achieve a depot effect, coating, antifungal agent, any preservative (including but not limited to Thimerosal™, benzalkonium chloride, or benzyl alcohol), antioxidant (including but not limited to ascorbic acid, sodium metabisulfite), tonicity controlling agent, absorption delaying agent, adjuvant, bulking agent (including but not limited to lactose, mannitol) and any other ingredient that may influence any parameter or characteristic of the oral dosage unit subject of the invention. A skilled person understands that one or more excipients may be used in the oral dosage unit on condition that the one or more excipient is compatible with the one or more pharmaceutical ingredient (i.e. in the context of the present invention at least the estetrol component) and that a pharmaceutically acceptable formulation is obtained.

[0160] In certain embodiments, the excipient may be an active pharmaceutical ingredient excipient, binder excipient, carrier excipient, co-processed excipient, coating system excipient, controlled release excipient, diluent excipient, disintegrant excipient, dry powder inhalation excipient, effervescent system excipient, emulsifier excipient, lipid excipient, lubricant excipient, modified release excipient, penetration enhancer excipient, permeation enhancer excipient, pH modifier excipient, plasticiser excipient, preservative excipient, preservative excipient, solubiliser excipient, solvent excipient, sustained release excipient, sweetener excipient, taste making excipient, thickener excipient, viscosity modifier excipient, filler excipient, compaction excipient, dry granulation excipient, hot melt extrusion excipient, wet granulation excipient, rapid release agent excipient, increased bioavailability excipient, dispersion excipient, solubility enhancement excipient, stabilizer excipient, capsule filling excipient, or any combination hereof. A skilled person is aware that use of such media and agents for pharmaceutical active substances is common practice and incorporation of these excipients is hence well known in the art. It is evident that all of the used ingredients should be non-toxic in the concentration contained in the final pharmaceutical composition and should not negatively interfere with the activity of the one or more pharmaceutically active ingredients, in this context at least the estetrol component.

[0161] Optionally, the composition is comprised in a tablet and comprises next to estetrol excipients fulfilling the functions of a first or a further filler, superdisintegrant, binder, disintegrant, and lubricant. An excipient can perform several of these functions. So can a binder also be a disintegrant. A tablet can also contain two or more excipients that perform the same function, such as two different binders. In preferred embodiments, the composition is comprised in a tablet comprising estetrol, lactose, sodium starch glycolate, maize / com starch, povidone, and magnesium stearate. Preferably, the composition is comprised in a tablet comprising estetrol monohydrate, lactose monohydrate, sodium starch glycolate type A, maize / com starch, povidone K30, and magnesium stearate. Optionally, the tablet is coated with a coating agent. In further optional embodiments, the coating agent comprises hypromellose, hydroxypropylcellulose, titanium dioxide, red iron oxide, hydrogenated cottonseed oil, and talc. By means of illustration and not limitation, suitable coating agents are AquaPolish orange 034.23 MS, AquaPolish P blue 064.65 MS, AquaPolish yellow 024.15 MS or AquaPolish pink 044.08 MS. The skilled person is aware that such coatings may be used in combination with a suitable amount of purified water. A skilled person further appreciates that any excipients present in any dosage unit such as an oral dosage unit should adhere to pharmaceutical grade industry quality standards such as Ph. Eur. and USP- NF.

[0162] The dosage unit may be suitable or even specifically manufactured for sublingual, buccal, and / or sublabial administration (i.e. implying a prolonged presence in the oral cavity of the subject). In such embodiments, the solid dosage unit is able to rapidly release the estetrol component when contacted with an aqueous solvent such as saliva. Hence, in these embodiments the solid dosage unit is an orodispersible dosage unit which releases at least about 50%, preferably at least about 60%, more preferably at least about 70%, yet more preferably at least about 80%, most preferably more than about 80% of the estetrol component within about 5 minutes, preferably within about 3 minutes, more preferably within about 2.5 minutes, more preferably within about 90 seconds, most preferably within about 90 seconds. The dosage unit may be an orodispersible dosage unit. In such embodiments, the dosage unit rapidly disintegrates in the oral cavity when it comes into contact with saliva and to disperse the estetrol component into the saliva so it may be absorbed through the mucosal lining of the oral cavity. A skilled person is aware of methods to determine the release rate of an estetrol component from a dosage unit. Non-limiting standardized tests generally accepted in the field include the 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.

[0163] The term “sublingual” as used herein refers to the pharmacological route of administration by which the estetrol component diffuses into the blood through tissues under the tongue.

[0164] The term “buccal” as used herein refers to the pharmacological route of administration by which the estetrol component diffuses into the blood through tissues of the buccal vestibule, the area inside the mouth between the lining of cheek (the buccal mucosa) and the teeth / gums. The term “sublabial” as used herein refers to the pharmacological route of administration by which the estetrol component is placed between the lip and the gingiva.

[0165] In certain embodiments, the estetrol component is comprised in an immediate release dosage unit or composition.

[0166] In certain embodiments, the estetrol component is formulated into a solid dosage unit, including but not limited to hard capsules, soft capsules, tablets, coated tablets such as lacquered tablets or sugar-coated tablets, granules, aqueous or oily solutions, syrups, emulsions, suspensions, ointments, pastes, lotions, gels, inhalants or suppositories.

[0167] As indicated above, the term “oral administration” intends to imply oral ingestion, i.e. Swallowing. In embodiments wherein the effective amount of an estetrol component is administered by means of oral administration, the oral dosage unit according to the invention is preferably a solid or semi-solid dosage unit such as tablets, capsules, cachets, pellets, pills, powders and granules. The term "solid or semisolid dosage unit " also encompasses capsules that contain a liquid, e.g. an oil, in which the present estetrol component and / or the optional progestogenic component is dissolved or dispersed. Tablets and equivalent solid and semi-solid dosage units can suitably contain materials such as binders (e.g. hydroxypropylmethyl cellulose, polyvinyl pyrrolidone, other cellulosic materials and starch), diluents (e.g. lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g. starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc). These tablets and equivalent solid dosage units may be prepared by any suitable means, which have been described in detail in the art (e.g. Kaur, Int Res J Pharm, 2012). Non-limiting examples of processing the estetrol component when manufacturing the dosage unit include wet granulation, e.g. using an aqueous solution or an organic solution, direct compression, 3D printing, or by coating carrier particles with the estetrol component using an organic or inorganic solvent.

[0168] By means of illustration and not limitation, an oral dosage unit comprising the composition subject of the present disclosure may be manufactured by a process involving wet granulation. A skilled person appreciates that a wet granulation process may suitable comprise the successive steps of: dispensing and sieving of the active ingredient(s) and excipients, blending the sieved materials in a processor, granulation, screening (i.e. further sieving) of the granules, and one or more blending steps of the sieved granules with one or more further excipients. Afterwards, if desired in view of the final dosage unit the granules may be compressed into for example a tablet, optionally involving a coating step of the tablets.

[0169] In certain embodiments, the estetrol component is the sole (i.e. single, only) pharmaceutically active ingredient part of the composition. The term “pharmaceutically active ingredient”, interchangeably used throughout the present disclosure with “pharmaceutically active agent” is to be interpreted according to the definition of the term by the World Health organisation: “a substance used in a finished pharmaceutical product (FPP), intended to display pharmacological activity or to otherwise have direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease, or to have direct effect in restoring, correcting or modifying physiological functions in human beings”. More particularly, in certain embodiments no progestogenic component is co-administered with the estetrol component (neither in the same composition or oral dosage unit nor in a further composition or dosage unit that is co-administered). In embodiments wherein the hepatically impaired subject underwent hysterectomy, the estetrol component is preferably administered as sole pharmaceutically active ingredient.

[0170] In alternative embodiments, the composition comprises at least one further pharmaceutically active ingredient in addition to the estetrol component.

[0171] Optionally, the dosage form comprises as a further pharmaceutically active ingredient a progestogenic component, or the method of treatment comprises a step of co-administering a progestogenic component to the subject. While embodiments wherein the progestogenic component is comprised in the same composition as the estetrol component are preferred, embodiments wherein the progestogenic component is administered by means of a separate composition or dosage unit are equally envisaged.

[0172] The terms “progestogen”, “gestagen”, or “gestogen” and derived hereof “progestogenic components” as used both herein and in the art refer to any molecule that produces effects similar to those of the natural female sex hormone progesterone in the body of a subject. Progestogens are considered to be agonists of the progesterone receptors and their functions have been thoroughly examined in the art (inter alia discussed in Kuhl, Climacteric, 2005). Progestins are a subgroup of progestogens that comprise synthetic progestogens. While the above terms may be used interchangeably in the art, there is a general understanding that when progestin is mentioned, synthetic progestogens are meant.

[0173] Examples of progestogenic components envisaged by the invention include without limitation: levonorgestrel, norgestimate, norethisterone, dydrogesterone, drospirenone, 3-beta- hydroxydesogestrel, 3-ketodesogestrel, 17-deacetylnorgestimate, 19-norprogesterone, acetoxypregnenolone, allylestrenol, amgestone, chlormadinone, cyproterone, demegestone, desogestrel, dienogest, dihydroge sterone, dimethisterone, ethisterone, ethynodiol diacetate, fluorogestone acetate, gastrinone, gestodene, gestrinone, hydroxymethylprogesterone, hydroxyprogesterone, lynestrenol, meciroge stone, medroxyprogesterone, megestrol, melengestrol, nomegestrol, norethindrone, norethynodrel, norgestrel (including d-norgestrel, and dl-norgestrel), norgestrienone, normethisterone, progesterone, quingestanol, ( 17a)- 17-hydroxy- 11 -methylene- 19- norpregna-4, 15-dien-20-yn-3-one, tibolone, trimegestone, algestone-acetophenide, nestorone, promegestone, 17-hydroxyprogesterone esters, 19-nor-17hydroxyprogesterone, 17alpha- ethynyltestosterone, 17alpha-ethynil- 19-nortestosterone, d- 17beta-acetoxy- 13beta-ethyl- 17alpha- ethynylgon-4-en-3-one oxime, 6beta, 7beta;15beta,16beta-dimethylene-3-oxo-17-pregna-4,9(ll)- diene-21, 17beta-carbolactone or tanaproget and precursors of these components that are capable of liberating these progestogens in vivo. The progestogenic components may be selected from the group comprising: progesterone, drospirenone, norethisterone, norethisteron-acetate (NETA), dydrogesterone, levonorgestrel (LNG), etonogestrel, norgestrel, nomegestrol, nomegestrol-acetate (NOMAC), trimegestone, nestorone, dydrogesterone, gestodene, desogestrel, norgestimate, cyproterone acetate, dienogest, and chlormadinone. Particularly preferred progestogens in the present context include without limitation drospirenone, progesterone and dydrogesterone.

[0174] In certain embodiments, the progestogen is a naturally occurring progestogen. In alternative embodiments, the progestogen is a progestin.

[0175] “Drospirenone” (abbreviated as DRSP, PubChem CID: 68873) is an example of a progestogenic component and enjoys a widespread use in Combined Oral Contraceptives (commonly abbreviated as COCs) due to its antimineralocorticoid and antiandrogenic activity combined with a general low off- target activity. In general, drospirenone-containing COCs are referred to as fourth generation COCs. Non-limiting examples of commercially available COCs comprising drospirenone are known as “Yaz®” and “Yasmin®”. An illustrative example of a drospirenone only progestogen pill is “Slynd®”, which is also commercially available. Additionally, hormone replacement therapy compositions comprising an estrogen such as estradiol and drospirenone are available such as “Angeliq®”. Drospirenone may alternatively be indicated in the art by its molecular formula C24H30O3, or by the structural formula (III):

[0176] Formula (III)

[0177] It is understood that when the term “drospirenone” is used herein, any drospirenone derivatives are also envisaged.

[0178] The methods described herein may include the administration of drospirenone to the subject receiving the estetrol component. In certain embodiments, the composition may comprise from about 0.25 mg to about 10 mg drospirenone, or a progestogenic component in an amount equivalent to a daily dose from about 0.25 mg to about 10 mg drospirenone. Preferably, the composition may comprise from about 1 mg to about 4 mg drospirenone or a progestogenic component in an amount equivalent to a daily dose from about 1 mg to about 4 mg drospirenone. More preferably, the composition may comprise from about 1 mg to about 3 mg of drospirenone, or a progestogenic component in an amount equivalent to a daily dose from about 1 mg to about 3 mg drospirenone. Yet more preferably, the composition may comprise from about 2.5 mg to about 3.5 mg of drospirenone, or a progestogenic component in an amount equivalent to a daily dose of from about 2.5 to about 3.5 mg of drospirenone.

[0179] Optionally, the composition comprises an estetrol component, preferably estetrol or estetrol monohydrate, in an amount from about 15 mg to about 25 mg and drospirenone in an amount of from about 0.25 mg to about 10 mg. In further optional embodiments, the composition comprises from about 15 mg to about 20 mg of an estetrol component, preferably estetrol or estetrol monohydrate, and from about 1 mg to about 4 mg drospirenone. In yet further optional embodiments, the composition comprises about 15 mg or about 20 mg of an estetrol component, preferably estetrol or estetrol monohydrate, and about 3 mg of drospirenone.

[0180] “Progesterone” (commonly abbreviated as “P4”; PubChem CID 5994) is an endogenous steroid and progestogen sex hormone involved in the menstrual cycle, pregnancy, and embryogenesis of women and constitutes the major progestogen in the body. Progesterone is a well-documented substance and has been used in the art for indications including but not limited to contraception, female hormone replacement therapy, and feminizing hormone therapy. Progesterone may be indicated in the art by reference to its structural formula C21H30O2, or by the structural formula (IV):

[0181] Formula (IV)

[0182] It is understood that when the term “progesterone” is used herein, any progesterone derivatives are also envisaged.

[0183] In certain embodiments, the composition may comprise from about 10 mg to about 500 mg, preferably from about 25 mg to about 300 mg progesterone, or a progestogenic component in an amount equivalent to a daily dose from about 10 mg to about 500 mg, preferably from about 25 mg to about 300 mg progesterone. Preferably, the composition may comprise from about 100 mg to about 200 mg progesterone or a progestogenic component in an amount equivalent to a daily dose from about 100 mg to about 200 mg progesterone. Optionally, the composition comprises an estetrol component, preferably estetrol or estetrol monohydrate, in an amount from about 15 mg to about 25 mg and progesterone in an amount of from about 25 mg to about 300 mg. In further optional embodiments, the composition comprises from about 15 mg to about 20 mg of an estetrol component, preferably estetrol or estetrol monohydrate, and from about 100 mg to about 200 mg progesterone. In yet further optional embodiments, the composition comprises about 15 mg or about 20 mg of an estetrol component, preferably estetrol or estetrol monohydrate, and from about 100 mg to about 200 mg of progesterone. In another embodiment, progesterone is used at a daily dose of 100 mg to 200 mg when it is used sequentially, for example when it is administered during about 14 days every month. In yet another embodiment, progesterone is used at a daily dose of 100 mg to 200 mg when it is used sequentially, for example when it is administered during about 14 days after the estetrol component has been administered for a minimum of 12 weeks and no longer than 13 weeks. Preferably, progesterone is used once daily for 14 consecutive days after completion of treatment with the estetrol component.

[0184] The progestin “dydrogesterone” (PubChem CID 9051), interchangeably indicated in the art such as “isopregnenone” and “didrogesteron”, has been used for numerous medical indications including dysfunctional bleeding, infertility, dysmenorrhea, endometriosis, and menopause hormone therapy. Dydrogesterone may be indicated in the art by reference to its structural formula C21H28O2 or by the structural formula (V):

[0185] It is understood that when the term “dydroge sterone” is used herein, any dydroge sterone derivatives are also envisaged.

[0186] In certain embodiments, the composition may comprise from about 1 mg to about 20 mg dydrogesterone, or a progestogenic component in an amount equivalent to a daily dose from about 5 mg to about 10 mg dydrogesterone. Preferably, the composition may comprise from about 1 mg to about 20 mg dydrogesterone or a progestogenic component in an amount equivalent to a daily dose from about 5 mg to about 10 mg dydrogesterone. Optionally, the composition comprises an estetrol component, preferably estetrol or estetrol monohydrate, in an amount from about 15 mg to about 25 mg and dydrogesterone in an amount of from about 1 mg to about 20 mg. In further optional embodiments, the composition comprises from about 15 mg to about 20 mg of an estetrol component, preferably estetrol or estetrol monohydrate, and from about 5 mg to about 10 mg dydrogesterone. In yet further optional embodiments, the composition comprises about 15 mg or about 20 mg of an estetrol component, preferably estetrol or estetrol monohydrate, and from about 5 mg to about 10 mg of dydrogesterone.

[0187] In the context of the present invention, other compounds (i.e. components, agents) may be used in conjunction with the estetrol component for administering to women who have an uterus. Selective Estrogen Receptor Modulators (SERMs) defines a category of such compounds, which are contemplated as useful complements to the estetrol component in the methods of the invention. A preferred SERM for use in the context of the present invention is bazedoxifene. In the methods and compositions further described herein, it has to be understood that when reference is made to a “progestogenic component”, such reference includes SERMs and in particular bazedoxifene. Preferably, the bazedoxifene is administered at a daily dose of from about 10 mg to 50 mg. More preferably, bazedoxifene is administered at a daily dose of from about 15 to about 25 mg. Most preferably, bazedoxifene is administered at a daily dose of about 20 mg.

[0188] The composition may be formulated into a dosage unit that is to be administered to a subject using any time interval deemed appropriate by a skilled person. A preferred administration scheme in the context of the present invention is a daily administration scheme (i.e. one administration every about 24 hours). In such embodiments, the composition described herein is therefore representative of a daily composition, a daily dosage unit. In alternative embodiments, the composition is formulated for a multiple dose per day administration scheme, which then accumulates to achieve the dose in line with the present invention, e.g. 15 to 25 mg of the estetrol component.

[0189] Optionally, the dosage unit is administered to a subject by means of a continuous administration schedule. The terms "continuous" and ’’continuously” as used herein, means that the dosage units are administered at relatively regular intervals, with no (therapeutically) significant interruptions. Naturally, minor interruptions may occur that do not affect the overall effectiveness of the present method, and indeed such aberrations are encompassed by the present invention. In a preferred embodiment, and more arithmetically, the administration regimen is deemed to be continuous if the longest interval between two subsequent administrations is not more than 3.5 times as long as the average interval. Even more preferably said longest interval is not more than 2.5 times, most preferably not more than 1.5 times as long as the average interval. By means of illustration and not limitation, the treatment strategies and method of treatments described herein preferably employ continuous administration of the estetrol component during a period of at least 10 days, preferably of at least 20 days. Alternatively, the dosage unit is administered to a subject by means of a sequential administration schedule. It is to be appreciated that the term “sequential” means an administration during, for example, 10 to 14 days each month or during 14 days every 3 months. Sequential administration schedules are particularly envisaged wherein a progestogenic component is part of the composition or treatment described herein.

[0190] A further aspect of the invention is directed to packaging units comprising the dosage units described herein. The packaging units may comprise at least 14, preferably at least 21, even more preferably at least 28, containers for holding separately packaged and individually removable dosage units, wherein each container comprises at least one dosage unit comprising of from about 15 to about 25 mg of an estetrol component. Preferably, the separately packaged and individually removable dosage units are oral dosage units. More preferably, each of the separately packaged and individually removable dosage units comprise about 15 or about 20 mg of estetrol component, preferably about 15 mg or 20 mg of estetrol or estetrol monohydrate.

[0191] Optionally, the packaging units additionally comprise at least 10, preferably 12, more preferably 14, additional containers for holding separately packaged and individually removable dosage units, wherein each additional container comprises at least one dosage unit comprising a progestogenic component. Optionally, each of the additional containers for holding the dosage units comprising the progestogenic component are individually visually arranged next to a container holding a dosage unit comprising the estetrol component when these two dosage units have to be administered on the same day. Optionally, the packaging unit additionally comprises the same number of additional containers for holding separately packaged and individually removable oral dosage forms, wherein each additional container comprises at least one daily, preferably solid, oral dosage form comprising a progestogen, preferably wherein said progestogen is selected from drospirenone, progesterone and dydrogesterone.

[0192] A skilled person will understand that the embodiments described above which are directed to packaging units may equivalently be presented as a kit-of-parts containing a first packaging unit, e.g. a blister pack, containing the daily oral dosage units comprising the estetrol component, and a second, distinct, packaging unit, e.g. a second, distinct, blister pack, containing the daily oral dosage units comprising the progestogen.

[0193] The skilled person will additionally know that, within the scope of the present invention, each packaging unit, e.g. blister pack, may be numbered or otherwise marked.

[0194] The packaging units may be provided in any suitable packaging means known in the art, non-limiting examples being troches, sachets, pouches, bottles, films, sprays, microcapsules, implants, rods or blister packs. By means of illustration and not limitation, each packaging unit may be a sealed blister pack with a cardboard, paperboard, foil plastic backing and enclosed in a suitable cover. Also envisaged in any one of the aspects defined herein are packaging units such as bottles. The material of the bottle is not particularly limiting. In preferred embodiments, the bottle is a glass bottle characterised by a colour capable of reducing or preventing degradation of the contents of the bottle by e.g. UV light while maintaining a degree of transparency that allows for visual inspection of the contents of said bottle. Suitable colours include without limitation amber, cobalt, or vintage green.

[0195] In a particular embodiment of the invention the packaging unit comprises 28 containers or a multiple of 28 containers, such as 2 to 12 multiple of 28 containers.

[0196] While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art in light of the foregoing description. Accordingly, it is intended to embrace all such alternatives, modifications, and variations as follows in the spirit and broad scope of the appended claims. The herein disclosed aspects and embodiments of the invention are further supported by the following non-limiting examples. The following specific experimental examples are provided in support of the claimed invention but are not to be seen as limiting the scope of the invention.

[0197] EXAMPLES

[0198] Example 1: Estetrol effect on neuronal survival upon growth factor withdrawal in primary cortical neurons from C57BL / 6 mice

[0199] 1. Introduction

[0200] 1.1 Aim of the Study

[0201] The aim of the study was to investigate the effect of estetrol (E4) at 5 concentrations and estradiol (E2) at 1 concentration on neuronal survival in primary cortical neurons from C57BL / 6 mice upon growth factor withdrawal.

[0202] 1.2 Study Design

[0203] Primary cortical neurons from E18 mouse pups were isolated and seeded in 96-well plates in phenol red free cell culture medium and B-27 supplement. On day in vitro (DIV) 8, a full medium change was carried out and B-27 free medium was added (growth factor withdrawal). Primary cortical neurons were then treated with E4 (Test Item; TI) at 5 different concentrations or E2 (Reference Item; RI) at 1 concentration for 28 hours. Vehicle control (VC) and lesion control (LC) were included in the experiment. Cell viability and cytotoxicity assays were performed after one time point (28 hours after growth factor removal, DIV9) by MTT assay and LDH assay, respectively. The experiment was performed in 6 technical replicates per condition (in total n=48). 1.3 Outcome summary

[0204] Growth factor withdrawal (LC) for 28 h led to a significant reduction of viability accompanied by a significant increase in LDH release (indicator of increased cell death) compared to the vehicle control (VC). Treatment with TI E4 led to a significant increase in cell viability at the 100 nM concentration, while significant reduction in LDH release was detected for 1 nM and 1 pM concentrations, when compared to the lesion control (LC). This is in favor of a neuroprotective effect for E4.

[0205] 2 Materials and Methods

[0206] 2.1 Preparation of primary mouse cortical neurons

[0207] Primary cortical neurons were prepared from timed pregnant wild-type C57BL / 6 mice at El 8. Animals were sacrificed and embryos were dissected in Calcium and Magnesium free Hanks Balanced Salt Solution (CMF-HBSS) containing 15 mM HEPES and 10 mM NaHCO3, pH 7.2. Embryos were decapitated, skin and skull gently removed and hemispheres were separated. After removing meninges and brain stem, the cortices were isolated, chopped with a sterile razor blade in Chop solution (Hibemate-E without Calcium containing 2% B-27) and digested in 2 mg / mL papain (Worthington) dissolved in Hibemate-E without Calcium for 30 minutes (± 5 min) at 30°C. Cortices were triturated for 10- 15 times with a fire-polished silanized Pasteur pipette in Hibemate-E without Calcium containing 2% B-27, 0.01% DNasel, 1 mg / mL BSA, and 1 mg / mL Ovomucoid Inhibitor. Undispersed pieces were allowed to settle by gravity for 1 min and the supernatant was centrifuged for 3 min at 228 g. The pellet was resuspended in Hibemate-E containing 2% B-27, 0.01% DNasel, 1 mg / ml BSA, 1 mg / mL Ovomucoid Inhibitor and diluted with Hibemate-E containing 2% B-27. After the second centrifugation step (3 min at 228 g), the pellet was resuspended in nutrition medium (Neurobasal with phenol red, 2% B-27, 0.5 mM glutamine, 1% Penicillin-Streptomycin). Cells were counted in a hemacytometer and seeded in nutrition medium on poly-D-lysine pre-coated 96-well plates. Cells were cultured at 37°C; 95% humidity and 5% CO2. All wells were handled the same way.

[0208] 2.2 Growth factor removal and treatment

[0209] On the day of preparation (DIV1), cortical neurons were seeded in nutrition medium (Neurobasal with phenol red, 2% B-27, 0.5 mM glutamine, 1% Penicillin-Streptomycin) on poly-D-lysine pre-coated 96- well plates at a density of 3 x 104cells per well.

[0210] After 4-6 days, a half medium exchange using nutrition medium was carried out so that 180 pl medium were present in each well thereafter.

[0211] On DIV8, a full medium exchange to 160 pl B-27 free medium (Neurobasal without phenol red, 0.5 mM glutamine, 1% Penicillin-Streptomycin) was performed and 10 pl of 17x TI / RI stock solutions were applied thereafter. The experiment was carried out with n=6 technical replicates per condition, vehicle treated cells with B-27 (VC) and without B-27 (LC) served as controls.

[0212] TI / RI stock solutions were prepared in DMSO and further diluted in culture medium so that a maximum final concentration of 0.1% DMSO was present in the wells. Compound dilutions were prepared so that the same final DMSO concentration was present in all wells. The vehicle was 0.1% DMSO in culture medium.

[0213] After 28 h on B-27 free medium, cells were subjected to MTT and LDH assays (see sections 2.3 and 2.4).

[0214] 2.3. MTT assay

[0215] For the cell viability assay, the colorimetric MTT assay was performed. This assay allows the measurement of the mitochondrial dehydrogenase activity, which reduces yellow MTT (3-(4,5- Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide) to dark blue formazan crystals. Since this reaction was catalyzed in living cells only this assay was used for the determination of cell viability. MTT solution was added to each well in a final concentration of 0.5 mg / ml. After 2 h, the MTT containing medium was aspirated. Cells were lysed in 3% SDS and the formazan crystals were dissolved in isopropanol / HCl. Optical density was measured with a plate-reader at wavelength 570 nm. Cell survival rate was expressed as optical density (OD). Values were calculated as percent of control values (VC).

[0216] 2.4. LDH assay

[0217] Cytotoxicity was assessed in the supernatant using the Lactate dehydrogenase (LDH) assay. LDH is a stable cytoplasmic enzyme present in all cells and rapidly released into the cell culture supernatant when the plasma membrane is damaged. The LDH activity in the cell culture supernatant was determined by a coupled enzymatic reaction in which the tetrazolium salt INT was reduced to formazan. The water- soluble formazan dye was used to determine the extent of cell death that occurred. 70 pl of cell culture supernatant was transferred to clear 96-well plates. 70 pl freshly prepared reaction mixture was added to each well and the mixture was incubated for approximately 1 h (± 30 min) at room temperature protected from light. Absorbance was measured at 492 nm and 620 nm as reference wavelength. Cell death was expressed as optical density (OD). Values of culture medium were subtracted as background control. Values were calculated as percent of control values (VC).

[0218] 3 Statistics

[0219] Basic statistical analysis was performed in Graph Pad Prism 9. Data are shown as % of vehicle control (%VC) and presented as mean + standard error of mean (SEM). Group differences were evaluated by One-way ANOVA followed by Dunnett's multiple comparisons test versus the lesion control (LC). 4 Results

[0220] As expected, growth factor withdrawal (LC) for 28 h led to a significant reduction of viability (Figure

[0221] IA) accompanied by a significant increase in LDH release (indicator of increased cell death) (Figure

[0222] IB) compared to the vehicle control (VC). Treatment with RI E2 revealed inconsistent results, with further reduced cell viability in the MTT assay compared to the lesion control (LC), while LDH release was also reduced. Treatment with TI E4 led to an increase in cell viability, reaching significance at the 100 nM concentration (Figure 1A), while significant reduction in LDH release was observed for 1 nM and 1 pM concentrations, when compared to the LC (Figure IB).

[0223] These results are in favor of a neuroprotective effect for E4.

[0224] Example 2: Estetrol effect on amyloid beta toxicity in SH-SY5Y cells

[0225] 1. Introduction

[0226] 1.1 Aim of the Study

[0227] The aim of the study was to analyze the effect of estetrol (E4) at 5 concentrations and estradiol (E2) at 3 concentrations on cell viability in SH-SY5Y cells (neuroblastoma cell line) upon amyloid beta toxicity induced by treatment with Api-42. The estetrol used in this experiment in in solution.

[0228] 1.2 Study Design

[0229] SH-SY5Y cells, seeded in 96-well plates, were treated with Ap 1-42 (5 pM) to induce amyloid beta toxicity. E4 (Test Item; TI) at 5 concentrations or E2 (Reference Item; RI) at 3 concentrations was added simultaneously with Ap 1-42. Vehicle control (VC) and Ap 1-42 lesion control (LC) were included in the experiment. Cell viability assay was performed after one time point (48 h) by MTT assay. The experiment was performed in 6 technical replicates per condition (in total n= 120).

[0230] 1.3 Outcome summary

[0231] Treatment with Ap 1-42 (LC) led to significantly reduced cell viability of approximately 35% compared to the vehicle control (VC). Treatment with the 2 lowest tested concentrations of TI E4 led to significantly increased cell viability compared to the LC, indicating a protection against amyloid beta toxicity.

[0232] 2 Materials and methods

[0233] 2.1 Cultivation and treatment of SH-SY5Y cells

[0234] SH-SH5Y cells were kept in culture medium (DMEM medium, 10% FCS, 1% NEAA, 1% L-Glutamine 200 mM, 100 pg / ml Gentamycin) until 80-90% confluency. Cells were maintained at 37°C, 95% humidity and 5% CO2. On day 1 (DIV1), cells were seeded in culture medium on 96-well plates at a cell density of 2.5 x 104cells per well.

[0235] On DIV3, medium was changed to 150 pl assay medium (DMEM medium without phenol red Gibco 31053028, 10% charcoal stripped FCS Gibco 12676029, 1% NEAA, 1% L-Glutamine 200 mM, 100 pg / ml Gentamycin) in all wells and treatments were performed.

[0236] TI / RI stock solutions were prepared in DMSO and further diluted in culture medium so that a maximum final concentration of 0.1% DMSO was present in the wells. Compound dilutions were prepared so that the same final DMSO concentration was present in all wells. The vehicle was 0.1% DMSO in culture medium.

[0237] For TI / RI treatment, 10 pl of 17x TI / RI stock solutions were added together with lOpl of pre-aggregated Api-42(see section 2.2).

[0238] Vehicle (0.1% DMSO) treated and Api-42 treated cells served as controls (vehicle control; VC and lesion control; LC, respectively).

[0239] Cells were maintained at 37°C, 95% humidity and 5% CO2 for 48 h when cell viability was determined by MTT assay (see Example 1).

[0240] 2.2 Afll-42 preparation and application

[0241] A stock solution of 170 pM Api-42 (Bachem, Cat. Number: H-8146) was prepared in 50 mM TrisBuffer, 150 mM NaCl, pH 7.4. After sonication for 2 minutes, 30 pl of Api-42 (170 pM) and 30 pl of ddH2O were combined to obtain a concentration of 85 pM. After aggregation at 4°C for 48 h, the solution was sonicated for another minute prior to application (10 pl to 160 pl). The aggregation degree or state was not measured.

[0242] The final concentration of Ap 1-42 on the SH-SH5Y cells was 5 pM.

[0243] The experiment was performed in n=6 technical replicates for all groups.

[0244] 3 Statistics

[0245] Basic statistical analysis was performed in Graph Pad Prism 9. Data are shown as % of vehicle control (%VC) and presented as mean + standard error of mean (SEM). Group differences were evaluated by One-way ANOVA followed by Dunnett's multiple comparisons test versus the lesion control (LC)

[0246] 4. Results

[0247] As expected, treatment with Ap 1-42 (LC) led to significantly reduced cell viability of approximately 35% compared to the non-lesioned vehicle control (VC, Figure 2). Treatment with the 2 lowest tested concentrations of TI E4 led to significantly increased cell viability compared to the lesion control (LC, Figure 2A). RI E2 was not able to significantly rescue the Api-42 treatment effect (Figure 2B).

[0248] These results indicate a protection against amyloid beta toxicity after treatment with E4.

[0249] Example 3: Estetrol effect on amyloid beta toxicity in primary hippocampal neurons from C57BL / 6 mice

[0250] 1. Introduction

[0251] 1.1 Aim of the Study

[0252] The aim of the study was to investigate the effect of estetrol (E4) at 5 concentrations and estradiol (E2) at 1 concentration in primary hippocampal neurons of C57BL / 6 mice upon amyloid beta toxicity induced with A 1-42 lesion.

[0253] 1.2 Study Design

[0254] Primary hippocampal neurons from E18 mouse pups were isolated and seeded in 96-well plates in phenol red free cell culture medium and B-27 supplement. On day in vitro (DIV) 8, Api-42 was preaggregated. On DIVIO, cells were treated with E4 (Test Item; TI) at 5 different concentrations or E2 at

[0255] 1 concentration (Reference Item; RI) and pre-aggregated Ap 1-42 mix was added simultaneously. Cells were cultivated until DIV 16. Vehicle control (VC) and lesion control (LC) were included in the experiment. Cell viability and cytotoxicity assays were performed after one time point (DIV 16) by MTT assay and LDH assay, respectively. The experiment was performed in 6 technical replicates per condition (in total n=48).

[0256] 1.3 Outcome summary

[0257] Treatment with Api-42 (LC) led to significantly reduced cell viability of approximately 35% compared to the vehicle control (VC). Treatment with 10 nM and 1 pM of TI E4 led to significantly increased cell viability compared to the lesion control (LC), as well as well as a trend towards reduction of LDH release, indicating a protection against amyloid beta toxicity.

[0258] 2 Materials and Methods

[0259] 2.1 Preparation of primary mouse hippocampal neurons

[0260] Primary hippocampal neurons were prepared from timed pregnant wild-type C57BL / 6 mice at E18. Animals were sacrificed and embryos were dissected in Calcium and Magnesium free Hanks Balanced Salt Solution (CMF-HBSS) containing 15 mM HEPES and 10 mM NaHCO3, pH 7.2. Embryos were decapitated, skin and skull gently removed and hemispheres were separated. After removing meninges and brain stem, the hippocampi were isolated, chopped with a sterile razor blade in Chop solution (Hibemate-E without Calcium containing 2% B-27) and digested in 2 mg / mL papain (Worthington) dissolved in Hibemate-E without Calcium for 30 minutes (± 5 min) at 30°C. Hippocampi were triturated for 10- 15 times with a fire-polished silanized Pasteur pipette in Hibemate-E without Calcium containing 2% B-27, 0.01% DNasel, 1 mg / mL BSA, and 1 mg / mL Ovomucoid Inhibitor. Undispersed pieces were allowed to settle by gravity for 1 min and the supernatant was centrifuged for 3 min at 228 g. The pellet was resuspended in Hibemate-E containing 2% B-27, 0.01% DNasel, 1 mg / ml BSA, 1 mg / mL Ovomucoid Inhibitor and diluted with Hibemate-E containing 2% B-27. After the second centrifugation step (5 min at 228 g), the pellet was resuspended in nutrition medium with glutamate (Neurobasal with phenol red, 2% B-27, 0.5 mM glutamine, 25 pM glutamate, 1% Penicillin-Streptomycin).

[0261] Cells were counted in a hemacytometer and seeded in nutrition medium with glutamate on poly-D- lysine pre-coated 96-well plates at a density of 4x104 cells / well. Cells were cultured at 37°C; 95% humidity and 5% CO2. All wells were handled the same way.

[0262] 2.2 Abeta aggregation and treatment

[0263] On the day of preparation (DIV1), hippocampal neurons were seeded in nutrition medium with glutamate (Neurobasal with phenol red, 2% B-27, 0.5 mM glutamine, 25 pM glutamate, 1% Penicillin- Streptomycin) on poly-D-lysine pre-coated 96-well plates at a density of 4 x 104cells per well.

[0264] After 4-6 days, a full medium exchange, using nutrition medium without glutamate and phenol red free neurobasal was carried out so that 200 pl medium were present in each well thereafter.

[0265] On DIV8, a stock solution of 340 pM Api-42 was prepared in 50 mM Tris-Buffer, 150 mM NaCl, pH 7.4. After sonication for 2 minutes, 30 pl of Api-42 (340 pM) and 30 pl of ddH2O were combined to obtain a concentration of 170 pM. After aggregation at 4°C for 48 h, the solution was sonicated for another minute prior to application. The aggregation degree or state was not measured.

[0266] On DIVIO, 120 pl medium per well were removed and 70 pl fresh medium were added to have 150 pl medium per well. Next, 10 pl of the TI / RI 17x stock solutions were applied, directly thereafter 10 pl of the pre-aggregated Api-42 were added to the cells. The final concentration of Api-42 on the cells was 10 pM. Vehicle (0.1% DMSO) treated and Api-42 treated cultures, containing 0.1% DMSO, served as vehicle control (VC) and lesion control (LC), respectively.

[0267] On DIV16, cells were subjected to MTT and LDH assays (see Example 1).

[0268] The experiment was performed in n=6 technical replicates for all groups.

[0269] 3 Statistics

[0270] Basic statistical analysis was performed in Graph Pad Prism 9. Data are shown as % of vehicle control (%VC) and presented as mean + standard error of mean (SEM). Group differences were evaluated by One-way ANOVA followed by Dunnett's multiple comparisons test versus the lesion control (LC). 4 Results

[0271] As expected, treatment with Ap 1-42 (LC)led to significantly reduced cell viability of approximately 35% compared to the vehicle control (VC, Figure 3A). As observed previously in internal studies of the test site, LDH release upon A i-42 addition is only slightly increased, pointing towards apoptotic, rather than necrotic processes. Still LDH assay was performed to monitor TI or RI related impacts. Interestingly, treatment with 10 nM and 1 pM of TI E4 led to significantly increased cell viability compared to the lesion control (LC). At these TI concentrations also LDH release showed a trend towards reduction but lacking statistical significance (p=0.13; Figure 3B). RI E2 was not able to significantly rescue the A i-42 lesion effect.

[0272] These results indicate a protection against amyloid beta toxicity after treatment with E4.

[0273] Example 4: Estetrol effect on Tau phosphorylation in SH-SY5Y-hTau441 Cells

[0274] 1. Introduction

[0275] 1.1 Aim of the Study

[0276] The aim of the study was to analyze the effect of Estetrol (E4) at 5 concentrations and Estradiol (E2) at 1 concentration on Tau phosphorylation in SH-SY5Y-hTau441 (P301L) cells.

[0277] 1.2 Study Design

[0278] SH-SY5Y-hTau441 (P301L) cells stably overexpress the longest Tau isoform 441 carrying the P301L mutation. SH-SY5Y-hTau441 (P301L) cells were cultivated and differentiated with Retinoic acid five days prior to the treatment. Afterwards, cells were seeded in 24 well plates and treated with E4 (Test Item; TI) at 5 concentrations or E2 (Reference Item 1; RI.l) at 1 concentration for 24 hours. Vehicle control (VC) and Preference iltem 2 (RI.2, CHIR99021, a GSK3 kinase inhibitor)were included in the experiment as controls. Evaluation of protein levels of total Tau and six phospho-Tau (pTau) sites (pTau 181, 202, 214, 231, 262 and 396) were performed by MSD analysis. The experiment was performed in 5-6 technical replicates per condition (in total n=42).

[0279] 1.3 Outcome summary

[0280] Total Tau levels were not significantly affected by any treatment compared to the vehicle control (VC). RI.2 CHIR, a GSK3P inhibitor, significantly reduced the levels of pTaul81, pTau231 and pTau396, and tended to reduce the level of pTau214, compared to the VC. Treatment with TI E4 led to a significant reduction of the levels of pTaul81, pTau202, pTau262 and pTau396 at 10 pM concentration. This is in favor of a reduced Tau phosphorylation after treatment with E4.

[0281] 2 Materials and Methods

[0282] 2.1 Cultivation and treatment of SH-SY5Y-hTau441 cells SH-SY5Y-hTau441(P301L) cells were kept in culture medium (DMEM medium, 10% FCS, 1% NEAA, 1% L-Glutamine, 100 pg / ml Gentamycin, 300 pg / ml Geneticin G-418) for ~2 days until 80-90% confluency. Next, cells were differentiated in culture medium supplemented with 10 pM retinoic acid (RA) for 5 days with medium changing every 2 to 3 days.

[0283] Prior to the treatment, cells were seeded onto 24-well plates at a cell density of 2 x 105cells per well. On the next day, medium was changed to 320 pl assay medium (phenolred free DMEM, 10% charcoal stripped FCS Gibco 12676029, 1% NEAA, 1% L-Glutamine, 100 pg / ml Gentamycin, 300 pg / ml Geneticin G-418). Thereafter, cells in 24 well plates were treated with TI, RIs or vehicle by addition of 20 pl from 17x stock. After 24 h of incubation, cells on 24-well plates were harvested. For this purpose, cells were washed once with cold PBS and harvested in 60 pl RIPA-Buffer [50 mM Tris pH 7.4, 1% Nonidet P40, 0.25% Nadeoxy-cholate, 150 mM NaCl, 1 mM EDTA supplemented with freshly added 1 pM NaF, 0.2 mM Na-orthovanadate, 80 pM Glycerophosphate, protease (Calbiochem) and phosphatase (Sigma) inhibitor cocktail] .

[0284] TI / RIs stock solutions were prepared in DMSO and further diluted in culture medium so that a final concentration of 0. 1% DMSO was present in the wells. Compound dilutions were prepared so that the same final DMSO concentration was present in all wells. The vehicle was 0.1% DMSO in culture medium.

[0285] Protein concentration was determined by BCA assay and samples were adjusted to a uniform total protein concentration. Total Tau and phosphorylated Tau levels were determined by Mesoscale Discovery (MSD, see sections 2.2 and 2.3). The experiment was performed in n=5 technical replicates for all groups (VC and RI.2 n=6).

[0286] 2.2 Total Tau, pTau231 and pTau262 MSD

[0287] Levels of total Tau as well as phosphorylated Tau at residues 231 and 262 in RIPA extracts from the SHSY5Y-hTau441(P301L) cells were determined by immunosorbent assay (Mesoscale Discovery) according to the instructions of the manufacturer. Data were evaluated in comparison to calibration curves provided in the kit and are expressed as pg per mb for total Tau or arbitrary units (AU) for pTau231 and pTau262. Total protein concentrations were determined using the BCA protein assay kit from Thermo Scientific, according to manufacturer’s protocol for normalization purposes.

[0288] For total Tau / pTau231 duplex assay, samples were applied at 3 pg / mL total protein concentration, and for pTau262 assay at 20 pg / mL (25 pl per well).

[0289] 2.3 pTau!81, pTau202, pTau214 and pTau396 MSD

[0290] 5 pL anti-human Tau (Tau-13, Biolegend; Cat.No 835201; QPS ID478) diluted at 1:200 in PBS was added to the electrode spot in each well of a 96 well HighBind plate and incubated for 1 h at room temperature (RT) without shaking. After the coating step, the plate was washed once with 150 pL Wash buffer (IxTBS + 0,02 % Tween 20) per well and blocked with blocking buffer (IxTBS + 5 % BSA) for 1 h at RT with shaking (400 rpm). After a washing step, 50 pL of diluted sample was added to each well and incubated for 2 h at RT with shaking (400 rpm), followed by washing of the plate three times. 25 pL of diluted detection antibody for pTau 181 (rabbit anti-Phospho-Tau (Thr 181), Cell Signaling, Cat.No 12885; QPS ID723; 1: 1000), pTau202 (rabbit anti-Phospho-Tau (Ser202), Abeam, Cat.No abl08387; QPS ID398; 1: 1000), pTau214 (rabbit anti-Phospho-Tau (Ser214)[DlQ2X]; Cell Signaling Cat.No. 77348S, QPS ID578; 1: 1000), or pTau396 (rabbit anti-phosphoTAU(Ser396) [EPR2731]; Abeam Cat.No. abl09390, QPS ID604; 1: 1000) was added to each well and incubated for 2 h at RT with shaking (400 rpm). After three times washing of the plate, 25 pL of diluted Sulfo-Tag antibody (goat anti-rabbit-IgG-SulfoTag) was added to the well and incubated for 1 h at RT with shaking (400 rpm). Thereafter, the plate was washed three times and 150 pL of 2x Read Buffer T with surfactant was added and pTau levels were read on Sector Imager. As a standard, protein lysates of untreated cells at various protein concentrations were used. pTau levels were given as arbitrary units. For pTaul81, pTau202 and pTau396, samples were applied at 10 pg / mL total protein concentration, and for pTau214 at 40 pg / mL (50 pl per well).

[0291] 3. Statistics

[0292] Basic statistical analysis was performed in Graph Pad Prism 9. Data are presented as mean + standard error of mean (SEM) and group differences were evaluated by one-way ANOVA versus vehicle control group (VC). Outliers were assessed using Grubbs test

[0293] 4. Results

[0294] Total Tau levels were not significantly affected by any treatment compared to the vehicle control (VC) (Figure 5A). pTaul81 levels were significantly reduced by RI.2 CHIR treatment as well as 10 pM and 10 nM TI E4 (Figure 5B). pTau202 levels were also significantly reduced by 10 pM and 10 nM TI E4, as well as RI. 1 E2 (Figure 5C).

[0295] No significant effects on pTau214 levels were detected, even though a strong tendency towards reduction were observed for 100 nM and 1 pM TI E4 as well as RI.2 CHIR (Figure 5D).

[0296] Phosphorylation of Tau on pTau231 site was significantly reduced upon RI.2 CHIR treatment compared to the VC (Figure 5E). No effect of TI E4 or RI.l E2 were observed.

[0297] In contrast, pTau262 levels were not affected by any RI, but significantly reduced levels were detected in 100 nM and 10 pM TI E4 treated cell lysates compared to the VC (Figure 5F). Phosphorylation of Tau on pTau396 site was significantly reduced upon RI.2 CHIR treatment compared to the VC (Figure 5G). Treatment with 10 nM and 10 pM TI E4 also led to a significant reduction of pTau396 levels.

[0298] These results are in favor of a reduced Tau phosphorylation after treatment with E4.

[0299] Example 5: Estetrol effect on Tau phosphorylation in primary cortical neurons from hTau mice

[0300] 1. Introduction

[0301] 1.1 Aim of the Study

[0302] The aim of the study was to analyze the effect of Estetrol (E4) at 5 concentrations and Estradiol (E2) at 1 concentration on Tau phosphorylation in primary cortical neurons obtained from the hTau mouse model.

[0303] 1.2 Study Design

[0304] Primary cortical neurons from El 8 hTau transgenic mouse pups were isolated. hTau mice express human Tau derived from a human PAC, Hl haplotype, known as 8c mice, while murine tau was knocked out by a targeted disruption of exon 1. Mice have a hybrid background of DBA, Swiss Webster, C57BL / 6 and 129 / SvJae.

[0305] Primary cortical neurons were seeded in 24-well plates in phenol red free DMEM media and B-27 supplement and treated with E4 (Test Item; TI) at 5 concentrations or E2(Reference Item 1; RI. l) at 1 concentration for 24 hours. Vehicle control (VC) and Reference Item 2 (RI.2, CHIR99021, a GSK3 kinase inhibitor) were included in the experiment as controls. Evaluation of protein levels of total Tau and six phospho-Tau (pTau) sites (pTau 181, 202, 214, 231, 262 and 396) were performed by MSD analysis. The experiment was performed in 5 technical replicates per condition (n=40).

[0306] 1.3 Outcome Summary

[0307] Total Tau levels were significantly increased after treatment with the highest concentration of TI E4, RI. l E2 and RI.2 CHIR compared to the vehicle control (VC). RI.2 CHIR, a GSK3P inhibitor, significantly reduced the levels of all six pTau sites compared to the VC.

[0308] Treatment with TI E4 led to a significant reduction in the levels of pTau214, pTau231 and pTau262 at 10 nM and 10 pM, but also to a significant increase in the levels of pTaul81, pTau202 and pTau396 at 1 nM. This is in favor of a concentration-dependent modulation of Tau phosphorylation after treatment with. 12

[0309] 2. Materials and Methods

[0310] 2.1 Isolation and Cultivation of Primary Mouse Cortical Neurons Primary cortical neurons were prepared from timed mated hTau mice at El 8. Animals were sacrificed and embryos were dissected in Calcium and Magnesium free Hanks Balanced Salt Solution (CMF- HBSS) containing 15 mM HEPES and 10 mM NaHCO3, pH 7.2. Embryos were decapitated, skin and skull gently removed and cortical hemispheres were separated.

[0311] Before further extraction steps, a small piece of tissue per embryo was collected to assess the genotype by PCR (see 2.2). Each embryonic brain was stored in clearly marked tubes at 4°C in dissection medium until genotyping results were available (max. 3 hours). Thereafter, the following steps were carried out whereas only transgenic embryos were pooled.

[0312] After removing meninges and brain stem, the cortex was isolated, chopped with a sterile razor blade in chop solution (Hibemate-E without Calcium containing 2% B-27) and digested in 2 mg / ml papain (Worthington) dissolved in Hibemate-E without Calcium for 30 minutes at 30°C. Cortices were triturated for 10-15 times with a fire-polished silanized Pasteur pipette in Hibemate-E without Calcium containing 2% B-27, 0.01% DNasel, 1 mg / ml BSA, and 1 mg / ml Ovomucoid Inhibitor. Undispersed pieces were allowed to settle by gravity for 1 min and the supernatant was centrifuged for 2 min at 228 g. The pellet was triturated in Hibemate-E containing 2% B-27, 0.01% DNasel, 1 mg / ml BSA, 1 mg / ml Ovomucoid Inhibitor and diluted with Hibemate-E containing 2% B-27. After the second centrifugation step, the pellet was resuspended in culture medium (Neurobasal phenol red free, 2% B-27, 0.5 mM glutamine, 1% Penicillin-Streptomycin).

[0313] 2.2 Genotyping of embryos

[0314] Genotyping of embryos was carried out by extracting DNA according to a quick protocol using 25 mM NaOH / O.2 mM EDTA, 98°C and 40 mM Tris-HCl pH 5.5 for neutralization. Thereafter, samples were diluted 1: 10 and subjected to PCR using strain specific primer.

[0315] 2.3 Cultivation and treatment ofhTAU cortical neurons

[0316] Cells were seeded onto 4x 24-well plates at a cell density of 1.5 x 105cells per well (DIV1) in complete, phenol red free medium (Neurobasal phenol red free, 2% B-27, 0.5 mM glutamine, 1% Penicillin- Streptomycin). On DIV4-6 a half medium exchange was performed so that 320 pl complete medium were present in each well thereafter.

[0317] TI / RIs stock solutions were prepared in DMSO and further diluted in culture medium so that a maximum final concentration of 0.1% DMSO was present in the wells. Compound dilutions were prepared so that the same final DMSO concentration was present in all wells. The vehicle was 0.1% DMSO in culture medium. In DIV8, cells in 24 well plates were treated with TI, RIs or vehicle by addition of 20 pl from 17x stocks. After 24 h of incubation (DIV9), cells on 24-well plates were harvested. For this purpose, cells were washed once with cold PBS and harvested in 60 pl RIPA-Buffer [50 mM Tris pH 7.4, 1% Nonidet P40, 0.25% Na-deoxy-cholate, 150 mM NaCl, 1 mM EDTA supplemented with freshly added 1 pM NaF, 0.2 mM Na-ortho-vanadate, 80 pM Glycerophosphate, protease (Calbiochem) and phosphatase (Sigma) inhibitor cocktail]. Protein concentration was determined by BCA assay and samples were adjusted to a uniform total protein concentration. Total Tau and phosphorylated Tau were determined by Mesoscale Discovery (MSD). Levels of pTau were expressed as normalized to the total Tau level in the respective samples.

[0318] The experiment was performed in n=5 technical replicates for all groups (VC and RI.2 n=6).

[0319] 2.4 Total Tau and pTau231, pTau262 MSD

[0320] Total Tau as well as phosphorylated Tau at residues 231 and 262 were determined as described in Example 4, with the following modifications. Samples were applied at a uniform total protein concentration of 15 pg / mL for total Tau / pTau231 Duplex assay, and 50 pg / mL for pTau262 assay. Data were evaluated in comparison to calibration curves provided in the kit and expressed as pg per pg for total Tau or normalized to the total Tau level in the respective samples for pTau231 and pTau262.

[0321] 2.5 pTau!81, pTau202, pTau214 and pTau396 MSD

[0322] Levels of phosphorylated Tau at residues 181, 202, 214 and 396 were determined as described in Example 4, with the following modification. Data were expressed as normalized to the total Tau level in the respective samples.

[0323] 3 Statistics

[0324] Basic statistical analysis was performed. Data are presented as mean + standard error of mean (SEM) and group differences were evaluated for each time point separately by One-way ANOVA with Dunnett's multiple comparisons test (post hoc test) compared to vehicle control (VC).

[0325] 4 Results

[0326] Assessment of total Tau levels revealed a significant increase after treatment with RI.l E2 and RI.2 CHIR, and also with the highest concentration of TI E4 compared to the vehicle control (VC, Figure 6A).

[0327] Analysis of pTaul81 levels revealed a significant reduction after RI.2 CHIR treatment compared to the respective VC, while treatment with TI E4 at 1 nM led to a significant increase (Figure 6B).

[0328] The levels of pTau202 were significantly reduced after treatment with RI.2 CHIR, while treatment with TI E4 at 1 nM led to a slight, but significant, increase (Figure 6C). pTau214 levels were significantly reduced after treatment with RI. l E2 and RI.2 CHIR, and also with TI E4 at 10 nM (Figure 6D).

[0329] Phosphorylation at Tau231 site was significantly reduced after treatment with RI.1 E2 and RI.2 CHIR, and with TI E4 at 10 pM and 10 nM (Figure 6E). pTau262 levels were significantly reduced after treatment with RI.2 CHIR and TI E4 at 10 pM (Figure 6F).

[0330] For pTau396 levels, a significant reduction was observed after treatment with RI.2 CHIR while treatment with TI E4 at 1 nM led to a significant increase (Figure 6G).

[0331] These results are in favor of a concentration-dependent modulation of Tau phosphorylation after treatment with E4.

[0332] Example 6: Estetrol effect on reactive oxygen species (ROS) production upon H2O2 treatment in SH- SY5Y cells

[0333] 1. Introduction

[0334] 1.1 Aim of the Study

[0335] The aim of the study was to analyze the effect of estetrol (E4) at 5 concentrations and estradiol (E2) at 1 concentration on oxidative stress and reactive oxygen species (ROS) production in SH-SY5Y cells (neuroblastoma cell line) upon treatment with H2O2.

[0336] 1.2 Study Design

[0337] SH-SY 5Y cells, seeded in 96-well plates, were treated with E4 (Test Item; TI) at 5 concentrations or E2 (Reference Item; RI) at 1 concentration in assay medium (phenol red-free DMEM media with charcoal stripped FCS) for three hours then treated with H2O2 (100 pM). Vehicle control (VC) and lesion control (LC) were included in the experiment. Oxidative stress assay (DCDFA assay) was performed at one time point after the treatment with H2O2 (3h). The experiment was performed in 6 technical replicates per condition (in total n=48).

[0338] 1.3 Outcome summary

[0339] Treatment with H2O2 (LC) led to a significant increase in ROS production compared to the vehicle control (VC). Treatment with TI E4 led to a significant reduction of ROS generation at the highest tested concentrations when compared to the lesion control (LC). This is in favor of a reduced production of ROS after treatment with E4.2 Materials and Methods - Cultivation and treatment of SH-SY5Y cells

[0340] SH-SH5Y cells were kept in culture medium (DMEM medium, 10% FCS, 1% NEAA, 1% L-Glutamine 200 mM, 100 pg / ml Gentamycin) until 80-90% confluency. Cells were maintained at 37°C, 95% humidity and 5% CO2.

[0341] On day 1 (DIV1), cells were seeded in culture medium on 96-well plates at a cell density of 2.5 x 104cells per well.

[0342] On DIV3, medium was changed to 160 pl assay medium (DMEM medium without phenol red Gibco 31053028, 10% charcoal stripped FCS Gibco 12676029, 1% NEAA, 1% L-Glutamine 200 mM, 100 pg / ml Gentamycin) in all wells and treatments were performed by addition of 10 pl 17x TI / RI stock. After 2.25 h treatment, cells were washed once in lx DCFDA assay buffer and thereafter incubated for 45 min at 37°C with 80 pL / well DCFDA solution at 10 pM (abl 13851, Abeam). During this time also 5 pl of 17x TI / RI stocks were added.

[0343] Thereafter, cells were again washed with lx DCFDA assay buffer and medium was changed to 150 pl assay medium (DMEM medium without phenol red Gibco 31053028, 10% charcoal stripped FCS Gibco 12676029, 1% NEAA, 1% L-Glutamine 200 mM, 100 pg / ml Gentamycin) in all wells and treatments were performed by addition of 10 pl 17x TI / RI stock. Directly thereafter 100 pM H2O2 lesion was applied by addition of 10 pl 17x stock.

[0344] After 3 h incubation with TI / RI+ H2O2, cells were submitted to DCFDA assay. This assay is based on the oxidation of 2’, 7’ -dichlorofluorescin diacetate (DCFDA) into the highly fluorescent 2’, 7’ - dichlorofluorescein (DCF) by ROS. The fluorescent signal was measured at 485 nm / 535 nm (Cytation 5, BioTek; SOPNEQU201).

[0345] TI / RI stock solutions were prepared in DMSO and further diluted in culture medium so that a final concentration of 0. 1% DMSO was present in the wells. Compound dilutions were prepared so that the same final DMSO concentration was present in all wells. The vehicle was 0.1% DMSO in culture medium.

[0346] 3 Statistics

[0347] Basic statistical analysis was performed in Graph Pad Prism 9. Data are presented as mean + standard error of mean (SEM) and group differences were evaluated by one-way ANOVA versus lesion control (LC). Outliers were assessed using Grubbs test.

[0348] 4 Results

[0349] As expected, treatment with H2O2 (LC) led to a significant increase in fluorescent signal compared to the vehicle control (VC), indicating an increased production of ROS (Figure 4). Treatment with TI E4 led to a significant reduction of ROS generation at the two highest tested concentrations when compared to the lesion control (LC). No effect of the treatment with RI E2 was detected.

[0350] These results are in favor of a reduced production of ROS after treatment with E4.

[0351] Example 7: Estetrol effect on neuronal bioenergetics in cellular models of Alzheimer’s disease

[0352] 1. Introduction

[0353] 1.1 Aim of the study Neuronal bioenergetics and mitochondrial function are disrupted early in the development of Alzheimer’s disease (AD), and maintaining normal glucose metabolism can be very beneficial in preventing the development of the disease (Mosconi et al., 2009, J Nucl Med Mol Imaging. May;36(5):811-22, Mosconi et al., 2021, Sci Rep. Jun 9;11(1): 10867).

[0354] Estrogens are known to regulate cell bioenergetic metabolism, including glucose homeostasis, mitochondrial function, as well as antioxidant defenses. Especially, previous studies have shown that estrogens (estradiol (E2) and estrone (El)) are able to boost mitochondrial function in vitro in neuronal cells (Grimm et al., Sci Rep. 2021 Jun 9;11(1): 10867; Grimm et al., Molecular Basis of Disease. 2014;1842(12):2427-2438).The purpose of this study was to assess the effect of the estrogen estetrol (E4) on neuronal bioenergetics in vitro under physiological condition or in cellular models of AD.

[0355] 1.2 Study design

[0356] The effect of E4 on neuronal bioenergetics were analyzed using three cellular models:

[0357] - SH-SY 5Y cells as control cells

[0358] - P30 IL mutant tau overexpressing cells as model of AD-related tauopathy

[0359] - Amyloid precursor protein (APP)-overexpressing SH-SY5Y cells as model of amyloid beta pathology

[0360] The following readouts were assessed using the indicated assays::

[0361] - Cell viability / metabolism: MTT assay

[0362] - ATP level: ATP bioluminescence assay

[0363] - Mitochondrial membrane potential (MMP): staining with fluorescent dye tetramethylrhodamine, methyl ester, and perchlorate (TMRM)

[0364] - Reactive oxygen species (ROS) level: staining with dihydroethdium (DHE) for assessment of total superoxide anion radicals level, and MitoSOX for assessment of mitochondria superoxide anion radicals level

[0365] - Mitochondrial respiration and cellular glycolysis: simultaneous real-time measurement of oxygen consumption rate (OCR, mitochondrial respiration) and extracellular acidification rate (ECAR, glycolysis) using the Seahorse Bioscience Analyser.

[0366] A first screening was performed to determine the potential toxic concentration range of E4 using a MTT assay, as well as the most efficient concentration(s) and treatment duration increasing cellular ATP level. The two best concentrations and the best treatment duration were selected and then used in all assays in the three cellular models. E2 was used as positive control.

[0367] 1.3 Outcome summary The initial screening revealed that a repeated treatment with E4 (2 x 24h) significantly increased both cell viability and ATP level in SH-SY 5Y cells (control cells). In control cells, ATP level and MMP were increased by E4 treatment, and the level of total and mitochondrial ROS was decreased. In a cellular model of AD-related tauopathy, E4 treatment increased cell viability, ATP level and MMP. All the remaining readouts are still under investigation.

[0368] The current results are in favor of a promotion of neuronal bioenergetics after treatment with E4 in healthy condition and in AD-related tauopathy. Similar results are predicted in amyloid beta pathology.

[0369] 2. Materials and Methods

[0370] 2.1 Cell lines

[0371] SH-SH5Y neuroblastoma cells is a “neuron-like” cell line widely used in neuroscience and suitable for drug screening. They were used in the initial screening experiments and to assess the effect of E4 on the bioenergetic phenotype of cells in normal (healthy) condition.

[0372] P301LTau-expressing SH-SY5Y human neuroblastoma cells were used as cellular model of AD-related tauopathy (Fairley et al., Spermidine Rescues Bioenergetic and Mitophagy Deficits Induced by Disease- Associated Tau Protein. International Journal of Molecular Sciences, 2023). Indeed, abnormal Tau hyperphosphorylation and aggregation in neurofibrillary tangles (NFTs) is a hallmark of AD brain. Tau mutations, such as P301Lhave been identified in the Tau (MAPT) gene and render Tau more susceptible to hyperphosphorylation, favor dissociation from microtubules, and further convey a greater propensity in promoting NFT aggregation, similarly to what is observed in AD where no MAPT mutations have been identified. Amyloid precursor protein (APP)-overexpressing SH-SY5Y cells were used as a cellular model of amyloid beta pathology.2.2 Cell culture conditions

[0373] Cells were grown at 37 °C in a humidified incubator chamber under an atmosphere of 5% CO2 in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% (v / v) heat inactivated fetal calf serum (FCS), 5% (v / v) horse serum (HS), 1% GlutaMax and 1% (v / v) penicillin / streptomycin. Cells were passaged 1-2 times per week and plated for treatment when they reached 80-90% confluence.

[0374] 2.3 Treatment paradigmCells were plated one day before treatment in 96 wells plate at a density of 1.5 x io4cells / well, with at least 5 replicates per condition. To limit cell growth and to optimize mitochondrial respiration, treatment medium contained only 5% FCS, and was supplemented with 3 mM pyruvate. Each assay was repeated at least 3-5 times.

[0375] In the initial screening, assessment of cell viability was performed on SH-SY 5Y neuroblastoma cells to determine the potential toxic concentration range of E4 (from 10 nM to 50.000 nM) using a MTT assay (see 2.4). Three treatment durations were tested: 24h, 48h, and 72h. In the following assays, cells were treated with a final concentration of E4 of 100 nM and 1000 nM. The treatment was repeated after 24 hrs (total treatment duration: 48 hrs). Estradiol (E2) at 100 nM was used as positive control in all experiments.

[0376] Stock solution of the E4 and E2 were prepared in DMSO (stock solution concentration = 10 mM). A treatment with DMSO alone was used as vehicle (Veh) control condition (final assay concentration of DMSO = 0.001%, which is the concentration of DMSO present in 1000 nM E4 and E2).

[0377] 2.4 Cell Viability Assay

[0378] Cell viability was investigated using a MTT assay. After treatment, the cells were incubated with 5 mg / ml MTT (3-(4,5-dimethylthyazol-2-yl)-2,5-diphenyl-tetrazolium bromide) in DMEM for 2 hours. MTT is reduced to a violet formazan derivative by mitochondrial enzymatic activity. At the end of the reaction, the cells were dissolved in 100% DMSO. MTT absorbance was measured at 550 nm using the multiplate reader Cytation 3 (BioTek).

[0379] 2.5 ATP levels

[0380] Total ATP content was determined using a bioluminescence assay (ATPlite Istep) according to the instructions of the manufacturer. Briefly, cells were plated in at least 5 replicates into white 96-well cell culture plates at a density of 1.5 x 104cells / well. The method measures the formation of light from ATP and luciferin catalyzed by the enzyme luciferase. The emitted light is linearly correlated to the ATP concentration and was measured using the multiplate reader Cytation 3 (BioTek).

[0381] 2. 6 Determination of mitochondrial membrane potential (MMP)

[0382] The MMP was measured using the fluorescent dye tetramethylrhodamine, methyl ester, and perchlorate (TMRM). Cells were plated in at least 5 replicates into a black 96-well cell culture plate at a density of 1.5 x io4cells / well. Cells were loaded with the dye at a concentration of 0.4 pM for 20 min. After washing twice with HBSS, the fluorescence was detected using the multiplate reader Cytation 3 (BioTek) at 530 nm (excitation) / 590 nm (emission). Fluorescence intensity of the dye is dependent on MMP.

[0383] 2. 7 Determination of reactive oxygen species (ROS) level

[0384] Total and mitochondrial superoxide anion radical levels were assessed using the dihydroethdium (DHE), and the red mitochondrial superoxide indicator (MitoSOX), respectively. Cells were plated in at least 5 replicates into black 96-well cell culture plates at a density of 1.5 x 104cells / well. After treatment, cells were incubated with 10 pM of DHE for 20 min or 5 pM of MitoSOX for 90 min at room temperature in the dark on an orbital shaker. After washing the cells three times with HBSS, the formation of red fluorescent products were detected at 531 nm (excitation) / 595 nm (emission). The intensity of fluorescence was proportional to the total and mitochondrial superoxide anion levels. The fluorescence was measured using the multiplate reader Cytation 3 (BioTek).

[0385] 3. Results

[0386] 3.1 Initial screening

[0387] In a first set of experiments, the effect of a broad range of E4 concentration (from 10 to 10.000 nM) was assessed on cell viability. MTT assays were therefore performed 24h, 48h and 72h after a single treatment with E4. In this condition, none of the E4 concentrations or treatment duration affected cell viability (Figure 7A left).

[0388] Next, the effectof a single treatment with E4 on ATP level was assessed after 24h, 48h or 72h. A significant increase in ATP level (+4 to 5%) was detected after a 48h treatment with all the tested doses (Figure 7A right).

[0389] Then, to test whether repeated E4 treatments influenced cell viability, cells were again treated for 24h, 48h, 72h, but this time treatment was renewed every 24h. The concentration of 50.000 nM E4 was also added to assess whether a higher E4 concentration had an impact on cell viability. In this condition, the E4 concentrations of 10, 100, and 1000 nM significantly increased cell viability after 48 h of treatment (+3 to 4% increase) (Figure 15B left). In contrast, the 50.000 nM concentration decreased cell viability already after 24h of treatment (-5%, -13% and -35% decrease after 24h, 48h and 72h, respectively).

[0390] Similar data were obtained when ATP level was assessed after repeated E4 treatment. The E4 concentrations of 100, and 1000 nM significantly increased ATP level after 48 h of treatment (+3 to 4% increase) (Figure 7B right). In contrast, the 50.000 nM concentration decreased ATP level already after 24h of treatment (-3%, -8% and -28% decrease after 24h, 48h and 72h, respectively).

[0391] Of note, the decrease in cell viability and ATP level after a 50.000 nM treatment with E4 could be attributed to the high DMSO concentration in this preparation (0.5%). Indeed, a treatment with 5% DMSO alone induced a decrease in cell viability and ATP level in the same range that the E4 treatment at 50.000 nM (data not shown). The concentration of DMSO present in the Vehicle group is 0.01% and correspond to the concentration present in the E4 group 1000 nM.

[0392] Taken together, these data indicate that a repeated E4 treatment of 2 x 24h significantly increased both cell survival and ATP level, while a single treatment with E4 increased only ATP level after 48h. According to both MTT and ATP test, the E4 concentrations of 100 nM and 1000 nM seemed to be the most efficient to increase both cell viability and ATP level after 2 x 24 h of treatment.

[0393] Similar experiments were performed with the positive control E2 (Figure 7C left). E2 at 100 nM and 1000 nM increased cell viability after 24h of treatment (+6 and + 7% respectively). E2 at 10 nM increased cell viability after 2 x 24 h of treatment (+5%). E2 at 10.000 nM decreased cell viability after 48 and 72 h of treatment (-18% and -16% respectively).

[0394] Regarding the effects of E2 on ATP level (Figure 7C right), results were the following: E2 at 10 nM, 100 nM and 1000 nM increased ATP level after 24h of treatment (+4%, +6% and +5% respectively). E2 at 100 nM increased ATP level after 2 x 24 h of treatment (+5%). E2 at 100 nM increased ATP level after 3 x 24 h of treatment (+5%).

[0395] 3.2 Control cellsln order to directly compare the effects of E4 and E2 on mitochondrial bioenergetics, ATP tests were repeated with the best E4 concentrations (100 nM and 1000 nM) and the best E2 concentration (100 nM). A 2 x 24h treatment was applied, based on the previous data obtained with E4, with a higher number of replicates per conditions. In this new batch of experiments, a 10% and 9% increase was detected in ATP level with 100 nM and 1000 nM E4, respectively, as well as a 10% increase with 100 nM E2 (Figure 8A).

[0396] MMP was measured using the same treatment conditions. Both E4 concentration induced a 23% increase in MMP, while E2 induced a 26% increase in MMP (Figure 8B). Of note, the effects of E2 were similar to those observed in former studies (Grimm et al., Sex hormone-related neurosteroids differentially rescue bioenergetic deficits induced by amyloid-P or hyperphosphorylated Tau protein, Cellular and Molecular Life Sciences 2015; Grimm et al., Improvement of neuronal bioenergetics by neurosteroids: Implications for age-related neurodegenerative disorders, Biochimica et Biophy sica Acta (BBA) - Molecular Basis of Disease, 2014).

[0397] An increase in mitochondrial bioenergetic activity is often coupled with an increase in ROS production, especially superoxide anion radicals, which are by-products of mitochondrial respiration. Therefore, the level of total and mitochondrial superoxide anion radicals was assessed after treatment with E4 and E2 (9). No significant change in total and mitochondrial superoxide anion radicals’ level was observed after treatment with 100 nM E4 nor with 100 nM E2. A significant decrease in total and mitochondrial superoxide anion radicals’ level was observed after treatment with 1000 nM E4 (-5% and -7%, respectively). These data indicate that the increase in ATP level and MMP induced by E4 and E2 is not coupled with an increase in ROS level, and that E4 may even have antioxidant effects decreasing ROS levels with a treatment concentration of 1000 nM.

[0398] 3.3 P 30 IL cells

[0399] To analyze whether E4 and E2 had a similar effect on cell viability and mitochondrial bioenergetics in a cellular model of AD-related tauopathy, the same parameters were assessed in SH-SY5Y cells expressing the P301LTau mutation (P301L cells). A significant increase in cell viability was observed in cells treated with E4 (100 nM and 1000 nM) or E2 (100 nM) when compared to Veh-treated cells (Figure 10A). Namely, P301L cell viability was increased by 4% with 100 nM E4 and by 5% with 1000 nM E4 and 100 nM E2, when compared to Veh-treated cells.

[0400] Regarding ATP level, a 5% increase was observed in P301L cells treated with 100 nM E4, 6% increase with 1000 nM E4, and only 2% increase with 100 nM E2, when compared to Veh-treated cells (Figure 10B).

[0401] The assessment of the MMP revealed a 11% increase in P301L cells treated with 100 nM E4, 38% increase with 1000 nM E4, and 50% increase with 100 nM E2, when compared to Veh-treated cells (Figure 10C).

[0402] Together, these data indicate that E4 increases cell viability and mitochondria bioenergetics in P301L cells. Even if the percentage of increase was different between E2 and E4 regarding their effects on ATP level and MMP, their efficacy was still comparable.

[0403] 4. Discussion and perspectives

[0404] The initial screening experiments revealed that a repeated treatment with E4 (2 x 24h) significantly increased both cell viability and ATP level in SH-SY5Y cells.

[0405] In control cells, ATP level and MMP were increased after treatment with E4 and E2 with similar efficacy. Treatment with E4 1000 nM but not with E2 could decrease the level of total and mitochondrial ROS.

[0406] In P301L cells, treatment with both E4 (100 nM and 1000 nM) and E2 increased cell viability, ATP level and MMP.

[0407] The following readouts are still under investigation:

[0408] - All previously described readouts in the cellular model of amyloid beta pathology

[0409] - ROS level in the P301L cells

[0410] Mitochondrial respiration and cellular glycolysis in all three cell lines. The current results indicate that a treatment with E4 could promote neuronal bioenergetics in healthy condition and in AD-related tauopathy. We expect to observe similar results in amyloid beta pathology.

[0411] Example 8: Estetrol effect in a zebrafish model of tauopathy

[0412] 1. Aim of the study

[0413] The aim of the study will be to test the impact of a treatment with estetrol (E4) on several aspects of pathological Tau behavior using the zebrafish TgA152T dendra-tau (A152T) expressing the pathological human variant A152T. This Tau mutation is associated with increased risk of Alzheimer's disease and frontotemporal dementia. 2. Background

[0414] The zebrafish is increasingly recognized as a model organism for translational research into human neuropathology (Chia et al., 2022, Front Mol Neurosci 15:940484). This vertebrate model allows the rapid assessment of drug actions and effects, and is used to study human neurodegenerative disease mechanisms. Advantages of this model are the ease of producing genetic mutants and expressing fluorescent markers that can be easily observed in vivo during the transparent larval stages over time. In this species, the nervous system develops rapidly and is already functional by 72h post-fertilization. Importantly, neural circuitries and neurochemical signal transduction pathways show similarities with the mammalian brain, and the zebrafish shows a high degree of genetic conservation.

[0415] Recently, a zebrafish line that expresses mutant Al 52T human tau (hTau-A152T) pan-neuronally has been generated. In humans, this tau variant increases the risk of Alzheimer's disease and frontotemporal dementia (Coppola et al., 2012, Hum Mol Genet 21:3500-3512; Lopez et al., 2017, Brain 140: 1128- 1146). In zebrafish larvae, the A152T mutation results in increased cellular apoptosis, neurodegeneration, and impaired locomotor behavior in response to stimuli when compared to wildtype hTau (hTau-WT). Moreover, Tau is hyperphosphorylated at multiple sites, shows conformational changes and forms aggregates in hTau-A152T fish. This zebrafish line thus provides a good model for studying the effects of protective agents in tau-associated pathology and obtaining mechanistic data (Lopez et al., 2017, Brain 140: 1128-1146).

[0416] A particular advantage is that both hTau-A152T and hTau-WT have been subcloned into pDendra2, which codes for the photoactivatable fluorescent protein Dendra2, allowing protein tracking in living larvae.

[0417] 3. Study design

[0418] Control and A152T embryos will be treated with E4 or E2 at different concentrations. DMSO will be used as vehicle to dilute the compounds and as a control (0.1% DMSO). Pharmacological inhibitors of estrogen receptors (ERs) will also be used to identify the targets of E4.

[0419] The first main task will be to optimize the treatment regarding the timing, the concentration and the duration of treatment.A likely scenario would be to treat embryos for 24 hours starting from 22 hours post fertilization (hpf) until 48 hpf. This would cover the time of neural development, axonal growth and neuronal circuit formation.

[0420] Once these parameters are optimized, the following outcomes of Tau pathology will be assessed:

[0421] - Tau hyperphosphorylation detection and quantification

[0422] - Neurodegeneration by assessment of neuronal death in the spinal cord

[0423] - Tau degradation by following the clearance of the photoconvertible Dendra-tau protein Motor neurons development and outgrowth

[0424] Locomotor behavior by assessment of the escape response to stimuli

[0425] 4. _ Predicted outcome

[0426] We predict to observe beneficial effect of the treatment with E4 on all studied parameters:

[0427] - Reduced tau phosphorylation

[0428] - Reduced neuronal death

[0429] - Improved tau degradation

[0430] - Improved motor neurons development and outgrowth

[0431] - Improved locomotor behavior

[0432] Altogether, these results will be in favour of a prevention of tauopathy development upon treatment with E4.

[0433] Example 9: Estetrol Effect in the E4FAD transgenic mouse model of AD

[0434] L _ Aim of the study

[0435] The purpose of this study will be to investigate the effect of a treatment with E4 on the development of symptoms associated with AD in the E4FAD transgenic mouse model of AD. This mouse model is a cross between the widely used 5xFAD (mutations associated with the familial form of AD) mouse and APOE- s4 targeted replacement mouse (the mice will have the human APO E-s4 allele). APOE-s4 is the strongest genetic risk factor for Alzheimer’s disease (AD), and is associated with an increase in the levels of amyloid deposition and an early age of onset (Di Battista AM et al., Curr Alzheimer Res. 2016;13(l 1): 1200-1207). _ Study design

[0436] Ovariectomized female mice will be treated with E4 or E2, and several read-outs will be assessed such as behavioral outcomes (cognition, memory), Ap deposition and insoluble / soluble A , gliosis, dosage of plasma markers (NfL, Ap).

[0437] 2 _ Predicted outcome

[0438] It will be observed that cognition and memory improve in mice treated with either E2 or E4. There will also be a positive influence on the other readouts. The effect with E4 will be at least comparable to E2 or even better.

[0439] Example 10: Analysis of the influence of E4 menopausal hormone therapy on the Alzheimer’s disease biomarkers pathophysiology.

[0440] L _ Objective of the analysis: To evaluate the effect of E4 on AD biomarkers (A[3 pathway, tau pathophysiology, neuronal loss, axonal damage and neurodegeneration) in postmenopausal women with or without APOE e4 allele carriers serum samples are analyzed, that were collected during a clinical study. Said study was designed to evaluate the effect of 15 or 20 mg estetrol (E4) monohydrate, or placebo on the severity and frequency of vasomotor symptoms (VMS) and the safety of E4 monohydrate 20 mg.

[0441] 2. _ Material and methods:

[0442] A randomized phase III clinical trial was designed to evaluate the effect of Estetrol (E4) monohydrate 15 or 20 mg, or placebo on the severity and frequency of vasomotor symptoms (VMS) (Efficacy Study Part) and the safety of E4 monohydrate 20 mg (Safety Study Part).

[0443] The first part was the Efficacy Study mainly designed to evaluate the frequency and severity of vasomotor symptoms [VMS] in both hysterectomized and non-hysterectomized postmenopausal participants after treatment with two doses of E4 monohydrate (15 mg or 20 mg) or placebo for 12 consecutive weeks. Thereafter, treatment proceeded for a total duration of up to 53 weeks, to continue the evaluation of secondary efficacy, safety and the effect on the endometrium. For endometrial protection, all non-hysterectomized subjects received treatment with 200 mg progesterone (P4) once daily for 14 consecutive days, after completion of the E4 / placebo treatment.

[0444] The second part was the Safety Study designed to evaluate the general safety, secondary efficacy (lipid and glucose metabolism, health-related quality of life [HRQoL] and treatment satisfaction [TS] ) after treatment with E4 20 mg for up to 53 weeks in hysterectomized and non-hysterectomized postmenopausal participants. For endometrial protection, all non-hysterectomized subjects received treatment with 200 mg progesterone (P4) once daily for 14 consecutive days, after completion of the E4 treatment.

[0445] 3. _ Eligibility Criteria

[0446] Ages Eligible for Study: 40 Years to 65 Years (Adult, Older Adult)

[0447] Sexes Eligible for Study: Female

[0448] Accepts Healthy Volunteers: Yes

[0449] 4. _ Inclusion Criteria:

[0450] 1. Signed and dated written informed consent form and any required privacy authorization prior to the initiation of any trial procedure, after the nature of the trial has been explained according to local regulatory requirements;

[0451] 2. Females > 40 up to < 65 years of age at randomization / treatment allocation;

[0452] 3. For hysterectomized subjects: documented hysterectomy must have occurred at least 6 weeks prior to the start of screening. Hysterectomy can be total or subtotal (i.e., cervix was not removed).

[0453] 4. For non-hysterectomized subjects: uterus with bi-layer endometrial thickness < 4 mm on TVUS

[0454] 5. For non-hysterectomized subjects: endometrial biopsy taken during screening that reveals no abnormal result, i.e., presence of hyperplasia (simple or complex, with or without atypia), presence of carcinoma, and presence of disordered proliferative endometrium findings. The screening biopsy should have sufficient endometrial tissue for diagnosis. Biopsies without tissue or with insufficient tissue may be repeated once;

[0455] 6. Seeking treatment for relief of VMS associated with menopause; a. For the Efficacy Study part: at least 7 moderate to severe bothersome VMS per day or at least 50 moderate to severe bothersome VMS per week in the last 7 consecutive days during the Screening period; b. For the Safety Study part: at least 1 moderate to severe VMS per week;

[0456] 7. Body mass index > 18.0 kg / m2up to < 38.0 kg / m2;

[0457] 8. A mammogram that shows no sign of significant disease performed during screening or within 9 months prior to the start of screening;

[0458] 9. Post-menopausal status defined as any of the following: a. For non-hysterectomized subjects:

[0459] ■At least 12 months of spontaneous amenorrhea with serum follicle stimulating hormone (FSH) >40 mIU / mL (value obtained after washout of estrogen / progestin containing drugs, see exclusion criteria 18 and 20);

[0460] ■ or at least 6 months of spontaneous amenorrhea with serum FSH >40 mIU

[0461] / mb and E2 <20 pg / mL (value obtained after washout of estrogen / progestin containing drugs, see exclusion criteria 18 and 20);

[0462] ■ or at least 6 weeks postsurgical bilateral oophorectomy; b. For hysterectomized subjects:

[0463] ■ serum FSH >40 mIU / mL and E2 <20 pg / mL (values obtained after washout of estrogen / progestin containing drug, see exclusion criteria 18 and 20);

[0464] ■ or at least 6 weeks post-surgical bilateral oophorectomy;

[0465] 10. Good physical and mental health, in the judgement of the Investigator as based on medical history, physical and gynecological examination, and clinical assessments performed prior to Visit 1 ;

[0466] 11. Able to understand and comply with the protocol requirements, instructions, and protocol- stated restrictions;

[0467] 12. Able and willing to complete trial daily diaries and questionnaires. Exclusion Criteria:

[0468] 1. History of malignancy, with the exception of basal cell or squamous cell carcinoma of the skin if diagnosed more than 1 year prior to the Screening visit; . Any clinically significant findings found by the Investigator at the breast examination and / or on mammography suspicious of breast malignancy that would require additional clinical testing to rule out breast cancer (however, simple cysts confirmed by ultrasound are allowed);

[0469] 3. Papanicolaou (PAP) test with atypical squamous cells undetermined significance (ASC-US) or higher (low-grade squamous intraepithelial lesion [LSIL], atypical squamous cells- cannot exclude high-grade squamous intraepithelial lesion [HSIL] [ASC-H], HSIL dysplastic or malignant cells) in sub-totally hysterectomized and non-hysterectomized subjects. Note: ASC-US is allowed if a reflex human papilloma virus (HPV) testing is performed and is negative for high risk oncogene HPV subtypes 16 and 18; . For non-hysterectomized subjects: a. History or presence of uterine cancer, endometrial hyperplasia, or disordered proliferative endometrium; b. Presence of endometrial polyp; c. Undiagnosed vaginal bleeding or undiagnosed abnormal uterine bleeding; d. Endometrial ablation; e. Any uterine / endometrial abnormality that in the judgment of the investigator contraindicates the use of estrogen and / or progestin therapy. This includes presence or history of adenomyosis or significant myoma;

[0470] 5. Systolic blood pressure (BP) higher than 130 mmHg, diastolic BP higher than 80 mmHg during screening;

[0471] 6. History of venous or arterial thromboembolic disease (e.g., superficial or deep vein thrombosis, pulmonary embolism, stroke, myocardial infarction, angina pectoris, etc.), or first-degree family history of venous thromboembolism (VTE);

[0472] 7. History of known acquired of congenital coagulopathy or abnormal coagulation factors, including known thrombophilia's;

[0473] 8. Laboratory values of fasting glucose above 125 mg / dL and / or glycated hemoglobin above 70 / .

[0474] / / o,

[0475] 9. Dyslipoproteinaemia (LDL >190 mg / dL and / or triglycerides >300 mg / dL);

[0476] 10. Subjects smoking >15 cigarettes per day;

[0477] 11. Presence or history of gallbladder disease, unless cholecystectomy has been performed;

[0478] 12. Systemic lupus erythematosus;

[0479] 13. Any malabsorption disorders including gastric bypass surgery;

[0480] 14. History of acute liver disease in the preceding 12 months before the start of screening or presence or history of chronic or severe liver disease [alanine transaminase (ALT) or aspartate transaminase (AST) >2 x upper limit of normal (ULN), bilirubin >1.5 ULN], or liver tumors;

[0481] 15. Chronic or current acute renal impairment (estimated glomerular filtration rate <60 ml / min);

[0482] 16. Porphyria;

[0483] 17. Diagnosis or treatment of major psychiatric disorder (e.g., schizophrenia, bipolar disorder, etc.) in the judgement of the Investigator;

[0484] 18. Use of estrogen / progestin containing drug(s) up to: a. 1 week before screening start for vaginal non-systemic hormonal products (rings, creams, gels); b. 4 weeks before screening start for vaginal or transdermal estrogen or estrogen / progestin products; c. 8 weeks before screening start for oral estrogen and / or progestin products and / or selective estrogen receptor modulator therapy; d. 8 weeks before screening start for intrauterine progestin therapy; e. 3 months before screening start for progestin implants or estrogen alone injectable drug therapy; f. 6 months before screening start for estrogen pellet therapy or progestin injectable drug therapy;

[0485] 19. Use of androgen / dehydroepiandrosterone (DHEA) containing drugs: a. 8 weeks before screening start for oral, topical, vaginal or transdermal androgen; b. 6 months before screening start for implantable or injectable androgen therapy;

[0486] 20. Use of phytoestrogens or black cohosh for treatment of VMS up to 2 weeks before the start of screening;

[0487] 21. For the women participating in the Efficacy Study part: use of prescription or over-the- counter products used for the treatment of VMS, e.g., anti-depressants: paroxetine, escitalopram, methyldopa, opioid and clonidine up to 4 weeks before the start of screening, and venlafaxine and desvenlafaxine up to 3 months before the start of screening , and not willing to stop these during their participation in the trial;

[0488] 22. Not willing to stop any hormonal products as described in exclusion criteria 18, 19 and 20 during their participation in the trial;

[0489] 23. Inadequately treated hyperthyroidism with abnormal TSH and free T4 at screening. Subjects with low or high TSH are allowed if free T4 at screening is within normal range;

[0490] 24. History or presence of allergy / intolerance to the investigational product or drugs of this class or any component of it, or history of drug or other allergy that, in the opinion of the Investigator contraindicates subject participation;

[0491] 25. For non-hysterectomized subjects: history or presence of allergy to peanuts;

[0492] 26. History of alcohol or substance abuse (including marijuana, even if legally allowed) or dependence in the previous 12 months before the start of screening as determined by the Investigator, based on reported observations; 27. Sponsor or contract research organization (CRO) employees or employees under the direct supervision of the Investigator and / or involved directly in the trial;

[0493] 28. Subjects with known or suspected history of a clinically significant systemic disease, unstable medical disorders, life-threatening disease or current malignancies that would pose a risk to the subject in the opinion of the Investigator;

[0494] 29. Participation in another investigational drug clinical trial within 1 month (30 days) or having received an investigational drug within the last month (30 days) before the start of screening;

[0495] 30. Is judged by the Investigator to be unsuitable for any reason.

[0496] 6. _ Blood collection, pre-analytical processing, and AD serum biomarkers assessment:

[0497] Blood withdrawal was performed at baseline and at the end of treatment (duration of treatment ranges from 6 days to 405 days, mean duration= 269 days). All blood samples were handled in a standardized way and centrifuged at 2000 x g during 15 min. After that, the serum was extracted, aliquoted per 1 ml, and frozen at -20°C (long term storage at -80°C). Aliquoting was done using standard procedures. The subsequent neurological serum parameters will all be determined in one run to exclude inter-assay variation.

[0498] Serum t-tau and NfL concentrations will be measured using the Single molecule array (Simoa) Tau2.0 and the NF -Light Advantage assays, respectively (Quanterix, Billerica, MA).

[0499] The p-tau231 concentration will be measured using an in-house Simoa assay on an HD-X Analyzer (Quanterix, Billerica, MA), as previously described.

[0500] BACE1 levels will be measured as described by Vergallo et al. (Vergallo, Andrea et al. “Brain Ap load association and sexual dimorphism of plasma BACE1 concentrations in cognitively normal individuals at risk for AD.” Alzheimer's & dementia : the journal of the Alzheimer's Association vol. 15,10 (2019): 1274-1285. doi: 10. 1016 / j .jalz.2019.07.001). The previously described amyblood assay will be used to measure A i-42 and A i-40 (Thijssen, Elisabeth H et al. “Highly specific and ultrasensitive plasma test detects Abeta( 1-42) and Abeta( 1-40) in Alzheimer's disease.” Scientific reports vol. 11,1 9736. 6 May. 2021, doi: 10.1038 / s41598-021-89004-x).

[0501] 7. _ APOE genotyping

[0502] APOE variants will be genotyped by sequencing in line with previous works of Adamczuk et al. (Adamczuk, Katarzyna et al. “Polymorphism of brain derived neurotrophic factor influences amyloid load in cognitively intact apolipoprotein E s4 carriers.” NeuroImage. Clinical vol. 2 512-20. 11 Apr. 2013, doi: 10.1016 / j.nicl.2013.04.001).

[0503] 8. Statistical analysis The statistical analyses will be conducted on all participants who have no missing data on sample biomarkers collected at the two timepoints, education, MMSE, and APOE genotype as for example previously described by Depypere et al. (Depypere, Herman et al. “Menopause hormone therapy significantly alters pathophysiological biomarkers of Alzheimer's disease.” Alzheimer's & dementia: the journal of the Alzheimer's Association vol. 19,4 (2023): 1320-1330. doi: 10.1002 / alz.12759).. The Gaussian distribution and homoskedasticity will be checked visually through histograms and density plots with normal probability density function curve overlaid as well as Q-Q plots.

[0504] To perform group-wise comparisons for categorical and continuous variable, Pearson’s chi-squared test and Student’s t-test will be used, respectively.

[0505] Outliers will be visually inspected for each single biomarker and not for the composite ratio values.

[0506] We will use linear models (LM) to assess the potential difference of sample biomarkers concentrations at VI (baseline) between the study groups (treatment vs. non-treatment with both doses).

[0507] Age and APOE s4 carrier status (presence vs. absence) will be included as covariates to rule out potential confounding effect and to better weight in on treatment effect size in case of significant results.

[0508] For longitudinal analysis, the annual rate of change (ARC) will be calculated as the difference in sample concentration between the two visits (VI and V2) divided by the delay in years.

[0509] We will use LM to test whether the two study groups may have a different ARC. Age and APOE s4 status will be set as covariates.

[0510] To follow, an additional subgroup analysis will be performed to examine whether APOE s4 status impacts the treatment effect on sample biomarkers. We will use a LM with an interaction term between the group (MHT arm or control arm) and APOE s4 status (APOE s4 status* group).

[0511] Eventually, post-hoc analysis will be performed to breakdown significant results and capture the difference between each pair of subgroups created by the interaction term.

[0512] For all models, regression coefficients ( ) and standard error (SE) will be reported. In addition, effect sizes will be estimated using partial Cohen’s f2, which represent the amount of variance of the response variables (outcome) that is explained by an explanatory variable (predictor) after accounting for other predictors in the regression model

[0513] All tests were two-sided and p-values, p < .05 will be considered significant in all statistical elaboration. Influent data points on model outputs were inspected through Cook’s distance. When the distance is equal or higher than 1, models will be refitted with a robust regression to consider the presence of influent data point (Depypere, Herman et al. “Menopause hormone therapy significantly alters pathophysiological biomarkers of Alzheimer's disease.” Alzheimer's & dementia: the journal of the Alzheimer's Association vol. 19,4 (2023): 1320-1330. doi: 10.1002 / alz. 12759). Since the sample biomarker investigated track partially independent molecular pathways, separate LM for each biomarker will be run and no correction of p will be applied.

[0514] Statistical analyses will be performed using R software, version 4.0.5.

[0515] 9. Predicted outcome

[0516] The effect of treatment will differ according to APOE s4 allele status with the pairwise subgroups comparison indicating that controls with APOE s4 show greater reduction in Api-42 / p-tau231 ratio levels than both E4 group individuals APOE s4 allele carriers and noncarriers. Although Api-42 / p- tau231 ratio will not differ between the E4 subgroups split by APOE s4 status, the E4 APOE 84-positive individuals will exhibit a significantly greater reduction of Ap 1-42 levels than the E4 APOE 84-negative individuals.

[0517] Moreover, APOE s4 carrier control individuals will have significantly different levels ofApi-42 / p- tau231 ratio than non-carriers. E4 group individuals carrying at least one APOE s4 allele will have smaller reduction of Api-42 than APOE s4-negative treated women. Of note, E4 APOE 84-positive women will have at least a mild within-group increment of sample Api-42 over time, while E4 APOE 84-negative women will show a longitudinal reduction of the same biomarker, that might be significant. The E4 subgroups will show opposite directions, albeit with at least slight changes, in p-tau231 levels with either an increase or a decrease in APOE 84-positive and s4-negative women, respectively. Notably, control APOE 84-positive individuals will display an average higher increment rate of p- tau231 levels than each E4 subgroup (and preferably the control APOE s4-negative individuals as well). Hence, it is conceivable to infer that the effect of MHT in APOE s4-positive women is on the Ap pathway.

[0518] In summary, a prominent response to E4 at both doses on all AD biomarkers measured in this study such as t-tau, NfL, p-tau231 BACE1 and, in particular, the critical Ap pathway biomarker level will be demonstrated. The effect will be present at both doses and may be more pronounced with the higher dose. Women at genetic risk for AD (carrying at least oneAPOEe4 allele) will be shown to particularly benefit from menopausal hormone replacement therapy with E4. The effect of E4 will be similar to the effect of estradiol or even exceeds the effect of estradiol.

[0519] Example 11: A Randomized, Double-Blind, Placebo-Controlled, Phase Ila Study with Planned Interim Analysis to Evaluate the Effects of 20 mg Estetrol (E4) monohydrate in Early Postmenopausal Women with Vasomotor Symptoms, Memory Decline, and preclinical Alzheimer’s Disease Biomarkers

[0520] In early postmenopausal women, vasomotor symptoms (VMS) such as hot flushes are considered a debilitating condition that can lead to significant additional health concerns. For some women, it is indeed associated with altered sleeping quality and efficiency and, secondarily, to cognitive decline and to brain markers associated with the development of Alzheimer’s disease (AD). This study is a 12-week proof-of-concept (PoC) phase Ila study aimed at obtaining preliminary evidence of efficacy of E4 in preventing or decreasing sleeping problems, related verbal memory decline, and brain functional connectivity problems, as well as abnormal levels of related preclinical AD biomarkers. After testing 12 subjects, enrolment will be paused; a planned interim analysis will be performed to decide whether to terminate enrolment / study, continue dosing, or changing to a higher or lower dose.

[0521] 2. _ Study Rationale

[0522] AD is the only neurodegenerative disease (NDD) that affects women more than men. The hormonal shifts that occur with menopause is increasingly recognized to promote the risks of brain changes associated with AD. Brain changes generally associated with AD onset or early preclinical development of AD have been found indeed in ‘normal’ (asymptomatic) perimenopausal or menopausal women - so-called preclinical AD biomarkers because the latter may develop during a 20-year prodromal phase originating in midlife, thus proximate with the hormonal transitions of endocrine aging characteristic of the menopause transition in women (Jett et al., 2022). AD preclinical stage is specifically linked with verbal memory decline - its earliest sign (Howieson et al., 1997; Qin et al., 2022).

[0523] Evidence suggests that estrogen exposure or estrogen therapies (ETs) may reduce cognition and memory problems, microbiome gut-brain axis imbalance, as well as preclinical AD biomarkers and / or corresponding surrogate endpoints (‘preclinical AD is the earliest stage with measurable biomarkers before clinical diagnosis, onset, and clinical symptoms) (Dubois et al., 2016; Schelbaum et al., 2021; Mosconi et al., 2018; Duan et al., 2021; Castillo et al., 2020; Zeydan et al., 2021). ETs have recently been reported to positively regulate the gut microbiome composition, cognitive function, and general health of menopausal women (Vieira et al., 2017). Suigical menopause-induced memory impairment was reversed by ETs (Maki et al., 2008; Phillips and Sherwin, 1992). ETs were generally associated with reduced incidence of AD onset and AD-related deaths (Manson et al., 2017; Song et al., 2020). Estrogens were reported to enhance blood flow in impaired memory-related cortical areas, dendritic spine density, synaptic plasticity, hippocampal neurogenesis, oxidative protection, and antiinflammatory actions (Boyle et al., 2021; Hara et al., 2015; Morrison et al., 2006).

[0524] Estetrol, also known as E4, is a natural estrogen produced by the fetal liver. It differs in various ways both from other natural estrogens like estradiol and synthetic estrogens like ethinylestradiol, with implications for tolerability and safety. For instance, it appears to have minimal estrogenic effects in the breasts and liver (Gerard et al., 2015; Mawet et al., 2015). Oral E4 reduces VMS in postmenopausal women (currently studied with two phase III studies in North America (MIT-Do001-C302, NCT04090957) and Europe (MIT-Do001-C301, NCT04209543). Although some ETs (e.g., estradiol, CEE) are associated with promising clinically-relevant effects on cognitive impairment in AD patients (Birge 1997; Asthana et al., 1999), we do not have any clinical data on whether E4 has similar attributes (e.g., early signs of memory declines such as SMC or mild cognitive impairment (MCI), and brain markers of AD (Najar et al., 2021; Fagan et al., 2006, Asthon et al., 2021; Mosconi et al., 2021). Serum AD markers have been recently recognized by the Geneva AD Biomarker Roadmap Initiative (2019) as valid biomarkers (as for cerebrospinal fluid (CSF) markers) in the early-stage preclinical development of AD (Asthon et al., 2021, Simren et al., 2021; Nakamura et al., 2018). Accordingly, we intend to explore corresponding biomarker level changes induced by E4 in Apolipoprotein E4 (APOE4) carrier and non-APOE4 carrier postmenopausal women - APOE4 protein is generally expressed in people developing AD and, as such, is considered the most important genetic risk factor of the disease (https: / / www.nia.nih.gov / news / study-reveals-how-apoe4-gene-may-increase- risk-dementia). Since E4 has been associated with a better safety profde than the other ETs, it is postulated that E4 could become a particularly well-suited hormonal therapy for postmenopausal women seeking additional protection and / or health benefits against cognitive decline or memory complaints and the early signs of AD development or their surrogate endpoints (i.e., corresponding biomarkers such as phosphorylated tau (P-tau), amyloid betal-40 (Ap40), and amyloid betal-42 (AP42).

[0525] 3. _ Study Design

[0526] Two arms, monocentric, single dose, randomized, double-blind, placebo-controlled, parallel design, interventional proof-of-concept (PoC) phase Ila study with planned interim analysis.

[0527] Once testing of the first 12 subjects is completed, enrolment will be paused for a planned interim analysis. The latter will be performed to decide whether, 1) 20 mg dosage of E4 should be maintained; 2) dosage should be changed to a higher or lower dose. Only data from the first and main primary endpoint will be analysed by an independent data analyst - i.e., change from baseline to week 12 in episodic verbal memory decline values assessed with a list-learning verbal memory task and functional magnetic resonance imaging (fMRI) in regions of interest.

[0528] 4. _ Study Objectives

[0529] • Primary objective: To assess the effects of 12-week treatment on verbal memory decline (SMC or MCI) assessed with a list-learning verbal memory task and fMRI and at least one the underlying physiological changes - i.e., brain activity or sleep efficiency.

[0530] • Secondary objectives: 1) To assess the effects of 6- and 12-week treatment on treatment emergent adverse events (TEAEs) and serious adverse events (SAEs); 2) To assess the effects of 6- or 12- week treatment on verbal memory decline (Subjective memory complaints - SMC or mild cognitive impairment - MCI) assessed with several complimentary method and fMRI and at least one the following physiological changes - i.e., brain activity, sleep quality, sleep efficiency, hot flush frequency assessed with complementary methods (e.g., Pittsburgh Sleep Quality Index, ambulatory skin conductance monitoring device).

[0531] • Exploratory objectives: To assess the effects of 6- and 12-week treatment on at least one corresponding pre-pathophysiological change (e.g., reduction of amyloid accumulation or change in P-tau, total tau (T-tau), or A 4O levels) generally associated with SMC or MCI, microbiome imbalance, or early preclinical signs of AD development or onset in early postmenopausal women.

[0532] Primary endpoints:

[0533] • Change from baseline to week 12 in episodic verbal memory decline values assessed with a listlearning verbal memory task and fMRI in regions of interest (ROIs) [Time Frame: Baseline up to Week 12],

[0534] • Change from baseline to week 12 in sleep efficiency levels (i.e., ratio between the time a person spends asleep, and the total time dedicated to sleep reported as a percentage) using actigraphy [Time Frame: Baseline up to Week 12],

[0535] • Change from baseline to week 12 in fMRI levels (magnitude of activation) of network functional connectivity (prefrontal and hippocampal) [Time Frame: Baseline up to Week 12],

[0536] Secondary endpoints:

[0537] • Change from baseline to week 6 in episodic verbal memory decline values assessed with a listlearning verbal memory task and fMRI in ROIs [Time Frame: Baseline up to Week 6],

[0538] • Change at week 12 in episodic verbal memory decline values assessed with a list-learning verbal memory task and fMRI in ROIs compared to placebo [Time Frame: Week 12],

[0539] • Change from baseline to week 6 in sleep efficiency levels using actigraphy [Time Frame: Baseline up to Week 6] .

[0540] • Change at week 12 in sleep efficiency levels using actigraphy compared to placebo [Time Frame: Week 12],

[0541] • Change from baseline to week 6 in fMRI levels (magnitude of activation) of network functional connectivity (prefrontal and hippocampal) [Time Frame: Baseline up to Week 6],

[0542] • Change at week 12 in fMRI levels (magnitude of activation) of network functional connectivity (prefrontal and hippocampal) compared to placebo [Time Frame: Week 12],

[0543] • Change from baseline to week 6 in verbal memory decline values assessed with the Wechsler Memory Scale-Revised (WMS-R) and fMRI (activity level in frontal cortical areas) [Time Frame: Baseline up to Week 6], • Change from baseline to week 12 in verbal memory decline values assessed with the WMS-R and fMRI (activity level in frontal cortical areas) [Time Frame: Baseline up to Week 12],

[0544] • Change at week 12 in verbal memory decline values assessed with the WMS-R and fMRI (activity level in frontal cortical areas) compared to placebo [Time Frame: Week 12],

[0545] • Change from baseline to week 6 in sleep quality levels (i.e., self-satisfaction with all aspects of the sleep experience) levels using the modified Pittsburgh Sleep Quality Index (mPSQI) [Time Frame: Baseline up to Week 6],

[0546] • Change from baseline to week 12 in sleep quality levels using mPSQI [Time Frame: Baseline up to Week 12],

[0547] • Change at week 12 in sleep quality levels using the mPSQI compared to placebo [Time Frame: Baseline up to Week 12],

[0548] • Change from baseline to week 6 in frequency of ambulatory physiological VMS (hot flushes) monitored values assessed with ambulatory skin conductance (Biolog Model 3991 x / 2-HFI, UFI, Morro Bay, CA) [Time Frame: 6 weeks].

[0549] • Change from baseline to week 12 in frequency of ambulatory physiological VMS (hot flushes) monitored values assessed with ambulatory skin conductance [Time Frame: 12 weeks].

[0550] • Change at week 12 in frequency of ambulatory physiological VMS (hot flushes) monitored values assessed with ambulatory skin conductance compared to placebo [Time Frame: 12 weeks].

[0551] • Change from baseline to week 6 in frequency of TEAs and SAEs (i.e., physical, gynecological, breast, vital sign, ECG exam results).

[0552] • Change from baseline to week 12 in frequency of TEAEs and SAEs.

[0553] • Change from baseline to week 12 in plasma levels of neurofilament light (NfL), P-Tau (181, 217), T-Tau, A 42, A[340, A|342 / 40 ratio. Chitinase 3 -like 1 (YKL-40), [3-secretase (BACE 1), and neurogranin [Time Frame: Baseline up to Week 12],

[0554] • Change at week 12 in levels of at least one of the preclinical AD biomarkers (NfL, P-Tau (181, 217), T-Tau, A|342, A|340, A[342 / 40 ratio, YKL-40, BACE 1, or neurogranin) in APOE4 carriers compared to non-APOE4 carriers [Time Frame: Week 12],

[0555] • Change from baseline to week 6 in plasma concentration of Indoxyl Sulphate (IS) measured with High Performance Liquid Chromatography (HPLC) [Time Frame: Baseline to Week 6],

[0556] • Change from baseline to week 12 in plasma concentration of IS measured with HPLC [Time Frame: Baseline to Week 12],

[0557] • Change from baseline to week 6 in plasma concentration of P-Cresyl Sulphate (PCS) measured with HPLC [Time Frame: Baseline to Week 6], • Change from baseline to week 12 in plasma concentration of PCS measured with HPLC [Time Frame: Baseline to Week 12],

[0558] • Change from baseline to week 6 in plasma concentration of glial fibrillary acidic protein (GFAP) measured with enzyme-linked immunoassay (Elisa kit) [Time Frame: Baseline to Week 12],

[0559] • Change from baseline to week 12 in plasma concentration of GFAP [Time Frame: Baseline to Week 12].

[0560] 6. _ Safety assessment:

[0561] • Medical and gynecologic history

[0562] • Physical examination, including breast and gynecologic examinations

[0563] • Vital signs

[0564] • ECG

[0565] • Papanicolaou test (PAP test)

[0566] • Endometrial assessment using transvaginal ultra-sound sonography (TVUS), when indicated, endometrial biopsy

[0567] • Mammography

[0568] • Laboratory examinations:

[0569] Fasted blood sampling for o Hematology / chemistry o Lipid / glucose parameters for inclusion

[0570] Blood sampling for: o thyroid-stimulating hormone (FSH), and follicle-stimulating ormcmQ (TSH) for inclusion

[0571] • Urine pregnancy test

[0572] • APOE4 allele genotyping test

[0573] • Prior and concomitant medications

[0574] • TEAEs, SAEs, and adverse events (AEs) will be assessed during the entire trial

[0575] Endometrial Safety Assessment and Stopping Rule (non-hysterectomized subjects only):

[0576] All non-hysterectomized (NH) subjects will have TVUS during the screening period to assess bi-layer endometrial thickness. Only subjects with endometrial thickness < 4 mm will be eligible for the trial. TVUS will be repeated at 6 weeks, end of treatment (EoT), and any time after 6 weeks if a subject presents persistent and / or recurrent bleeding. For NH subjects who report some endometrial-related bleeding towards the end of the 12-week study, 200 mg progesterone (P4) for two weeks after completion the E4 / placebo treatment will be offered. If TVUS demonstrates an endometrial thickness >10 mm, an endometrial biopsy will be performed. If the biopsy shows hyperplasia, the study drug will be discontinued, the subject will be withdrawn from treatment (if not at EoT), and the subjects will be treated with progesterone (P4) 200 mg once daily for 14 days. At approximately 10-14 days after completion of the progestin treatment, TVUS assessment of endometrial thickness and a biopsy will be performed. If the biopsy does not demonstrate resolution, the subject will be further treated according to local practice / guidelines and followed until resolution.

[0577] 7. _ Subjects Population

[0578] • Description: Early postmenopausal women (i.e., more than 12 consecutive months without menses but no more than 8 years after onset of menopause), aged between 40 and 65 years (inclusive), hysterectomized or non-hysterectomized, experiencing early signs of memory decline (SMC or MCI), VMS (i.e., more than 5 episodes per day), sleep quality problems, and detectable levels of relevant early surrogate endpoints such as preclinical AD biomarkers (at least one of them, e.g., NfL, P-tau 181, P-tau 217, T-tau, A042, A04O, A042 / 4O ratio, YKL-40, BACE 1, and neurogranin).

[0579] • Number of subjects to be enrolled: 40, i.e., 20 subjects per arm (arm 1: drug treatment; arm 2: placebo treatment).

[0580] • Number of subjects expected to complete the trial: 34 or more (15% of dropout rate).

[0581] 8. _ Study Products

[0582] • Investigational product: 20 mg E4 monohydrate oral tablet once daily

[0583] • Comparator product: Placebo oral tablet once daily

[0584] • Other product (non-hysterectomized subjects only): P4200 mg orally once daily for 14 consecutive days (after completion of E4 treatment). Subjects randomized to placebo will receive P4.

[0585] 9. _ Duration of Treatment

[0586] • Individual subject: Twelve (12) weeks

[0587] • Entire study: Forty-two (42) months (3.5 years) including recruitment, preparation, tests per se with 40 subjects, data analysis, and clinical study report writing. An interim analysis is planned as soon as 12 subjects have completed testing (i.e., approximately 10 or 12 months after study onset) in order to tentatively get preliminary evidence of efficacy (verbal memory decline and corresponding fMRI changes) compared to baseline with 20 mg E4.

[0588] 10. Inclusion Criteria

[0589] Subjects will be included in the study if they meet all of the following inclusion criteria:

[0590] 1. Female, age 40-65 years, inclusive, at randomization / treatment allocation;

[0591] 2. Early postmenopausal status based on the Stages of Reproductive Aging Workshop (STRAW +10, Stage la, lb, 1c) criteria (i.e., absence of menses for at least 12 consecutive months and no later than 8 years after onset); a. For non-hysterectomized subjects'.

[0592] - at least 12 months of spontaneous amenorrhea with serum FSH >40 mIU / mL and E2 <20 pg / mL (value obtained after washout of estrogen / progestin containing drugs).

[0593] - or at least 6 months postsurgical bilateral oophorectomy. b. For hysterectomized subjects'.

[0594] - serum FSH >40 mIU / mL and E2 <20 pg / mL (values obtained after washout of estrogen / progestin containing drug).

[0595] - postsurgical bilateral oophorectomy.

[0596] 3. For subjects who have undergone hysterectomy and / or bilateral oophorectomy, documented surgery must have occurred at least 6 months prior to the start of screening. Hysterectomy can be total or subtotal (i.e., cervix was not removed);

[0597] 4. For non-hysterectomized subjects', uterus with bi-layer endometrial thickness <4 mm on transvaginal ultrasound;

[0598] 5. Body mass index (BMI) at screening between 18.0 and 39.0 kg / m2, inclusive;

[0599] 6. A mammogram that shows no sign of significant disease performed during screening or within 9 months prior to the start of screening;

[0600] 7. Good physical and mental health, in the judgement of the investigator as based on medical history, physical and gynecological examination, and clinical assessments performed prior to Visit 1;

[0601] 8. Signed and dated written informed consent form and any required privacy authorization prior to the initiation of any trial procedure, after the nature of the trial has been explained according to local regulatory requirements;

[0602] 9. Able to understand and comply with the protocol requirements, instructions, and protocol-stated restrictions;

[0603] 10. Able and willing to complete trial diaries and questionnaires;

[0604] 11. At least 5 self-reported hot flushes per day on average during at least the last 4 weeks;

[0605] 12. Episodic verbal memory problems specifically associated SMC and / or MCI;

[0606] 13. Sleeping quality problems excluding insomnia and apnea;

[0607] Detectable plasma levels of preclinical AD biomarkers (at least one among the following: neurofilament light (NfL), P-Tau 181, P-Tau 217, T-tau, A042, A04O, A042 / 4O ratio, YKL-40, BACE 1, and / or neurogranin);

[0608] 11. Exclusion Criteria

[0609] Subjects will not be included in the study if they meet one of the following exclusion criteria:

[0610] 1. A confirmed diagnosis of AD;

[0611] 2. More than 8 years with menopause; 3. Any clinically significant findings found by the Investigator at the breast examination and / or on mammography suspicious of breast malignancy that would require additional clinical testing to rule out breast cancer (however, simple cysts confirmed by ultrasound are allowed);

[0612] 4. PAP test with atypical squamous cells undetermined significance (ASC-US) or higher (low grade squamous intraepithelial lesion [LSIL], atypical squamous cells cannot exclude high grade squamous intraepithelial lesion [HSIL] [ASC-H], HSIL dysplastic or malignant cells) in sub-totally hysterectomized and non-hysterectomized subjects. Note: ASC-US is allowed if a reflex human papilloma virus (HPV) testing is performed and is negative for high risk oncogene HPV subtype 16 and 18;

[0613] 5. For non-hysterectomized subjects: a. History or presence of uterine cancer, endometrial hyperplasia; b. Presence of endometrial polyp(s); c. Undiagnosed vaginal bleeding or undiagnosed abnormal uterine bleeding; d. Endometrial ablation; e. Any uterine / endometrial abnormality that in the judgment of the investigator contraindicates the use of estrogen; and / or progestin therapy. This includes presence or history of adenomyosis or significant myoma;

[0614] 6. Systolic blood pressure (BP) higher than 130 mmHg, diastolic BP higher than 80 mmHg during screening;

[0615] 7. Any clinically significant abnormalities identified on the screening 12-lead ECG;

[0616] 8. History of venous or arterial thromboembolic disease (e.g., superficial or deep vein thrombosis, pulmonary embolism, stroke, myocardial infarction, angina pectoris, etc.), or first-degree family history of venous thromboembolism (VTE);

[0617] 9. History of known acquired of congenital coagulopathy or abnormal coagulation factors, including known thrombophilia’s;

[0618] 10. Laboratory values of fasting glucose above 125 mg / dL and / or glycated hemoglobin above 7.5%;

[0619] 11. Are current smokers;

[0620] 12. Presence or history of gallbladder disease, unless cholecystectomy has been performed;

[0621] 13. Systemic lupus erythematosus;

[0622] 14. Any malabsorption disorders including gastric bypass surgery; 15. History of acute liver disease in the preceding 12 months before the start of screening or presence or history of chronic or severe liver disease [alanine transaminase (ALT) or aspartate transaminase (AST) >2x upper limit of normal (ULN), bilirubin >1.5 ULN], or liver tumors;

[0623] 16. Chronic or current acute renal impairment (estimated glomerular filtration rate <60 ml / min);

[0624] 17. Inadequately treated hyperthyroidism with abnormal TSH and free T4 at screening. Subjects with low or high TSH are allowed if free T4 at screening is within normal range;

[0625] 18. Porphyria;

[0626] 19. Diagnosis or treatment of major psychiatric disorder (e.g., schizophrenia, bipolar disorder, epilepsia and gabapentin users, major depression disorder, general anxiety disorder, mania, substance use disorder, alcohol use disorder, etc.) in the judgement of the Investigator;

[0627] 20. Contraindications to MRI for safety reasons (e.g., MRI-incompatible implant or claustrophobia);

[0628] 21. Use of estrogen / progestin containing drug(s) up to: a. 8 weeks before screening start for vaginal non-systemic hormonal products (rings, creams, gels), vaginal or transdermal estrogen or estrogen / progestin products, oral estrogen and / or selective estrogen receptor modulator therapy, or intrauterine progestin therapy. b. 3 months before screening start for progestin implants or estrogen alone injectable drug therapy; c. 6 months before screening start for estrogen pellet therapy or progestin injectable drug therapy;

[0629] 22. Use of estrogen agonist / antagonist, e.g., ospemifene, within 8 weeks of screening;

[0630] 23. Use of androgen / dehydroepiandrosterone (DHEA)-containing drugs: a. 8 weeks before screening start for oral, topical, vaginal or transdermal androgen; b. 6 months before screening start for implantable or injectable androgen therapy;

[0631] 24. Use of phytoestrogens or black cohosh for treatment of VMS up to 2 weeks before the start of screening;

[0632] 25. Not willing to stop any hormonal products (as listed above - i.e., estrogen therapy, androgen, DHEA, phytoestrogen) during their participation in the trial;

[0633] 26. History or presence of allergy / intolerance to the investigational product or drugs of this class or any component of it, or history of drug or other allergy that, in the opinion of the Investigator contraindicates subject participation; 27. For non-hysterectomized subjects', history or presence of allergy to progestagens or peanuts;

[0634] 28. History of alcohol or substance abuse (including marijuana, even if legally allowed) or dependence in the previous 12 months before the start of screening as determined by the Investigator, based on reported observations (drug screens before recruitment confirmation will be performed);

[0635] 29. Sponsor or CRO employees or employees under the direct supervision of the Investigator and / or involved directly in the trial;

[0636] 30. Subjects with known or suspected history of a clinically significant systemic disease, unstable medical disorders, life-threatening disease or current malignancies that would pose a risk to the subject in the opinion of the Investigator;

[0637] 31. Participation in another investigational drug clinical trial within 1 month (30 days) or having received an investigational drug within the last month (30 days) before the start of screening;

[0638] 32. History of head injury association with loss of consciousness for more than 15 minutes.

[0639] Is judged by the Investigator to be unsuitable for any other reason

[0640] 12. Visit Schedule

[0641] At screening, baseline, 6 weeks, 12 weeks, and follow-up (2 weeks after EoT)

[0642] 13. Statistical Methods:

[0643] Analyses will be performed on an intention-to-treat population. Categorical factors will be summarized using frequencies and / or percentages, while continuous measures will be described using means and standard deviations. Comparisons of the changes between groups at 12 weeks will be performed using linear models with change as the outcome. Results will be presented as the mean and 95 % confidence interval. Analyses will be performed using SAS software (Stat version 15.2; SAS Institute, Cary, NC, USA) with a significance level of 0.05. In this exploratory POC trial, no adjustment for multiplicity of testing will be performed.

[0644] 14. Predicted outcome

[0645] The treatment is expected to result in a positive effect on at least one, several or all parameters. The safety profile is expected to be at least reasonable or even excellent.

[0646] REFERENCES (alphabetical order)

[0647] Asthana et al., Cognitive and neuroendocrine response to transdermal estrogen in postmenopausal women with Alzheimer’s disease: results of a placebo-controlled, double-blind, pilot study. Psychoneuroendocrinology 24, 1999. Asthon et al., The validation status of blood biomarkers of amyloid and phospho-tau assessed with the 5-phase development framework for AD biomarkers. Eur J Nucl Med Mol Imaging 48, 2021.

[0648] Asthon et al., Effects of pre-analytical procedures on blood biomarkers for Alzheimer's pathophysiology, glial activation, and neurodegeneration. Alzheimers Dement 13, 2021.

[0649] Birge, The role of estrogen in the treatment of Alzheimer’s disease. Neurology 48, 1997.

[0650] Boyle et al., Estrogen, brain structure, and cognition in postmenopausal women. Human Brain Mapp

[0651] 42.2021.

[0652] Castillo et al., Genistein and Galantamine Combinations Decrease P-Amyloid Peptide (^-Induced Genotoxicity and Cell Death in SH-SY5Y Cell Line: an In Vitro and In Silico Approach for Mimic of Alzheimer's Disease. Neurotox Res 38, 2020.

[0653] Duan et al., Study on the neuroprotective effects of Genistein on Alzheimer's disease. Brain Behav

[0654] 11. 2021.

[0655] Dubois et al., Preclinical Alzheimer's disease: Definition, natural history, and diagnostic criteria.

[0656] Alzheimer Dement 12, 2016.

[0657] Fagan et al., Inverse relation between in vivo amyloid imaging load and cerebrospinal fluid Abeta42 in humans.Annals Neurol 59, 2006.

[0658] Gerard et al., Estetrol is a weak estrogen antagonizing estradiol-dependent mammary gland proliferation. J Endocrinol 224, 2015.

[0659] Hara et al., Estrogen Effects on Cognitive and Synaptic Health Over the Lifecourse. Physiol Rev 95, 2015.

[0660] Howieson et al., Cognitive markers preceding Alzheimer's dementia in the healthy oldest old. J Am Geriatr Soc 45, 1997.

[0661] Jett et al, Endogenous and Exogenous Estrogen Exposures: How Women's Reproductive Health Can Drive Brain Aging and Inform Alzheimer's Prevention. Front Aging Neurosci 14, 2022

[0662] Maki et al., The timing of estrogen therapy after ovariectomy— implications for neurocognitive function. Nat Clin Pract Endocrinol Metab 4, 2008.

[0663] Manson et al., Menopausal Hormone Therapy and Long-term All-Cause and Cause-Specific Mortality: The Women's Health Initiative Randomized Trials. JAMA 18, 2017.

[0664] Mawet et al. , Unique effects on hepatic function, lipid metabolism, bone and growth endocrine parameters of estetrol in combined oral contraceptives. Eur J Contracept Reprod Health Care 20, 2015.

[0665] Morrison et al., Estrogen, menopause, and the aging brain: how basic neuroscience can inform hormone therapy in women. J Neurosci 26, 2006

[0666] Mosconi et al., Increased Alzheimer's risk during the menopause transition: A 3 -year longitudinal brain imaging study, PLoS One 13, 2018.

[0667] Mosconi et al., Menopause impacts human brain structure, connectivity-, energy metabolism, and amyloid-beta deposition. Sci Rep 11, 2021.

[0668] Najar et al., Reproductive period and preclinical cerebrospinal fluid markers for Alzheimer disease: a 25-year study. Menopause 28, 2021.

[0669] Nakamura et al., High performance plasma amyloid-P biomarkers for Alzheimer's disease. Nature 554, 2018.

[0670] Phillips and Sherwin, Effects of estrogen on memory-’ function in surgically menopausal women.

[0671] Psychoneuroendocrinol 17, 1992.

[0672] Qin et al., Phosphory lated Tau 181 Serum Levels Predict Alzheimer's Disease in the Preclinical Stage. Front Aging Neurosci 14, 2022.

[0673] Schelbaum et al., Association of Reproductive History with Brain MRI Biomarkers of Dementia Risk in Midlife. Neurology 97, 2021.

[0674] Simren et al., The diagnostic and prognostic capabilities of plasma biomarkers in Alzheimer's disease.

[0675] Alzheimers Dement 17, 2021.

[0676] Song et al., Reproductive and hormonal factors and risk of cognitive impairment among Singapore Chinese women. Am J Obstet Gynecol 223, 2020.

[0677] Vieira et al, Influence of Oral and Gut Microbiota in the Health of Menopausal Women. Front Microbiol 8, 2017.

[0678] Zeydan et al., Sleep quality and cortical amyloid-P deposition in postmenopausal women of the Kronos early estrogen prevention study. Neuroreport 32, 2021.

Claims

CLAIMS1. A composition for use in the prevention and / or treatment of Alzheimer’s disease symptoms in a subject, wherein said composition comprises from about 15 mg to about 25 mg of an estetrol component.

2. The composition for use according to claim 1, wherein the composition is administered once daily.

3. The composition for use according to claim 1 or 2, wherein the composition comprises about 15 mg of an estetrol component, preferably wherein the composition comprises about 15 mg of estetrol or estetrol monohydrate.

4. The composition for use according to claim 1 or 2, wherein the composition comprises about 20 mg of estetrol component, preferably wherein the composition comprises about 20 mg of estetrol or estetrol monohydrate.

5. The composition for use according to any one of claims 1 to 4, wherein the subject has a genetic predisposition to develop Alzheimer’s disease.

6. The composition for use according to any one of claims 1 to 5, wherein the subject is a carrier of the APOEs4 allele, has a mutation in the APP gene, a mutation in the PSEN 1 gene, a mutation in the PSEN2 gene, or any combination thereof.

7. The composition for use according to any one of claims 1 to 6, wherein the subject is a carrier of the APOEs4 allele.

8. The composition for use according to any one of claims 1 to 7, wherein the Alzheimer’s disease is menopause-associated Alzheimer’s disease or early-stage Alzheimer's disease, preferably wherein the early Alzheimer’s disease is pre-clinical stage Alzheimer’s disease or earliest clinical stage of Alzheimer's disease.

9. The composition for use according to any one of claims 1 to 8, wherein the subject is a menopausal subject.

10. The composition for use according to any one of claims 1 to 9, wherein the Alzheimer’s disease symptoms are selected from the group consisting of: impaired memory, verbal memory decline, subjective memory complaints (SMC), mild cognitive impairment (MCI), depression, anxiety, anger, irritability, insomnia, paranoia, or any combination thereof.

11. The composition for use according to any one of claims 1 to 10, wherein the composition is formulated as an oral, sublingual, buccal, or sublabial dosage unit, preferably wherein the compositionis formulated as an oral dosage unit, more preferably an oral dosage unit that is specifically formulated for oral ingestion.

12. The composition for use according to any one of claims 1 to 11, wherein the composition has a neuroprotective effect against amyloid beta toxicity.

13. The composition for use according to any one of claims 1 to 12, wherein the composition is formulated to correspond to an oral dosage unit.

14. The composition for use according to any one of claims I to 13, wherein the composition further comprises a progestogenic component.

15. The composition for use according to claim 14, wherein the progestogenic component is drospirenone, preferably of from about 0.25 mg to about 10 mg of drospirenone, more preferably of from about 1 mg to about 4 mg.

Images